Sample records for hamster oocyte membrane

  1. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes (United States)

    Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronucl...

  2. Changes of spontaneous parthenogenetic activation and development potential of golden hamster oocytes during the aging process. (United States)

    Jiang, Han; Wang, Ce; Guan, Jiyu; Wang, Lingyan; Li, Ziyi


    The golden hamster is an excellent animal experimental model for oocyte research. The hamster oocytes are very useful in clinical examination of human spermatozoan activity. Non-fertile oocytes can lead to time-dependent processes of aging, which will affect the results of human spermatozoa examination. As a consequence there is a need to investigate the aging and anti-aging processes of golden hamster oocytes. In order to study the aging processes and parthenogenetic activation of golden hamster oocytes, in vivo oocytes, oocytes cultured with or without cumulus cells, and oocytes treated with Trichostatin A (TSA) or caffeine were collected and investigated. We found that: (1) spontaneous parthenogenetic activation, developmental potential (cleavage rate), and zona pellucida (ZP) hardening undergo age-dependent changes in in vivo, in vitro, and after TSA or caffeine treatment; (2) in vivo, oocytes became spontaneously parthenogenetic 25 h post-hCG treatment; (3) in vitro, cumulus cells did not significantly increase the parthenogenetic activation rate of cultured hamster oocytes; and (4) TSA or caffeine could delay spontaneous oocyte parthenogenetic activation and the aging processes by at least 5h, but also accelerated the hardening of the ZP. These results define the conditions for the aging and anti-aging processes in golden hamster oocytes. TSA and caffeine play roles in controlling spontaneous activation, which could facilitate the storage and use of golden hamster oocytes for studying processes relevant to human reproduction. Copyright © 2014 Elsevier GmbH. All rights reserved.

  3. Age-associated metabolic and morphologic changes in mitochondria of individual mouse and hamster oocytes.

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    Fatma Simsek-Duran

    Full Text Available BACKGROUND: In human oocytes, as in other mammalian ova, there is a significant variation in the pregnancy potential, with approximately 20% of oocyte-sperm meetings resulting in pregnancies. This frequency of successful fertilization decreases as the oocytes age. This low proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. In this study, we have examined mitochondrial biogenesis in both hamster (Mesocricetus auratus and mouse (Mus musculus ova as models for understanding the effects of aging on mitochondrial structure and energy production within the mammalian oocyte. METHODOLOGY/PRINCIPAL FINDINGS: Individual metaphase II oocytes from a total of 25 young and old mice and hamsters were collected from ovarian follicles after hormone stimulation and prepared for biochemical or structural analysis. Adenosine triphosphate levels and mitochondrial DNA number were determined within individual oocytes from young and old animals. In aged hamsters, oocyte adenosine triphosphate levels and mitochondrial DNA molecules were reduced 35.4% and 51.8%, respectively. Reductions of 38.4% and 44% in adenosine triphosphate and mitochondrial genomes, respectively, were also seen in aged mouse oocytes. Transmission electron microscopic (TEM analysis showed that aged rodent oocytes had significant alterations in mitochondrial and cytoplasmic lamellae structure. CONCLUSIONS/SIGNIFICANCE: In both mice and hamsters, decreased adenosine triphosphate in aged oocytes is correlated with a similar decrease in mtDNA molecules and number of mitochondria. Mitochondria in mice and hamsters undergo significant morphological change with aging including mitochondrial vacuolization, cristae alterations, and changes in cytoplasmic lamellae.

  4. Down-regulation of membrana granulosa cell gap junctions is correlated with irreversible commitment to resume meiosis in golden Syrian hamster oocytes. (United States)

    Racowsky, C; Baldwin, K V; Larabell, C A; DeMarais, A A; Kazilek, C J


    One of the currently popular hypotheses for the regulation of meiotic resumption in mammalian oocytes proposes that the preovulatory surge of luteinizing hormone causes down-regulation of follicular gap junctions, which in turn disrupts transfer of a meiotic arrester from the somatic cells into the oocyte. The present study has investigated this hypothesis by examining the integrity of membrana granulosa cell gap junctions during the period of irreversible commitment to maturation of golden Syrian hamster oocytes in vivo. Our results have revealed a significant progressive decrease in the fractional area of cell surface occupied by gap junction membrane with increasing percentage of oocytes irreversibly committed to mature (1.946% and 0.921% fractional gap junction area at 0% and 100% oocytes irreversibly committed to mature, respectively, P less than 0.05). This net loss of membrana granulosa cell gap junctions from the cell surface was accompanied by a significant decrease in density of gap junction particles, whether they were arranged in rectilinear or non-rectilinear packing patterns. Furthermore, the number of gap junction particles per unit area of surface membrane scanned also underwent a significant progressive decrease with increasing percentage of oocytes irreversibly committed to mature. These data with the hamster are consistent with the hypothesis that down-regulation of membrana granulosa cell gap junctions may be of central importance in the regulation of gonadotropic stimulation of meiotic resumption in mammalian oocytes.

  5. Human sperm chromosome analysis after subzonal sperm insemination of hamster oocytes

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    Cozzi, J. [Medical School of Grenoble (France)


    Sperm microinjection techniques, subzonal sperm insemination (SUZI) and intracytoplasmic sperm injection (ICSI), have achieved a wide spread clinical application for the treatment of male infertility. To date, only one study has focused on sperm karyotypes after microinjection. Martin et al. reported a very high incidence of abnormal human sperm complements after ICSI into hamster oocytes. In the present study, are reported the first human sperm karyotypes after SUZI of hamster oocytes. Spermatozoa from two control donors were treated by calcium ionophore A23187 and injected under the zona of hamster eggs. The microinjected eggs were then cultured for cytogenetic analysis of the pronuclei. Out of 47 analyzed sperm chromosome metaphases, 5 (10.6%) were abnormal, 4 (8.5%) were hypohaploid and 1 (2.1%) had a structural abnormality. The sex ratio was not significantly different from the expected 1:1 ratio. Rates of chromosomal abnormalities in microinjected spermatozoa were similar to those observed in spermatozoa inseminated with zona free eggs, suggesting that SUZI procedure per se does not increase sperm chromosomal abnormalities.

  6. Cholesterol depletion disorganizes oocyte membrane rafts altering mouse fertilization.

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    Jorgelina Buschiazzo

    Full Text Available Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1 a decrease of the fertilization rate and index; and (2 a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.

  7. Effect of two ram sperm capacitating media on acrosome reaction and zona-free hamster oocyte penetration test

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    Silvia Ferrari


    Full Text Available An in vitro zona-free hamster oocyte penetration test was utilized in 24 trials in order to evaluate the capacitation media used for ram sperm. A pool of fresh semen was collected from three crossed breed rams. Two semen drops were washed by centrifugation and incubated in high ionic strength treatment (HIS or in a defined medium with HEPES, on heat ewe serum and heparin. After the incubation to promote capacitation, simplified triple-stain technique was used to evaluate the spontaneous acrosome reaction of the capacitated sperm. Superovulation in 96 golden hamsters was induced by PMSG and hCG. The oocytes were treated with hyaluronidase and trypsin to remove, respectively, the cumulus cells and the zona pellucida. Oocytes and capacitated sperm were incubated during 3 hours for further penetration. Then, oocytes were fixed and stained, being evaluated under phase contrast microscope. No significant statistical difference (p >; 0.05 was found between media, concerning the penetration rate of the capacitated sperm and between number of sperm viable with acrosome reaction after the capacitation treatment using two different media. It was concluded that both media utilized were effective in capacitating ram sperm.

  8. Requirement of sperm-oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes. (United States)

    Sengoku, K; Tamate, K; Takaoka, Y; Horikawa, M; Goishi, K; Okada, R; Tsuchiya, K; Ishikawa, M


    We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida-free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5+/-0.6 and 4.3+/-0.6 for ICSI; 3.0+/-0.3 and 3.8+/-0.4 for activated and 0.2+/-0.1 and 0.6+/-0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore-puromycin-activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes.

  9. Recombinant hamster oviductin is biologically active and exerts positive effects on sperm functions and sperm-oocyte binding.

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    Xiaojing Yang

    Full Text Available Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1 in human embryonic kidney 293 (HEK293 cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function.

  10. In vitro and in vivo studies reveal that hamster oocyte meiotic arrest is maintained only transiently by follicular fluid, but persistently by membrana/cumulus granulosa cell contact. (United States)

    Racowsky, C; Baldwin, K V


    Studies were carried out with the golden Syrian hamster to investigate the capacity of follicular fluid to maintain oocyte meiotic arrest and to determine the importance of cumulus-membrana granulosa cell contact in the regulation of meiotic status. The follicular fluid studies were conducted by cytological assessment of meiotic stage up to 6 hr after transferring cumulus-free oocytes into antra of explanted "host" follicles in vitro or into follicles of anesthetized animals prior to the gonadotropin surge at proestrus in vivo. The cumulus-membrana granulosa contact studies were undertaken with explanted follicles in which the oocyte-cumulus complex was dislodged from the underlying membrana granulosa, released into the antrum, and subsequently allowed to reestablish contact during 6 hr of incubation within the follicle. The extent of recontact of the dislodged complex with the underlying membrana granulosa was assessed visually at the end of incubation and was classified as close, moderate, or none. These various degrees of contact typically involved the following number of cumulus cells, as determined by serial sectioning of a representative sample of follicles after dislodgement and subsequent incubation: close, 32.7 +/- 1.78; moderate, 9.0 +/- 2.1; and no contact, 0. After 6 hr of incubation either in vitro or in vivo, few transferred oocytes remained at the germinal vesicle (GV) stage (18.8 +/- 8.7 and 17.3 +/- 4.0% GV, respectively). However, time course experiments revealed that meiotic resumption was significantly delayed in transferred oocytes compared with either liberated oocytes, spontaneously maturing oocytes, or follicle-enclosed oocytes induced to mature by luteinizing hormone in vitro (after 4 hr, transferred, 31.3 +/- 6.0% GV; liberated, 0% GV; follicle-enclosed, 0% GV; after 6 hr, 0% transferred oocytes exhibited a GV). In the dislodgement studies, after 6 hr of incubation, 26% of complexes reestablished close contact with the underlying membrana

  11. Antral follicle development influences plasma membrane organization but not cortical granule distribution in mouse oocytes. (United States)

    Cecconi, S; Focarelli, R; Rossi, G; Talevi, R; Colonna, R


    In the present study, we evaluated the contributions of antral follicle development and antral granulosa cell-released factor(s) to the acquisition of a mature mouse oocyte plasma membrane organization and cortical granule distribution. This has been performed by comparing in-vitro matured oocytes derived from early antral follicles (here referred to as denuded oocytes) or from pre-ovulatory follicles, and cultured either as cumulus-intact or cumulus-free oocytes, with in-vivo ovulated eggs. By using scanning and transmission electron microscopy, the denuded oocyte surface appears to be characterized by the presence of long microvilli, while that of pre-ovulatory oocytes and of ovulated eggs by shorter microvilli. However, denuded oocytes can acquire a pre-ovulatory-like plasma membrane configuration when matured in vitro in the presence of early antral granulosa or cumulus cells, but not of NIH-3T3 fibroblasts. On the contrary, fluorescence and confocal microscopy analyses after labelling with fluorescent Lens culinaris agglutinin show that all the oocyte classes analysed are characterized by similar cortical granule distribution and density. Thus, complete antral follicle development plays an important role in the process of oocyte surface differentiation, probably through the action of antral granulosa cell-released factor(s), but it does not affect oocyte capacity to normally distribute cortical granules.

  12. Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4. (United States)

    Orsini, Francesco; Santacroce, Massimo; Cremona, Andrea; Gosvami, Nitya N; Lascialfari, Alessandro; Hoogenboom, Bart W


    Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.

  13. Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation. (United States)

    Buschiazzo, Jorgelina; Ríos, Glenda L; Canizo, Jesica R; Antollini, Silvia S; Alberio, Ricardo H


    Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD) did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane) and cholesteryl esters (stored in lipid droplets), revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs) among seasons and a dynamic organizational structure of cholesterol

  14. Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation.

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    Jorgelina Buschiazzo

    Full Text Available Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane and cholesteryl esters (stored in lipid droplets, revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs among seasons and a dynamic organizational structure

  15. Dynamics of intracellular phospholipid membrane organization during oocyte maturation and successful vitrification of immature oocytes retrieved by ovum pick-up in cattle. (United States)

    Aono, Akira; Nagatomo, Hiroaki; Takuma, Tetsuya; Nonaka, Rika; Ono, Yoshitaka; Wada, Yasuhiko; Abe, Yasuyuki; Takahashi, Masashi; Watanabe, Tomomasa; Kawahara, Manabu


    The objective was to determine if immature bovine oocytes with cumulus cells at the germinal vesicle (GV) stage could be vitrified by aluminum sheets (AS; pieces of sheet-like aluminum foil). Cleavage rates in fertilized oocytes previously vitrified by the AS procedure were higher than those vitrified by a nylon-mesh holder (NM) procedure (89.3 ± 2.1% vs. 65.0 ± 3.7%). Cleaved embryos derived from the AS but not from the NM procedures developed to blastocysts. Furthermore, to investigate the effects of vitrifying GV oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes, the intracellular phospholipid membrane (IM) was stained with the lipophilic fluorescent dye, 3,3'-dioctadecyloxa-carbocyanine perchlorate. After vitrification by AS, the IM remained intact relative to that of oocytes vitrified by NM. During in vitro maturation, reorganization of the IM was also undamaged in oocytes vitrified by AS before oocyte maturation, and the IM within oocytes vitrified by the NM procedure was evidently impaired. Finally, vitrification (AS) was used for GV oocytes collected using the ovum pick-up method. A bull calf was born after in vitro production and subsequent embryo transfer. The vitrification techniques described herein should facilitate generation of viable in vitro production bovine blastocysts using oocytes recovered using the ovum pick-up method. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Plasma membrane block to polyspermy in human oocytes and preimplantation embryos. (United States)

    Sengoku, K; Tamate, K; Horikawa, M; Takaoka, Y; Ishikawa, M; Dukelow, W R


    The existence and time course of the human plasma membrane block to polyspermy were investigated by an in vitro fertilization assay using zona pellucida-free unfertilized oocytes, pronuclear oocytes and embryos. In the time course study using a high sperm concentration (10(5) spermatozoa ml-1), the number of penetrating spermatozoa at 30 and 60 min after insemination were 1.3 +/- 0.3 and 2.9 +/- 0.4, respectively. By 2 h after insemination, spermatozoa penetration reached a maximum. A lower maximum number of penetrating spermatozoa was observed at a low sperm concentration (10(4) spermatozoa ml-1), but the number of penetrating spermatozoa still reached a maximum by 2 h after insemination. A reinsemination experiment demonstrated that the number of penetrating spermatozoa was not significantly different between control and reinseminated oocytes, while sperm penetration was not observed in the oocyte beyond the two-cell stage. Furthermore, the number of binding spermatozoa decreased after fertilization and most of the four-cell stage embryos displayed no sperm binding. These results suggest that the plasma membrane block plays an important role in the prevention of polyspermy in the human oocyte, and that the plasma membrane block may involve permanent changes in the binding or fusion ability of spermatozoa in the oolemma after fertilization.

  17. Prophase I Mouse Oocytes Are Deficient in the Ability to Respond to Fertilization by Decreasing Membrane Receptivity to Sperm and Establishing a Membrane Block to Polyspermy1 (United States)

    Kryzak, Cassie A.; Moraine, Maia M.; Kyle, Diane D.; Lee, Hyo J.; Cubeñas-Potts, Caelin; Robinson, Douglas N.; Evans, Janice P.


    ABSTRACT Changes occurring as the prophase I oocyte matures to metaphase II are critical for the acquisition of competence for normal egg activation and early embryogenesis. A prophase I oocyte cannot respond to a fertilizing sperm as a metaphase II egg does, including the ability to prevent polyspermic fertilization. Studies here demonstrate that the competence for the membrane block to polyspermy is deficient in prophase I mouse oocytes. In vitro fertilization experiments using identical insemination conditions result in monospermy in 87% of zona pellucida (ZP)-free metaphase II eggs, while 92% of ZP-free prophase I oocytes have four or more fused sperm. The membrane block is associated with a postfertilization reduction in the capacity to support sperm binding, but this reduction in sperm-binding capacity is both less robust and slower to develop in fertilized prophase I oocytes. Fertilization of oocytes is dependent on the tetraspanin CD9, but little to no release of CD9 from the oocyte membrane is detected, suggesting that release of CD9-containing vesicles is not essential for fertilization. The deficiency in membrane block establishment in prophase I oocytes correlates with abnormalities in two postfertilization cytoskeletal changes: sperm-induced cortical remodeling that results in fertilization cone formation and a postfertilization increase in effective cortical tension. These data indicate that cortical maturation is a component of cytoplasmic maturation during the oocyte-to-egg transition and that the egg cortex has to be appropriately primed and tuned to be responsive to a fertilizing sperm. PMID:23863404

  18. Prophase I mouse oocytes are deficient in the ability to respond to fertilization by decreasing membrane receptivity to sperm and establishing a membrane block to polyspermy. (United States)

    Kryzak, Cassie A; Moraine, Maia M; Kyle, Diane D; Lee, Hyo J; Cubeñas-Potts, Caelin; Robinson, Douglas N; Evans, Janice P


    Changes occurring as the prophase I oocyte matures to metaphase II are critical for the acquisition of competence for normal egg activation and early embryogenesis. A prophase I oocyte cannot respond to a fertilizing sperm as a metaphase II egg does, including the ability to prevent polyspermic fertilization. Studies here demonstrate that the competence for the membrane block to polyspermy is deficient in prophase I mouse oocytes. In vitro fertilization experiments using identical insemination conditions result in monospermy in 87% of zona pellucida (ZP)-free metaphase II eggs, while 92% of ZP-free prophase I oocytes have four or more fused sperm. The membrane block is associated with a postfertilization reduction in the capacity to support sperm binding, but this reduction in sperm-binding capacity is both less robust and slower to develop in fertilized prophase I oocytes. Fertilization of oocytes is dependent on the tetraspanin CD9, but little to no release of CD9 from the oocyte membrane is detected, suggesting that release of CD9-containing vesicles is not essential for fertilization. The deficiency in membrane block establishment in prophase I oocytes correlates with abnormalities in two postfertilization cytoskeletal changes: sperm-induced cortical remodeling that results in fertilization cone formation and a postfertilization increase in effective cortical tension. These data indicate that cortical maturation is a component of cytoplasmic maturation during the oocyte-to-egg transition and that the egg cortex has to be appropriately primed and tuned to be responsive to a fertilizing sperm.


    Abstract Glutathione (GSH) is thought to play critical roles in oocyte function including spindle maintenance and provision of reducing power needed to initiate sperm chromatin decondensation. Previous observations that GSH concentrations are higher in mature than immature o...

  20. A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes. (United States)

    Chan, P J; Corselli, J U; Patton, W C; Jacobson, J D; Chan, S R; King, A


    To correlate sperm variables with sperm DNA fragmentation, as assessed by using a modified alkaline comet assay for sperm smears. The comet assay was adapted for fixed sperm smears (59 cases), and the level of DNA fragmentation was determined. Clinical and academic research environment. 59 patients undergoing fertility treatment. Sperm samples leftover from IVF procedures were fixed and processed for the comet assay. Sperm head DNA density and sperm variables. A correlation was observed between increased sperm head DNA fragmentation and decreased penetration of zona-free hamster oocytes. Heat-induced hyperactive motility decreased as DNA fragmentation increased. The DNA fragmentation did not correlate with percentages of intact acrosome, normality, maturity, and strict normal morphology. The advantages of the comet assay for archived cells include simplicity, low intraassay coefficient of variation, and low performance cost; in addition, DNA analysis can be carried out at leisure. Low DNA damage was associated with higher hyperactivation and oocyte penetration, suggesting that failed fertilization was linked to compromised DNA integrity in the sperm. Exploration of compounds to repair damaged DNA is warranted.

  1. Effects of Different Maturation Systems on Bovine Oocyte Quality, Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming. (United States)

    Sprícigo, José F W; Diógenes, Mateus N; Leme, Ligiane O; Guimarães, Ana L; Muterlle, Carolle V; Silva, Bianca Damiani Marques; Solà-Oriol, David; Pivato, Ivo; Silva, Luciano Paulino; Dode, Margot A N


    The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (Pvitrification because the blastocyst rate at D7 was similar (P>0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]+), which was more highly expressed in MII compared to FSH (Pbovine oocytes to vitrification.

  2. Xenopus laevis Oocytes as a Model System for Studying the Interaction Between Asbestos Fibres and Cell Membranes. (United States)

    Bernareggi, Annalisa; Ren, Elisa; Borelli, Violetta; Vita, Francesca; Constanti, Andrew; Zabucchi, Giuliano


    The mode of interaction of asbestos fibres with cell membranes is still debatable. One reason is the lack of a suitable and convenient cellular model to investigate the causes of asbestos toxicity. We studied the interaction of asbestos fibres with Xenopus laevis oocytes, using electrophysiological and morphological methods. Oocytes are large single cells, with a limited ability to endocytose molecular ligands; we therefore considered these cells to be a good model for investigating the nature of asbestos/membrane interactions. Electrophysiological recordings were performed to compare the passive electrical membrane properties, and those induced by applying positive or negative voltage steps, in untreated oocytes and those exposed to asbestos fibre suspensions. Ultrastructural analysis visualized in detail, any morphological changes of the surface membrane caused by the fibre treatment. Our results demonstrate that Amosite and Crocidolite-type asbestos fibres significantly modify the properties of the membrane, starting soon after exposure. Cells were routinely depolarized, their input resistance decreased, and the slow outward currents evoked by step depolarizations were dramatically enhanced. Reducing the availability of surface iron contained in the structure of the fibres with cation chelators, abolished these effects. Ultrastructural analysis of the fibre-exposed oocytes showed no evidence of phagocytic events. Our results demonstrate that asbestos fibres modify the oocyte membrane, and we propose that these cells represent a viable model for studying the asbestos/cell membrane interaction. Our findings also open the possibly for finding specific competitors capable of hindering the asbestos-cell membrane interaction as a means of tackling the long-standing asbestos toxicity problem. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail:

  3. Binding kinetics of Clostridium difficile toxins A and B to intestinal brush border membranes from infant and adult hamsters

    Energy Technology Data Exchange (ETDEWEB)

    Rolfe, R.D. (Texas Tech Univ. Health Sciences Center, Lubbock (USA))


    This study was undertaken to determine if the relative resistance of neonates and infants to Clostridium difficile-associated intestinal disease can be related to age-dependent differences in intestinal receptors for C. difficile toxins A and B. Brush border membranes (BBMs) from the small intestines of adult and infant hamsters were examined for their ability to bind radiolabeled toxins A and B. (125I)toxin A bound to both infant and adult hamster BBMs at physiological temperature, whereas (125I)toxin B did not bind to the BBMs under any of the conditions examined. The number of (125I)toxin A molecules bound at saturation was approximately 4 x 10(10) per micrograms of membrane protein for adult BBMs and 1 x 10(11) per micrograms of membrane protein for infant BBMs. Scatchard plot analysis suggested the presence of a single class of toxin A binding sites on both infant and adult hamster BBMs. Maximal binding capacity and Kd values were 0.63 pmol/mg of protein and 66.7 nM, respectively, for the infant BBMs, and 0.24 pmol/mg of protein and 27 nM, respectively, for the adult BBMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of extracted BBM proteins revealed differences in the proteins of infant and adult BBMs. However, there were not any detectable differences in the protein bands which bound (125I)toxin A between infant and adult hamsters. The results from these investigations indicate that differences in the binding kinetics of toxins A and/or B to infant and adult hamster BBMs do not account for the observed differences in their susceptibility to C. difficile-associated intestinal disease.

  4. Effects of Different Maturation Systems on Bovine Oocyte Quality, Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming.

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    José F W Sprícigo

    Full Text Available The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM. Four different maturation systems were tested: 1 in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136; 2 in vitro maturation using immature oocytes obtained by ovum pick-up (OPU from unstimulated heifers (IMA; n = 433; 3 in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444; and 4 in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658. A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT, while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P0.05 for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%. MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1 + H]+, which was more highly expressed in MII compared to FSH (P<0.05. The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification.

  5. Membranes during yolk-platelet development in oocytes of the toad Bufo marinus. (United States)

    Richter, H -P


    Oocytes of the toad Bufo marinus have been studied by means of thin section and particularly freeze-fracture electron microscopy to characterize the cytoplasmic membranes around the yolk organelle, and the storage of yolk material in precursors and platelets. This appears to be a previously unknown type of yolk-platelet formation. During yolk-organelle development from the primordial precursor to the bi-partite fully grown yolk platelet, numerous lipoid droplets are attached to the periphery of the platelet, indicating an intense uptake of lipids. As is typical for amphibians, the fully grown yolk platelet has a crystalline internum covered by a dense osmiophilic externum, and the whole organelle is enveloped by a plasma membrane that shows no direct connection or fusion with endocytotic vesicles. The yolk membrane exhibits few intramembraneous particles (IMPs) at the core areas and some more where it borders fields of lipoid droplets. Here the IMPs show a net-like arrangement in the furrows between adjacent droplets.

  6. Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling. (United States)

    Zhang, T; Rawson, D M; Tosti, L; Carnevali, O


    This study investigated enzymatic activity of cathepsins and the membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling. Stage III oocytes (>0.5mm), obtained through dissection of anaesthetised female fish and desegregation of ovarian cumulus, were exposed to 2M methanol or 2M DMSO (both prepared in Hank's medium) for 30min at 22 degrees C before being loaded into 0.5ml plastic straws and placed into a programmable cooler. After controlled slow freezing, samples were plunged into liquid nitrogen (LN) and held for at least 10min, and thawed by immersing straws into a 27 degrees C water bath for 10s. Thawed oocytes were washed twice in Hank's medium. Cathepsin activity and membrane integrity of oocytes were assessed both after cryoprotectant treatment at 22 degrees C and after freezing in LN. Cathepsin B and L colorimetric analyses were performed using substrates Z-Arg-ArgNNap and Z-Phe-Arg-4MbetaNA-HCl, respectively, and 2-naphthylamine and 4-methoxy-2-naphthylamine were used as standards. Cathepsin D activity was performed by analysing the level of hydrolytic action on haemoglobin. Oocytes membrane integrity was assessed using 0.2% Trypan blue staining for 5min. Analysis of cathepsin activities showed that whilst the activity of cathepsin B and D was not affected by 2M DMSO treatment, their activity was lowered when treated with 2M methanol. Following freezing to -196 degrees C, the activity of all cathepsins (B, D and L) was significantly decreased in both 2M DMSO and 2M methanol. Trypan blue staining showed that 63.0+/-11.3% and 72.7+/-5.2% oocytes membrane stayed intact after DMSO and methanol treatment for 30min at 22 degrees C, respectively, whilst 14.9+/-2.6% and 1.4+/-0.8% stayed intact after freezing in DMSO and methanol to -196 degrees C. The results indicate that cryoprotectant treatment and freezing modified the activities of lysosomal enzymes involved in oocyte maturation and yolk

  7. Membrane phospholipid fatty acid composition regulates cardiac SERCA activity in a hibernator, the Syrian hamster (Mesocricetus auratus.

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    Sylvain Giroud

    Full Text Available Polyunsaturated fatty acids (PUFA have strong effects on hibernation and daily torpor. Increased dietary uptake of PUFA of the n-6 class, particularly of Linoleic acid (LA, C18:2 n-6 lengthens torpor bout duration and enables animals to reach lower body temperatures (T(b and metabolic rates. As previously hypothesized, this well-known influence of PUFA may be mediated via effects of the membrane fatty acid composition on sarcoplasmic reticulum (SR Ca(2+-ATPase 2a (SERCA in the heart of hibernators. We tested the hypotheses that high proportions of n-6 PUFA in general, or specifically high proportions of LA (C18:2 n-6 in SR phospholipids (PL should be associated with increased cardiac SERCA activity, and should allow animals to reach lower minimum T(b in torpor. We measured activity of SERCA from hearts of hibernating and non-hibernating Syrian hamsters (Mesocricetus auratus in vitro at 35 °C. Further, we determined the PL fatty acid composition of the SR membrane of these hearts. We found that SERCA activity strongly increased as the proportion of LA in SR PL increased but was negatively affected by the content of Docosahexaenoic acid (DHA; C22:6 n-3. SR PL from hibernating hamsters were characterized by high proportions of LA and low proportions of DHA. As a result, SERCA activity was significantly higher during entrance into torpor and in torpor compared to inter-bout arousal. Also, animals with increased SERCA activity reached lower T(b during torpor. Interestingly, a subgroup of hamsters which never entered torpor but remained euthermic throughout winter displayed a phenotype similar to animals in summer. This was characterized by lower proportions of LA and increased proportions of DHA in SR membranes, which is apparently incompatible with torpor. We conclude that the PUFA composition of SR membranes affects cardiac function via modulating SERCA activity, and hence determines the minimum T(b tolerated by hibernators.

  8. Spumiform basement membrane aberrations in the microvasculature of the midbrain periaqueductal gray region in hamster: rostro-caudal pathogenesis? (United States)

    Gerrits, P O; Kortekaas, R; de Weerd, H; Luiten, P G M; van der Want, J J L; Veening, J G


    Spumiform basement membrane degeneration (sbmd) is a specific kind of aberration present in the capillaries of the midbrain periaqueductal gray (PAG) region of the senescent hamster. These capillaries, separated by the ependymal cell layer, are bordering the Sylvian cerebral aqueduct. The aqueduct, connecting the 3rd and 4th ventricle, may be crucial for local homeostatic as well as general autonomic functions of the PAG. Local pressure effects of the flowing and pulsating cerebrospinal fluid on the PAG-vasculature are probably different for the rostral 'entrance' and the caudal 'exit' of the aqueduct. In view of the different functions of the various divisions of the PAG, the frequency and extent of the aberrations in the rostral, intermediate and caudal dl/vlPAG-microvasculature could shed some light on the causal factors involved in the regional distribution of the particular microvascular aberrations found in the PAG during aging. In the present study we investigated the ultrastructure of capillaries in dorsal and ventral subdivisions of anterior and posterior regions of the PAG of young and old female Syrian hamsters. Sbmds were classified into four stages of spumiform severity and for each stage the frequency was determined in the rostral PAG, at two levels in the intermediate PAG and in a dorsal and a ventral part of the caudal PAG. Results of our quantitative studies showed that in aged hamster PAG various stages of sbmd were present in 91.6 ± 0.6% of all capillaries. No clear evidence was found for regional differentiation between rostral, intermediate and caudal parts of the PAG. Next to sbmd, capillary split basement membrane (sbm) and vacuolization were common features at all five PAG locations. 84.3 ± 2.3% of all screened PAG capillaries displayed sbm. In agreement with our previous findings, several other types of microvascular aberrations were observed in addition to general aspects of aging and some ependymal structural peculiarities. We conclude


    Here we use a hamster animal model to identify early pregnancy loss due to an acute chemical exposure to the female during the perifertilization interval. The fungicide carbendazim (methyl 1H-benzimidazole-2-carbamate), a microtubule poison with antimitotic activity, was selected...

  10. The movement of water and cryoprotectants across the plasma membrane of mammalian oocytes and embryos and its relevance to vitrification. (United States)

    Edashige, Keisuke


    The permeability of the plasma membrane to water and cryoprotectants is one of the most important factors for determining suitable conditions for vitrification of mammalian oocytes and embryos. In mouse oocytes and early stage embryos, water and cryoprotectants move slowly, principally by simple diffusion. In contrast, in morulae (and probably blastocysts), water, glycerol, and ethylene glycerol move rapidly, principally by facilitated diffusion via aquaporin 3, and DMSO moves rapidly via channels other than aquaporin 3. However, propylene glycol moves principally by simple diffusion. In cows and pigs, similar results were obtained. However, in bovine morulae, DMSO moves principally by simple diffusion. In pigs, permeability to water, glycerol, and ethylene glycol increases not at the morula stage but at the blastocyst stage, and increases further at the expanded blastocyst stage. Therefore, in general, the permeability of mammalian oocytes and early stage embryos to water and cryoprotectants is low. Then, at later stages, the permeability to water and some cryoprotectants markedly increases and occurs by facilitated diffusion via channels, although there are some species-specific differences.

  11. Plasma membrane events associated with the meiotic divisions in the amphibian oocyte: insights into the evolution of insulin transduction systems and cell signaling

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    Morrill Gene A


    Full Text Available Abstract Background Insulin and its plasma membrane receptor constitute an ancient response system critical to cell growth and differentiation. Studies using intact Rana pipiens oocytes have shown that insulin can act at receptors on the oocyte surface to initiate resumption of the first meiotic division. We have reexamined the insulin-induced cascade of electrical and ion transport-related plasma membrane events using both oocytes and intact plasma membranes in order to characterize the insulin receptor-steroid response system associated with the meiotic divisions. Results [125I]Insulin binding (Kd = 54 ± 6 nM at the oocyte plasma membrane activates membrane serine protease(s, followed by the loss of low affinity ouabain binding sites, with a concomitant 3–4 fold increase in high affinity ouabain binding sites. The changes in protease activity and ouabain binding are associated with increased Na+/Ca2+ exchange, increased endocytosis, decreased Na+ conductance resulting in membrane hyperpolarization, increased 2-deoxy-D-glucose uptake and a sustained elevation of intracellular pH (pHi. Hyperpolarization is largely due to Na+-channel inactivation and is the main driving force for glucose uptake by the oocyte via Na+/glucose cotransport. The Na+ sym- and antiporter systems are driven by the Na+ free energy gradient generated by Na+/K+-ATPase. Shifts in α and/or β Na+-pump subunits to caveolar (lipid raft membrane regions may activate Na/K-ATPase and contribute to the Na+ free energy gradient and the increase in both Na+/glucose co-transport and pHi. Conclusions Under physiological conditions, resumption of meiosis results from the concerted action of insulin and progesterone at the cell membrane. Insulin inactivates Na+ channels and mobilizes fully functional Na+-pumps, generating a Na+ free energy gradient which serves as the energy source for several membrane anti- and symporter systems.

  12. Single-molecule motions of oligoarginine transporter conjugates on the plasma membrane of Chinese hamster ovary cells. (United States)

    Lee, H-L; Dubikovskaya, E A; Hwang, H; Semyonov, A N; Wang, H; Jones, L R; Twieg, R J; Moerner, W E; Wender, P A


    To explore the real-time dynamic behavior of molecular transporters of the cell-penetrating-peptide (CPP) type on a biological membrane, single fluorescently labeled oligoarginine conjugates were imaged interacting with the plasma membrane of Chinese hamster ovary (CHO) cells. The diffusional motion on the membrane, characterized by single-molecule diffusion coefficient and residence time (tau R), defined as the time from the initial appearance of a single-molecule spot on the membrane (from the solution) to the time the single molecule disappears from the imaging focal plane, was observed for a fluorophore-labeled octaarginine (a model guanidinium-rich CPP) and compared with the corresponding values observed for a tetraarginine conjugate (negative control), a lipid analogue, and a fluorescently labeled protein conjugate (transferrin-Alexa594) known to enter the cell through endocytosis. Imaging of the oligoarginine conjugates was enabled by the use of a new high-contrast fluorophore in the dicyanomethylenedihydrofuran family, which brightens upon interaction with the membrane at normal oxygen concentrations. Taken as a whole, the motions of the octaarginine conjugate single molecules are highly heterogeneous and cannot be described as Brownian motion with a single diffusion coefficient. The observed behavior is also different from that of lipids, known to penetrate cellular membranes through passive diffusion, conventionally involving lateral diffusion followed by membrane bilayer flip-flop. Furthermore, while the octaarginine conjugate behavior shares some common features with transferrin uptake (endocytotic) processes, the two systems also exhibit dissimilar traits when diffusional motions and residence times of single constructs are compared. Additionally, pretreatment of cells with cytochalasin D, a known actin filament disruptor, produces no significant effect, which further rules out unimodal endocytosis as the mechanism of uptake. Also, the involvement of

  13. Evidence from mathematical modeling that carbonic anhydrase II and IV enhance CO2 fluxes across Xenopus oocyte plasma membranes. (United States)

    Occhipinti, Rossana; Musa-Aziz, Raif; Boron, Walter F


    Exposing an oocyte to CO2/HCO3 (-) causes intracellular pH (pHi) to decline and extracellular-surface pH (pHS) to rise to a peak and decay. The two companion papers showed that oocytes injected with cytosolic carbonic anhydrase II (CA II) or expressing surface CA IV exhibit increased maximal rate of pHi change (dpHi/dt)max, increased maximal pHS changes (ΔpHS), and decreased time constants for pHi decline and pHS decay. Here we investigate these results using refinements of an earlier mathematical model of CO2 influx into a spherical cell. Refinements include 1) reduced cytosolic water content, 2) reduced cytosolic diffusion constants, 3) refined CA II activity, 4) layer of intracellular vesicles, 5) reduced membrane CO2 permeability, 6) microvilli, 7) refined CA IV activity, 8) a vitelline membrane, and 9) a new simulation protocol for delivering and removing the bulk extracellular CO2/HCO3 (-) solution. We show how these features affect the simulated pHi and pHS transients and use the refined model with the experimental data for 1.5% CO2/10 mM HCO3 (-) (pHo = 7.5) to find parameter values that approximate ΔpHS, the time to peak pHS, the time delay to the start of the pHi change, (dpHi/dt)max, and the change in steady-state pHi. We validate the revised model against data collected as we vary levels of CO2/HCO3 (-) or of extracellular HEPES buffer. The model confirms the hypothesis that CA II and CA IV enhance transmembrane CO2 fluxes by maximizing CO2 gradients across the plasma membrane, and it predicts that the pH effects of simultaneously implementing intracellular and extracellular-surface CA are supra-additive. Copyright © 2014 the American Physiological Society.

  14. Direct Microscale Measurement of Mouse Oocyte Membrane Permeability to Water and Ethylene Glycol at Subzero Temperatures Using Cryomicroscopy. (United States)

    Han, X

      BACKGROUND: Investigation of cell osmotic behavior at subzero temperatures is of critical importance to the optimization of cooling procedures for cryopreservation. Based on established thermodynamic models, plasma membrane permeability coefficients for water and cryoprotectant agent (CPA) (Lcpa, Pp) and their activation energies (EaLp, EaPcpa) are essential to predict the change of cell volume and composition of intracellular solutions corresponding to different cooling procedures. However, currently available methods to measure Lp at subzero temperatures suffer from technical difficulties due to ice formation and there are no generalized methods to measure Pcpa at subzero temperatures. The present study aims to investigate cell osmotic behavior at subzero temperatures without ice formation. In the study cells were directly injected into super-cooled CPA solutions mounted on a cryomicroscope, and the corresponding osmotic properties were measured. Using ethylene glycol (EG), the value of PEG for mouse (CD-1) metaphase II oocytes at 0, -5, -10 degree C was determined to be 8.451.20, 7.430.91, 6.401.10, x10-6 cm/min, respectively, and EaPEG was calculated to be 3.9 kCal/mol. Lp in the presence of EG (LpEG) at 0, -5, -10 , -15 degree C was determined to be 7.0 1.15, 4.90 1.20, 2.44 0.31, 1.200.24, x 10-2 µm/min/atm, respectively, and EaLp was calculated to be 15.5 kCal/mol. Comparing these values with those previously measured at superzero temperatures, we concluded that for mouse oocytes, the Arrhenius relationship for LpEG is consistent at superzero and subzero temperatures, but the values of PEG at subzero temperatures are much lower than the extrapolated values from the Arrhenius relationship at superzero temperatures, possibly caused by membrane phase transition at low temperatures.

  15. Leishmania donovani: immunostimulatory cellular responses of membrane and soluble protein fractions of splenic amastigotes in cured patient and hamsters.

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    Shraddha Kumari

    Full Text Available Visceral leishmaniasis (VL, caused by the intracellular parasite Leishmania donovani, L. chagasi and L. infantum is characterized by defective cell-mediated immunity (CMI and is usually fatal if not treated properly. An estimated 350 million people worldwide are at risk of acquiring infection with Leishmania parasites with approximately 500,000 cases of VL being reported each year. In the absence of an efficient and cost-effective antileishmanial drug, development of an appropriate long-lasting vaccine against VL is the need of the day. In VL, the development of a CMI, capable of mounting Th1-type of immune responses, play an important role as it correlate with recovery from and resistance to disease. Resolution of infection results in lifelong immunity against the disease which indicates towards the feasibility of a vaccine against the disease. Most of the vaccination studies in Leishmaniasis have been focused on promastigote--an infective stage of parasite with less exploration of pathogenic amastigote form, due to the cumbersome process of its purified isolation. In the present study, we have isolated and purified splenic amastigotes of L. donovani, following the traditional protocol with slight modification. These were fractionated into five membranous and soluble subfractions each i.e MAF1-5 and SAF1-5 and were subjected for evaluation of their ability to induce cellular responses. Out of five sub-fractions from each of membrane and soluble, only four viz. MAF2, MAF3, SAF2 and SAF3 were observed to stimulate remarkable lymphoproliferative, IFN-γ, IL-12 responses and Nitric Oxide production, in Leishmania-infected cured/exposed patients and hamsters. Results suggest the presence of Th-1 type immunostimulatory molecules in these sub-fractions which may further be exploited for developing a successful subunit vaccine from the less explored pathogenic stage against VL.

  16. Injection of insect membrane in Xenopus oocyte: An original method for the pharmacological characterization of neonicotinoid insecticides. (United States)

    Crespin, Lucille; Legros, Christian; List, Olivier; Tricoire-Leignel, Hélène; Mattei, César


    Insect nicotinic acetylcholine receptors (nAChRs) represent a major target of insecticides, belonging to the neonicotinoid family. However, the pharmacological profile of native nAChRs is poorly documented, mainly because of a lack of knowledge of their subunit stoichiometry, their tissue distribution and the weak access to nAChR-expressing cells. In addition, the expression of insect nAChRs in heterologous systems remains hard to achieve. Therefore, the structure-activity characterization of nAChR-targeting insecticides is made difficult. The objective of the present study was to characterize insect nAChRs by an electrophysiological approach in a heterologous system naturally devoid of these receptors to allow a molecular/cellular investigation of the mode of action of neonicotinoids. Methods To overcome impediments linked to the expression of insect nAChR mRNA or cDNA, we chose to inject insect membranes from the pea aphid (Acyrthosiphon pisum) into Xenopus oocytes. This microtransplantation technique was designed to gain access to native nAChRs embedded in their membrane, through direct stimulation with nicotinic agonists. Results We provide evidence that an enriched-nAChR membrane allows us to characterize native receptors. The presence of such receptors was confirmed with fluorescent α-BgTX labeling. Electrophysiological recordings of nicotine-induced inward currents allowed us to challenge the presence of functional nAChR. We compared the effect of nicotine (NIC) with clothianidin (CLO) and we assessed the effect of thiamethoxam (TMX). Discussion This technique has been recently highlighted with mammalian and human material as a powerful functional approach, but has, to our knowledge, never been used with insect membrane. In addition, the use of the insect membrane microtransplantation opens a new and original way for pharmacological screening of neurotoxic insecticides, including neonicotinoids. Moreover, it might also be a powerful tool to investigate the

  17. Oocyte Activation and Fertilisation: Crucial Contributors from the Sperm and Oocyte. (United States)

    Yeste, Marc; Jones, Celine; Amdani, Siti Nornadhirah; Coward, Kevin


    This chapter intends to summarise the importance of sperm- and oocyte-derived factors in the processes of sperm-oocyte binding and oocyte activation. First, we describe the initial interaction between sperm and the zona pellucida, with particular regard to acrosome exocytosis. We then describe how sperm and oocyte membranes fuse, with special reference to the discovery of the sperm protein IZUMO1 and its interaction with the oocyte membrane receptor JUNO. We then focus specifically upon oocyte activation, the fundamental process by which the oocyte is alleviated from metaphase II arrest by a sperm-soluble factor. The identity of this sperm factor has been the source of much debate recently, although mounting evidence, from several different laboratories, provides strong support for phospholipase C ζ (PLCζ), a sperm-specific phospholipase. Herein, we discuss the evidence in support of PLCζ and evaluate the potential role of other candidate proteins, such as post-acrosomal WW-binding domain protein (PAWP/WBP2NL). Since the cascade of downstream events triggered by the sperm-borne oocyte activation factor heavily relies upon specialised cellular machinery within the oocyte, we also discuss the critical role of oocyte-borne factors, such as the inositol trisphosphate receptor (IP3R), protein kinase C (PKC), store-operated calcium entry (SOCE) and calcium/calmodulin-dependent protein kinase II (CaMKII), during the process of oocyte activation. In order to place the implications of these various factors and processes into a clinical context, we proceed to describe their potential association with oocyte activation failure and discuss how clinical techniques such as the in vitro maturation of oocytes may affect oocyte activation ability. Finally, we contemplate the role of artificial oocyte activating agents in the clinical rescue of oocyte activation deficiency and discuss options for more endogenous alternatives.

  18. Human Acyl-Coenzyme A:Cholesterol Acyltransferase Expressed in Chinese Hamster Ovary Cells: Membrane Topology and Active Site Location


    Lin, Song; Lu, Xiaohui; Chang, Catherine C.Y.; Chang, Ta-Yuan


    Acyl-CoA:cholesterol acyltransferase (ACAT) is a membrane-bound enzyme that produces cholesteryl esters intracellularly. Two ACAT genes (ACAT1 and ACAT2) have been identified. The expression of ACAT1 is ubiquitous, whereas that of ACAT2 is tissue restricted. Previous research indicates that ACAT1 may contain seven transmembrane domains (TMDs). To study ACAT2 topology, we inserted two different antigenic tags (hemagglutinin, monoclonal antibody Mab1) at various hydrophi...

  19. Overleeft de hamster?

    NARCIS (Netherlands)

    Apeldoorn, van R.C.; Klein Douwel, C.; Thomas, P.


    Een analyse van de achteruitgang van de hamster (Cricetus cricetus) in Europa en Limburg, de oorzaken (veranderingen in de landbouw; versnippering van leefgebieden), en oplossingsrichtingen voor een duurzaam overleven van de hamster in Limburg (kernpopulaties in duurzame populatienetwerken)

  20. Age-Associated Lipidome Changes in Metaphase II Mouse Oocytes.

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    Hyuck Jun Mok

    Full Text Available The quality of mammalian oocytes declines with age, which negatively affects fertilization and developmental potential. The aging process often accompanies damages to macromolecules such as proteins, DNA, and lipids. To investigate if aged oocytes display an altered lipidome compared to young oocytes, we performed a global lipidomic analysis between oocytes from 4-week-old and 42 to 50-week-old mice. Increased oxidative stress is often considered as one of the main causes of cellular aging. Thus, we set up a group of 4-week-old oocytes treated with hydrogen peroxide (H2O2, a commonly used oxidative stressor, to compare if similar lipid species are altered between aged and oxidative-stressed oocytes. Between young and aged oocytes, we identified 26 decreased and 6 increased lipids in aged oocytes; and between young and H2O2-treated oocytes, we identified 35 decreased and 26 increased lipids in H2O2-treated oocytes. The decreased lipid species in these two comparisons were overlapped, whereas the increased lipid species were distinct. Multiple phospholipid classes, phosphatidic acid (PA, phosphatidylinositol (PI, phosphatidylserine (PS, and lysophosphatidylserine (LPS significantly decreased both in H2O2-treated and aged oocytes, suggesting that the integrity of plasma membrane is similarly affected under these conditions. In contrast, a dramatic increase in diacylglycerol (DG was only noted in H2O2-treated oocytes, indicating that the acute effect of H2O2-caused oxidative stress is distinct from aging-associated lipidome alteration. In H2O2-treated oocytes, the expression of lysophosphatidylcholine acyltransferase 1 increased along with increases in phosphatidylcholine. Overall, our data reveal that several classes of phospholipids are affected in aged oocytes, suggesting that the integrity of plasma membrane is associated with maintaining fertilization and developmental potential of mouse oocytes.

  1. Fractal organization of feline oocyte cytoplasm

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    G De Vico


    Full Text Available The present study aimed at verifying whether immature cat oocytes with morphologic irregular cytoplasm display selfsimilar features which can be analytically described by fractal analysis. Original images of oocytes collected by ovariectomy were acquired at a final magnification of 400 X with a CCD video camera connected to an optic microscope. After greyscale thresholding segmentation of cytoplasm, image profiles were submitted to fractal analysis using FANAL++, a program which provided an analytical standard procedure for determining the fractal dimension (FD. The presentation of the oocyte influenced the magnitude of the fractal dimension with the highest FD of 1.91 measured on grey-dark cytoplasm characterized by a highly connected network of lipid droplets and intracellular membranes. Fractal analysis provides an effective quantitative descriptor of the real cytoplasm morphology, which can influence the acquirement of in vitro developmental competence, without introducing any bias or shape approximation and thus contributes to an objective and reliable classification of feline oocytes.

  2. Fine structure of Egyptian buffalo oocytes ( Bubalus bubalis ) during ...

    African Journals Online (AJOL)

    Results showed that the cumulus cells were close to each other and zona plucida (ZP) in the first group than the second and third group, lipid droplets (LD) appeared normal and nearly from plasma membrane in the group of oocytes matured in vitro for 8 h than oocytes matured in vitro for 24 h, microvilli (Mv) appeared with ...

  3. Evaluation of different factors affecting the efficiency of oocytes cryopreservation in the bovine model


    De Blasi, Marina


    Interest in oocyte cryopreservation has recently increased. Cattle oocytes are sensitive to low temperatures, and despite the efforts of numerous research groups cryopreservation of oocytes remains a difficult task. This problem may be in part due to the large size of bovine oocytes, which consequently have a low surface to volume ratio, making it more difficult for water and cryoprotectants (CP) to move across the cell plasma membranes. Several attempts to improve the survival rate of oocy...

  4. Membrane proteins associated with sperm-oocyte interaction: A proteomic comparison between Kedah Kelantan (Bos indicus) and Mafriwal (Bos taurus × Bos indicus) sperm (United States)

    Ashrafzadeh, Ali; Nathan, Sheila; Othman, Iekhsan; Yee, Tee Ting; Karsani, Saiful Anuar


    Production performance of European cattle breeds has significantly improved through various breeding programs. However, European breeds are more susceptible to heat stress compared to zebu cattle (Bos indicus) as their conception rate can range between 20 to 30% in hot seasons compared to winter. To identify cattle sperm proteins associated with zebu cattle higher fertility and heat tolerance in tropical environments, we utilised a proteomics-based approach to compare sperm from the highly fertile Malaysian indigenous breed, Kedah Kelantan (Bos indicus), with sperm from the sub-fertile crossbreed, Mafriwal (Bos taurus × Bos indicus). Frozen semen of three high performance bulls from each breed was processed to obtain live and pure sperm. Proteins were separated and gel bands were processed by in-gel tryptic digestion. For each breed, mass spectrometry data was acquired over 11 replicates. The analyzed data identified peptides with different expression levels (99% confidence level) and protein identification was determined by targeted MS/MS. Among the identified proteins associated with sperm-oocyte interaction, two proteins were up-regulated in Kedah Kelantan sperm and 7 proteins were up-regulated in or specific to Mafriwal. Our results suggest that the higher fertility of zebu cattle in tropical areas may not be related to more efficient sperm-oocyte interaction. Further analysis of the other regulated proteins in these two breeds may contribute further knowledge on the physiological reason/s for higher fertility and heat tolerance of Zebu cattle in tropical areas.

  5. Beschermingsplan hamster 2005-2010

    NARCIS (Netherlands)

    Haye, la M.J.J.; Jansman, H.A.H.


    Alterra-Concept van het beschermingsplan hamster 2005-2010. De hamster is in het meest westelijke deel van het Europese verspreidingsgebied bedreigd. De kennis die in de afgelopen periode is opgedaan van de hamster en de maatregelen die in het veld zijn uitgevoerd vormen de basis voor dit tweede

  6. Cryopreservation of zebrafish (Danio rerio) oocytes by vitrification. (United States)

    Guan, M; Rawson, D M; Zhang, T


    Cryopreservation of fish oocytes is challenging because these oocytes have low membrane permeability to water and cryoprotectant and are highly chilling sensitive. Vitrification is considered to be a promising approach for their cryopreservation as it involves rapid freezing and thawing of the oocytes and therefore minimising the chilling injury. In the present study, vitrification properties and the toxicity of a range of vitrification solutions containing different concentrations of Me2SO, methanol, propylene glycol and ethylene glycol were investigated. Two different base media and vitrification methods were compared. The effect of different post-thaw dilution solutions together with incubation periods on oocyte viability were also investigated. Stage III zebrafish oocytes were equilibrated in increasing concentrations of cryoprotectants for 30 min in 3 steps. Oocytes were thawed rapidly in a water bath and cryoprotectants were removed in 4 steps. Oocyte viability was assessed using trypan blue staining. The results showed that vitrification solutions V3 and V4 in KCl buffer had low toxicity and vitrified well. The survivals of oocytes after stepwise dilution using solutions containing permeable cryoprotectants were significant higher than those diluted in 0.5M glucose, and the use of CVA65 vitrification system improved oocyte survival when compared with plastic straws after 30 min at 22 degrees C post-thawing. Cryopreservation of zebrafish oocytes by vitrification is reported here for the first time, although oocyte survivals after cryopreservation assessed by trypan blue staining were relatively high shortly after thawing, they became swollen and translucent after incubation in KCl buffer. Further studies are needed to optimise the post-thaw culturing conditions.

  7. Human oocyte cryopreservation. (United States)

    Tao, Tao; Zhang, Wenling; Del Valle, Alfonso


    This review summarized the clinical breakthroughs in the human oocyte cryopreservation field in the past 2 years and gave special emphasis on the role of vitrification method. Human oocyte cryopreservation is an attractive strategy to preserve female fertility, as it offers more opportunities to the future destination of the female gametes and also raises fewer legal and ethical questions compared with embryo cryopreservation. It became promising in recent years because of dramatic improvement in cryopreservation technologies. Human oocyte cryopreservation would not become a clinical routine until the availability of reliable cryopreservation methods and long-term follow-up results of the babies born by this technique. Oocyte cryopreservation produced very exciting results with pregnancy and implantation rates comparable to embryo cryopreservation and in some cases comparable to fresh in-vitro fertilization cycles with both modified slow-freezing and vitrification methods. A cancer patient conceived and delivered her own babies by this technology after recovery from the disease. Oocyte cryopreservation became a new focus in assisted reproductive technology. We witnessed the advanced development of human oocyte cryopreservation in the past years because of increasing demand, medically, legally and ethically, and also because of the dramatic improvement of the freezing technique. There is still a long way to go to integrate it into a routine clinical procedure to benefit more patients and encourage clinicians to follow the standard protocols.

  8. Gait Disturbances in Dystrophic Hamsters

    Directory of Open Access Journals (Sweden)

    Thomas G. Hampton


    Full Text Available The delta-sarcoglycan-deficient hamster is an excellent model to study muscular dystrophy. Gait disturbances, important clinically, have not been described in this animal model. We applied ventral plane videography (DigiGait to analyze gait in BIO TO-2 dystrophic and BIO F1B control hamsters walking on a transparent treadmill belt. Stride length was ~13% shorter (<.05 in TO-2 hamsters at 9 months of age compared to F1B hamsters. Hindlimb propulsion duration, an indicator of muscle strength, was shorter in 9-month-old TO-2 (247±8 ms compared to F1B hamsters (272±11 ms; <.05. Braking duration, reflecting generation of ground reaction forces, was delayed in 9-month-old TO-2 (147±6 ms compared to F1B hamsters (126±8 ms; <.05. Hindpaw eversion, evidence of muscle weakness, was greater in 9-month-old TO-2 than in F1B hamsters (17.7±1.2∘ versus 8.7±1.6∘; <.05. Incline and decline walking aggravated gait disturbances in TO-2 hamsters at 3 months of age. Several gait deficits were apparent in TO-2 hamsters at 1 month of age. Quantitative gait analysis demonstrates that dystrophic TO-2 hamsters recapitulate functional aspects of human muscular dystrophy. Early detection of gait abnormalities in a convenient animal model may accelerate the development of therapies for muscular dystrophy.

  9. Coenzyme Q10 supplementation during in vitro maturation of bovine oocytes (Bos taurus) helps to preserve oocyte integrity after vitrification. (United States)

    Ruiz-Conca, M; Vendrell, M; Sabés-Alsina, M; Mogas, T; Lopez-Bejar, M


    Oocyte vitrification causes less cell stress than slow cooling, but cytoskeletal and spindle alterations may occur affecting the oocyte competence. In vitro maturation (IVM) supplementation with different antioxidant molecules has been performed to attenuate this harmful stress. Coenzyme Q10 (CoQ10 ) supplementation has previously shown to have positive effects in bovine and mouse in vitro embryo development. The aim of this study was to evaluate the effects of CoQ10 during bovine oocyte IVM and vitrification. Cumulus-oocyte complexes (COCs) (n = 311) were cultured under standard maturation conditions with 0 μM (control), 25 μM and 50 μM CoQ10 supplementation. After 22 hr, a cohort of 170 oocytes both from the control and from CoQ10 -supplemented groups were vitrified, warmed and returned to incubation until 24 hr of maturation, while the rest of the oocytes (n = 141) remained fresh. Then, oocyte survival was assessed morphologically by stereomicroscopy. Oocytes from all groups were then fixed and stained for assessing cortical granules (CG) migration and nuclear stage. High rates of oocyte MII progression and appropriate CG migration as a continuous layer beneath the plasma membrane were obtained both in control and in CoQ10 groups. Results showed that although vitrification has great impact in survival of IVM bovine oocytes, 50 μM CoQ10 supplementation significantly improved oocyte survival (p = .045) and reduced the premature CG exocytosis, helping to preserve the CG migration pattern (31.3% control vs. 54.5% in 50 μM CoQ10 ; p = .039), attenuating the negative effects of vitrification. © 2017 Blackwell Verlag GmbH.

  10. Expression of estrogen receptor α 36 (ESR36 in the hamster ovary throughout the estrous cycle: effects of gonadotropins.

    Directory of Open Access Journals (Sweden)

    Prabuddha Chakraborty

    Full Text Available Estradiol-17β (E plays an important role in ovarian follicular development. Evidence indicates that some of the effect of E is mediated by the transmembrane estrogen receptor. In this study, we examined the spatio-temporal expression of recently discovered ERα36 (ESR36, a splice variant of Esr1 and a receptor for non-genomic E signaling, in the hamster ovary during the estrous cycle and the role of gonadotropins and ovarian steroid hormones in ESR36 expression. ESR36 expression was high on estrus (D1:0900 h and declined precipitously by proestrus (D4:0900 h and remained low up to D4:1600 h. Immunofluorescence findings corroborated immunoblot findings and revealed that ESR36 was expressed only in the cell membrane of both follicular and non-follicular cells, except the oocytes. Ovarian ESR36 was capable of binding to the E-affinity matrix, and have different molecular weight than that of the ESR1 or GPER. Hypophysectomy (Hx resulted in a marked decline in ESR36 protein levels. FSH and LH, alone or combined, markedly upregulated ESR36 protein in Hx hamsters to the levels observed in D1 hamsters, but neither E nor P had any effect. Inhibition of the gonadotropin surge by phenobarbital treatment on D4:1100 h attenuated ESR36 expression in D1:0900 h ovaries, but the decline was restored by either FSH or LH replacement on D4 afternoon. This is the first report to show that ESR36, which is distinct from ESR1 or GPER is expressed in the plasma membrane of ovarian follicular and non-follicular cells, binds to E and its expression is regulated directly by the gonadotropins. In light of our previous findings, the results suggest that ovarian cells contain at least two distinct membrane estrogen receptors, such as GPER and ESR36, and strongly suggest for a non-genomic action of E regulating ovarian follicular functions.

  11. Role of animal pole protuberance and microtubules during meiosis in sea cucumber Apostichopus japonicus oocytes (United States)

    Pang, Zhenguo; Chang, Yaqing; Sun, Huiling; Yu, Jiaping


    Fully grown oocytes of Apostichopus japonicus have a cytoplasmic protuberance where the oocyte attaches to the follicle. The protuberance and the oolamina located on the opposite side of the oocyte indicate the animal-vegetal axis. Two pre-meiotic centrosomes are anchored to the protuberance by microtubules between centrosomes and protuberance. After meiosis reinitiation induced by DTT solution, the germinal vesicle (GV) migrates towards the protuberance. The GV breaks down after it migrates to the oocyte membrane on the protuberance side. The protuberance then contracts back into the oocyte and the first polar body extrudes from the site of the former protuberance. The second polar body forms beneath the first. Thus the oocyte protuberance indicates the presumptive animal pole well before maturation of the oocyte.

  12. Membrane lipid profile of in vitro-produced embryos is affected by vitrification but not by long-term dietary supplementation of polyunsaturated fatty acids for oocyte donor beef heifers. (United States)

    Leão, Beatriz C S; Rocha-Frigoni, Nathália A S; Nogueira, Ériklis; Cabral, Elaine C; Ferreira, Christina R; Eberlin, Marcos N; Accorsi, Mônica F; Neves, Thiago V; Mingoti, Gisele Z


    Dietary rumen-protected polyunsaturated fatty acids (PUFAs) rich in linoleic acid (LA) may affect embryo yield, and LA can modulate the molecular mechanisms of lipid uptake in bovine blastocysts produced in vitro. In embryos, membrane lipids, such as phosphatidylcholines (PCs) and sphingomyelins (SMs), affect cryopreservation success. The aim of the present study was to evaluate embryonic developmental rates after the IVF of oocytes retrieved from Nellore heifers fed for approximately 90 days with rumen-protected PUFAs rich in LA. In addition, we evaluated embryo cryotolerance and the membrane structure lipid composition using matrix-assisted laser desorption ionisation mass spectrometry of fresh and vitrified embryos. Embryo development to the blastocyst stage (mean 43.2%) and embryo survival after vitrification and warming (mean 79.3%) were unaffected by diet. The relative abundance of one lipid species (PC ether (PCe; 38:2, which means that this lipid has 38 carbon atoms and 2 double bonds in the fatty acyl residues) was increased after PUFAs supplementation. However, 10 ions were affected by cryopreservation; ions consistent with PC 32:0, PC 34:1, SM 24:1, PC 40:6 or PC 42:9, PC plasmalogen (PCp) 44:10 or PC 42:7, triacylglycerol (TAG) 54:9 and a not assigned ion (m/z 833.2) were lower in blastocysts that survived to the cryopreservation process compared with fresh blastocysts, whereas the abundance of the ions PC 36:3 or PC 34:0, PCe 38:2 or PC 36:6 and PC 36:5 or PCe 38:1 were increased after cryopreservation. Thus, the results demonstrate that the mass spectrometry profiles of PC, SM and TAG species differ significantly in bovine blastocysts upon cryopreservation. Because the lipid ion abundances of fresh and vitrified-warmed embryos were distinct, they can be used as potential markers of post-cryopreservation embryonic survival.

  13. Conjugated linoleic acid improves oocyte cryosurvival through modulation of the cryoprotectants influx rate. (United States)

    Matos, Joana E; Marques, Carla C; Moura, Teresa F; Baptista, Maria C; Horta, Antonio E M; Soveral, Graça; Pereira, Rosa M L N


    In cryopreservation, oocytes are subjected to extreme hyperosmotic conditions, inducing large volume changes that, along with an abrupt temperature drop, interfere with their developmental competence. Our objectives in this work were to find conditions enabling an increase in oocyte cryosurvival and subsequent development. Abattoir-derived bovine oocytes were cultured without (Control group) or with trans-10,cis-12 conjugated linoleic acid isomer (CLA group). Comparative observations were made for 1) the oocyte developmental competence after exposure to cryoprotectants followed or not by vitrification/warming, 2) the oocyte membrane permeability to water (using the non-permeant cryoprotectant sucrose) and 3) the oocyte membrane permeability to two cryoprotectants (ethylene glycol, EG, and dimethyl sulfoxide, DMSO). Mature oocytes cultured with or without CLA and vitrified/warmed or only exposed to cryoprotectants without vitrification were subjected to in vitro fertilization; embryo culture proceeded until the blastocyst stage. The oocyte membrane permeabilities to water and cryoprotectants were estimated using mature oocytes subjected to hyperosmotic challenges. For water permeability, 200 mM sucrose was used, whereas for the cryoprotectant permeability, a 10 % solution of both EG and DMSO was used. The data were analyzed using the MIXED procedure and Student's T-test. CLA supplementation improves the developmental competence of vitrified/warmed and cryoprotectants exposed oocytes (p < 0.01) and reduces their membrane permeability to water (37 %, p < 0.001) and to cryoprotectants (42 %, p < 0.001). By slowing the fluxes of water and of permeant cryoprotectants, CLA contributed to improved oocyte cryosurvival and post-thawed viability. This isomer supplementation to the maturation media should be considered when designing new protocols for oocyte cryopreservation.

  14. Mouse Oocytes Acquire Mechanisms That Permit Independent Cell Volume Regulation at the End of Oogenesis. (United States)

    Richard, Samantha; Tartia, Alina P; Boison, Detlev; Baltz, Jay M


    Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1. In fully-grown oocytes, release of adhesion and GLYT1 activation also occur spontaneously in oocytes removed from the follicle. It is unknown, however, whether the capacity to release oocyte-ZP adhesion or activate GLYT1 first arises in the oocyte after ovulation is triggered or instead growing oocytes already possess these capabilities but they are suppressed in the follicle. Here, we assessed when during oogenesis oocyte-ZP adhesion can be released and when GLYT1 can be activated, with adhesion assessed by an osmotic assay and GLYT1 activity determined by [ 3 H]-glycine uptake. Oocyte-ZP adhesion could not be released by growing oocytes until they were nearly fully grown. Similarly, the amount of GLYT1 activity that can be elicited in oocytes increased sharply at the end of oogenesis. The SLC6A9 protein that is responsible for GLYT1 activity and Slc6a9 transcripts are present in growing oocytes and increased over the course of oogenesis. Furthermore, SLC6A9 becomes localized to the oocyte plasma membrane as the oocyte grows. Thus, oocytes acquire the ability to regulate their cell volume by releasing adhesion to the ZP and activating GLYT1 as they approach the end of oogenesis. J. Cell. Physiol. 232: 2436-2446, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Light and transmission electron microscopy of immature camelus dromedarius oocyte. (United States)

    Nili, H; Mesbah, F; Kafi, M; Nasr Esfahani, M H


    In order to provide a consistent system for laboratory production of embryos, the characteristics of immature camel oocyte must first be described. The objective of this study was to define ultrastructural features of immature camel oocyte. Ovaries were obtained from camels at a local abattoir, and then transported to the laboratory within 2 h. Camelus cumulus oocyte complexes (COCs) were aspirated from 2-6 mm follicles using a 22-gauge needle. Excellent and good quality COCs were selected and prepared for transmission electron microscopy study using a cavity slide. The fine structure of camel oocyte is morphologically similar to that of other mammalian oocytes. However, some minor differences exist between COC of camel and other mammalian species. Different size and shape of membrane-bound vesicles, lipid droplet, mitochondria and cortical granules were distributed throughout the ooplasm. Discrete or in association with endoplasmic reticulum, Golgi complexes were observed in the periphery of the oocytes. The majority of the oocytes were in the germinal vesicle stage.

  16. Development capacity of pre- and postpubertal pig oocytes evaluated by somatic cell nuclear transfer and parthenogenetic activation

    DEFF Research Database (Denmark)

    Skovsgaard, Hanne; Li, Rong; Liu, Ying


    Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear...... transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible...... for the development of good oocytes after PA. The results show a low CL% of small-gilts compared with the other groups. The BL% increased with gilt-oocyte-diameter; however, sow oocytes reached the highest BL%. Total cell number was higher in sow than in gilt blastocysts. The SCNT experiments showed no differences...

  17. Acquisition of Oocyte Polarity. (United States)

    Clapp, Mara; Marlow, Florence L


    Acquisition of oocyte polarity involves complex translocation and aggregation of intracellular organelles, RNAs, and proteins, along with strict posttranscriptional regulation. While much is still unknown regarding the formation of the animal-vegetal axis, an early marker of polarity, animal models have contributed to our understanding of these early processes controlling normal oogenesis and embryo development. In recent years, it has become clear that proteins with self-assembling properties are involved in assembling discrete subcellular compartments or domains underlying subcellular asymmetries in the early mitotic and meiotic cells of the female germline. These include asymmetries in duplication of the centrioles and formation of centrosomes and assembly of the organelle and RNA-rich Balbiani body, which plays a critical role in oocyte polarity. Notably, at specific stages of germline development, these transient structures in oocytes are temporally coincident and align with asymmetries in the position and arrangement of nuclear components, such as the nuclear pore and the chromosomal bouquet and the centrioles and cytoskeleton in the cytoplasm. Formation of these critical, transient structures and arrangements involves microtubule pathways, intrinsically disordered proteins (proteins with domains that tend to be fluid or lack a rigid ordered three-dimensional structure ranging from random coils, globular domains, to completely unstructured proteins), and translational repressors and activators. This review aims to examine recent literature and key players in oocyte polarity.

  18. Meiotic maturation and developmental capability of ovine oocytes at germinal vesicle stage following vitrification using different cryodevices. (United States)

    Quan, Guo Bo; Wu, Guo Quan; Wang, Ya Jing; Ma, Yuan; Lv, Chun Rong; Hong, Qiong Hua


    In order to assess effects of vitrification on ovine oocytes at the germinal vesicle (GV) stage, the conventional plastic straw (CS), the open-pulled straw (OPS), and Cryoloop were used to vitrify ovine oocytes. Oocytes were randomly divided into five groups: (1) Control; (2) Oocytes exposed to vitrification and dilution solutions without any cryopreservation (toxicity); (3) Oocytes vitrified using CS (CS); (4) Oocytes vitrified using OPS (OPS), and (5) Oocytes vitrified using Cryoloop (Cryoloop). The viability, cumulus cell expansion, nuclear maturation after in vitro maturation (IVM), and developmental capability of vitrified oocytes following parthenogenetic activation (PA) or in vitro fertilization (IVF) were assessed. The pretreatment in the vitrification and dilution solutions without any freezing or thawing did not adversely influence oocytes. The viability of vitrified oocytes were significantly declined compared to unfrozen oocytes (P straws or Cryoloop was significantly higher than that in the CS group (P plastic straws was significantly less than those of the other freezing groups (P straws. However, the cleavage rate of vitrified oocytes in the CS group was significantly less than that in the OPS or Cryoloop group (P plastic straw developed to the blastocyst stage following IVF. There was no significant difference existing between OPS and Cryoloop with respect to the blastocyst rate. After staining with cFDA and PI, cumulus cells surrounding oocytes were partly damaged by vitrification and thawing while the membrane of vitrified oocyte still remained intact. In conclusion, vitrification can seriously damage ovine immature oocytes and cumulus cells surrounding oocytes, which may subsequently affect their developmental capability. Finally, this study further proves that increasing the freezing and thawing velocity benefits survival of vitrified immature oocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn


    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  20. Development block of golden hamster ICSI embryos is associated with decreased expression of HDAC1, HSPA1A and MYC. (United States)

    Pan, Xiaoyan; Kong, Delong; Liu, Limei; Gao, Fei; Zhang, Xueming; Tang, Bo; Li, Ziyi


    We have investigated the mechanism for embryo development block in vitro and to improve the development rate of golden hamster embryos in vitro. Intracytoplasmic sperm injection (ICSI) technique was used to produce golden hamster ICSI embryos. The changes in the histone acetylation and the expression of histone deacetylase and related genes were analyzed by immunocytochemical staining and real-time PCR both in golden hamster in vivo embryos and in ICSI embryos. Aged oocytes significantly increased the oocyte spontaneous activation rate. In vitro cultured ICSI embryos suffered from severe development block in M199TE medium. Expression of histone deacetylase 1 (HDAC1) was significantly decreased in the nuclei of the arrested ICSI 2-cell embryos, and its nuclear and cytoplasmic expression pattern was also markedly altered. The acetylation level of H4K5, however, was not significantly changed between golden hamster in vivo embryos and ICSI embryos. HSPA1A and MYC, the marker genes for zygotic genome activation (ZGA), were transcriptionally decreased in arrested ICSI 2-cell embryos. Transcription of HDAC1 was also downregulated in these embryos, whereas the mRNA expression of the proapoptotic gene, BAX, was not changed. These results indicate that the golden hamster ICSI embryo development block during ZGA is associated with decreased nuclear expression and altered expression of HDAC1. HSPA1A, MYC, and HDAC1 mRNA levels, which decrease, resulting in ZGA failure. © 2014 International Federation for Cell Biology.

  1. Observations Regardin Oocyte in Vitro Maturation after Recovery from Slaughter House Females

    Directory of Open Access Journals (Sweden)

    Valeriu Carabă


    Full Text Available The oocytes viability must be taken as an important selection parameter for successful in vitro cultivation. The ovaries were collected from the slaughterhouse and maintained at 4°C for 7 days. Fallowing cumulus -oocytes complexes recovery the viability was tested using two staining methods. For the first experiment we used 27 cumulus - oocytes complexes, stained with Neutral red and for the second experiment we used 11 cumulus - oocytes complexes stained with Trypan blue. Fallowing staining with Neutral red 23 cumulus - oocytes complexes were assessed as viable (were stained in red – enzymatic activity within the cells and for the Trypan blue staining 11 cumulus - oocytes complexes were assessed as viable (remained unstained – integers cellular membranes.

  2. Cholesterol added prior to vitrification on the cryotolerance of immature and in vitro matured bovine oocytes. (United States)

    Arcarons, Núria; Morató, Roser; Vendrell, Meritxell; Yeste, Marc; López-Bejar, Manel; Rajapaksha, Kosala; Anzar, Muhammad; Mogas, Teresa


    This study examines whether incorporating cholesterol-loaded methyl-β-cyclodextrin (CLC) in the bovine oocyte plasma membrane improves oocyte tolerance to vitrification. In vitro matured oocytes were incubated with 2 mg/ml BODIPY-labeled CLC for different time intervals in FCS or PVA supplemented medium or exposed to different CLC concentrations to examine the subcellular localization of cholesterol by confocal microscopy live-cell imaging. Subsequently, the effects of optimized CLC concentrations and incubation times prior to vitrification on early embryo development were assessed. Then, we evaluated the effects of pretreatment with 2 mg/ml CLC for 30 min before the vitrification of immature (GV) and in vitro matured (MII) oocytes on developmental competence and gene expression. Our results indicate a high plasma membrane labeling intensity after 30 min of incubation with 2 mg/ml CLC for 30 min, regardless of the holding medium used. When oocytes were incubated with 1 mg/ml, 2 mg/ml and 3 mg/ml of CLC, intense labeling was observed at the plasma membrane after 40, 30 and 20 min, respectively. CLC pre-treatment before the vitrification of bovine oocytes did not affect subsequent cleavage and embryo development rates irrespective of CLC concentrations, incubation times or meiotic stage. However, pretreatment seems to improve the quality of embryos derived from vitrified oocytes, mainly when oocytes were vitrified at the GV stage.

  3. Cholesterol added prior to vitrification on the cryotolerance of immature and in vitro matured bovine oocytes.

    Directory of Open Access Journals (Sweden)

    Núria Arcarons

    Full Text Available This study examines whether incorporating cholesterol-loaded methyl-β-cyclodextrin (CLC in the bovine oocyte plasma membrane improves oocyte tolerance to vitrification. In vitro matured oocytes were incubated with 2 mg/ml BODIPY-labeled CLC for different time intervals in FCS or PVA supplemented medium or exposed to different CLC concentrations to examine the subcellular localization of cholesterol by confocal microscopy live-cell imaging. Subsequently, the effects of optimized CLC concentrations and incubation times prior to vitrification on early embryo development were assessed. Then, we evaluated the effects of pretreatment with 2 mg/ml CLC for 30 min before the vitrification of immature (GV and in vitro matured (MII oocytes on developmental competence and gene expression. Our results indicate a high plasma membrane labeling intensity after 30 min of incubation with 2 mg/ml CLC for 30 min, regardless of the holding medium used. When oocytes were incubated with 1 mg/ml, 2 mg/ml and 3 mg/ml of CLC, intense labeling was observed at the plasma membrane after 40, 30 and 20 min, respectively. CLC pre-treatment before the vitrification of bovine oocytes did not affect subsequent cleavage and embryo development rates irrespective of CLC concentrations, incubation times or meiotic stage. However, pretreatment seems to improve the quality of embryos derived from vitrified oocytes, mainly when oocytes were vitrified at the GV stage.

  4. Diving into the oocyte pool

    DEFF Research Database (Denmark)

    Kristensen, Stine G; Pors, Susanne E; Andersen, Claus Y


    PURPOSE OF REVIEW: The ovarian reserve comprises an enormous surplus of follicles. Despite this, some women produce insufficient numbers of oocytes by conventional fertility treatments. However, recent technical accomplishments may transform assisted reproductive technology (ART) in such a way...... of the signaling pathways activating dormant follicles and breakthroughs in techniques for autologous transfer of mitochondria have opened new doors to unexploited sources of oocytes and attractive ways of revitalizing oocytes. Extended numbers of mature oocytes may be obtained by in-vitro activation of dormant...... follicles in cortical biopsies or in-vitro maturation of immature oocytes during the natural or stimulated cycle, and used directly for fertility treatment or as a source of autologous mitochondria. SUMMARY: New approaches utilizing the abundant resources of immature oocytes combined with techniques...

  5. Finite element model to study calcium distribution in oocytes ...

    African Journals Online (AJOL)

    Parvaiz Ahmad Naik


    Mar 20, 2015 ... RyR & VGCC can significantly raise the calcium concentration level in the oocyte cell in order to initiate, sustain and terminate ... Ca2+ waves are dependent on the diffusion of Ca2+ ions both within and possibly between the .... Vm is the membrane potential. R is gas constant and T is absolute temperature.

  6. Finite element model to study calcium distribution in oocytes ...

    African Journals Online (AJOL)

    Parvaiz Ahmad Naik


    Mar 20, 2015 ... ing species studied to date from plants to humans.4,6 The fer- tilization ... mathematical model of simulation of spontaneous Ca2+ oscil- lations in ..... Membrane potential. А0:05 V. zCa. Valency of calcium. 2. VOocyte. Volume of oocyte cytosol. 5:48 В 10А11 l. F. Faraday's constant. 96487 C=mole. R.

  7. In vivo induction of oocyte maturation and ovulation in zebrafish.

    Directory of Open Access Journals (Sweden)

    Toshinobu Tokumoto

    Full Text Available The maturation of fish oocytes is a well-characterized system induced by progestins via non-genomic actions. In a previous study, we demonstrated that diethylstilbestrol (DES, a non-steroidal estrogen, induces fish oocyte maturation via the membrane progestin receptor (mPR. Here, we attempted to evaluate the effect of DES as an environmental endocrine disrupting chemical (EDC upon fish oocyte maturation using live zebrafish. DES triggered oocyte maturation within several hours in vivo when administrated directly into the surrounding water. The natural teleost maturation-inducing hormone, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP also induced oocyte maturation in vivo. Steroids such as testosterone, progesterone or 17alpha-hydroxyprogesterone were also effective in vivo. Further studies indicated that externally applied 17,20beta-DHP even induced ovulation. In contrast to 17,20beta -DHP, DES induced maturation but not ovulation. Theoretically this assay system provides a means to distinguish pathways involved in the induction of ovulation, which are known to be induced by genomic actions from the pathway normally involved in the induction of oocyte maturation, a typical non-genomic action-dependent pathway. In summary, we have demonstrated the effect of EDCs on fish oocyte maturation in vivo. To address the effects, we have explored a conceptually new approach to distinguish between the genomic and non-genomic actions induced by steroids. The assay can be applied to screens of progestin-like effects upon oocyte maturation and ovulation for small molecules of pharmacological agents or EDCs.

  8. G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio. (United States)

    Majumder, Suravi; Das, Sumana; Moulik, Sujata Roy; Mallick, Buddhadev; Pal, Puja; Mukherjee, Dilip


    GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17β-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20β-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Molecular and Cellular Mechanisms of Sperm-Oocyte Interactions Opinions Relative to in Vitro Fertilization (IVF

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    George Anifandis


    Full Text Available One of the biggest prerequisites for pregnancy is the fertilization step, where a human haploid spermatozoon interacts and penetrates one haploid oocyte in order to produce the diploid zygote. Although fertilization is defined by the presence of two pronuclei and the extraction of the second polar body the process itself requires preparation of both gametes for fertilization to take place at a specific time. These preparations include a number of consecutive biochemical and molecular events with the help of specific molecules and with the consequential interaction between the two gametes. These events take place at three different levels and in a precise order, where the moving spermatozoon penetrates (a the outer vestments of the oocyte, known as the cumulus cell layer; (b the zona pellucida (ZP; where exocytosis of the acrosome contents take place and (c direct interaction of the spermatozoon with the plasma membrane of the oocyte, which involves a firm adhesion of the head of the spermatozoon with the oocyte plasma membrane that culminates with the fusion of both sperm and oocyte membranes (Part I. After the above interactions, a cascade of molecular signal transductions is initiated which results in oocyte activation. Soon after the entry of the first spermatozoon into the oocyte and oocyte activation, the oocyte’s coat (the ZP and the oocyte’s plasma membrane seem to change quickly in order to initiate a fast block to a second spermatozoon (Part II. Sometimes, two spermatozoa fuse with one oocyte, an incidence of 1%–2%, resulting in polyploid fetuses that account for up to 10%–20% of spontaneously aborted human conceptuses. The present review aims to focus on the first part of the human sperm and oocyte interactions, emphasizing the latest molecular and cellular mechanisms controlling this process.

  10. Subcellular distribution of the Xenopus p58/lamin B receptor in oocytes and eggs. (United States)

    Gajewski, A; Krohne, G


    The p58/lamin B receptor of vertebrates is localized in the inner nuclear membrane. Antibodies raised against the bacterially expressed amino-terminal half of Xenopus p58 (Xp58) revealed that in Xenopus oocytes the vast majority of this membrane protein is localized in cytoplasmic membranes. Only very small amounts of p58 not detectable by immunofluorescence microscopy were contained in the oocyte nuclear envelope. In contrast, nuclear membranes of 2-cell stage embryos were successfully stained with p58 antibodies, nuclei reconstituted in vitro in Xenopus egg extracts contained p58, and the nucleoplasmic domain of Xp58 could be specifically bound to sperm chromatin in vitro. One major difference between oocytes and early embryonic cells is that no chromatin is associated with the oocyte inner nuclear membrane whereas the complement of lamins is identical in both cell types. To gain insight into the properties of oocyte p58 we microinjected isolated nuclei of cultured rat cells into the cytoplasm of Xenopus oocytes. The oocyte p58 was detectable by immunofluorescence microscopy within 16-20 hours in the nuclear membrane of rat nuclei. Our data indicate that the peripheral chromatin but not lamins are required for the retention of p58 in the inner nuclear membrane. Sucrose step gradient centrifugation of total oocyte membranes revealed that the oocyte p58 was predominantly recovered in membrane fractions that did not contain lamins whereas membrane associated lamins and p58 of unfertilized eggs were found in the same fractions. By electron microscopical immunolocalizations one major population of meiotic p58 vesicles was identified that contained exclusively p58 and a second minor population (ca. 11% of p58 vesicles) contained in addition to p58 membrane bound B-type lamins. Egg vesicles containing pore membrane proteins were predominantly recovered in gradient fractions that did not contain p58 and B-type lamins. Our data indicate that the targeting of p58 to

  11. Ultrastructure of previtellogene oocytes in the neotenic cave salamander Proteus anguinus anguinus (Amphibia, Urodela, Proteidae). (United States)

    Mali, Lilijana Bizjak; Bulog, Boris


    Oogenesis in the neotenic, cave dwelling salamander Proteus anguinus anguinus has not been studied yet, and this study provides a detailed description of the early growth of the oocytes. Early previtellogene oocytes ranging from 100 to 600 µm in diameter were examined by light and transmission electron microscopy. The oocytes were divided into two stages based on size, color, and histology. Stage I oocytes can be identified by their transparent cytoplasm and a homogenous juxtanuclear mass, composed of numerous lipid droplets and mitochondria. Stage II oocytes are no longer transparent and have increased in diameter to 300- 600 µm, and many cortical alveoli differing in size have appeared. The common and most predominant ultrastructural characteristics of both stages of previtellogene oocytes are extensive quantities of smooth membrane, numerous mitochondria, and lipid droplets, as well as abundant free ribosomes. Myeline-like structures and remarkable annulate lamellae of closely packed membrane stacks are also frequently observed. Previtellogenic oocytes are the most predominant oocytes in the ovaries of Proteus, and while they possess certain structural characteristics typical for other amphibians, some features are unique and could result from adaptation to the subterranean environment.

  12. Obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy. (United States)

    Boudoures, Anna L; Saben, Jessica; Drury, Andrea; Scheaffer, Suzanne; Modi, Zeel; Zhang, Wendy; Moley, Kelle H


    Mitochondria are the most prominent organelle in the oocyte. Somatic cells maintain a healthy population of mitochondria by degrading damaged mitochondria via mitophagy, a specialized autophagy pathway. However, evidence from previous work investigating the more general macroautophagy pathway in oocytes suggests that mitophagy may not be active in the oocyte. This would leave the vast numbers of mitochondria - poised to be inherited by the offspring - vulnerable to damage. Here we test the hypothesis that inactive mitophagy in the oocyte underlies maternal transmission of dysfunctional mitochondria. To determine whether oocytes can complete mitophagy, we used either CCCP or AntimycinA to depolarize mitochondria and trigger mitophagy. After depolarization, we did not detect co-localization of mitochondria with autophagosomes and mitochondrial DNA copy number remained unchanged, indicating the non-functional mitochondrial population was not removed. To investigate the impact of an absence of mitophagy in oocytes with damaged mitochondria on offspring mitochondrial function, we utilized in vitro fertilization of high fat high sugar (HF/HS)-exposed oocytes, which have lower mitochondrial membrane potential and damaged mitochondria. Here, we demonstrate that blastocysts generated from HF/HS oocytes have decreased mitochondrial membrane potential, lower metabolites involved in ATP generation, and accumulation of PINK1, a mitophagy marker protein. This mitochondrial phenotype in the blastocyst mirrors the phenotype we show in HF/HS exposed oocytes. Taken together, these data suggest that the mechanisms governing oocyte mitophagy are fundamentally distinct from those governing somatic cell mitophagy and that the absence of mitophagy in the setting of HF/HS exposure contributes to the oocyte-to-blastocyst transmission of dysfunctional mitochondria. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Regulatory contribution of heterotrimeric G-proteins to oocyte maturation in the sea urchin. (United States)

    Voronina, Ekaterina; Wessel, Gary M


    Regulation of animal oocyte maturation is hypothesized to involve heterotrimeric G-proteins. It is difficult to test this hypothesis though without knowing what G-proteins are present in these cells and where are they localized. We set out to test the hypothesis that G-proteins regulate maturation in the sea urchin oocyte by identifying resident G-proteins in oocytes and eggs, and then investigating their function. We find four families of G-protein alpha-subunits (Galphai, Galphaq, Galphas, and Galpha12) present in both oocytes and eggs of the sea urchin. Three of them, Galphai, Galphaq, and Galphas are present on the plasma membrane of the oocyte, while the fourth is located on cytoplasmic vesicles. Upon oocyte maturation, these proteins remain in eggs, and continue to be expressed in embryonic tissues. To test the functional contribution of the G-proteins to the regulation of oocyte maturation, we employ specific intervening reagents, including antibodies and competitor peptides to each Galpha subunit, and specific Galpha toxins. We find that Gi is a main candidate for a positive regulator of sea urchin oocyte maturation. These studies provide a foundation to further test specific hypotheses of the G-protein mediated regulation of oocyte maturation, fertilization, and early development in the sea urchin.

  14. Dimethyl Sulfoxide Perturbs Cell Cycle Progression and Spindle Organization in Porcine Meiotic Oocytes.

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    Xuan Li

    Full Text Available Meiotic maturation of mammalian oocytes is a precisely orchestrated and complex process. Dimethyl sulfoxide (DMSO, a widely used solvent, drug, and cryoprotectant, is capable of disturbing asymmetric cytokinesis of oocyte meiosis in mice. However, in pigs, DMSO's effect on oocyte meiosis still remains unknown. We aimed to evaluate if DMSO treatment will affect porcine oocyte meiosis and the underlying molecular changes as well. Interestingly, we did not observe the formation of the large first polar body and symmetric division for porcine oocytes treated with DMSO, contrary to findings reported in mice. 3% DMSO treatment could inhibit cumulus expansion, increase nuclear abnormality, disturb spindle organization, decrease reactive oxygen species level, and elevate mitochondrial membrane potential of porcine oocytes. There was no effect on germinal vesicle breakdown rate regardless of DMSO concentration. 3% DMSO treatment did not affect expression of genes involved in spindle organization (Bub1 and Mad2 and apoptosis (NF-κB, Pten, Bcl2, Caspase3 and Caspase9, however, it significantly decreased expression levels of pluripotency genes (Oct4, Sox2 and Lin28 in mature oocytes. Therefore, we demonstrated that disturbed cumulus expansion, chromosome alignment, spindle organization and pluripotency gene expression could be responsible for DMSO-induced porcine oocyte meiotic arrest and the lower capacity of subsequent embryo development. Our results provide new insights on DMSO's effect on porcine oocyte meiosis and raise safety concerns over DMSO's usage on female reproduction in both farm animals and humans.

  15. Significant Down-Regulation of “Biological Adhesion” Genes in Porcine Oocytes after IVM

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    Joanna Budna


    Full Text Available Proper maturation of the mammalian oocyte is a compound processes determining successful monospermic fertilization, however the number of fully mature porcine oocytes is still unsatisfactory. Since oocytes’ maturation and fertilization involve cellular adhesion and membranous contact, the aim was to investigate cell adhesion ontology group in porcine oocytes. The oocytes were collected from ovaries of 45 pubertal crossbred Landrace gilts and subjected to two BCB tests. After the first test, only granulosa cell-free BCB+ oocytes were directly exposed to microarray assays and RT-qPCR (“before IVM” group, or first in vitro matured and then if classified as BCB+ passed to molecular analyses (“after IVM” group. As a result, we have discovered substantial down-regulation of genes involved in adhesion processes, such as: organization of actin cytoskeleton, migration, proliferation, differentiation, apoptosis, survival or angiogenesis in porcine oocytes after IVM, compared to oocytes analyzed before IVM. In conclusion, we found that biological adhesion may be recognized as the process involved in porcine oocytes’ successful IVM. Down-regulation of genes included in this ontology group in immature oocytes after IVM points to their unique function in oocyte’s achievement of fully mature stages. Thus, results indicated new molecular markers involved in porcine oocyte IVM, displaying essential roles in biological adhesion processes.

  16. Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes. (United States)

    Nakhoul, N L; Davis, B A; Romero, M F; Boron, W F


    It is generally accepted that gases such as CO2 cross cell membranes by dissolving in the membrane lipid. No role for channels or pores in gas transport has ever been demonstrated. Here we ask whether expression of the water channel aquaporin-1 (AQP1) enhances the CO2 permeability of Xenopus oocytes. We expressed AQP1 in Xenopus oocytes by injecting AQP1 cRNA, and we assessed CO2 permeability by using microelectrodes to monitor the changes in intracellular pH (pHi) produced by adding 1.5% CO2/10 mM HCO3- to (or removing it from) the extracellular solution. Oocytes normally have an undetectably low level of carbonic anhydrase (CA), which eliminates the CO2 hydration reaction as a rate-limiting step. We found that expressing AQP1 (vs. injecting water) had no measurable effect on the rate of CO2-induced pHi changes in such low-CA oocytes: adding CO2 caused pHi to fall at a mean initial rate of 11.3 x 10(-4) pH units/s in control oocytes and 13.3 x 10(-4) pH units/s in oocytes expressing AQP1. When we injected oocytes with water, and a few days later with CA, the CO2-induced pHi changes in these water/CA oocytes were more than fourfold faster than in water-injected oocytes (acidification rate, 53 x 10(-4) pH units/s). Ethoxzolamide (ETX; 10 microM), a membrane-permeant CA inhibitor, greatly slowed the pHi changes (16.5 x 10(-4) pH units/s). When we injected oocytes with AQP1 cRNA and then CA, the CO2-induced pHi changes in these AQP1/CA oocytes were approximately 40% faster than in the water/CA oocytes (75 x 10(-4) pH units/s), and ETX reduced the rates substantially (14.7 x 10(-4) pH units/s). Thus, in the presence of CA, AQP1 expression significantly increases the CO2 permeability of oocyte membranes. Possible explanations include 1) AQP1 expression alters the lipid composition of the cell membrane, 2) AQP1 expression causes overexpression of a native gas channel, and/or 3) AQP1 acts as a channel through which CO2 can permeate. Even if AQP1 should mediate a CO2 flux

  17. Mutation induction in synchronous hamster cells

    Energy Technology Data Exchange (ETDEWEB)

    Aebersold, P.M.


    Mutagenesis of synchronous Mutahinese hamster cells by 5-bromodeoxyuridine (BUdR) shows pronounced cell cycle dependency. Resistance to 6-thioguanine (6-TG) and ouabain are induced maximally by BUdR at different times early in the DNA synthesis period, suggesting that the genes coding for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and the (Na/sup +/K/sup +/)-associated ATPase of the plasma membrane are replicated early in the DNA synthesis period. Although BUdR induces mutations in specific genes only when present during their replication, the rate of mutation induction is not linearly related to the amount of BUdR incorporated into DNA. The data show a BUdR concentration threshold for mutation induction, suggesting that BUdR exerts some deterimental allosteric effect on DNA synthesis enzymes.

  18. The beneficial effects of antifreeze proteins in the vitrification of immature mouse oocytes.

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    Jun Woo Jo

    Full Text Available Antifreeze proteins (AFPs are a class of polypeptides that permit organismal survival in sub-freezing environments. The purpose of this study was to investigate the effect of AFP supplementation on immature mouse oocyte vitrification. Germinal vesicle-stage oocytes were vitrified using a two-step exposure to equilibrium and vitrification solution in the presence or absence of 500 ng/mL of AFP III. After warming, oocyte survival, in vitro maturation, fertilization, and embryonic development up to the blastocyst stage were assessed. Spindle and chromosome morphology, membrane integrity, and the expression levels of several genes were assessed in in vitro matured oocytes. The rate of blastocyst formation was significantly higher and the number of caspase-positive blastomeres was significantly lower in the AFP-treated group compared with the untreated group. The proportion of oocytes with intact spindles/chromosomes and stable membranes was also significantly higher in the AFP group. The AFP group showed increased Mad2, Hook-1, Zar1, Zp1, and Bcl2 expression and lower Eg5, Zp2, Caspase6, and Rbm3 expression compared with the untreated group. Supplementation of the vitrification medium with AFP has a protective effect on immature mouse oocytes, promoting their resistance to chilling injury. AFPs may preserve spindle forming ability and membrane integrity at GV stage. The fertilization and subsequent developmental competence of oocytes may be associated with the modulation of Zar1, Zp1/Zp2, Bcl2, Caspase6, and Rbm3.

  19. Vitrification of Germinal Vesicle Stage Oocytes


    ABE, Yasuyuki; AONO, Nobuya; Hara, Kenshiro; Matsumoto, Hiromichi; BAKHTIYARI, Mehrdad; Sasada, Hiroshi; Sato, Eimei


    In order to cryopreserve germinal vesicle (GV) stage oocytes, we first need to develop a novel container for keeping large quantities of GV oocytes, because of collecting them as cumulus oocytes complexes (COCs) that have bigger size and larger volume than oocytes themselves, and second modify a protocol for optimizing vitrification of them. In this mini-review, we describe our recent progress for attaining these objectives. When 65 bovine COCs having GV oocytes could be placed on a sheet of ...

  20. In vivo versus in vitro oviductal glycoprotein (OGP) association with the zona pellucida (ZP) in the hamster and baboon. (United States)

    O'Day-Bowman, M B; Mavrogianis, P A; Minshall, R D; Verhage, H G


    The goal of this study was to determine if differences exist between in vivo vs. in vitro OGP association with the ZP and to quantitate those differences. Ovarian oocytes were harvested 12.5 or 27 hr post-hCG from hyperstimulated hamsters or baboons, respectively. Hamster and baboon ovarian oocytes were incubated in vitro in media +/- homologous OGP (100 or 200 microg/100 microl) or in some studies with 100 microl oviductal fluid for 3, 6, or 24 hr at 37 degrees C. Some of the baboon ovarian oocytes were transferred immediately after harvesting to the ampulla of both oviducts using a tom cat catheter and retrieved after a 3 hr in situ incubation. Hamster oviductal oocytes were collected 3, 6, and 24 hr following ovulation. After incubation or oocyte retrieval from the oviduct, cumulus cells were removed, oocytes were washed extensively and binding of OGP to the ZP was examined by immunofluorescence. Fluorescence intensity was quantified using densitometric scanning of photographic negatives with the background of each negative as an internal control. In all studies, OGP association with the ZP was significantly greater in vivo than in vitro (P ZP did not significantly increase with incubation time or OGP concentration; however, a small nonsignificant increase in OGP association with the ZP in the oviduct was detected over time. Differences did not appear to be due to depletion of OGP from the in vitro incubation media, since Western blot analysis of the media showed that OGP was still present. Although OGP concentration in vivo is unknown, Western blots showed similar intensity comparing 100 microg of OGP media and oviductal fluid. Immunolocalization of OGP using laser confocal microscopy showed regional differences in OGP binding. The outer half of the zona pellucida had significantly more OGP bound than the inner half on oviductal oocytes. No regional differences were detected for in vitro incubated oocytes. In conclusion, OGP association with the ZP is greater

  1. Analysis of the phospholipid profile of metaphase II mouse oocytes undergoing vitrification.

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    Jaehun Jung

    Full Text Available Oocyte freezing confers thermal and chemical stress upon the oolemma and various other intracellular structures due to the formation of ice crystals. The lipid profiles of oocytes and embryos are closely associated with both, the degrees of their membrane fluidity, as well as the degree of chilling and freezing injuries that may occur during cryopreservation. In spite of the importance of lipids in the process of cryopreservation, the phospholipid status in oocytes and embryos before and after freezing has not been investigated. In this study, we employed mass spectrometric analysis to examine if vitrification has an effect on the phospholipid profiles of mouse oocytes. Freshly prepared metaphase II mouse oocytes were vitrified using copper grids and stored in liquid nitrogen for 2 weeks. Fresh and vitrified-warmed oocytes were subjected to phospholipid extraction procedure. Mass spectrometric analyses revealed that multiple species of phospholipids are reduced in vitrified-warmed oocytes. LIFT analyses identified 31 underexpressed and 5 overexpressed phospholipids in vitrified mouse oocytes. The intensities of phosphatidylinositol (PI {18∶2/16∶0} [M-H]- and phosphatidylglycerol (PG {14∶0/18∶2} [M-H]- were decreased the most with fold changes of 30.5 and 19.1 in negative ion mode, respectively. Several sphingomyelins (SM including SM {d38∶3} [M+H]+ and SM {d34∶0} [M+K]+ were decreased significantly in positive ion mode. Overall, the declining trend of multiple phospholipids demonstrates that vitrification has a marked effect on phospholipid profiles of oocytes. These results show that the identified phospholipids can be used as potential biomarkers of oocyte undergoing vitrification and will allow for the development of strategies to preserve phospholipids during oocyte cryopreservation.

  2. Reconstitution of the Golgi apparatus after microinjection of rat liver Golgi fragments into Xenopus oocytes

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    Paiement, J.; Jolicoeur, M.; Fazel, A.; Bergeron, J.J.


    We have studied the reconstitution of the Golgi apparatus in vivo using an heterologous membrane transplant system. Endogenous glycopeptides of rat hepatic Golgi fragments were radiolabeled in vitro with (3H)sialic acid using detergent-free conditions. The Golgi fragments consisting of dispersed vesicles and tubules with intraluminal lipoprotein-like particles were then microinjected into Xenopus oocytes and their fate studied by light (LM) and electron microscope (EM) radioautography. 3 h after microinjection, radiolabel was observed by LM radioautography over yolk platelet-free cytoplasmic regions near the injection site. EM radioautography revealed label over Golgi stacked saccules containing the hepatic marker of intraluminal lipoprotein-like particles. At 14 h after injection, LM radioautographs revealed label in the superficial cortex of the oocytes between the yolk platelets and at the oocyte surface. EM radioautography identified the labeled structures as the stacked saccules of the Golgi apparatus, the oocyte cortical granules, and the plasmalemma, indicating that a proportion of microinjected material was transferred to the surface via the secretion pathway of the oocyte. The efficiency of transport was low, however, as biochemical studies failed to show extensive secretion of radiolabel into the extracellular medium by 14 h with approximately half the microinjected radiolabeled constituents degraded. Vinblastine (50 microM) administered to oocytes led to the formation of tubulin paracrystals. Although microinjected Golgi fragments were able to effect the formation of stacked saccules in vinblastine-treated oocytes, negligible transfer of heterologous material to the oocyte surface could be detected by radioautography.

  3. Possible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography. (United States)

    Mio, Yasuyuki; Iwata, Kyoko; Yumoto, Keitaro; Kai, Yoshiteru; Sargant, Haruka C; Mizoguchi, Chizuru; Ueda, Minako; Tsuchie, Yuka; Imajo, Akifumi; Iba, Yumiko; Nishikori, Kyoko


    To analyze the fertilization process related to polyspermy block in human oocytes using an in vitro culturing system for time-lapse cinematography. We had 122 oocytes donated for this study from couples that provided informed consent. We recorded human oocytes at 2,000 to 2,800 frames every 10 s during the fertilization process and thereafter every 2 min using a new in vitro culture system originally developed by the authors for time-lapse cinematography. We displayed 30 frames per second for analysis of the polyspermy block during fertilization. Three oocytes showed the leading and following sperm within the zona pellucida in the same microscopic field. The dynamic images obtained during the fertilization process using this new system revealed that once a leading sperm penetrated the zona pellucida and attached to the oocyte membrane, a following sperm was arrested from further penetration into the zona pellucida within 10 s. The present results strongly suggest the existence of a novel mechanism of polyspermy block that takes place at the zona pellucida immediately after fertilization. These findings are clearly different from previous mechanisms describing polyspermy block as the oocyte membrane block to sperm penetration and the zona reaction. The finding presented herein thus represents a novel discovery about the highly complicated polyspermy block mechanism occurring in human oocytes.

  4. Immunophotoaffinity labeling of binders of 1-methyladenine, the oocyte maturation-inducing hormone of starfish. (United States)

    Toraya, Tetsuo; Kida, Tetsuo; Kuyama, Atsushi; Matsuda, Shinjiro; Tanaka, Seiichi; Komatsu, Yo; Tsurukai, Taro


    Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary and resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative 1-MeAde receptors on the oocyte surface have been suggested, but not yet been biochemically characterized. Immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection, was attempted to detect unknown 1-MeAde binders including putative maturation-inducing hormone receptors in starfish oocytes. When the oocyte crude membrane fraction or its Triton X-100/EDTA extract was incubated with N(6)-[6-(5-azido-2-nitrobenzoyl)aminohexyl]carboxamidomethyl-1-methyladenine and then photo-irradiated, followed by western blotting with antibody that was raised against a 1-MeAde hapten, a single band with Mr of 47.5 K was detected. The band was lost when extract was heated at 100 °C. A similar 47.5 K band was detected in the crude membrane fraction of testis as well. Upon labeling with whole cells, this band was detected in immature and maturing oocytes, but only faintly in mature oocytes. As judged from these results, this 1-MeAde binder might be a possible candidate of the starfish maturation-inducing hormone receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Membranes


    Junbo Hou; Min Yang


    Lithium ion batteries have proven themselves the main choice of power sources for portable electronics. Besides consumer electronics, lithium ion batteries are also growing in popularity for military, electric vehicle, and aerospace applications. The present review attempts to summarize the knowledge about some selected membranes in lithium ion batteries. Based on the type of electrolyte used, literature concerning ceramic-glass and polymer solid ion conductors, microporous filter type separa...

  6. Effect of soybean phosphatidylcholine on lipid profile of bovine oocytes matured in vitro. (United States)

    Pitangui-Molina, Caroline P; Vireque, Alessandra A; Tata, Alessandra; Belaz, Katia Roberta A; Santos, Vanessa G; Ferreira, Christina R; Eberlin, Marcos N; Silva-de-Sá, Marcos Felipe; Ferriani, Rui A; Rosa-E-Silva, Ana Carolina J S


    The phospholipid (PL) composition of embryo and oocyte membranes affects thermal phase behavior and several physicochemical properties such as fluidity and permeability. The characterization of PL profiles and the development of suitable in vitro maturation (IVM) protocols, that are able to modify membrane's composition, may result in significant improvements in oocyte developmental potential and cryotolerance. Using soybean phosphatidylcholine (PC) as a model supplement, we evaluated the effect of PL supplementation during IVM on bovine cumulus-oocyte-complex (COC). Substantial changes in the lipid profiles of oocyte membrane were observed and associated with pre-implantation data. The propensity of the PC supplement to become soluble in the maturation medium and/or diffuse into mineral oil was also assessed. Oocytes were matured in TCM without supplementation, i.e. control, (n=922) or supplemented with 50 or 100μM PC (n=994). The maturation media and mineral oil pre- and post- IVM, along with control and PC-treated oocytes were then analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and the lipid profiles were compared via principal component analysis (PCA). Soybean PCs are bioavailable and stable in IVM medium; further, PCs did not diffuse to the mineral oil, which also remained unaltered by the metabolism of treated oocytes. PC supplementation at 100μM resulted in substantially greater relative abundances of polyunsatured PL, namely PC (32:1), PC (34:2), PC (36:6), PC (36:4), and PC (38:6), in oocyte membrane. These differences indicated that short-term exposure to the PC supplement could indeed modify the lipid composition of IVM-oocytes in a dose-dependent manner. Membrane incorporation of polyunsaturated molecular species of PC was favored, and does so without compromising the viability of the subsequent embryo in regards to cleavage, blastocyst development and hatching rate. The reported approach will allow for the

  7. Diaphragm Structure and Function in Emphysematous Hamsters


    Reid WD; RK Wilton


    OBJECTIVE: To determine if muscle fibre injury, reduced force output and fatigue of the diaphragm accompanied hyperinflation induced by experimental emphysema.DESIGN: Controlled and randomized.ANIMALS: Adult male golden Syrian hamsters: seven control and seven experimental emphysema hamsters were studied.INTERVENTIONS: Following anesthesia, experimental emphysema hamsters received a transtracheal injection of elastase. They also received injections of β-aminopropionitrile every other day for ...

  8. Evolutionary conservation of the mature oocyte proteome

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    Tamar Lotan


    Significance: The current study provides the first proteomic profile of an oocyte of a cnidarian organism the starlet sea anemone N. vectensis and gives new insights on the ancient origin of an oocyte proteome template. The comparative analysis with a chordate oocyte suggests that the oocyte proteome predates the divergence of the cnidarian and bilaterian lineages. In addition, the data generated in the study will serve as a valuable resource for further developmental and evolutional studies.

  9. Oocyte cryopreservation: where are we now? (United States)

    Argyle, Catrin E; Harper, Joyce C; Davies, Melanie C


    Since the first live birth from oocyte cryopreservation three decades ago, oocyte cryopreservation has become an important component of ART. Cryopreservation techniques have evolved, leading to higher success rates and the introduction of oocyte cryopreservation into IVF clinics worldwide. Concurrently, there has been an increase in patient demand, especially for so-called 'social egg freezing' that allows women to preserve their fertility in anticipation of age-related fertility decline. This review addresses a need to evaluate the current status of oocyte cryopreservation. It explores current techniques and success rates, clinical applications, the rise of elective oocyte cryopreservation, and future implications. A search was performed using Web of Science and PubMed databases for publications between January 1980 and December 2015. Keywords used included 'egg freezing', 'oocyte freezing', 'oocyte cryopreservation', 'oocyte vitrification', and 'fertility preservation'. The success rate of oocyte cryopreservation has risen, and the increasing use of vitrification offers has improved outcomes, with IVF pregnancy rates now similar to those achieved with fresh oocytes. There are conflicting opinions about the comparative success rates of open and closed vitrification. Patients are accessing and receiving oocyte cryopreservation for a wide range of indications, and there has been a marked increase in patient numbers and oocyte cryopreservation cycles. Oocyte cryopreservation for circumventing age-related infertility is becoming more widely accepted. Oocyte cryopreservation is an established component of ART, with vitrification now being the cryopreservation technique of choice. Increasing numbers of women undergo oocyte cryopreservation for both medical and social reasons. It is important to continue auditing outcomes and reporting long-term follow-up of children born from frozen-thawed oocytes. © The Author 2016. Published by Oxford University Press on behalf of

  10. Regulation of hamster sperm hyperactivation by extracellular Na. (United States)

    Takei, Gen L; Fujinoki, Masakatsu


    Mammalian sperm motility has to be hyperactivated to be fertilization-competent. Hyperactivation is regulated by extracellular environment. Osmolality of mammalian semen is higher than that in female reproductive tract; however, the effect of them on hyperactivation has not been investigated. So we investigated the effect of osmotic environment on hyperactivation using hamster spermatozoa at first. Increase in the osmolality of the media (∼370 mOsm) by increasing the concentration of NaCl (∼150 mmol/L) caused the delay of the expression of hyperactivation. When NaCl concentration varied in the same range (75-150 mmol/L) whereas the osmolality was fixed at 370 mOsm by adding mannitol, the delay of hyperactivation occurred dependent on NaCl concentration. Increase in NaCl concentration also caused suppression of curvilinear velocity, bend angle, and sliding velocity of the flagellum at the onset of incubation, suggesting that NaCl concentration affect both activation and hyperactivation in hamster spermatozoa. Hamster sperm intracellular Ca(2+) concentration decreased as extracellular NaCl concentration increased, whereas membrane potential and intracellular pH were unaffected by extracellular NaCl concentration. SN-6 and SEA0400, inhibitors of Na(+)-Ca(2+) exchanger (NCX), increased intracellular Ca(2+) and accelerated hyperactivation in the presence of 150 mmol/L NaCl. Tyrosine phosphorylation on fibrous sheath proteins was unaffected by extracellular NaCl concentration. These results suggest that extracellular Na(+) suppresses hamster sperm hyperactivation by reducing intracellular Ca(2+) via an action of NCX in a tyrosine phosphorylation-independent manner. It seems that the removal of suppression by extracellular Na(+) leads to the expression of hyperactivated motility. © 2016 Society for Reproduction and Fertility.

  11. Directed Student Inquiry: Modeling in Roborovsky Hamsters (United States)

    Elwess, Nancy L.; Bouchard, Adam


    In this inquiry-based activity, Roborovsky hamsters are used to provide students with an opportunity to develop their skills of analysis, inquiry, and design. These hamsters are easy to maintain, yet offer students a means to use conventional techniques and those of their own design to make further observations through measuring, assessing, and…

  12. Recent Progress in Cryopreservation of Bovine Oocytes

    Directory of Open Access Journals (Sweden)

    In-Sul Hwang


    Full Text Available Principle of oocyte cryoinjury is first overviewed and then research history of cryopreservation using bovine oocytes is summarized for the last two decades with a few special references to recent progresses. Various types of cryodevices have been developed to accelerate the cooling rate and applied to the oocytes from large domestic species enriched with cytoplasmic lipid droplets. Two recent approaches include the qualitative improvement of IVM oocytes prior to the vitrification and the short-term recovery culture of vitrified-warmed oocytes prior to the subsequent IVF. Supplementation of L-carnitine to IVM medium of bovine oocytes has been reported to reduce the amount of cytoplasmic lipid droplets and improve the cryotolerance of the oocytes, but it is still controversial whether the positive effect of L-carnitine is reproducible. Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes, was inhibited by a short-term culture of the postwarm oocytes in the presence of Rho-associated coiled-coil kinase (ROCK inhibitor. Use of an antioxidant α-tocopherol, instead of the ROCK inhibitor, also supported the revivability of the postwarm bovine oocytes. Further improvements of the vitrification procedure, combined with pre- and postvitrification chemical treatment, would overcome the high sensitivity of bovine oocytes to cryopreservation.

  13. Caffeine delays oocyte aging and maintains the quality of aged oocytes safely in mouse. (United States)

    Zhang, Xia; Liu, Xiaoyan; Chen, Li; Wu, Dan-Ya; Nie, Zheng-Wen; Gao, Ying-Ying; Miao, Yi-Liang


    Caffeine, as an oocyte aging inhibitor, was used in many different species to control or delay oocyte aging. However, the safety of caffeine and developmental competence of aged oocytes inhibited by caffeine has not been studied systematically. So we detected the spindle morphology, distribution of cortical granules, zona pellucida hardening and pronucleus formation to assess oocyte quality of caffeine treated oocytes. We found that aged oocytes treated by caffeine maintained weak susceptibility to activating stimuli and regained normal competent after aged further 6 hr. Caffeine maintained the spindle morphology, changed cortical granules distribution of aged oocytes and could not prevent zona pellucida hardening. Furthermore, caffeine increased pronucleus formation of aged oocytes and decreased fragmentation after fertilization. These results suggested that caffeine could maintain the quality of aged oocytes safely in mouse.

  14. Melatonin inhibits apoptosis and improves the developmental potential of vitrified bovine oocytes. (United States)

    Zhao, Xue-Ming; Hao, Hai-Sheng; Du, Wei-Hua; Zhao, Shan-Jiang; Wang, Hao-Yu; Wang, Na; Wang, Dong; Liu, Yan; Qin, Tong; Zhu, Hua-Bin


    Vitrification of oocytes has been shown to be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. However, little information is available the effect of melatonin on the ROS levels and apoptotic events in vitrified oocytes. Therefore, we studied the effect of melatonin on ROS and apoptotic events in vitrified bovine oocytes by supplementing vitrification solution or in vitro maturation (IVM) and vitrification solution with 10(-9) m melatonin. We analyzed the ROS, mitochondrial Ca(2+) (mCa(2+) ) and membrane potential (ΔΨm), externalization of phosphatidylserine (PS), caspase-3 activation, DNA fragmentation, mRNA expression levels of Bax and Bcl2 l1, and developmental potential of vitrified bovine oocytes. Vitrified bovine oocytes exhibited increased levels of ROS, mCa(2+) , Bax mRNA, and caspase-3 protein and higher rates of PS externalization and DNA fragmentation, and decreased ΔΨm and Bcl2 l1 mRNA expression level. However, melatonin supplementation in vitrification solution or IVM and vitrification solution significantly decreased the levels of ROS, mCa(2+) , Bax mRNA expression, and caspase-3 protein, and PS externalization and DNA fragmentation rates, and increased the ΔΨm and Bcl2 l1 mRNA expression level in vitrified oocytes, resulting in an increased developmental ability of vitrified bovine oocytes after parthenogenetic activation. The developmental ability of vitrified oocytes with melatonin supplementation in IVM and vitrification solution was similar to that of fresh ones. This study showed that supplementing the IVM and vitrification medium or vitrification medium with 10(-9) m melatonin significantly decreased the ROS level and inhibited apoptotic events of vitrified bovine oocytes, consequently increasing their developmental potential. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. [Recent knowledge on follicle and oocyte maturation. 2. Oocyte development and maturation]. (United States)

    Sudik, R; Fliess, F R


    A review is given about the present knowledge in oocyte development and oocyte maturation. The four parts of the review contain: development of the oocyte in the fetal ovary, morphology and metabolism during meiotic arrest, oocyte maturation, and the relations between oocyte maturation and in vitro-fertilization in the human. The morphological and biochemical changes in the maturation process and present hypotheses about maturation regulation are described especially. The increasing knowledge in this field supports the progress of in vitro-fertilization in the human. On the other hand this technique contributes importantly to new directions in oocyte research.

  16. Calcium and actin in the saga of awakening oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Santella, Luigia, E-mail:; Limatola, Nunzia; Chun, Jong T.


    The interaction of the spermatozoon with the egg at fertilization remains one of the most fascinating mysteries of life. Much of our scientific knowledge on fertilization comes from studies on sea urchin and starfish, which provide plenty of gametes. Large and transparent, these eggs have served as excellent model systems for studying egg activation and embryo development in seawater, a plain natural medium. Starfish oocytes allow the study of the cortical, cytoplasmic and nuclear changes during the meiotic maturation process, which can also be triggered in vitro by hormonal stimulation. These morphological and biochemical changes ensure successful fertilization of the eggs at the first metaphase. On the other hand, sea urchin eggs are fertilized after the completion of meiosis, and are particularly suitable for the study of sperm–egg interaction, early events of egg activation, and embryonic development, as a large number of mature eggs can be fertilized synchronously. Starfish and sea urchin eggs undergo abrupt changes in the cytoskeleton and ion fluxes in response to the fertilizing spermatozoon. The plasma membrane and cortex of an egg thus represent “excitable media” that quickly respond to the stimulus with the Ca{sup 2+} swings and structural changes. In this article, we review some of the key findings on the rapid dynamic rearrangements of the actin cytoskeleton in the oocyte/egg cortex upon hormonal or sperm stimulation and their roles in the modulation of the Ca{sup 2+} signals and in the control of monospermic fertilization. - Highlights: • Besides microtubules, microfilaments may anchor the nucleus to oocyte surface. • The cortical Ca{sup 2+} flash and wave at fertilization mirror electrical membrane change. • Artificial egg activation lacks microvilli extension in the perivitelline space. • Calcium is necessary but not sufficient for cortical granules exocytosis. • Actin cytoskeleton modulates Ca{sup 2+} release at oocyte maturation

  17. Cortical Granule Exocytosis Is Mediated by Alpha-SNAP and N-Ethilmaleimide Sensitive Factor in Mouse Oocytes. (United States)

    de Paola, Matilde; Bello, Oscar Daniel; Michaut, Marcela Alejandra


    Cortical granule exocytosis (CGE), also known as cortical reaction, is a calcium- regulated secretion that represents a membrane fusion process during meiotic cell division of oocytes. The molecular mechanism of membrane fusion during CGE is still poorly understood and is thought to be mediated by the SNARE pathway; nevertheless, it is unkown if SNAP (acronym for soluble NSF attachment protein) and NSF (acronym for N-ethilmaleimide sensitive factor), two key proteins in the SNARE pathway, mediate CGE in any oocyte model. In this paper, we documented the gene expression of α-SNAP, γ-SNAP and NSF in mouse oocytes. Western blot analysis showed that the expression of these proteins maintains a similar level during oocyte maturation and early activation. Their localization was mainly observed at the cortical region of metaphase II oocytes, which is enriched in cortical granules. To evaluate the function of these proteins in CGE we set up a functional assay based on the quantification of cortical granules metaphase II oocytes activated parthenogenetically with strontium. Endogenous α-SNAP and NSF proteins were perturbed by microinjection of recombinant proteins or antibodies prior to CGE activation. The microinjection of wild type α-SNAP and the negative mutant of α-SNAP L294A in metaphase II oocytes inhibited CGE stimulated by strontium. NEM, an irreversibly inhibitor of NSF, and the microinjection of the negative mutant NSF D1EQ inhibited cortical reaction. The microinjection of anti-α-SNAP and anti-NSF antibodies was able to abolish CGE in activated metaphase II oocytes. The microinjection of anti-γ SNAP antibody had no effect on CGE. Our findings indicate, for the first time in any oocyte model, that α-SNAP, γ-SNAP, and NSF are expressed in mouse oocytes. We demonstrate that α-SNAP and NSF have an active role in CGE and propose a working model.

  18. Dietary lipid saturation influences environmental temperature preference but not resting metabolic rate in the Djungarian Hamster (Phodopus sungorus). (United States)

    Pannorfi, Ryan; Zee, Barry M; Vatnick, Itzick; Berner, Nancy; Hiebert, Sara M


    Heterothermic rodents increase self-selection of diets rich in polyunsaturated fatty acids (PUFAs) when exposed to cold, short days, or short-day melatonin profiles, and Djungarian hamsters (Phodopus sungorus) do so in long days in response to cold exposure alone. To determine whether Djungarian hamsters are also capable of selecting a thermal environment in response to dietary lipid composition, continuously normothermic hamsters were fed either a PUFA-rich diet or a diet rich in saturated fatty acids (SFAs) for 6-10 wk and given a choice of thermal environments. As predicted, SFA-fed hamsters were more likely than PUFA-fed hamsters to occupy the single heated corner of their cage ([Formula: see text]) and were most likely to show this diet-related difference in behavior when T(a) fell within the thermal neutral zone. Respirometry revealed no effect of diet on whole-animal or mass-specific resting metabolic rate or on lower critical temperature. The results are more consistent with the homeoviscous adaptation hypothesis, which predicts that organisms should make physiological and/or behavioral adjustments that preserve membrane fluidity within a relatively small range, than with the membrane pacemaker hypothesis, which predicts that high PUFA content in membrane phospholipids should increase basal metabolic rate.

  19. The beneficial effect of repaglinide on in vitro maturation and development ability of immature mouse oocytes. (United States)

    Kalehoei, Eshrat; Azadbakht, Mehri


    Repaglinide is a hypoglycemic drug, causing depolarization of the cell membrane, opening the voltage-gated calcium channels, and then increasing intracellular calcium in the pancreatic B cells by inhibition of the K-ATP-sensitive channels. Oocyte in vitro maturation (IVM) is influenced by different factors such as calcium signaling. In this study, we examined the effects of repaglinide on in vitro maturation and fertilization ability of mouse oocyte. Immature oocytes were isolated from female Naval Medical Research Institute mice which are 6-8 wk old mechanically and then cultured in 30 μl droplets of T6 medium with different concentrations of repaglinide. The control group did not receive repaglinide (R0). Treatment groups received different concentrations (5, 10, and 100 nM and 1 and 10 μM) of repaglinide (R1, R2, R3, R4, and R5, respectively). Oocyte in vitro maturation rate was assessed after 24 h. In vitro fertilization was performed using metaphase II oocytes obtained from R0 and R4 treatments. Embryo cleavage rate was calculated at 48 h post-IVF. Chi-square test was used for evaluating difference between control and treatment groups (p vitro oocyte maturation and embryo cleavage.

  20. Lipid content and composition of oocytes from five coral species: potential implications for future cryopreservation efforts.

    Directory of Open Access Journals (Sweden)

    Chiahsin Lin

    Full Text Available Given the previously documented importance of lipid concentration and composition in the successful cryopreservation of gorgonian corals, these parameters were assessed in oocytes of five species of scleractinian coral; Platygyra daedalea, Echinopora gemmacea, Echinophyllia aspera, Oxypora lacera and Astreopora expansa. Wax esters, phosphatidylethanolamine, phosphatidylcholine, and fatty acids were all measured at detectable levels, and the latter were produced at significantly elevated quantities in E. gemmacea, E. aspera, and O. lacera. On the other hand, phosphatidylethanolamine, phosphatidylcholine, and wax ester were found at significantly higher concentrations in A. expansa oocytes. Triacylglycerol was not present in any species. Interestingly, the total lipid content of oocytes from all five scleractinians was significantly lower than that of oocytes of two gorgonian species, Junceella juncea and Junceella fragilis. As higher total lipid concentrations may be correlated with greater degrees of cellular membrane fluidity at lower temperatures, it stands to reason that gorgonian coral oocytes may be more likely to survive the cryopreservation process than oocytes of scleractinian corals.

  1. [Oocyte vitrification in an ART laboratory]. (United States)

    Boyer, P; Montjean, D; Tourame, P; Gervoise-Boyer, M


    Oocyte vitrification has been authorized in France after the modification of French bioethics law in July 2011. This evolution will bring a dramatic change in patients' management since, from 2011, infertile couples, oocyte donation and fertility preservation programs will benefit this technique in France. We have introduced oocyte vitrification in our ART laboratory through a validation of the method using Evidence-Based Medicine model: open system Cryotop, Ethylène-glycol 15% and DMSO 15%. Based on our 1-year experience, oocyte vitrification upgrades our daily practice while also minimizing embryo cryoconservation as recommended by the law. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  2. Polymyopathy in a Syrian golden hamster

    NARCIS (Netherlands)

    Wijnands, M.V.W.; Woutersen, R.A.


    A Syrian golden hamster suffered from general swelling of skeletal muscles. At microscopical observation the muscle tissue exhibited degeneration and necrosis, as well as regenerative features. The inflammatory response was very slight. The histopathological lesions were diagnosed as polymyopathy.

  3. Effect of vitrification on the mRNA transcriptome of bovine oocytes. (United States)

    Wang, N; Li, C-Y; Zhu, H-B; Hao, H-S; Wang, H-Y; Yan, C-L; Zhao, S-J; Du, W-H; Wang, D; Liu, Y; Pang, Y-W; Zhao, X-M


    Vitrification has been shown to decrease the developmental capacity of mammalian oocytes, and this is closely associated with the abnormal mRNA expressions of vitrified oocytes. However, the effect of vitrification on transcriptional machinery of oocytes examined by RNA sequencing (RNA-seq) has yet to be defined. In the present study, the mRNA transcriptomes of fresh and vitrified bovine oocytes were analysed by Smart-seq2 with the differently expressed genes determined by DEseq2 (an adjusted p-value of .05 and a minimum fold change of 2). The differentially expressed mRNAs were then searched against the Gene Ontology (GO) and Genomes (KEGG) database. Finally, the mRNA expressions of 10 candidate genes were validated using quantitative real-time PCR (qRT-PCR). Approximately 12,000 genes were detected in each sample of fresh or vitrified oocytes. Of these, the expression levels of 102 genes differed significantly in vitrified groups: 12 genes mainly involved in cell cycle, fertilization and glucose metabolism were upregulated, and 90 genes mainly involved in mitochondria, ribosomal protein, cytoskeleton, transmembrane protein, cell cycle and calcium ions were downregulated. GO analysis showed that these genes were mainly enriched in terms of membrane-bounded organelles, macromolecular complex, and intracellular part. The mRNA expression levels of 10 candidate genes selected randomly were in agreement with the results of the RNA-seq. In conclusion, our results showed that vitrification affected the mRNA transcriptome of bovine oocytes by downregulating genes, which contributed to the decreased developmental capacity of vitrified oocytes. Our findings will be useful in determining approaches to improve the efficiency of vitrified oocytes. © 2017 Blackwell Verlag GmbH.

  4. Hypotonic regulation of mouse epithelial sodium channel in Xenopus laevis oocytes. (United States)

    Galizia, Luciano; Marino, Gabriela I; Ojea, Alejandro; Kotsias, Basilio A


    The regulation of the epithelial Na⁺ channel (ENaC) during cell swelling is relevant in cellular processes in which cell volume changes occur, i.e., migration, proliferation and cell absorption. Its sensitivity to hypotonically induced swelling was investigated in the Xenopus oocyte expression system with the injection of the three subunits of mouse ENaC. We used voltage-clamp techniques to study the amiloride-sensitive Na⁺ currents (INa(amil)) and video microscopic methodologies to assess oocyte volume changes. Under conditions of mild swelling (25 % reduced hypotonicity) inward current amplitude decreased rapidly over 1.5 min. In contrast, there was no change in current amplitude of H₂O-injected oocytes to the osmotic insult. INa(amil) kinetics analysis revealed a decrease in the slower inactivation time constant during the hypotonic stimuli. Currents from ENaC-injected oocytes were not sensitive to external Cl⁻ reduction. Neither short- nor long-term cytochalasin D treatment affected the observed response. Oocytes expressing a DEG mutant β-ENaC subunit (β-S518K) with an open probability of 1 had reduced INa(amil) hypotonic response compared to oocytes injected with wild-type ENaC subunits. Finally, during the hypotonic response ENaC-injected oocytes did not show a cell volume difference compared with water-injected oocytes. On this basis we suggest that hypotonicity-dependent ENaC inhibition is principally mediated through an effect on open probability of channels in the membrane.

  5. Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes. (United States)

    Jarecki, Brian W; Makino, Shin-ichi; Beebe, Emily T; Fox, Brian G; Chanda, Baron


    Voltage-gated ion channels are a class of membrane proteins that temporally orchestrate the ion flux critical for chemical and electrical signaling in excitable cells. Current methods to investigate the function of these channels rely on heterologous expression in living systems or reconstitution into artificial membranes; however these approaches have inherent drawbacks which limit potential biophysical applications. Here, we describe a new integrated approach combining cell-free translation of membrane proteins and in vivo expression using Xenopus laevis oocytes. In this method, proteoliposomes containing Shaker potassium channels are synthesized in vitro and injected into the oocytes, yielding functional preparations as shown by electrophysiological and fluorescence measurements within few hours. This strategy for studying eukaryotic ion channels is contrasted with existing, laborious procedures that require membrane protein extraction and reconstitution into synthetic lipid systems.

  6. Proteomic Analysis of Chinese Hamster Ovary Cells

    DEFF Research Database (Denmark)

    Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama


    To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimens......To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis...

  7. Recurrent G-to-A substitution in a single codon of SREBP cleavage-activating protein causes sterol resistance in three mutant Chinese hamster ovary cell lines


    Nohturfft, Axel; Hua, Xianxin; Brown, Michael S.; Goldstein, Joseph L.


    Oxygenated sterols such as 25-hydroxycholesterol kill Chinese hamster ovary cells because they inhibit the proteolytic processing of sterol regulatory element binding proteins (SREBPs), a pair of membrane-bound transcription factors that activate genes controlling cholesterol synthesis and uptake from lipoproteins. The unprocessed SREBPs remain membrane-bound, they cannot activate the cholesterol biosynthetic pathway, and the cells die of cholesterol deprivation. S...

  8. VHA-19 is essential in Caenorhabditis elegans oocytes for embryogenesis and is involved in trafficking in oocytes.

    Directory of Open Access Journals (Sweden)

    Alison J Knight

    Full Text Available There is an urgent need to develop new drugs against parasitic nematodes, which are a significant burden on human health and agriculture. Information about the function of essential nematode-specific genes provides insight to key nematode-specific processes that could be targeted with drugs. We have characterized the function of a novel, nematode-specific Caenorhabditis elegans protein, VHA-19, and show that VHA-19 is essential in the germline and, specifically, the oocytes, for the completion of embryogenesis. VHA-19 is also involved in trafficking the oocyte receptor RME-2 to the oocyte plasma membrane and is essential for osmoregulation in the embryo, probably because VHA-19 is required for proper eggshell formation via exocytosis of cortical granules or other essential components of the eggshell. VHA-19 may also have a role in cytokinesis, either directly or as an indirect effect of its role in osmoregulation. Critically, VHA-19 is expressed in the excretory cell in both larvae and adults, suggesting that it may have a role in osmoregulation in C. elegans more generally, probably in trafficking or secretion pathways. This is the first time a role for VHA-19 has been described.


    INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22. SC Jeffay*, SD Perreault, KL Bobseine*, JE Welch*, GR Klinefelter, US EPA, Research Triangle Park, NC. SP22, a rat sperm membrane protein that is highly-correlated w...

  10. Glycosylation profile of a recombinant urokinase-type plasminogen activator receptor expressed in Chinese hamster ovary cells

    DEFF Research Database (Denmark)

    Ploug, M; Rahbek-Nielsen, H; Nielsen, P F


    migration and tissue remodeling. The uPA receptor is a glycolipid-anchored membrane protein belonging to the Ly-6/uPAR superfamily and is the only multidomain member identified so far. We have now purified the three individual domains of a recombinant soluble uPAR variant, expressed in Chinese hamster ovary...

  11. The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum

    Directory of Open Access Journals (Sweden)

    Sánchez Mercedes


    Full Text Available Abstract A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1 when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE, 2 after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE, and 3 after oocyte activation (surrounded by the fertilization envelope, (FE. The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP shows proteolytic activity on gp75 of the VE.

  12. Comparison of in-vitro outcomes from cryopreserved oocytes and sibling fresh oocytes. (United States)

    Chamayou, S; Alecci, C; Ragolia, C; Storaci, G; Maglia, E; Russo, E; Guglielmino, A


    In Italy, the restrictive IVF law generalizes the indication for oocyte freezing for surplus oocytes in 78.5% of in-vitro assisted reproductive cycles. With a view to understanding better what the prospects for intracytoplasmic sperm injection (ICSI) on frozen-thawed oocytes might be, the consequences of freeze-thaw procedures on fertilization, cleavage rates and embryo quality obtained from frozen-thawed oocytes were studied and compared with the results obtained from sibling fresh oocytes. Eleven IVF and 29 ICSI on 76 and 169 fresh oocytes were performed and the corresponding 40 ICSI on 221 sibling frozen-thawed oocytes. There was no difference in terms of fertilization rate between fresh and sibling frozen-thawed oocytes. The cleavage rate (98.0 and 94.4% with fresh oocytes in IVF and ICSI; 77.3% with frozen-thawed oocytes in ICSI; P cryoconservation induces irreversible damage to microtubule repolymerization. The consequences of oocyte cryopreservation procedures on embryo development are reviewed.

  13. Apoptosis in mammalian oocytes: a review. (United States)

    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K


    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.

  14. Closed system for bovine oocyte vitrification

    Directory of Open Access Journals (Sweden)

    Helena Ševelová


    Full Text Available The aim of our study was to develop a vitrification carrier for bovine oocyte cryopreservation. The carrier was to be cheap enough, elementary in its construction and meet contemporary requirements for a safe closed system. In a closed system, a cell is prevented from direct exposure to liquid nitrogen, thus minimizing the risk of cross-contamination. Furthermore, two questions regarding the proper vitrification technique were resolved: if it is necessary to partially denude the oocytes before the vitrification process or whether intact cumulus oocyte complexes should be frozen; and if it is more advantageous to preheat the vitrification solutions to female body temperature (39 °C or to keep them at room temperature. Our results show that it is better to partially denude the oocytes prior to vitrification because cryopreserved intact cumulus oocyte complexes often proved dark, non-homogeneous or fragmented cytoplasm after warming, with many of them having visibly widened perivitelline spaces or fractured zonae pellucidae as a result of extensive damage during vitrification. Consequently, intact cumulus oocyte complexes showed significantly lower numbers of cleavage stage embryos on Day 3 compared to partially denuded oocytes (7.4% and 26%, respectively. On the other hand, the survival rate and following development of fertilized oocytes in preheated vitrification solution were equal to results reached at room temperature conditions. In conclusion, results achieved with the newly developed carrier were comparable to previously published studies and therefore they could be recommended for common use.

  15. Molecular and structural aspects of oocyte maturation

    NARCIS (Netherlands)

    Hölzenspies, J.J.


    In the mammalian ovary, oocytes are contained within follicles, specialized structures that facilitate oocyte growth and development. During the reproductive cycle, several follicles are recruited into growth, and through a process of selection, one (human, cow) or several (mouse, pig) of these

  16. Successful ongoing pregnancies after vitrification of oocytes. (United States)

    Lucena, Elkin; Bernal, Diana Patricia; Lucena, Carolina; Rojas, Alejandro; Moran, Abby; Lucena, Andrés


    To demonstrate the efficiency of vitrifying mature human oocytes for different clinical indications. Descriptive case series. Cryobiology laboratory, Centro Colombiano de Fertilidad y Esterilidad-CECOLFES LTDA. (Bogotá, Colombia). Oocyte vitrification was offered as an alternative management for patients undergoing infertility treatment because of ovarian hyperstimulation syndrome, premature ovarian failure, natural ovarian failure, male factor, poor response, or oocyte donation. Mature oocytes were obtained from 33 donor women and 40 patients undergoing infertility treatment. Oocytes were retrieved by ultrasound-guided transvaginal aspiration and vitrified with the Cryotops method, with 30% ethylene glycol, 30% dimethyl sulfoxide, and 0.5 mol/L sucrose. Viability was assessed 3 hours after thawing. The surviving oocytes were inseminated by intracytoplasmic sperm injection. Fertilization was evaluated after 24 hours. The zygotes were further cultured in vitro for up to 72 hours until time of embryo transfer. Recovery, viability, fertilization, and pregnancy rates. Oocyte vitrification with the Cryotop method resulted in high rates of recovery, viability, fertilization, cleavage, and ongoing pregnancy. Vitrification with the Cryotop method is an efficient, fast, and economical method for oocyte cryopreservation that offers high rates of survival, fertilization, embryo development, and ongoing normal pregnancies, providing a new alternative for the management of female infertility.

  17. Control of Oocyte Reawakening by Kit.

    Directory of Open Access Journals (Sweden)

    Hatice Duygu Saatcioglu


    Full Text Available In mammals, females are born with finite numbers of oocytes stockpiled as primordial follicles. Oocytes are "reawakened" via an ovarian-intrinsic process that initiates their growth. The forkhead transcription factor Foxo3 controls reawakening downstream of PI3K-AKT signaling. However, the identity of the presumptive upstream cell surface receptor controlling the PI3K-AKT-Foxo3 axis has been questioned. Here we show that the receptor tyrosine kinase Kit controls reawakening. Oocyte-specific expression of a novel constitutively-active KitD818V allele resulted in female sterility and ovarian failure due to global oocyte reawakening. To confirm this result, we engineered a novel loss-of-function allele, KitL. Kit inactivation within oocytes also led to premature ovarian failure, albeit via a contrasting phenotype. Despite normal initial complements of primordial follicles, oocytes remained dormant with arrested oocyte maturation. Foxo3 protein localization in the nucleus versus cytoplasm explained both mutant phenotypes. These genetic studies provide formal genetic proof that Kit controls oocyte reawakening, focusing future investigations into the causes of primary ovarian insufficiency and ovarian aging.

  18. The Role of Oocyte Organelles in Determining Developmental Competence. (United States)

    Reader, Karen L; Stanton, Jo-Ann L; Juengel, Jennifer L


    The ability of an oocyte to undergo successful cytoplasmic and nuclear maturation, fertilization and embryo development is referred to as the oocyte's quality or developmental competence. Quality is dependent on the accumulation of organelles, metabolites and maternal RNAs during the growth and maturation of the oocyte. Various models of good and poor oocyte quality have been used to understand the essential contributors to developmental success. This review covers the current knowledge of how oocyte organelle quantity, distribution and morphology differ between good and poor quality oocytes. The models of oocyte quality are also described and their usefulness for studying the intrinsic quality of an oocyte discussed. Understanding the key critical features of cytoplasmic organelles and metabolites driving oocyte quality will lead to methods for identifying high quality oocytes and improving oocyte competence, both in vitro and in vivo.

  19. The equine oocyte: factors affecting meiotic and developmental competence. (United States)

    Hinrichs, Katrin


    There is currently much interest in assisted reproduction techniques in the horse, however, many aspects of oocyte maturation, fertilization, and embryo development in the horse differ from those in other species. Because of the close attachment of the equine oocyte to the follicle wall, scraping of the follicle is the most effective method for oocyte recovery. A notable feature of equine oocytes is that those with expanded cumuli (Ex oocytes), which originate from atretic follicles, have higher meiotic competence (ability to mature to metaphase II in vitro) than do oocytes with compact cumuli (Cp oocytes). Cp oocytes originate in viable follicles but are largely juvenile. Recovery and culture of equine oocytes immediately after slaughter yields a higher maturation rate than that obtained from oocytes after ovary storage; this is related to damage to chromatin in Cp oocytes during storage. In contrast, developmental competence (rate of blastocyst development in vitro) is higher in oocytes recovered from the ovary after a delay. The optimum duration of maturation varies based on cumulus morphology and time of recovery from the ovary, but there is no difference in developmental competence between Ex and Cp oocytes. Because standard in vitro fertilization is not repeatable in the horse, oocyte transfer (surgical transfer of oocytes to the oviducts of inseminated mares) has been developed to allow fertilization of isolated oocytes. Fertilization in vitro may be achieved using intracytoplasmic sperm injection; culture of injected oocytes in a medium with high glucose can yield over 30% blastocyst development. (c) 2010 Wiley-Liss, Inc.

  20. Methods for modeling chinese hamster ovary (cho) cell metabolism

    DEFF Research Database (Denmark)


    Embodiments of the present invention generally relate to the computational analysis and characterization biological networks at the cellular level in Chinese Hamster Ovary (CHO) cells. Based on computational methods utilizing a hamster reference genome, the invention provides methods...

  1. Enteritis caused by Pasteurella pneumotropica infection in hamsters.


    Lesher, R J; Jeszenka, E V; Swan, M E


    Pasteurella pneumotropica was isolated in essentially pure cultures from the bowels of hamsters with enteritis 7 days after parturition. Newly received hamsters showed presence of P. pneumotropica in their nasal cavities but not in their uteri, lungs, spleens, or bowels.

  2. Effect of temperature on oxidative stress, antioxidant levels and uncoupling protein expression in striped hamsters. (United States)

    Zhou, Si-Si; Cao, Li-Li; Xu, Wei-Dong; Cao, Jing; Zhao, Zhi-Jun


    According to the rate of living-free radical hypothesis, higher metabolic rates should increase reactive oxygen species (ROS) production. However, the "uncoupling to survive" hypothesis postulates that uncoupling proteins (UCPs) can decrease ROS production by lowering the potential of the inner mitochondrial membrane, in which case the correlation between metabolic rate and ROS levels would be a negative rather than positive. In this study, we examined energy intake, oxidative stress levels, antioxidant activity and the expression of UCPs in brown adipose tissue (BAT), and in the liver, heart, skeletal muscle and brain, of striped hamsters (Cricetulus barabensis) acclimated to either 5 °C or 32.5 °C. The energy intake of hamsters acclimated to 5 °C increased by 70.7%, whereas the energy intake of hamsters acclimated to 32.5 °C decreased by 31.3%, relative to hamsters kept at room temperature (21 °C) (Phamsters acclimated to 5 °C. These results suggest that the relationship between ROS levels and metabolic rate was negative, rather than positive. UCP1 expression in BAT may have played a role in lowering ROS levels. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Strain-Specific Barriers against Bovine Prions in Hamsters


    Nicot, Simon; Baron, Thierry


    We investigated the susceptibilities of Syrian golden hamsters to transmissible spongiform encephalopathy agents from cattle. We report efficient transmission of the L-type atypical bovine spongiform encephalopathy (BSE) agent into hamsters. Importantly, hamsters were also susceptible to the transmissible mink encephalopathy agent from cattle, which has molecular features similar to those of the L-type BSE agent, as also shown in bovinized transgenic mice. In sharp contrast, hamsters could no...

  4. Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Schmalzing, G.; Eckard, P.; Kroener, S.P.; Passow, H. (Max-Planck-Institut fuer Biophysik, Frankfurt (Germany, F.R.))


    During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by (gamma-32P)ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from (3H)ouabain bound to the cell surface before maturation could be phosphorylated with inorganic (32P)phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.

  5. How is plasminogen/plasmin system contributing to regulate sperm entry into the oocyte? (United States)

    Grullón, Luis A; Gadea, Joaquín; Mondéjar, Irene; Matás, Carmen; Romar, Raquel; Coy, Pilar


    Plasminogen is present in the oviduct, on the zona pellucida (ZP) and on oolemma, and reduces the number of sperm penetrating the oocyte during in vitro fertilization in pig and cow. It is unknown how this reduction occurs. We tested whether plasminogen (1) changed the ZP resistance to enzymatic digestion thus making the passage of the spermatozoa across it difficult; (2) reduced the sperm functionality, assessed by sperm viability, motility, spontaneous acrosome reaction and membrane lipid disorder; or (3) affected the sperm-ZP binding before or after sperm-ZP interaction. The mechanism by which plasminogen/plasmin system contributes to regulate sperm entry into the oocyte is not inducing a ZP hardening or a decrease in sperm functionality but detaching more than 50% of sperm bound to the ZP. It is suggested that the fertilizing spermatozoon activates plasminogen into plasmin at the oocyte surface and that plasmin removes additional spermatozoa attached to the ZP.

  6. How Is Plasminogen/Plasmin System Contributing to Regulate Sperm Entry Into the Oocyte? (United States)

    Grullón, Luis A.; Gadea, Joaquín; Mondéjar, Irene; Matás, Carmen; Romar, Raquel


    Plasminogen is present in the oviduct, on the zona pellucida (ZP) and on oolemma, and reduces the number of sperm penetrating the oocyte during in vitro fertilization in pig and cow. It is unknown how this reduction occurs. We tested whether plasminogen (1) changed the ZP resistance to enzymatic digestion thus making the passage of the spermatozoa across it difficult; (2) reduced the sperm functionality, assessed by sperm viability, motility, spontaneous acrosome reaction and membrane lipid disorder; or (3) affected the sperm–ZP binding before or after sperm–ZP interaction. The mechanism by which plasminogen/plasmin system contributes to regulate sperm entry into the oocyte is not inducing a ZP hardening or a decrease in sperm functionality but detaching more than 50% of sperm bound to the ZP. It is suggested that the fertilizing spermatozoon activates plasminogen into plasmin at the oocyte surface and that plasmin removes additional spermatozoa attached to the ZP. PMID:23420828

  7. Size of lethality target in mouse immature oocytes determined with accelerated heavy ions. (United States)

    Straume, T; Dobson, R L; Kwan, T C


    Mouse immature oocytes were irradiated in vivo with highly charged, heavy ions from the Bevalac accelerator at the Lawrence Berkeley Laboratory. The particles used were 670-MeV/nucleon Si14+, 570-MeV/nucleon Ar18+, and 450-MeV/nucleon Fe26+. The cross-sectional area of the lethality target in these extremely radiosensitive cells was determined from fluence-response curves and information on energy deposition by delta rays. Results indicate a target cross-section larger than that of the nucleus, one which closely approximates the cross-sectional area of the entire oocyte. For 450-MeV/nucleon Fe26+ particles, the predicted target cross-sectional area is 120 +/- 16 microns2, comparing well with the microscopically determined cross-sectional area of 111 +/- 12 microns2 for these cells. The present results are in agreement with our previous target studies which implicate the oocyte plasma membrane.

  8. Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes

    Directory of Open Access Journals (Sweden)

    Halkier Barbara A


    Full Text Available Abstract Background We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector. Results Injection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized. Conclusion Expression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified.

  9. Immunophotoaffinity labeling of the binding proteins for 1-methyladenine, an oocyte maturation-inducing hormone of starfish. (United States)

    Kida, Tetsuo; Matsuda, Shinjiro; Kuyama, Atsushi; Toraya, Tetsuo


    Starfish oocytes are naturally arrested at the prophase stage of the first meiotic division and resume meiosis in response to 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative receptors for 1-MeAde have not yet been characterized biochemically, although the specific binding of 1-MeAde to the isolated cortices of starfish oocytes was reported so far. Based on the structure-activity relationship of 1-MeAde analogs, we have designed a photoaffinity labeling reagent. The photoaffinity labeling of oocyte membrane fractions, followed by immunoblotting analysis with anti-1-MeAde antibody, results in the detection of an almost single protein band. This 1-MeAde-binding protein might be a possible candidate of the maturation-inducing hormone receptor of starfish.

  10. Scrambled and fried: Cigarette smoke exposure causes antral follicle destruction and oocyte dysfunction through oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Sobinoff, A.P. [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308 (Australia); Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia); Beckett, E.L.; Jarnicki, A.G. [Centre for Asthma and Respiratory Disease, The University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308 (Australia); Sutherland, J.M. [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308 (Australia); Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia); McCluskey, A. [Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia); Hansbro, P.M. [Centre for Asthma and Respiratory Disease, The University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308 (Australia); McLaughlin, E.A., E-mail: [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308 (Australia); Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia)


    Cigarette smoke is a reproductive hazard associated with pre-mature reproductive senescence and reduced clinical pregnancy rates in female smokers. Despite an increased awareness of the adverse effects of cigarette smoke exposure on systemic health, many women remain unaware of the adverse effects of cigarette smoke on female fertility. This issue is compounded by our limited understanding of the molecular mechanisms behind cigarette smoke induced infertility. In this study we used a direct nasal exposure mouse model of cigarette smoke-induced chronic obstructive pulmonary disease to characterise mechanisms of cigarette-smoke induced ovotoxicity. Cigarette smoke exposure caused increased levels of primordial follicle depletion, antral follicle oocyte apoptosis and oxidative stress in exposed ovaries, resulting in fewer follicles available for ovulation. Evidence of oxidative stress also persisted in ovulated oocytes which escaped destruction, with increased levels of mitochondrial ROS and lipid peroxidation resulting in reduced fertilisation potential. Microarray analysis of ovarian tissue correlated these insults with a complex mechanism of ovotoxicity involving genes associated with detoxification, inflammation, follicular activation, immune cell mediated apoptosis and membrane organisation. In particular, the phase I detoxifying enzyme cyp2e1 was found to be significantly up-regulated in developing oocytes; an enzyme known to cause molecular bioactivation resulting in oxidative stress. Our results provide a preliminary model of cigarette smoke induced sub-fertility through cyp2e1 bioactivation and oxidative stress, resulting in developing follicle depletion and oocyte dysfunction. - Highlights: • Cigarette smoke exposure targets developing follicle oocytes. • The antral follicle oocyte is a primary site of ovarian cigarette smoke metabolism. • Cyp2e1 is a major enzyme involved in ameliorating smoke-induced ovotoxicity. • Cigarette smoke causes oocyte

  11. PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis. (United States)

    Terzaghi, L; Tessaro, I; Raucci, F; Merico, V; Mazzini, G; Garagna, S; Zuccotti, M; Franciosi, F; Lodde, V


    Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.

  12. Inhibiting sperm pyruvate dehydrogenase complex and its E3 subunit, dihydrolipoamide dehydrogenase affects fertilization in Syrian hamsters.

    Directory of Open Access Journals (Sweden)

    Archana B Siva

    Full Text Available BACKGROUND/AIMS: The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc and its E3 subunit, dihydrolipoamide dehydrogenase (DLD in hamster in vitro fertilization (IVF via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. METHODOLOGY AND PRINCIPAL FINDINGS: Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid. Oocytes fertilized with MICA-treated (MT [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. CONCLUSIONS: This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In

  13. Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit, Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters (United States)

    Sailasree, Purnima; Singh, Durgesh K.; Kameshwari, Duvurri B.; Shivaji, Sisinthy


    Background/Aims The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. Methodology and Principal Findings Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. Conclusions This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the

  14. Current trends and progress in clinical applications of oocyte cryopreservation (United States)

    Cil, Aylin P.; Seli, Emre


    Purpose of review To delineate the current trends in the clinical application of oocyte cryopreservation. Recent findings Although the first live birth from oocyte cryopreservation was reported approximately three decades ago, significant improvement in the clinical application of oocyte cryopreservation took place only over the past decade. On the basis of the available evidence suggesting that success rates with donor oocyte vitrification are similar to that of IVF with fresh donor oocytes, the American Society of Reproductive Medicine has recently stated that oocyte cryopreservation should no longer be considered experimental for medical indications, outlying elective oocyte cryopreservation. Meanwhile, a few surveys on the attitudes toward oocyte cryopreservation revealed that elective use for the postponement of fertility is currently the most common indication for oocyte cryopreservation. Most recently, a randomized controlled trial revealed important evidence on the safety of nondonor oocyte cryopreservation, and confirmed that the clinical success of vitrification is comparable to that of IVF with fresh oocytes. Summary The evidence suggesting similar IVF success rates with both donor and nondonor cryopreserved oocytes compared with fresh oocytes will increase the utilization of elective oocyte cryopreservation. Appropriate counseling of women for oocyte cryopreservation requires the establishment of age-based clinical success rates with cryopreserved oocytes for various indications. PMID:23562954

  15. The DNA damage response in mammalian oocytes

    Directory of Open Access Journals (Sweden)

    John eCarroll


    Full Text Available DNA damage is one of the most common insults that challenge all cells. To cope, an elaborate molecular and cellular response has evolved to sense, respond to and correct the damage. This allows the maintenance of DNA fidelity essential for normal cell viability and the prevention of genomic instability that can lead to tumour formation. In the context of oocytes, the impact of DNA damage is not one of tumour formation but of the maintenance of fertility. Mammalian oocytes are particularly vulnerable to DNA damage because physiologically they may lie dormant in the ovary for many years (>40 in humans until they receive the stimulus to grow and acquire the competence to become fertilized. The implication of this is that in some organisms, such as humans, oocytes face the danger of cumulative genetic damage for decades. Thus, the ability to detect and repair DNA damage is essential to maintain the supply of oocytes necessary for reproduction. Therefore, failure to confront DNA damage in oocytes could cause serious anomalies in the embryo that may be propagated in the form of mutations to the next generation allowing the appearance of hereditary disease. Despite the potential impact of DNA damage on reproductive capacity and genetic fidelity of embryos, the mechanisms available to the oocyte for monitoring and repairing such insults have remained largely unexplored until recently. Here, we review the different aspects of the response to DNA damage in mammalian oocytes. Specifically, we address the oocyte DNA damage response from embryonic life to adulthood and throughout oocyte development.

  16. Histopathology of Lyme arthritis in LSH hamsters

    Energy Technology Data Exchange (ETDEWEB)

    Hejka, A.; Schmitz, J.L.; England, D.M.; Callister, S.M.; Schell, R.F.


    The authors studied the histopathologic evolution of arthritis in nonirradiated and irradiated hamsters infected with Borrelia burgdorferi. Nonirradiated hamsters injected in the hind paws with B. burgdorferi developed an acute inflammatory reaction involving the synovium, periarticular soft tissues, and dermis. This acute inflammatory reaction was short-lived and was replaced by a mild chronic synovitis as the number of detectable spirochetes in the synovium, periarticular soft tissues, and perineurovascular areas diminished. Exposing hamsters to radiation before inoculation with B. burgdorferi exacerbated and prolonged the acute inflammatory phase. Spirochetes also persisted longer in the periarticular soft tissues. A major histopathologic finding was destructive and erosive bone changes of the hind paws, which resulted in deformation of the joints. These studies should be helpful in defining the immune mechanism participating in the onset, progression, and resolution of Lyme arthritis.

  17. Expression of FSH receptor in the hamster ovary during perinatal development (United States)

    Chakraborty, Prabuddha; Roy, Shyamal K.


    FSH plays an important role in ovarian follicular development, and it functions via the G-protein coupled FSH receptor. The objectives of the present study were to determine if full-length FSHR mRNA and corresponding protein were expressed in fetal through postnatal hamster ovaries to explain the FSH-induced primordial follicle formation, and if FSH or estrogen (E) would affect the expression. A full-length and two alternately spliced FSHR transcripts were expressed from E14 through P20. The level of the full-length FSHR mRNA increased markedly through P7 before stabilizing at a lower level with the formation and activation of primordial follicles. A predicted 87kDa FSHR protein band was detected in fetal through P4 ovaries, but additional bands appeared as ovary developed. FSHR immunosignal was present in undifferentiated somatic cells and oocytes in early postnatal ovaries, but was granulosa cells specific after follicles formed. Both eCG and E significantly up-regulated full-length FSHR mRNA levels. Therefore, FSHR is expressed in the hamster ovary from the fetal life to account for FSH-induced primordial follicle formation and cAMP production. Further, FSH or E regulates the receptor expression. PMID:25462586

  18. Improving oocyte quality by transfer of autologous mitochondria from fully grown oocytes. (United States)

    Kristensen, Stine Gry; Pors, Susanne Elisabeth; Andersen, Claus Yding


    Older women are often the most challenging group of patients in fertility clinics due to a decline in both number and overall quality of oocytes. The quality of oocytes has been linked to mitochondrial dysfunction. In this mini-review, we discuss this hypothesis and suggest alternative treatment options using autologous mitochondria to potentially augment pregnancy potential in ART. Autologous transfer of mitochondria from the patient's own germline cells has attracted much attention as a possible new treatment to revitalize deficient oocytes. IVF births have been reported after transfer of oogonial precursor cell-derived mitochondria; however, the source and quality of the mitochondria are still unclear. In contrast, fully grown oocytes are loaded with mitochondria which have passed the genetic bottleneck and are likely to be of high quality. An increased supply of such oocytes could potentially be obtained by in vitro follicle activation of ovarian cortical biopsies or from surplus immature oocytes collected from women undergoing ART or fertility preservation of ovarian tissue. Taken together, autologous oocytes are not necessarily a limiting resource in ART as fully grown oocytes with high quality mitochondria can be obtained from natural or stimulated ovaries and potentially be used to improve both quality and quantity of oocytes available for fertility treatment. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail:

  19. Congenital Transmission of Experimental Leishmaniasis in a Hamster Model (United States)

    Osorio, Yaneth; Rodriguez, Luz D.; Bonilla, Diana L.; Peniche, Alex G.; Henao, Hector; Saldarriaga, Omar; Travi, Bruno L.


    Little information is available on transplacental transmission of Leishmania spp. We determined the frequency and impact of congenital infection caused by Leishmania panamensis or L. donovani in experimentally infected hamsters. A polymerase chain reaction showed that congenital transmission occurred in 25.8% (24 of 93) of offspring born to L. panamensis-infected hamsters and 14.6% (11 of 75) offspring born to L. donovani-infected hamsters. Mortality during lactation was higher in offspring born to L. panamensis-infected hamsters and offspring born to L. donovani-infected hamsters than controls, and lymphoproliferation to Leishmania was more frequent in offspring born to L. panamensis-infected hamsters (17.4%, 11 of 63) than in offspring born to L. donovani-infected hamsters (8.5%, 3 of 35). After weaning, only offspring born to L. donovani-infected hamsters had lower weight gain (P < 0.001) and hematocrit levels (P = 0.0045) than controls. Challenge of offspring born to L. panamensis-infected hamsters with L. panamensis showed no differences in lesion evolution, and offspring born to L. donovani-infected hamsters were more susceptible to L. donovani challenge than controls. Consequently, prenatal exposure of hamsters to L. donovani significantly increased the mortality risk and susceptibility to secondary homologous infection. PMID:22556079

  20. Assisted oocyte activation following ICSI fertilization failure. (United States)

    Vanden Meerschaut, Frauke; Nikiforaki, Dimitra; Heindryckx, Björn; De Sutter, Petra


    The capacity of intracytoplasmic sperm injection (ICSI) to permit almost any type of spermatozoa to fertilize oocytes has made it the most successful treatment for male factor infertility. Despite its high success rates, fertilization failure following ICSI still occurs in 1-3% of couples. Assisted oocyte activation (AOA) is being increasingly applied in human assisted reproduction to restore fertilization and pregnancy rates in couples with a history of ICSI fertilization failure. However, controversy still exists mainly because the artificial activating agents do not mimic precisely the initial physiological processes of mammalian oocyte activation, which has led to safety concerns. This review addresses the mechanism of human oocyte activation and the relatively rare phenomenon of fertilization failure after ICSI. Next, it describes the current diagnostic approaches and focuses on the application, efficiency and safety of AOA in human assisted reproduction. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  1. Bovine cumulus-oocyte disconnection in vitro

    DEFF Research Database (Denmark)

    Maddox-Hyttel, Poul


    Cumulus-oocyte complexes were obtained from cows by aspiration of small (1-6 mm in diameter) antral follicles after slaughter. Complexes with a compact multilayered cumulus investment were cultured and processed for transmission electron microscopy after different periods of culture including a 0...... frequency of gap junctions was maintained until 12-18 h of culture where the junctional contact was completely disrupted. This decrease in intercellular communication was parallelled by resumption of oocyte meiosis....

  2. Ultrastructure of human mature oocytes after vitrification

    Directory of Open Access Journals (Sweden)

    M.A. Khalili


    Full Text Available Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morphofunctional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microscopy has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.

  3. How does polyspermy happen in mammalian oocytes? (United States)

    Wang, Wei-Hua; Day, Billy N; Wu, Guang-Ming


    Polyspermy is one of the most commonly observed abnormal types of fertilization in mammalian oocytes. In vitro fertilization (IVF) provides approaches to study the mechanisms by which oocytes block polyspermic fertilization. Accumulated data indicate that oocyte, sperm and insemination conditions are all related to the occurrence of polyspermic fertilization. A high proportion of immature and aged oocytes showed polyspermy as compared with mature oocytes. Preincubation of oocytes and/or sperm with oviductal epithelial cells or collected oviductal fluid before IVF reduces polyspermic penetration. Recently, it was found that an abnormal zona pellucida is one of main causes of polyspermy in human eggs. A high proportion of polyspermy has resulted from the use of a high concentration of capacitated spermatozoa at the site of fertilization, irrespective of in the in vivo or in vitro environment. Oviductal secretions or oviductal epithelial cells themselves can regulate the number of spermatozoa reaching or binding to the zona pellucida thus reducing multiple sperm penetration. Suboptimal in vitro conditions, such as supplementations in IVF media, pH, and temperature during IVF, also induce polyspermic fertilization in some mammals. Species-specific differences are present regarding the relationship between insemination conditions and polyspermy. Copyright 2003 Wiley-Liss, Inc.

  4. Oocyte glutathione and fertilisation outcome of Macaca nemestrina and Macaca fascicularis in in vivo- and in vitro-matured oocytes. (United States)

    Curnow, E C; Ryan, J P; Saunders, D M; Hayes, E S


    Fertilisation and development of IVM non-human primate oocytes is limited compared with that of in vivo-matured (IVO) oocytes. The present study describes the IVM of macaque oocytes with reference to oocyte glutathione (GSH). Timing of maturation, comparison of IVM media and cysteamine (CYS) supplementation as a modulator of GSH were investigated. A significantly greater proportion of oocytes reached MII after 30 h compared with 24 h of IVM. Following insemination, IVM oocytes had a significantly lower incidence of normal fertilisation (i.e. 2PN = two pronuclei and at least one polar body) and a higher rate of abnormal fertilisation (1PN = one pronucleus and at least one polar body) compared with IVO oocytes. Immunofluorescence of 1PN zygotes identified incomplete sperm head decondensation and failure of male pronucleus formation as the principal cause of abnormal fertilisation in IVM oocytes. The IVO oocytes had significantly higher GSH content than IVM oocytes. Cumulus-denuded oocytes had significantly lower GSH following IVM compared with immature oocytes at collection. Cysteamine supplementation of the IVM medium significantly increased the GSH level of cumulus-intact oocytes and reduced the incidence of 1PN formation, but did not improve GSH levels of the denuded oocyte. Suboptimal GSH levels in macaque IVM oocytes may be related to reduced fertilisation outcomes.

  5. Ahne hamster lõikuskuul/ Tambet Kaugema

    Index Scriptorium Estoniae

    Kaugema, Tambet


    Eesti Nuku- ja Noorsoteatri jõululavastusest "Ahne hamster ja värvilised jäälilled", autor Miloš Macourek, tõlkija Leo Metsar, lavastaja ja muusikaline kujundaja Virko Annus, mängib Tarmo Männard. Esietendus 21. novembril Köismäe tornis

  6. Disparities in activity levels and learning ability between Djungarian hamster (Phodopus sungorus) and Roborovskii hamster (Phodopus roborovskii). (United States)

    Ikeda, Hiromi; Nagasawa, Mao; Yamaguchi, Takeshi; Minaminaka, Kimie; Goda, Ryosei; Chowdhury, Vishwajit S; Yasuo, Shinobu; Furuse, Mitsuhiro


    The Djungarian hamster and the Roborovskii hamster belong to the same genus of Phodopus. However, the Djungarian hamster is tame and shows sedative behavior, while Roborovskii hamster is not tame and shows high levels of locomotor activity. Hyperactivity occurs in animals with tameless behavior. Tameness or tamelessness behavior is very important because tameness helps for breeding and controlling as well as it enables a strong human-animal bond. In the present study, we examined the relationships between activity levels and cognitive function in Djungarian and Roborovskii hamsters. Three types of behavioral tests were performed to analyze their activity levels, memory and leaning ability. The levels of L- and D-amino acids and monoamines in the brain were then determined. Roborovskii hamsters showed significantly higher locomotor activity than Djungarian hamsters. Memory ability was not significantly different between the two hamsters, but Roborovskii hamsters showed lower learning ability. Brain levels of D-serine which is related to enhancement in memory and learning ability, were significantly higher in Djungarian hamsters, but the reverse was true for brain dopamine and serotonin levels. These results suggest that these differences in brain metabolism may be related to the behavioral differences between the two hamsters. © 2016 Japanese Society of Animal Science.

  7. A lack of coordination between sister-chromatids segregation and cytokinesis in the oocytes of B6.YTIR (XY) sex-reversed female mice. (United States)

    Zhu, Jia-Qiao; Tan, Seang Lin; Taketo, Teruko


    The B6.YTIR (XY) mouse develops bilateral ovaries despite the expression of the testis-determining gene Sry during gonadal differentiation. We reported that the oocytes of the XY female are defective in their cytoplasm, resulting in a failure in the second meiotic division after activation or fertilization in vitro. However, the mechanism of meiotic failure or the cause of infertility remained to be clarified. In the present study, we obtained mature oocytes from XY females by superovulation and confirmed that these oocytes also fail in zygotic development. By using confocal microscopy 3D-analysis, we demonstrated that meiotic spindles were properly positioned and oriented in the MII-oocytes from XY females. After parthenogenic activation, fewer oocytes from XY females extruded the second polar body, and in those oocytes, sister-chromatids were often separated but neither set entered the second polar body. ARP2, F-actin, and ORC4, known to play roles in asymmetric meiotic division, were initially localized along the ooplasmic membrane and concentrated over the MII-spindle but lost their cortical polarity after activation while the sister-chromatids moved away from the oolemma in the oocytes from XY females. Our results indicate that the second polar body extrusion is uncoupled from the sister-chromatids separation in the oocytes from XY female mouse.

  8. A new vision of the origin and the oocyte development in the Ostariophysi applied to Gymnotus sylvius (Teleostei: Gymnotiformes

    Directory of Open Access Journals (Sweden)

    Gisleine Fernanda França

    Full Text Available Based on new knowledge coming from marine perciform species, the origin of oocytes and their development in the Ostariophysi, Gymnotus sylvius is described. In both Gymnotus sylvius and marine perciform fish, oogonia are found in the germinal epithelium that forms the surface of the ovarian lamellae. At the commencement of folliculogenesis, proliferation of oogonia and their entrance into meiosis gives rise to germ cell nests that extend into the stroma from the germinal epithelium. Both cell nests and the germinal epithelium are supported by the same basement membrane that separates them from the stroma. At the time of meiotic arrest, oocytes in a cell nest become separated one from the other as processes of prefollicle cells, these being derived from epithelial cells in the germinal epithelium, gradually encompass and individualize them while also synthesizing a basement membrane around themselves during folliculogenesis. The oocyte enters primary growth while still within the cell nest. At the completion of folliculogenesis, the oocyte and follicle cells, composing the follicle, are encompassed by a basement membrane. The follicle remains connected to the germinal epithelium as the both share a portion of common basement membrane. Cells originating from the stroma encompass the ovarian follicle, except where there is a shared basement membrane, to form the theca. The follicle, basement membrane and theca form the follicular complex. Oocyte development occurs inside the follicular complex. Development is divided into the stages primary and secondary growth, oocyte maturation and ovulation. Cortical alveoli appear in the ooplasm just prior to the beginning of secondary growth, the vitellogenic stage that begins with yolk deposition and proceeds until the oocyte is full-grown and the ooplasm is filled with yolk globules. Maturation is characterized by the germinal vesicle or nuclear migration, germinal vesicle breakdown or nuclear envelop

  9. Ultrastructure and mitochondrial numbers in pre- and postpubertal pig oocytes

    DEFF Research Database (Denmark)

    Pedersen, Hanne Skovsgaard; Callesen, Henrik; Løvendahl, Peter


    Prepubertal pig oocytes are associated with lower developmental competence. The aim of this experiment was to conduct an exhaustive survey of oocyte ultrastructure and to use a design-unbiased stereological approach to quantify the numerical density and total number of mitochondria in oocytes...... granules and central localisation of mitochondria, vesicles and lipid droplets. Prepubertal oocytes displayed more variation. The ultrastructure of large pre- and postpubertal oocytes was compatible with higher developmental competence, whereas that of smaller prepubertal oocytes could explain...... with different diameters from pre- and postpubertal pigs. The ultrastructure of smaller prepubertal immature oocytes indicated active cells in close contact with cumulus cells. The postpubertal oocytes were more quiescent cell types. The small prepubertal oocytes had a lower total mitochondrial number...

  10. Improving oocyte quality by transfer of autologous mitochondria from fully grown oocytes

    DEFF Research Database (Denmark)

    Kristensen, Stine Gry; Pors, Susanne Elisabeth; Andersen, Claus Yding


    Older women are often the most challenging group of patients in fertility clinics due to a decline in both number and overall quality of oocytes. The quality of oocytes has been linked to mitochondrial dysfunction. In this mini-review, we discuss this hypothesis and suggest alternative treatment ...

  11. Simple and inexpensive hardware and software method to measure volume changes in Xenopus oocytes expressing aquaporins. (United States)

    Dorr, Ricardo; Ozu, Marcelo; Parisi, Mario


    Water channels (aquaporins) family members have been identified in central nervous system cells. A classic method to measure membrane water permeability and its regulation is to capture and analyse images of Xenopus laevis oocytes expressing them. Laboratories dedicated to the analysis of motion images usually have powerful equipment valued in thousands of dollars. However, some scientists consider that new approaches are needed to reduce costs in scientific labs, especially in developing countries. The objective of this work is to share a very low-cost hardware and software setup based on a well-selected webcam, a hand-made adapter to a microscope and the use of free software to measure membrane water permeability in Xenopus oocytes. One of the main purposes of this setup is to maintain a high level of quality in images obtained at brief intervals (shorter than 70 ms). The presented setup helps to economize without sacrificing image analysis requirements.

  12. What controls polyspermy in mammals, the oviduct or the oocyte? (United States)

    Coy, Pilar; Avilés, Manuel


    A block to polyspermy is required for successful fertilisation and embryo survival in mammals. A higher incidence of polyspermy is observed during in vitro fertilisation (IVF) compared with the in vivo situation in several species. Two groups of mechanisms have traditionally been proposed as contributing to the block to polyspermy in mammals: oviduct-based mechanisms, avoiding a massive arrival of spermatozoa in the proximity of the oocyte, and egg-based mechanisms, including changes in the membrane and zona pellucida (ZP) in reaction to the fertilising sperm. Additionally, a mechanism has been described recently which involves modifications of the ZP in the oviduct before the oocyte interacts with spermatozoa, termed "pre-fertilisation zona pellucida hardening". This mechanism is mediated by the oviductal-specific glycoprotein (OVGP1) secreted by the oviductal epithelial cells around the time of ovulation, and is reinforced by heparin-like glycosaminoglycans (S-GAGs) present in oviductal fluid. Identification of the molecules contributing to the ZP modifications in the oviduct will improve our knowledge of the mechanisms of sperm-egg interaction and could help to increase the success of IVF systems in domestic animals and humans.

  13. Effects of trehalose vitrification and artificial oocyte activation on the development competence of human immature oocytes. (United States)

    Zhang, Zhiguo; Wang, Tianjuan; Hao, Yan; Panhwar, Fazil; Chen, Zhongrong; Zou, Weiwei; Ji, Dongmei; Chen, Beili; Zhou, Ping; Zhao, Gang; Cao, Yunxia


    Sucrose and trehalose are conventional cryoprotectant additives for oocytes and embryos. Ethanol can artificially enhance activation of inseminated mature oocytes. This study aims to investigate whether artificial oocyte activation (AOA) with ethanol can promote the development competence of in vitro matured oocytes. A total of 810 human immature oocytes, obtained from 325 patients undergoing normal stimulated oocyte retrieval cycles, were in vitro maturated (IVM) either immediately after collection (Fresh group n = 291)) or after being vitrified as immature oocytes (Vitrified group n = 519). These groups were arbitrarily assigned. All fresh and vitrified oocytes which matured after a period of IVM then underwent intra-cytoplasmic sperm injection (ICSI). Half an hour following ICSI, they were either activated by 7% ethanol (AOA group) or left untreated (Non-AOA group). Fertilization, cleavage rate, blastocyst quality and aneuploidy rate were then evaluated. High-quality blastocysts were only obtained in both the fresh and vitrified groups which had undergone AOA after ICSI. Trehalose vitrification slightly, but not significantly, increased the formation rates of high-quality embryos (21.7% VS 15.4%, P > 0.05) and blastocysts (15.7% VS 7.69%, P > 0.05)) when compared with sucrose vitrification. Aneuploidy was observed in 12 of 24 (50%) of the AOA derived high quality blastocysts. High-quality blastocysts only developed from fresh or vitrified immature oocytes if the ICSI was followed by AOA. This information may be important for human immature oocytes commonly retrieved in normal stimulation cycles and may be particularly important for certain patient groups, such as cancer patients. AOA with an appropriate concentration of ethanol can enhance the developmental competence of embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Calcium influx factor (CIF) as a diffusible messenger for the activation of capacitative calcium entry in Xenopus oocytes. (United States)

    Kim, H Y; Hanley, M R


    Acid extracts of thapsigargin-treated Xenopus oocytes revealed Ca2(+)-dependent Cl- currents by microinjection into Xenopus oocytes. These currents were detected in highly purified fractions by carrying out a sequence of purification steps including gel filtration chromatography and high performance thin layer chromatography. The nature of the membrane currents evoked by the highly purified fractions were carried by chloride ions as blockade by the selective chloride channel blocker 1 mM niflumic acid. Injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) eradicated the current activities, indicating that the current responses are completely Ca2(+)-dependent. Moreover, the currents were sensitive to the removal of extracellular calcium, indicating the dependence on calcium entry through plasma membrane calcium entry channels. These results elucidate that the highly purified fractions aquired by thapsigargin-stimulated oocytes is an authentic calcium influx factor (CIF). Thus, the detection of increased CIF production from thapsigargin treatment in Xenopus oocytes would give strong support for the existence of CIF as a diffusible messenger for the activation of capacitative calcium entry pathways in Xenopus oocytes.

  15. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes




    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium from ...

  16. Apoptosis maintains oocyte quality in aging Caenorhabditis elegans females.

    Directory of Open Access Journals (Sweden)

    Sara Andux


    Full Text Available In women, oocytes arrest development at the end of prophase of meiosis I and remain quiescent for years. Over time, the quality and quantity of these oocytes decreases, resulting in fewer pregnancies and an increased occurrence of birth defects. We used the nematode Caenorhabditis elegans to study how oocyte quality is regulated during aging. To assay quality, we determine the fraction of oocytes that produce viable eggs after fertilization. Our results show that oocyte quality declines in aging nematodes, as in humans. This decline affects oocytes arrested in late prophase, waiting for a signal to mature, and also oocytes that develop later in life. Furthermore, mutations that block all cell deaths result in a severe, early decline in oocyte quality, and this effect increases with age. However, mutations that block only somatic cell deaths or DNA-damage-induced deaths do not lower oocyte quality. Two lines of evidence imply that most developmentally programmed germ cell deaths promote the proper allocation of resources among oocytes, rather than eliminate oocytes with damaged chromosomes. First, oocyte quality is lowered by mutations that do not prevent germ cell deaths but do block the engulfment and recycling of cell corpses. Second, the decrease in quality caused by apoptosis mutants is mirrored by a decrease in the size of many mature oocytes. We conclude that competition for resources is a serious problem in aging germ lines, and that apoptosis helps alleviate this problem.

  17. Polarized light microscopy in mammalian oocytes. (United States)

    Caamaño, J N; Muñoz, M; Diez, C; Gómez, E


    The meiotic spindle structure plays a key role in normal chromosome alignment and segregation during meiosis. Polarized light microscopy (PLM) allows non-invasive evaluation of the meiotic spindle of metaphase oocytes from different animal species. The purpose of this article is to review the use of PLM in animal reproduction, mainly in the assessment of the meiotic spindle in oocytes. A brief overview of the methods to assess the meiotic spindle is presented as well as the principles behind the PLM. The use of PLM to evaluate oocyte quality and spindle morphology is discussed and the results on the viability of the oocytes after being exposed to PLM are presented. Several researchers showed that PLM could be successfully implemented on cryopreservation, nuclear transfer and intracytoplasmic sperm injection procedures as a tool to improve the outcome of these procedures. In addition, PLM can be used to develop studies on oocyte maturation and spindle dynamics. However, the information on the practical use of this technology in farm animals is very limited and further studies are needed to assess the importance of PLM in animal reproduction.

  18. Melatonin regulates lipid metabolism in porcine oocytes. (United States)

    Jin, Jun-Xue; Lee, Sanghoon; Taweechaipaisankul, Anukul; Kim, Geon A; Lee, Byeong Chun


    It is being increasingly recognized that the processes of lipogenesis and lipolysis are important for providing an essential energy source during oocyte maturation and embryo development. Recent studies demonstrated that melatonin has a role in lipid metabolism regulation, including lipogenesis, lipolysis, and mitochondrial biogenesis. In this study, we attempted to investigate the effects of melatonin on lipid metabolism during porcine oocyte in vitro maturation. Melatonin treatment significantly enhanced the number of lipid droplets (LDs) and upregulated gene expression related to lipogenesis (ACACA, FASN, PPARγ, and SREBF1). Oocytes treated with melatonin formed smaller LDs and abundantly expressed several genes associated with lipolysis, including ATGL, CGI-58, HSL, and PLIN2. Moreover, melatonin significantly increased the content of fatty acids, mitochondria, and ATP, as indicated by fluorescent staining. Concomitantly, melatonin treatment upregulated gene expression related to fatty acid β-oxidation (CPT1a, CPT1b, CPT2, and ACADS) and mitochondrial biogenesis (PGC-1α, TFAM, and PRDX2). Overall, melatonin treatment not only altered both the morphology and amount of LDs, but also increased the content of fatty acids, mitochondria, and ATP. In addition, melatonin upregulated mRNA expression levels of lipogenesis, lipolysis, β-oxidation, and mitochondrial biogenesis-related genes in porcine oocytes. These results indicated that melatonin promoted lipid metabolism and thereby provided an essential energy source for oocyte maturation and subsequent embryonic development. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. DNA damage response during mouse oocyte maturation. (United States)

    Mayer, Alexandra; Baran, Vladimir; Sakakibara, Yogo; Brzakova, Adela; Ferencova, Ivana; Motlik, Jan; Kitajima, Tomoya S; Schultz, Richard M; Solc, Petr


    Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II.  Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation.

  20. Aminopeptidase-like protease released from oocytes affects oocyte surfaces and suppresses the acrosome reaction in establishment of polyspermy block in oocytes of the mussel Mytilus edulis. (United States)

    Togo, T; Morisawa, M


    Suppression of the acrosome reaction of sperm on fertilized oocytes inhibits sperm-oocyte binding and is considered one of the three mechanisms responsible for polyspermy block in oocytes of the mussel Mytilus edulis (Togo et al., 1995). When unfertilized oocytes were inseminated in the presence of aminopeptidase inhibitors and the fertilized oocytes were inseminated again, neither the acrosome reaction nor sperm binding to fertilized oocytes were suppressed, suggesting that aminopeptidase-like protease participates in suppression of the acrosome reaction. The supernatant solution obtained after centrifuging a suspension of fertilized oocytes hydrolyzed aminopeptidase substrates, and these activities were inhibited strongly or effectively by aminopeptidase inhibitors. When unfertilized oocytes were incubated with this supernatant solution and inseminated, both the number of sperm bound and the acrosome reaction rate decreased, and these effects were reversed by conducting this treatment in the presence of aminopeptidase inhibitors. These results suggest that aminopeptidase-like protease released from the oocyte at fertilization affects the oocyte surface to suppress the acrosome reaction and consequently inhibits sperm-oocyte binding.

  1. Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse. (United States)

    Bedford, Sylvia J; Kurokawa, Manabu; Hinrichs, Katrin; Fissore, Rafael A


    In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca2+]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca2+]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca2+]i oscillations to injection of stallion sperm, and [Ca2+]i monitoring was performed. Such pairs consistently displayed [Ca2+]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species.

  2. Congenital Transmission of Experimental Leishmaniasis in a Hamster Model


    Osorio, Yaneth; Rodriguez, Luz D.; Diana L Bonilla; Peniche, Alex G.; Henao, Hector; Saldarriaga, Omar; Bruno L. Travi


    Little information is available on transplacental transmission of Leishmania spp. We determined the frequency and impact of congenital infection caused by Leishmania panamensis or L. donovani in experimentally infected hamsters. A polymerase chain reaction showed that congenital transmission occurred in 25.8% (24 of 93) of offspring born to L. panamensis-infected hamsters and 14.6% (11 of 75) offspring born to L. donovani-infected hamsters. Mortality during lactation was higher in offspring b...

  3. Passive immunization prevents induction of Lyme arthritis in LSH hamsters.


    Schmitz, J L; Schell, R F; Hejka, A G; England, D M


    We determined that sera obtained from hamsters infected with Borrelia burgdorferi could prevent the induction of Lyme arthritis. When irradiated hamsters were administered immune serum and subsequently challenged with B. burgdorferi, no evidence of infection was detected. Recipients failed to develop swelling of the hind paws, and no histopathologic changes were detected. In addition, B. burgdorferi was not recovered from tissues of hamsters that were passively immunized. By contrast, irradia...

  4. Vitrification of mouse MII oocytes: Developmental competency using paclitaxel

    Directory of Open Access Journals (Sweden)

    Farzaneh Fesahat


    Conclusion: A high concentration of paclitaxel, an anticancer drug, interrupted the mouse oocyte competency when supplemented to vitrification media. Consequently, the optimal concentration of this cytoskeleton stabilizer may improve the post-thawed developmental abilities of oocytes.

  5. Xenopus oocyte electrophysiology in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans


    Deorphanization of the large group of G protein-coupled receptors (GPCRs) for which an endogenous activating ligand has not yet been identified (orphan GPCRs) has become increasingly difficult. A specialized technique that has been successfully applied to deorphanize some of these GPCRs involves...... two-electrode voltage-clamp recordings of currents through ion channels, which are activated by GPCRs heterologously expressed in Xenopus oocytes. The ion channels that couple to GPCR activation in Xenopus oocytes can be endogenous calcium-activated chloride channels (CaCCs) or heterologously...... expressed G protein-coupled inwardly rectifying potassium channels (GIRKs). We will describe a general approach for expression of GPCRs in Xenopus oocytes and characterization of these using electrophysiological recordings. We will focus on the detection of GPCR activation by recordings of currents through...



    R.M. Santos; G. D. A. Araguchi; G. R. S. Sluizas; M. C. Menezes; Lega, E.; B. Paludeto; V. Fardin


    The Hamster, rodent originating from the Middle East, is a species studied along with other laboratory animals as experimental models in scientific papers and currently is also created as a pet, by virtue of being docile, easy to handle and require little space for survival. The suppurative processes in domestic animals are relatively frequent. Due to infectious diseases or purulent course of aggressiveness of the environment in which they live. The habit of storing food in the cheeks with sh...

  7. Effect of mebendazole on fibrosarcoma in hamsters

    African Journals Online (AJOL)

    Original Research Article. Effect of mebendazole on fibrosarcoma in hamsters. Dušica J Popović1*, Dušan Lalošević1, Kosta J Popović2, Ivan Čapo1, Jovan K. Popović3 and Dejan Miljković1. 1Department of Histology and Embryology, 2Department of Pharmacy, 3Department of Pharmacology, Toxicology and Clinical.

  8. Calreticulin from suboolemmal vesicles affects membrane regulation of polyspermy. (United States)

    Saavedra, María Dolores; Mondéjar, Irene; Coy, Pilar; Betancourt, Miguel; González-Márquez, Humberto; Jiménez-Movilla, María; Avilés, Manuel; Romar, Raquel


    This study was designed to determine whether calreticulin (CRT), a chaperone protein, is present in in vitro-matured (IVM) pig oocytes and to study its potential role in the block to polyspermy. Western blot analysis, using an anti-CRT antibody, of oocyte lysate showed an immunoreactive band of ∼60  kDa. Simultaneous labeling of IVM oocytes with anti-CRT antibody and peanut agglutinin lectin (PNA lectin, a porcine cortical granules (CG)-specific binding lectin) revealed localization of CRT in the subplasmalemmal region with a 27.7% colocalization with PNA staining. After IVF, PNA labeling was not observed and anti-CRT labeling decreased significantly in zygotes and disappeared in two-cell embryos. Western blot analysis of oocyte exudate obtained from zona pellucida (ZP)-free oocytes activated with calcium ionophore confirmed the presence of a band that reacted with an anti-CRT antibody. Anti-CRT antibody and PNA labeling were not observed in activated oocytes despite being detectable in non-activated oocytes. The presence of CRT in vesicles located under the oolemma was demonstrated using immunogold cytochemistry at the ultrastructural level. To study the role of CRT in fertilization, ZP-enclosed and ZP-free oocytes were incubated with exogenous CRT and then inseminated. Whereas ZP-free oocytes showed fewer penetrating sperm and lower polyspermy rates than untreated oocytes, the opposite effect was observed in ZP-enclosed oocytes. In conclusion, CRT is confined to subplasmalemmal vesicles partially overlapping with CG contents. Its exocytosis after the oocyte activation seems to participate in the membrane block to polyspermy in pigs but is not involved in the ZP block.

  9. Effect of medium additives during liquid storage on developmental competence of in vitro matured bovine oocytes. (United States)

    Suttirojpattana, Tayita; Somfai, Tamas; Matoba, Satoko; Parnpai, Rangsun; Nagai, Takashi; Geshi, Masaya


    Our aim was to improve the developmental competence of bovine oocytes during their liquid storage by using additives. In vitro matured oocytes were stored for 20 h at 25°C in HEPES buffered TCM 199 medium (base medium). After storage, in vitro embryo development after in vitro fertilization was compared to those of non-stored (control) ones. Addition of 10% (v/v) newborn calf serum or 10.27 mmol/L pyruvate alone to the base medium did not improve blastocyst formation rates in stored oocytes; however, their simultaneous addition significantly improved the rate compared with those stored in base medium (P serum had a synergic effect to moderate the reduction of oocyte quality during storage, whereas mitochondrial membrane pore inhibitor CsA and the antioxidant DTT did not affect their developmental competence. © 2016 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.

  10. Case report: Goldenhar syndrome following donor oocyte IVF. (United States)

    Gittins, Victoria; Kasraie, Jason


    To describe a case of Goldenhar syndrome in a couple receiving donated oocytes in an 'egg sharing' IVF cycle where the recipient of donor oocytes had Turner syndrome, hypothyroidism and gestational diabetes. Case report Child born to oocyte recipient with Goldenhar syndrome We believe this is the first reported case of a child born with Goldenhar syndrome following use of donated oocytes in IVF by a woman with Turner syndrome, hypothyroidism and gestational diabetes.

  11. Molecular and immunological characterization of the first allergenic lipocalin in hamster: the major allergen from Siberian hamster (Phodopus sungorus). (United States)

    Torres, José Alberto; de Las Heras, Manuel; Maroto, Aroa Sanz; Vivanco, Fernando; Sastre, Joaquín; Pastor-Vargas, Carlos


    The most frequent pet allergy is to cat and dog, but in recent years, it has become increasingly popular to have other pets, and the risk of exposure to new allergens is more prevalent. The list of new pets includes hamsters, and one of the most popular hamsters is the Siberian hamster (Phodopus sungorus). The aim of this study was the characterization and cloning of the major allergen from this hamster. The study of its allergenicity and cross-reactivity could improve the specific diagnosis and treatment for hamster-allergic patients. Thirteen Siberian hamster-allergic patients were recruited at the outpatient clinic. Protein extracts were prepared from the hair, urine, and salivary glands of four hamster species (European, golden, Siberian, and Roborovski). IgE-binding proteins were detected by immunoblotting and identified by mass spectrometry. The recombinant protein was produced in Escherichia coli and then purified by metal chelate affinity chromatography. The allergenic properties of the recombinant protein were tested by ELISA and immunoblotting, and biological activity was tested according to capacity for basophil activation. Three IgE-binding proteins were identified in extracts obtained from Siberian hamster hair, urine, and salivary glands. All proteins corresponded to the same protein, which was identified as a lipocalin. This lipocalin had no cross-reactivity with common and golden hamsters. The recombinant allergen was cloned and purified, showing similar IgE reactivity in vitro to Siberian hamster protein extracts. Also, the recombinant allergen was capable of producing biological activation in vivo. The major Siberian hamster allergen was cloned, and allergenic properties were characterized, providing a new tool for specific diagnosis of allergy to Siberian hamster. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Human oocyte vitrification: the permeability of metaphase II oocytes to water and ethylene glycol and the appliance toward vitrification

    National Research Council Canada - National Science Library

    Mullen, Steven F; Li, Mei; Li, Yuan; Chen, Zi-Jiang; Critser, John K


    To determine the permeability of human metaphase II oocytes to ethylene glycol and water in the presence of ethylene glycol, and to use this information to develop a method to vitrify human oocytes...

  13. Effect of melatonin on in vitro maturation of bovine oocytes ...

    African Journals Online (AJOL)

    ... Vs 17.67, 15.68, 16.53). In conclusion in this experiment, melatonin cannot improve cumulus cell expansion and nuclear maturation of bovine oocytes. When concentrations is high, melatonin may affect bovine oocytes meiotic maturation at metaphase-1 stage, but it is improbable melatonin be toxic for bovine oocytes.

  14. In vitro maturation of sheep oocytes in different concentrations of ...

    African Journals Online (AJOL)



    Sep 17, 2008 ... respectively. Some reports indicate (Kharche et al., 2006) that the addition serum enhances maturation, and development of in vitro-matured oocytes, our results do no support this results. And maturation of follicular oocytes is normally arrested at the prophase-I of the first meiotic division and the oocyte ...

  15. Effect of the exposure to methyl-β-cyclodextrin prior to chilling or vitrification on the viability of bovine immature oocytes. (United States)

    Sprícigo, J F W; Morais, K S; Yang, B S; Dode, M A N


    The present study aimed to evaluate the effect of methyl-β-cyclodextrin (MβCD) as a cholesterol loader to change oocyte plasma membrane and increase its tolerance toward cryopreservation. The first and second experiments were conducted to investigate if MβCD could improve nuclear and cytoplasmic maturation after oocyte exposure to cold stress for 10 or 30 min, respectively. No differences (P>0.05) in either experiment in the metaphase II (MII) rate of oocytes exposed to MβCD and cold stress; but these oocytes presented lower maturation rates than control groups. In the second experiment, a lower percentage of oocytes showed degenerated chromatin (P0.05). Chromatin degeneration was higher in the vitrification groups. We conclude that MβCD improved nuclear maturation by reducing oocyte degeneration after cold stress or vitrification; however, more studies are required to clarify the usefulness of MβCD use in oocyte cryopreservation. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Asymmetric learning to avoid heterospecific males in Mesocricetus hamsters. (United States)

    delBarco-Trillo, Javier; Johnston, Robert E


    If a female mates with a male of a closely related species, her fitness is likely to decline. Consequently, females may develop behavioral mechanisms to avoid mating with heterospecific males. In some species, one such mechanism is for adult females to learn to discriminate against heterospecific males after exposure to such males. We have previously shown that adult, female Syrian hamsters (Mesocricetus auratus) learn to discriminate against male Turkish hamsters (Mesocricetus brandti) after exposure to a single heterospecific male during 8 days across a wire-mesh barrier. Here we repeated that experiment but this time we exposed female Turkish hamsters to a male Syrian hamster for 8 days and then measured sexual and aggressive behaviors towards that heterospecific male and towards a conspecific male. In contrast to female Syrian hamsters, female Turkish hamsters did not differ in their latency to go into lordosis or in any measure of aggression towards either type of male. Female Turkish hamsters spent less time in lordosis with the heterospecific male, but the percentage of trials in which females copulated with conspecific and heterospecific males did not differ. When comparing females from both species that had been exposed to a heterospecific male for 8days, female Syrian hamsters copulated less and were more aggressive towards the heterospecific male compared to the behavior of female Turkish hamsters. We discuss how this asymmetric response between females of the two species may be due to the much larger geographical range of Turkish hamsters compared to Syrian hamsters. Copyright © 2012 Elsevier GmbH. All rights reserved.

  17. Fbos, a novel oocyte-specific protein, interacts with proteins important for oocyte development in rainbow trout (Oncorhynchus mykiss) (United States)

    Oogenesis is characterized by a series of developmentally regulated events, which result in the matured oocyte that can give rise to a new organism after fertilization. Oocyte-specific genes play critical roles in oogenesis; however, the molecular details of oocyte-specific genes are poorly defined....

  18. [Wide support for oocyte donation and banking in the Netherlands]. (United States)

    Bos, Annelies M E; Klapwijk, Petra; Fauser, Bart C J M


    To assess the general consensus on the cryopreservation of oocytes and the introduction of oocyte banking facilities in the Netherlands. Poll investigation A poll with the use of an online questionnaire was conducted among nearly 19,000 participants of the Dutch EenVandaag opinion panel in May 2011. The poll results were adjusted to the Dutch population based on data from the Dutch Central Office for Statistics for age, gender, education, marital status, geographical area and political preference (measured according to the lower house elections of 2010). The primary endpoints were the percentages of supporters of oocyte freezing for own future use and of the concept of introducing oocyte banking facilities in The Netherlands. The secondary endpoints were the demographic differences between supporters and opponents. Approximately half of 18.911 participants supported oocyte freezing (47%). Fifty-percent of all participants supported oocyte banking in the Netherlands. Supporters of oocyte freezing were mainly women ≤ 45 years of age, who are highly educated and have no children. Four percent of the participating women aged ≤ 45 years would seriously consider obtaining donor oocytes from an available oocyte banking facility. Twelve percent of the participating women ≤ 45 years of age said they would definitely donate their oocytes or would seriously consider donating. Thirty-seven percent of all participants were against the introduction of oocyte banking facilities. The most important arguments against oocyte freezing were that women should reproduce during normal reproductive years and that it was not medically necessary. Poll results showed much support for oocyte freezing and for the introduction of oocyte banking facilities in the Netherlands. In addition, the poll shows that oocyte banking facilities would fulfil a need in the population.

  19. Facilitated Lactate Transport by MCT1 when Coexpressed with the Sodium Bicarbonate Cotransporter (NBC) in Xenopus Oocytes (United States)

    Becker, Holger M.; Bröer, Stefan; Deitmer, Joachim W.


    Monocarboxylate transporters (MCT) and sodium-bicarbonate cotransporters (NBC) transport acid/base equivalents and coexist in many epithelial and glial cells. In nervous systems, the electroneutral MCT1 isoform cotransports lactate and other monocarboxylates with H+, and is believed to be involved in the shuttling of energy-rich substrates between astrocytes and neurons. The NBC cotransports bicarbonate with sodium and generates a membrane current. We have expressed these transporter proteins, cloned from rat brain (MCT1) and human kidney (NBC), alone and together, by injecting the cRNA into oocytes of the frog Xenopus laevis, and measured intracellular pH changes and membrane currents under voltage-clamp with intracellular microelectrodes, and radiolabeled lactate uptake into the oocytes. We determined the cytosolic buffer capacity, the H+ and lactate fluxes as induced by 3 and 10 mM lactate in oocytes expressing MCT1 and/or NBC, and in water-injected oocytes, in salines buffered with 5 mM HEPES alone or with 5% CO2/10 mM HCO3− (pH 7.0). In MCT1 + NBC- but not in MCT1- or NBC-expressing oocytes, lactate activated a Na+- and HCO3−-dependent membrane current, indicating that lactate/H+ cotransport via MCT1, due to the induced pH change, stimulates NBC activity. Lactate/H+ cotransport by MCT1 was increased about twofold when MCT1 was expressed together with NBC. Our results suggest that the facilitation of MCT1 transport activity is mainly due to the increase in apparent buffer capacity contributed by the NBC, and thereby suppresses the build-up of intracellular H+ during the influx of lactate/H+, which would reduce MCT1 activity. Hence these membrane transporters functionally cooperate and are able to increase ion/metabolite transport activity. PMID:14695265

  20. The dormant and the fully competent oocyte

    DEFF Research Database (Denmark)

    Grøndahl, Marie Louise; Borup, Rehannah; Vikeså, Jonas


    Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human...

  1. Ca2+ homeostasis regulates Xenopus oocyte maturation. (United States)

    Sun, Lu; Hodeify, Rawad; Haun, Shirley; Charlesworth, Amanda; MacNicol, Angus M; Ponnappan, Subramaniam; Ponnappan, Usha; Prigent, Claude; Machaca, Khaled


    In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte's competence to undergo maturation in preparation for fertilization and embryonic development.

  2. Production and purification of polyclonal anti-hamster ...

    African Journals Online (AJOL)

    Polyclonal antibodies are mixtures of monoclonal antibodies that were produced against different epitops. The goal of this project is to know the production, purification and horseradish peroxidase (HRP) conjugation of polyclonal antibodies against hamster immunoglobulins in rabbits. 300 ìg/300 ìl of ten hamster ...

  3. Calcium signals and oocyte maturation in marine invertebrates. (United States)

    Deguchi, Ryusaku; Takeda, Noriyo; Stricker, Stephen A


    In various oocytes and eggs of animals, transient elevations in cytoplasmic calcium ion concentrations are known to regulate key processes during fertilization and the completion of meiosis. However, whether or not calcium transients also help to reinitiate meiotic progression at the onset of oocyte maturation remains controversial. This article summarizes reports of calcium signals playing essential roles during maturation onset (=germinal vesicle breakdown, GVBD) in several kinds of marine invertebrate oocytes. Conversely, other data from the literature, as well as previously unpublished findings for jellyfish oocytes, fail to support the view that calcium signals are required for GVBD. In addition to assessing the effects of calcium transients on GVBD in marine invertebrate oocytes, the ability of maturing oocytes to enhance their calcium-releasing capabilities after GVBD is also reviewed. Furthermore, possible explanations are proposed for the contradictory results that have been obtained regarding calcium signals during oocyte maturation in marine invertebrates.

  4. Collection of human oocytes at laparoscopy and laparotomy. (United States)

    Lopata, A; Johnston, I W; Leeton, J F; Muchnicki, D; Talbot, J M; Wood, C


    In order to determine whether ovarian follicular aspiration at laparoscopy would provide enough oocytes for adequate in vitro studies, oocytes were collected during the periovulatory stage of the normal menstrual cycle from 45 women undergoing laparoscopy and from 25 women undergoing laparotomy. A larger average number of oocytes was recovered per patient at laparotomy due to better oocyte recoveries from ovarian wedges. However, the average number of oocytes rocovered per patient was the same at both procedures, providing that a suction vacuum of 200 mm Hg was used at laparoscopy, and under these conditions a greater percentage of follicles yielded oocytes at laparoscopy. Overall, 498 follicles were aspirated and 217 oocytes collected; the average recovery was 3 per patient. Moreover, the mean follicular diameter was 9.1 mm in infertile and 8.0 mm in fertile patients (p less than .05).

  5. Characteristics of 263K Scrapie Agent in Multiple Hamster Species (United States)

    Barbian, Kent D.; Race, Brent; Favara, Cynthia; Gardner, Don; Taubner, Lara; Porcella, Stephen; Race, Richard


    Transmissible spongiform encephalopathy (TSE) diseases are known to cross species barriers, but the pathologic and biochemical changes that occur during transmission are not well understood. To better understand these changes, we infected 6 hamster species with 263K hamster scrapie strain and, after each of 3 successive passages in the new species, analyzed abnormal proteinase K (PK)–resistant prion protein (PrPres) glycoform ratios, PrPres PK sensitivity, incubation periods, and lesion profiles. Unique 263K molecular and biochemical profiles evolved in each of the infected hamster species. Characteristics of 263K in the new hamster species seemed to correlate best with host factors rather than agent strain. Furthermore, 2 polymorphic regions of the prion protein amino acid sequence correlated with profile differences in these TSE-infected hamster species. PMID:19193264

  6. Changes in ovarian protein expression during primordial follicle formation in the hamster (United States)

    Mukherjee, Anindit; Reisdorph, Nichole; Guda, Babu; Pandey, Sanjit; Roy, Shyamal K


    Although many proteins have been shown to affect the transition of primordial follicles to the primary stage, factors regulating the formation of primordial follicles remains sketchy at best. Differentiation of somatic cells into early granulosa cells during ovarian morphogenesis is the hallmark of primordial follicle formation; hence, critical changes are expected in protein expression. We wanted to identify proteins, the expression of which would correlate with the formation of primordial follicles as a first step to determine their biological function in folliculogenesis. Proteins were extracted from embryonic (E15) and 8-day old (P8) hamster ovaries and fractionated by two-dimensional gel electrophoresis. Gels were stained with Proteosilver, and images of protein profiles corresponding to E15 and P8 ovaries were overlayed to identify protein spots showing altered expression. Some of the protein spots were extracted from SyproRuby-stained preparative gels, digested with trypsin, and analyzed by mass spectrometry. Both E15 and P8 ovaries had high molecular weight proteins at acidic, basic, and neutral ranges; however, we focused on small molecular weight proteins at 4-7 pH range. Many of those spots might represent post-translational modification. Mass spectrometric analysis revealed the identity of these proteins. The formation of primordial follicles on P8 correlated with many differentially and newly expressed proteins. Whereas Ebp1 expression was downregulated in ovarian somatic cells, Sfrs3 expression was specifically upregulated in newly formed granulosa cells of primordial follicles on P8. The results show for the first time that the morphogenesis of primordial follicles in the hamster coincides with altered and novel expression of proteins involved in cell proliferation, transcriptional regulation and metabolism. Therefore, formation of primordial follicles is an active process requiring differentiation of somatic cells into early granulosa cells and

  7. Biochemical heterogeneity, migration, and pre-fertilization release of mouse oocyte cortical granules

    Directory of Open Access Journals (Sweden)

    Calarco Patricia


    Full Text Available Abstract Background Oocyte cortical granules are important in the fertilization of numerous species including mammals. Relatively little is known about the composition, migration, and pre-fertilization release of mammalian oocyte cortical granules. Results Results obtained with confocal scanning laser microscopy indicated that mouse oocytes have at least two populations of cortical granules, one that bound both the lectin LCA and the antibody ABL2 and one that bound only LCA. Both types of granules were synthesized at the same time during oocyte maturation suggesting that the ABL2 antigen is targeted to specific granules by a sorting sequence. The distribution of both populations of cortical granules was then studied during the germinal vesicle to metaphase II transition. As the oocytes entered metaphase I, the first cortical granule free domain, which was devoid of both populations of cortical granules, formed over the spindle. During first polar body extrusion, a subpopulation of LCA-binding granules became concentrated in the cleavage furrow and underwent exocytosis prior to fertilization. Granules that bound ABL2 were not exocytosed at this time. Much of the LCA-binding exudate from the release at the cleavage furrow was retained in the perivitelline space near the region of exocytosis and was deduced to contain at least three polypeptides with approximate molecular weights of 90, 62, and 56 kDa. A second cortical granule free domain developed following pre-fertilization exocytosis and subsequently continued to increase in area as both, LCA and LCA/ ABL2-binding granules near the spindle became redistributed toward the equator of the oocyte. The pre-fertilization release of cortical granules did not affect binding of sperm to the overlying zona pellucida. Conclusions Our data show that mouse oocytes contain at least two populations of cortical granules and that a subset of LCA-binding cortical granules is released at a specific time (during

  8. Two pathways regulate cortical granule translocation to prevent polyspermy in mouse oocytes. (United States)

    Cheeseman, Liam P; Boulanger, Jérôme; Bond, Lisa M; Schuh, Melina


    An egg must be fertilized by a single sperm only. To prevent polyspermy, the zona pellucida, a structure that surrounds mammalian eggs, becomes impermeable upon fertilization, preventing the entry of further sperm. The structural changes in the zona upon fertilization are driven by the exocytosis of cortical granules. These translocate from the oocyte's centre to the plasma membrane during meiosis. However, very little is known about the mechanism of cortical granule translocation. Here we investigate cortical granule transport and dynamics in live mammalian oocytes by using Rab27a as a marker. We show that two separate mechanisms drive their transport: myosin Va-dependent movement along actin filaments, and an unexpected vesicle hitchhiking mechanism by which cortical granules bind to Rab11a vesicles powered by myosin Vb. Inhibiting cortical granule translocation severely impaired the block to sperm entry, suggesting that translocation defects could contribute to miscarriages that are caused by polyspermy.

  9. The establishment of polarized membrane traffic in Xenopus laevis embryos. (United States)

    Roberts, S J; Leaf, D S; Moore, H P; Gerhart, J C


    Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin

  10. Oocyte cryopreservation for donor egg banking. (United States)

    Cobo, Ana; Remohí, José; Chang, Ching-Chien; Nagy, Zsolt Peter


    Oocyte donation is an efficient alternative to using own oocytes in IVF treatment for different indications. Unfortunately, 'traditional' (fresh) egg donations are challenged with inefficiency, difficulties of synchronization, very long waiting periods and lack of quarantine measures. Given the recent improvements in the efficiency of oocyte cryopreservation, it is reasonable to examine if egg donation through oocyte cryopreservation has merits. The objective of the current manuscript is to review existing literature on this topic and to report on the most recent outcomes from two established donor cryobank centres. Reports on egg donation using slow freezing are scarce and though results are encouraging, outcomes are not yet comparable to a fresh egg donation treatment. Vitrification on the other hand appears to provide high survival rates (90%) of donor oocytes and comparable fertilization, embryo development, implantation and pregnancy rates to traditional (fresh) egg donation. Besides the excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg cryobank and makes it likely that this approach becomes the future standard of care. Oocyte donation is one of the last resorts in IVF treatment for couples challenged with infertility problems. However, traditional (fresh) egg donation, as it is performed today, is not very efficient, as typically all eggs from one donor are given to only one recipient, it is arduous as it requires an excellent synchronization between the donor and recipient and there are months or years of waiting time. Because of the development of an efficient oocyte cryopreservation technique, it is now possible to cryo-store donor (as well as non-donor) eggs, maintaining their viability and allowing their use whenever there is demand. Therefore, creating a donor oocyte cryobank would carry many advantages

  11. How does vitrification affect oocyte viability in oocyte donation cycles? A prospective study to compare outcomes achieved with fresh versus vitrified sibling oocytes. (United States)

    Solé, M; Santaló, J; Boada, M; Clua, E; Rodríguez, I; Martínez, F; Coroleu, B; Barri, P N; Veiga, A


    How does vitrification affect oocyte viability? Vitrification does not affect oocyte viability in oocyte donation cycles. Oocyte vitrification is performed routinely and successfully in IVF and oocyte donation programs. This is a prospective study performed between June 2009 and February 2012 to compare ongoing pregnancy rates and other indices of viability between fresh and vitrified oocytes. A total of 99 donations with more than 16 oocytes (MII) in which oocytes were allocated both to a synchronous recipient (fresh oocytes) and to an asynchronous recipient (vitrified oocytes) were included. The participants were consenting couples (donors and recipients) from the oocyte donation program. On the day of retrieval, the oocytes allocated to the synchronous recipient were inseminated and those allocated for banking were denuded of cumulus and vitrified. Vitrified oocytes were microinjected with spermatozoa 2 h after warming. Embryo transfer was performed on Day 2 of development in both groups, and the remaining embryos were cryopreserved on Day 3. Clinical pregnancy was defined by a positive fetal heartbeat at 6 weeks. A total of 989 oocytes were warmed and 85.6% survived. No significant differences were observed between fresh and vitrified oocytes: fertilization rate (80.7 versus 78.2%), ongoing embryo rate (71.0 versus 68.2%) or good-quality embryo rate (54.1 versus 49.8%). The mean number of embryos transferred was similar in both groups (1.82 ± 0.44 versus 1.90 ± 0.34). The implantation rate (33.3 versus 34.0%) and the multiple pregnancy rate (27.7 versus 20.8) were also similar between both groups (P > 0.05). The live birth rate per cycle was 38.4% in the recipients of fresh oocytes and 43.4% in the recipients of vitrified oocytes (P > 0.05). Eighty five frozen embryo transfers were also evaluated. Comparing embryos from fresh and vitrified oocytes there were no significant differences in the embryo survival rate (70.1 versus 65.8%), clinical pregnancy rate

  12. Investigation of possible oncogenic action of zinc polycarboxylate cement by implantation in mice and hamsters. (United States)

    Main, J H; Mock, D; Beagrie, G S; Smith, D C


    Powdered, sterilized zinc polycarboxylate was implanted subcutaneously in 56 young hamsters and 54 mice, using equal numbers of each sex, with 24 sham operated hamsters and 26 mice as controls. The hamsters were sacrificed after 15 months and the mice after 12. Implant material was recovered from one of 42 surviving mice and from 16 of 43 surviving hamsters. Benign adenomas of the gut, thyroid, sebaceous glands and ovary were found in 3 experimental mice and in one control. Benign adrenal adenomas were found in 3 experimental hamsters and in one control. One control hamster developed a rhabdomyosarcoma in a limb. One experimental hamster developed a leiomysarcoma in close relation to an implant.

  13. Fertilization and Embryo Development of Fresh and Cryopreserved Sibling Oocytes

    Directory of Open Access Journals (Sweden)

    Robert F. Casper


    Full Text Available Background: Oocyte cryopreservation is potentially the best way to preserve female fertility forunmarried women or young girls at risk of losing ovarian function. The aim of this study was tocompare fertilization and embryo development in frozen-thawed oocytes to their fresh siblings inwomen undergoing in vitro fertilization (IVF and embryo transfer (ET.Materials and Methods: Eleven infertile women undergoing infertility treatment, between theages of 24 to 37 years (mean ± SD = 31.6 ± 3.5, were included in this study. Mature oocytesfrom each patient were randomized into cryopreserved and fresh groups prior to intracytoplasmicsperm injection (ICSI. One hundred and thirty nine oocytes were retrieved, of which 105 were atmetaphase II (MII. Forty- five fresh MII oocytes were kept in culture whereas their sibling 60 MIIoocytes were cryopreserved using a slow cooling protocol. The frozen oocytes remained in LN2for 2 hours before thawing. ICSI was performed 1-2 hours after thawing for frozen oocytes and 4-5hours after retrieval for fresh oocytes. Fertilization and embryo development were compared.Results: Following thawing, 31 oocytes (51.6 % survived and 22 fertilized (79% while 32 freshoocytes fertilized upon ICSI (71%. The mean ± SE scores for embryos developing from frozenthawedoocytes were significantly lower at 48 and 72 hours post-ICSI than for embryos resultingfrom fresh oocytes (p<0.05.Conclusion: Our data demonstrated that oocyte freezing resulted in acceptable survival ratesfollowing cryopreservation, and similar fertilization rates following ICSI as compared to the freshsibling oocytes. However the number of blastomeres and the embryo quality on day three wassuperior in embryos from fresh oocytes when compared to the frozen oocytes.

  14. Follicular steroid hormones as markers of oocyte quality and oocyte development potential

    Directory of Open Access Journals (Sweden)

    Nayara López Carpintero


    Full Text Available Context: Various components of follicular fluid are suggested as biochemical predictors of oocyte quality. Previous studies of follicular steroid hormone levels have shown disparate results when related with fertilization outcomes. Aim: The objective of the study was to relate the levels of steroid hormones of each individual follicle with oocyte maturation, fertilization results, embryo quality, and pregnancy rates. Settings and Design: Prospective cohort study in a university hospital. Methods: In 31 patients, who underwent intracytoplasmic sperm injection, it was performed an ultrasound guided aspiration of follicular fluid of the first two mature follicles from each ovary. Follicular levels of estradiol, progesterone, testosterone, and dehydroepiandrosterone sulfate were measured by chemiluminescent immunoassay. Statistical Analysis: Generalized estimating equation model. Results: In follicular fluids with mature oocyte presence, in normal as well as in failed fertilization, there was a positive correlation between follicular testosterone and progesterone (r = 0.794, P = 0.0001 and r = 0.829, P = 0.0001. Progesterone levels were higher in cases of normal fertilization compared to failed fertilization (P = 0.003. B quality embryos came from oocytes immersed in follicular fluids with higher estradiol values and higher estradiol/progesterone and estradiol/testosterone ratios than those of C quality (P = 0.01; P = 0.0009; P = 0.001. Estradiol levels were higher in patients who achieved pregnancy (P = 0.02. Conclusion: The analysis of follicular hormone composition could be considered as an additional tool in oocyte selection.

  15. Cytoplasmic Streaming in the Drosophila Oocyte. (United States)

    Quinlan, Margot E


    Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.

  16. Cyclooxygenase-2 Expression in Hamster and Human Pancreatic Neoplasia1 (United States)

    Yip-Schneider, Michele T; Savage, Jesse J; Hertzler, Dean A; Cummings, William O


    Abstract Cyclooxygenase-2 (COX-2) has been implicated in the development of gastrointestinal malignancies. The aim of the present study was to determine COX-2 expression/activity throughout stages of experimental and human pancreatic neoplasia. COX-2 immunohistochemistry was performed in pancreata of hamsters subjected to the carcinogen N-nitrosobis-(2-oxopropyl)amine (BOP) and in human pancreatic tumors. COX-2 activity was determined by prostaglandin E2 assay in tumor versus matched normal pancreatic tissues. The activity of the COX inhibitor sulindac was tested in the PC-1 hamster pancreatic cancer model. COX-2 expression was elevated in all pancreatic intraepithelial neoplasias (PanINs) and adenocarcinomas. In BOP-treated hamsters, there were significant progressive elevations in COX-2 expression throughout pancreatic tumorigenesis. In human samples, peak COX-2 expression occurred in PanIN2 lesions and remained moderately elevated in PanIN3 and adenocarcinoma tissues. COX-2 activity was significantly elevated in hamster and human pancreatic cancers compared to pair-matched normal pancreas. Furthermore, hamster pancreatic tumor engraftment/formation in the PC-1 hamster pancreatic cancer model was reduced 4.9-fold by oral administration of sulindac. Increased COX-2 expression is an early event in pancreatic carcinogeneses. The BOP-induced hamster carcinogenesis model is a representative model used to study the role of COX-2 in well-differentiated pancreatic tumorigenesis. COX inhibitors may have a role in preventing tumor engraftment/formation. PMID:16820089

  17. Circadian rhythms accelerate wound healing in female Siberian hamsters. (United States)

    Cable, Erin J; Onishi, Kenneth G; Prendergast, Brian J


    Circadian rhythms (CRs) provide temporal regulation and coordination of numerous physiological traits, including immune function. CRs in multiple aspects of immune function are impaired in rodents that have been rendered circadian-arrhythmic through various methods. In Siberian hamsters, circadian arrhythmia can be induced by disruptive light treatments (DPS). Here we examined CRs in wound healing, and the effects of circadian disruption on wound healing in DPS-arrhythmic hamsters. Circadian entrained/rhythmic (RHYTH) and behaviorally-arrhythmic (ARR) female hamsters were administered a cutaneous wound either 3h after light onset (ZT03) or 2h after dark onset (ZT18); wound size was quantified daily using image analyses. Among RHYTH hamsters, ZT03 wounds healed faster than ZT18 wounds, whereas in ARR hamsters, circadian phase did not affect wound healing. In addition, wounds healed slower in ARR hamsters. The results document a clear CR in wound healing, and indicate that the mere presence of organismal circadian organization enhances this aspect of immune function. Faster wound healing in CR-competent hamsters may be mediated by CR-driven coordination of the temporal order of mechanisms (inflammation, leukocyte trafficking, tissue remodeling) underlying cutaneous wound healing. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Pregnancy and birth rates after oocyte donation. (United States)

    Remohí, J; Gartner, B; Gallardo, E; Yalil, S; Simón, C; Pellicer, A


    To determine accumulated conception and live birth rates in ovum donation. Retrospective study from a computer database. Pregnancies with one gestational sac observed by ultrasound have been included as conceptional cycles and pregnancies that resulted in one live child were recorded for the analysis of the live birth rates. Life table analysis was applied. Oocyte donation program at the Instituto Valenciano de Infertilidad. Three hundred ninety-seven recipients undergoing a total of 627 ETs were analyzed. Ovarian stimulation and ovum pick-up in donors. Uterine ET in recipients after appropriate exogenous steroid replacement. Accumulated and estimated (95% confidence intervals [CI]) conception and live birth rates in the oocyte donation program as well as considering age and cause of infertility of the recipients. Pregnancy rate after one cycle was 53.4% (CI 50.9% to 55.9%), with a delivery rate of 42.6% (CI 40.1% to 45.1%). Accumulated pregnancy rate increased up to 94.8% (CI 90.6% to 99.0%) after four transfers. Similarly, live birth rates reached 88.7% (CI 88.1% to 89.3%) after four attempts of ET by ovum donation. Cycle fecundity rates were maintained at approximately 50% after each attempt. Implantation rate was 18.3% (430/2,340 replaced embryos). Age and cause of entering the program did not influence the overall results of ovum donation. Oocyte donation is a successful treatment modality for infertile couples that offers even higher success rates than natural conception. No difference in cumulative pregnancy rate was observed regardless of recipient age, indication for oocyte donation, or number of cycles attempted.

  19. In vitro maturation of cumulus-oocyte complexes for efficient isolation of oocytes from outbred deer mice.

    Directory of Open Access Journals (Sweden)

    Jung Kyu Choi

    Full Text Available The outbred (as with humans deer mice have been a useful animal model of research on human behavior and biology including that of the reproductive system. One of the major challenges in using this species is that the yield of oocyte isolation via superovulation is dismal according to the literature to date less than ∼5 oocytes per animal can be obtained so far.The goal of this study is to improve the yield of oocyte isolation from outbred deer mice close to that of most laboratory mice by in vitro maturation (IVM of cumulus-oocyte complexes (COCs.Oocytes were isolated by both superovulation and IVM. For the latter, COCs were obtained by follicular puncture of antral follicles in both the surface and inner cortical layers of ovaries. Immature oocytes in the COCs were then cultured in vitro under optimized conditions to obtain metaphase II (MII oocytes. Quality of the oocytes from IVM and superovulation was tested by in vitro fertilization (IVF and embryo development.Less than ∼5 oocytes per animal could be isolated by superovulation only. However, we successfully obtained 20.3±2.9 oocytes per animal by IVM (16.0±2.5 and superovulation (4.3±1.3 in this study. Moreover, IVF and embryo development studies suggest that IVM oocytes have even better quality than that from superovulation The latter never developed to beyond 2-cell stage as usual while 9% of the former developed to 4-cells.We have successfully established the protocol for isolating oocytes from deer mice with high yield by IVM. Moreover, this is the first ever success to develop in vitro fertilized deer mice oocytes beyond the 2-cell stage in vitro. Therefore, this study is of significance to the use of deer mice for reproductive biology research.

  20. Polyspermy in Bufo arenarum oocytes matured in vitro. (United States)

    Oterino, J; Sánchez Toranzo, G; Zelarayán, L; Bühler, M I


    Full-grown ovarian oocytes of the amphibian Bufo arenarum were induced to mature in vitro by removing the follicular layers (spontaneous maturation) or by treatment with progesterone (hormone-induced maturation). These oocytes were then treated with trypsin and inseminated with homologous spermatozoa. Oocytes matured in vivo that had not undergone any influence of the oviducts (coelomic oocytes), inseminated under the same experimental conditions, were used as controls. The results show that oocytes induced to mature in vitro and exhibiting apparently normal signs of activation were polyspermic. In fact, 2 h after insemination numerous functioning pronuclei could be observed in the animal hemisphere. These results suggest that even though the oocytes which matured in vitro were able to undergo activation after insemination, they were unable to establish an effective block to polyspermy.

  1. Oocyte surface in four teleost fish species postspawning and fertilization

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    Elizete Rizzo


    Full Text Available Cytological and cytochemical studies were carried out to investigate the surface characteristics of oocytes of four teleost species from the São Francisco river. The fishes were submitted to hypophysation at the Três Marias Hybrobiology and Fishculture Station, Minas Gerais, Brazil, in January 1996. Postspawning, oocytes of the curimatãs Prochilodus affinis, Prochilodus marggravii and dourado Salminus brasiliensis were surrounded by a thick, three-layered zona pellucida with radial striae. The surface of spawned oocytes of the surubim, Pseudoplatystoma coruscans, was comprised of mucous coat located externally to a thin, two-layered and striated zona pellucida. Oocyte activation during fertilization, lead to cortical reaction, formation of a perivitelline space, reduction of the thickness of the zona pellucida and increase in the oocyte diameter in the four species. Following fertilization, many spermatozoa were embedded in the mucous coat of the surubim oocytes. During embryogenesis, this later coating became thicker, diffuse and less viscous while the zona pellucida (chorion was thinner in all studied species. Cytochemical analyses indicated species-specific differences in the oocyte surface after spawning. It was suggested that the mucous coat of surubim oocytes play a functional role during fertilization. The knowledge of the morphology of the oocyte surface of teleost is important for our understanding of the interactions between their eggs and surrounding environment and may also contribute significantly to phylogenetic studies.

  2. Oocyte-like cells induced from mouse spermatogonial stem cells

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    Wang Lu


    Full Text Available Abstract Background During normal development primordial germ cells (PGCs derived from the epiblast are the precursors of spermatogonia and oogonia. In culture, PGCs can be induced to dedifferentiate to pluripotent embryonic germ (EG cells in the presence of various growth factors. Several recent studies have now demonstrated that spermatogonial stem cells (SSCs can also revert back to pluripotency as embryonic stem (ES-like cells under certain culture conditions. However, the potential dedifferentiation of SSCs into PGCs or the potential generation of oocytes from SSCs has not been demonstrated before. Results We report that mouse male SSCs can be converted into oocyte-like cells in culture. These SSCs-derived oocytes (SSC-Oocs were similar in size to normal mouse mature oocytes. They expressed oocyte-specific markers and gave rise to embryos through parthenogenesis. Interestingly, the Y- and X-linked testis-specific genes in these SSC-Oocs were significantly down-regulated or turned off, while oocyte-specific X-linked genes were activated. The gene expression profile appeared to switch to that of the oocyte across the X chromosome. Furthermore, these oocyte-like cells lost paternal imprinting but acquired maternal imprinting. Conclusions Our data demonstrate that SSCs might maintain the potential to be reprogrammed into oocytes with corresponding epigenetic reversals. This study provides not only further evidence for the remarkable plasticity of SSCs but also a potential system for dissecting molecular and epigenetic regulations in germ cell fate determination and imprinting establishment during gametogenesis.

  3. Successful birth of the first frozen oocyte baby in India

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    Priya Selvaraj


    Full Text Available We report the first pregnancy and birth in India after the transfer of embryos generated from frozen- thawed oocytes. A 29-year-old woman with previous bad obstetric history and an abnormal karyotype, necessitating donor oocyte programme. Embryos were generated by microinjection of frozen-thawed sperms into thawed human oocytes (intracytoplasmic sperm injection. This resulted in an healthy male baby with a birth weight of 2.54 kg which was born by cesarean section at 35-36 weeks of gestation with normal follow-up. Thus oocyte cryopreservation can be performed with reproducible success leading to a viable offspring.

  4. PTK2b function during fertilization of the mouse oocyte

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    Luo, Jinping [Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160 (United States); McGinnis, Lynda K. [Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Carlton, Carol [Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Beggs, Hilary E. [Department of Ophthalmology, University of California, San Francisco, CA (United States); Kinsey, William H., E-mail: [Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160 (United States)


    Highlights: • PTK2b is expressed in oocytes and is activated following fertilization. • PTK2b suppression in oocytes prevents fertilization, but not parthenogenetic activation. • PTK2b suppression prevents the oocyte from fusing with or incorporating bound sperm. • PTK2b suppressed oocytes that fail to fertilize do not exhibit calcium oscillations. - Abstract: Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.

  5. British women's attitudes towards oocyte donation: ethnic differences and altruism. (United States)

    Purewal, S; van den Akker, O B A


    This study assessed the importance of altruism and willingness to donate oocytes in British Asian and Caucasian samples. The Theory of Planned Behaviour (TPB) was used to test the importance of attitudes towards oocyte donation, normative and control beliefs to attitudes to donate oocytes. One hundred and one participants (55% Asian, 45% Caucasian) completed questionnaires measuring altruism and attitudes to Oocyte donation. There were no socio-demographic differences between ethnic groups. Few women were willing to donate oocytes, Asian women were least likely to donate oocytes, and altruism was not related to willingness to donate. Forty-one participants considered themselves 'possible' oocyte donors and 54 as definite 'non' donors. Possible donors reported significantly more positive attitudes towards egg donation; asking women to donate under various circumstances; to the consequences of donating their eggs; positively experiencing egg donation and to factors that would induce women to donate. Subjective norms and behavioural control also influenced intention to donate. A number of components of the TPB were able to predict possible oocyte donation, and non-oocyte donation. This study provides some empirical support for specific factors influencing cultural differences in gamete donation in the UK. A future culturally appropriate targeted approach to donation education could redress the present imbalance in supply and demand of gametes in infertility treatment.

  6. A role for glucose in hypothermic hamsters (United States)

    Resch, G. E.; Musacchia, X. J.


    Hypothermic hamsters at a rectal temperature of 7 C showed a fivefold increase in survival times from 20 to 100.5 hr when infused with glucose which maintained a blood level at about 45 mg/100 ml. A potential role for osmotic effects of the infusion was tested and eliminated. There was no improvement in survival of 3-O-methylglucose or dextran 40-infused animals. The fact that death eventually occurs even in the glucose-infused animal after about 4 days and that oxygen consumption undergoes a slow decrement in that period suggests that hypothermic survival is not wholly substrate limited. Radioactive tracer showed that localization of the C-14 was greatest in brain tissue and diaphragm, intermediate in heart and kidney, and lowest in skeletal muscle and liver. The significance of the label at sites important to respiration and circulation was presented.

  7. Ultrastructural study of primordial germ cells, oogonia and oocytes in goat fetus

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    S.M Banan Khojasteh


    Full Text Available According to morphological evidences, primordial germ cells (PGCs are derived from the caudal endoderm of the yolk sac and migrate to embryonic gonads. After entering the gonads, first they differentiate to oogonia and then to oocytes. In the present study, ultrastructure of PGCs, oogonia and oocytes has been examined. Tissue samples were collected from posterior parts of the yolk sacs of fetuses in the early stages of development (with age of less than 1 month, and also gonads of fetuses at later developmental stages (with age of more than 1 month. Samples were studied using transmission electron microscopy after fixation, washing with buffers, dehydration, embedding and staining with uranyl acetate and lead citrate. The Results indicated that PGCs were large with oval to spherical nuclei, reticular chromatin with nucleoli, and there were plenty of glycogen and also different organelles in their cytoplasm. Oogonia showed active mitotic divisions. These cells had regular plasma membranes and were observed as cellular clusters with spherical shape, euchromatin nucleus containing one or more nucleoli, round mitochondria and vacuoles with different sizes in cytoplasm. Oocytes had larger sizes in comparison with oogonia but didn't show cellular clusters.

  8. Effects of barium, lanthanum and gadolinium on endogenous chloride and potassium currents in Xenopus oocytes. (United States)

    Tokimasa, T; North, R A


    1. The effects of multivalent cations on membrane currents recorded from Xenopus oocytes were studied. 2. The hyperpolarization-activated chloride current was reversibly blocked by lanthanum; half-maximal block occurred at a concentration of 8 microM. Zinc, cadmium, cobalt and nickel were less potent than lanthanum, and gadolinium, manganese, barium and strontium had no effect at a concentration of 100 microM. 3. The calcium-activated chloride current was blocked by gadolinium (50 microM), and lanthanum, cadmium, cobalt, nickel and manganese were equally effective. The actions of gadolinium and lanthanum were almost irreversible, while partial (30-80%) recovery was observed with the other cations. Zinc (100 microM) had no effect. 4. In lanthanum (100 microM), membrane depolarizations from -70 mV activated an outward potassium current that was partially blocked by barium (0.1-2 mM). The barium-sensitive current was confined to potentials less negative than -70 mV. The current consisted of a time-independent as well as a time-dependent component, the latter of which had voltage dependence similar to the M-current. 5. It is proposed that lanthanum, gadolinium and barium can usefully separate these endogenous membrane currents in Xenopus oocytes. PMID:8930835

  9. Effects of dietary palmitoleic acid on plasma lipoprotein profile and aortic cholesterol accumulation are similar to those of other unsaturated fatty acids in the F1B golden Syrian hamster. (United States)

    Matthan, Nirupa R; Dillard, Alice; Lecker, Jaime L; Ip, Blanche; Lichtenstein, Alice H


    The lower susceptibility of palmitoleic acid (16:1) to oxidation compared to PUFA may confer functional advantages with respect to finding acceptable alternatives to partially hydrogenated fats, but limited data are available on its effect on cardiovascular risk factors. This study investigated the effect of diets (10% fat, 0.1% cholesterol, wt:wt) enriched with macadamia [monounsaturated fatty acid (MUFA)16:1], palm (SFA,16:0), canola (MUFA,18:1), or safflower (PUFA,18:2) oils on lipoprotein profiles and aortic cholesterol accumulation in F1B Golden Syrian hamsters (n = 16/group). After 12 wk, 8 hamsters in each group were killed (phase 1). The remaining hamsters fed palm oil were changed to a diet containing coconut oil, while hamsters in the other diet groups continued on their original diets for an additional 6 wk (phase 2). With minor exceptions, the time course and dietary SFA source did not alter the study outcomes. Macadamia oil-fed hamsters had lower non-HDL cholesterol and triglyceride concentrations compared with the palm and coconut oil-fed hamsters and higher HDL-cholesterol compared with the coconut, canola, and safflower oil-fed hamsters. The aortic cholesterol concentration was not affected by dietary fat type. The hepatic cholesterol concentration was higher in the unsaturated compared with the saturated oil-fed hamsters. RBC membrane and aortic cholesteryl ester, triglyceride, and phospholipid fatty acid profiles reflected that of the dietary oil. These data suggest that an oil relatively high in palmitoleic acid does not adversely affect plasma lipoprotein profiles or aortic cholesterol accumulation and was similar to other unsaturated fatty acid-rich oils.

  10. Effects of Dietary Palmitoleic Acid on Plasma Lipoprotein Profile and Aortic Cholesterol Accumulation Are Similar to Those of Other Unsaturated Fatty Acids in the F1B Golden Syrian Hamster 1–3 (United States)

    Matthan, Nirupa R.; Dillard, Alice; Lecker, Jaime L.; Ip, Blanche; Lichtenstein, Alice H.


    The lower susceptibility of palmitoleic acid (16:1) to oxidation compared to PUFA may confer functional advantages with respect to finding acceptable alternatives to partially hydrogenated fats, but limited data are available on its effect on cardiovascular risk factors. This study investigated the effect of diets (10% fat, 0.1% cholesterol, wt:wt) enriched with macadamia [monounsaturated fatty acid (MUFA)16:1], palm (SFA,16:0), canola (MUFA,18:1), or safflower (PUFA,18:2) oils on lipoprotein profiles and aortic cholesterol accumulation in F1B Golden Syrian hamsters (n = 16/group). After 12 wk, 8 hamsters in each group were killed (phase 1). The remaining hamsters fed palm oil were changed to a diet containing coconut oil, while hamsters in the other diet groups continued on their original diets for an additional 6 wk (phase 2). With minor exceptions, the time course and dietary SFA source did not alter the study outcomes. Macadamia oil-fed hamsters had lower non-HDL cholesterol and triglyceride concentrations compared with the palm and coconut oil-fed hamsters and higher HDL-cholesterol compared with the coconut, canola, and safflower oil-fed hamsters. The aortic cholesterol concentration was not affected by dietary fat type. The hepatic cholesterol concentration was higher in the unsaturated compared with the saturated oil-fed hamsters. RBC membrane and aortic cholesteryl ester, triglyceride, and phospholipid fatty acid profiles reflected that of the dietary oil. These data suggest that an oil relatively high in palmitoleic acid does not adversely affect plasma lipoprotein profiles or aortic cholesterol accumulation and was similar to other unsaturated fatty acid-rich oils. PMID:19106316

  11. Porcine oocyte maturation in vitro: role of cAMP and oocyte-secreted factors – A practical approach (United States)

    APPELTANT, Ruth; SOMFAI, Tamás; MAES, Dominiek; VAN SOOM, Ann; KIKUCHI, Kazuhiro


    Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling. PMID:27349308

  12. Porcine oocyte maturation in vitro: role of cAMP and oocyte-secreted factors - A practical approach. (United States)

    Appeltant, Ruth; Somfai, Tamás; Maes, Dominiek; VAN Soom, Ann; Kikuchi, Kazuhiro


    Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling.

  13. Fasting-induced daily torpor in desert hamsters (Phodopus roborovskii). (United States)

    Chi, Qing-Sheng; Wan, Xin-Rong; Geiser, Fritz; Wang, De-Hua


    Daily torpor is frequently expressed in small rodents when facing energetically unfavorable ambient conditions. Desert hamsters (Phodopus roborovskii, ~20g) appear to be an exception as they have been described as homeothermic. However, we hypothesized that they can use torpor because we observed reversible decreases of body temperature (Tb) in fasted hamsters. To test this hypothesis we (i) randomly exposed fasted summer-acclimated hamsters to ambient temperatures (Tas) ranging from 5 to 30°C or (ii) supplied them with different rations of food at Ta 23°C. All desert hamsters showed heterothermy with the lowest mean Tb of 31.4±1.9°C (minimum, 29.0°C) and 31.8±2.0°C (minimum, 29.0°C) when fasted at Ta of 23°C and 19°C, respectively. Below Ta 19°C, the lowest Tb and metabolic rate increased and the proportion of hamsters using heterothermy declined. At Ta 5°C, nearly all hamsters remained normothermic by increasing heat production, suggesting that the heterothermy only occurs in moderately cold conditions, perhaps to avoid freezing at extremely low Tas. During heterothermy, Tbs below 31°C with metabolic rates below 25% of those during normothermia were detected in four individuals at Ta of 19°C and 23°C. Consequently, by definition, our observations confirm that fasted desert hamsters are capable of shallow daily torpor. The negative correlation between the lowest Tbs and amount of food supply shows that heterothermy was mainly triggered by food shortage. Our data indicate that summer-acclimated desert hamsters can express fasting-induced shallow daily torpor, which may be of significance for energy conservation and survival in the wild. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Evidence for a metabolic limitation of survival in hypothermic hamsters. (United States)

    Prewitt, R. L.; Anderson, G. L.; Musacchia, X. J.


    The underlying factors limiting survival in the hypothermic state are studied. Hamsters of both sexes, clipped and unclipped, were inducted into profound hypothermia by the helium cold method until they reached a temperature between 7 and 10 C. It appears that the primary cause of death is failure of respiration due to the depletion of carbohydrate energy supplies and may explain why survival time in hypothermia is shorter than the normal hibernation time of the hamster.

  15. Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification. (United States)

    Lai, D; Ding, J; Smith, G W; Smith, G D; Takayama, S


    osmotic stress resulting in better morphology, higher cell quality and improved developmental competence. This microfluidic procedure resulted in murine zygotes with a significantly smoother cell surface (P bovine oocytes (P < 0.01), decreased membrane perforations and cytoplasmic leakage in CPA-exposed murine zygotes (P < 0.05) and improved developmental competence of vitrified and warmed murine zygotes (P < 0.05) than CPA exposure using the current clinically used manual pipetting method. It is necessary to design the microfluidic device to be more user-friendly for widespread use. The theory and approach of eliminating osmotic stress by decreasing shrinkage rate is complementary to the prevalent osmotic stress theory in cryobiology which focuses on a minimum cell volume at which the cells shrink. The auto-microfluidic protocol described here has immediate applications for improving animal and human oocyte, zygote and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sublethal damage should be broadly useful for cryobiology. This project was funded by the National Institutes of Health and the University of Michigan Reproductive Sciences Program. The authors declare no conflicts of interest. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:

  16. Ocular pathological changes in hamsters experimentally infected with Schistosoma mansoni. (United States)

    Ismail, H I H; Ashour, D S; Abou Rayia, D M; Ali, A L


    Ocular lesions have been reported in patients with schistosomiasis; however, the problem with studying schistosomal infection of the human eye is that biopsies are almost impossible to take, and histopathological examination of suspicious lesions can only be undertaken post-mortem or after enucleation. This work aimed to study the possible effects and pathogenesis of schistosomiasis on the eye. This study involved 55 hamsters; five hamsters remained non-infected and the remaining 50 hamsters were infected with Schistosoma mansoni cercariae. Infected hamsters were sacrificed on weeks 8, 12, 16 and 20 post-infection (pi). Eye sections were prepared and stained for histopathological and immunohistochemical studies. Histopathological changes detected in hamsters infected after 16 and 20 weeks included looseness and oedema of the innermost retinal layers together with hyperplastic polypoid growth. Neither eggs nor granulomata were detected in eye sections throughout the experimental period. Deposition of S. mansoni antigen was revealed in 35% of infected hamsters. Later, on weeks 16 and 20 pi, moderate subepithelial conjuctival deposits and marked subchoroidal and scleral deposition were detected. In conclusion, the deposition of schistosomal antigen and immune complexes may play a pivotal role in the ocular changes that occur in schistosomiasis, even in the absence of detectable Schistosoma eggs. Schistosomiasis should be suspected in cases with unexplained ophthalmological findings, especially in endemic areas.

  17. Regulation of tonic gonadotropin release in prepubertal female hamsters

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    Smith, S.G.; Matt, K.S.; Prestowitz, W.F.; Stetson, M.H.


    Basal serum gonadotropin levels were monitored weekly in female hamsters from birth to 10 weeks of age. Hamsters raised on three different photoperiods presented uniform pre- and postpubertal patterns of serum LH and FSH, suggesting that gonadotropin release in the young hamster occurs independently of ambient photoperiod. In all groups, serum LH levels increased gradually in animals up to 4 weeks of age, after which levels plateaued at 50--100 ng/ml. Serum FSH was markedly elevated in 2- and 3-week-old hamsters (800--1200 ng/ml), but remained at 200--400 ng/ml in all other groups. We next examined the change in the responsiveness of the pituitary to exogenous gonadotropin-releasing hormone (GnRH) challenge. Female hamsters 2 days of age failed to respond to any dose (0.025--1000 ng) of GnRH, while 10-day old females responded in typical dose-dependent fashion. GnRH-stimulated LH release first occurred in 6-day-old hamsters and was maximal by day 9, whereas FSH release first occurred on day 8 and was maximal by day 9. The prepubertal pattern of gonadotropin release can, in part, be explained on the basis of the development of pituitary GnRH sensitivity, which occurs independently of photoperiod.

  18. Pig membrana granulosa cells prevent resumption of meiosis in cattle oocytes. (United States)

    Kalous, J; Sutovsky, P; Rimkevicova, Z; Shioya, Y; Lie, B L; Motlik, J


    Membrana granulosa was isolated from healthy large antral follicles of prepubertal or cyclic gilts stimulated with PMSG or PMSG and hCG. Ultrastructural observations revealed that pieces of pig membrana granulosa were associated with the basement membrane. The cattle cumulus-enclosed oocytes (COC) were placed in the rolled pieces of the pig membrana granulosa (PMG). After 8 and 24 hr of coculture with PMG from prepubertal gilts, only 16% and 21% of oocytes underwent GVBD, respectively. PMG from PMSG-stimulated cyclic gilts blocked the resumption of meiosis in all COC. The inhibitory effect of heterologous granulosa cells was fully reversible. When COC were initially incubated for 2 and 4 hr, subsequent culture in PMG prevented GVBD in 100% and 36% of oocytes, respectively. This suggests that functional contact between COC and PMG was established during the first 2 hr of coculture. To follow metabolic cooperation between PMG and COC, PMG was prelabeled with 3H-uridine and cocultured with COC. Autoradiography on semithin sections revealed the intensive passage of 3H-uridine from PMG into the cumulus layer and an oocyte. COC placed in PMG after GVBD (8 and 12 hr of an initial incubation) did not extrude the first polar body. PMG isolated from cyclic gilts after PMSG and hCG stimulation also inhibited GVBD of COC. Since nearly all COC placed in PMG isolated 10 and 12 hr after hCG remained in the GV stage after 24 hr of coculture, the hCG stimulation did not substantially diminish the meiosis inhibiting activity of PMG.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Effect of lower than expected number of oocyte on the IVF results after oocyte-pickup. (United States)

    Gonca, Süheyla; Gün, Ismet; Ovayolu, Ali; Silfeler, Dilek; Sofuoğlu, Kenan; Ozdamar, Ozkan; Yilmaz, Ali; Tunali, Gülden


    To investigate whether a lower than expected number of oocyte after ≥14 mm follicle aspiration during OPU has any effect on pregnancy outcomes Methods: This is a retrospective study done between 2010 and 2013 at the IVF Unit of the Zeynep Kamil Women and Children Diseases Education and Research Hospital, dealing with the medical records of infertile patients who underwent IVF cycle and controlled ovarian stimulation with long agonist or fix antogonist protocol. The patients included into the study were those diagnosed with a primary infertility, aged between 23 and 39, at a BMI of 22-28 kg/m(2) and having received the first or second IVF treatment. Male factor, presence of uterine anomaly, patients with serious endometriosis and patients with low ovarian reserve were all excluded from the study. Typically, oocyte pick-up was performed in all the patients 35.5 hours after the hCG implementation. Single or double embryo transfer was performed, where available. Patients were classified into two groups. Group 1 consisted of those with no difference between ≥14 mm aspirated follicle number and expected number of oocyte or with 1 missing number of oocyte at the most. Group 2 consisted of those with at least ≥2 missing number of oocyte between aspirated follicle number and expected number of oocyte. Statistical analysis was performed using Student's t test for continuous variables and chi-square test for categorical variables. Additionally, a Linear regression analysis was conducted between the total number of oocyte and pregnancy. In total, 387 treatment cycles were included into the study. Group 1 consisted of 134 patients and Group 2 consisted of 252 patients. Antral follicle number (12.8 ± 4.3 and 14.5 ± 4.1, P = 0.0007), hCG day E2 value (1990.7 ± 1056.4 and 2515.2 ± 1332.7, P < 0.0001) and the the number of aspirated follicle during OPU (9.1 ± 4.4 and 13.7 ± 5.5, P < 0.0001) were significantly higher in Group 2; whereas on the other hand, daily

  20. Cytogenetic Analysis of Sperm Nucleous Components of Iranian Normal and Sub-Fertile Individuals Using Zona Free Hamster Oocytes


    Mozdarani, H.; Mohseni.Meybodi, A.


    Purpose: The purpose of this study was to investigate whether infertility is affected by sperm chromatin and cytogenetic abnormalities. To this purpose, the frequency of sperm premature chromosome condensation (PCC) induction and numerical chromosome abnormalities in the sperm of normal and sub-fertile men were analyzed. PCC rate was studied for evaluating the role of sperm chromatin abnormalities in the process of nuclear decondensation.

  1. Oxygen tension and oocyte density during in vitro maturation affect the in vitro fertilization of bovine oocytes

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    Angelo Bertani Giotto


    Full Text Available Oocyte maturation is the key factor affecting the fertilization and embryonic development. Factors such as oocyte density and oxygen tension can directly influence the IMV. Thus, the objective of this study was to evaluate the effect of the association of oxygen tensions (5% or 20% with different oocyte densities (1:10?l or 1:20?l in the in vitro maturation (IVM of bovine oocytes on maturation and fertilization rates, ROS production and antioxidant activity. Three experiments were performed with bovine oocytes that were obtained from slaughterhouse ovaries. After selection, the oocytes were randomly distributed in four treatments: 1:10/5%; 1:10/20%; 1:20/5%and 1:20/20% for each experiment. In experiment I, nuclear maturation status and cytoplasmic maturation were evaluated through detection of the first polar body by immunofluorescence and the mitochondrial reorganization assay. In experiment II, ROS production and antioxidant activity were analyzed in oocytes and IVM medium after 24 h of maturation through detection of ROS, reduced glutathione (GSH and Superoxide dismutase activity by spectrofluorimetric methods. In experiment III, fertilization was evaluated through pronucleus formation, sperm penetration with or without decondensation and polyspermy rates by immunofluorescence. In experiment I, the nuclear maturation and cytoplasmic maturation were similar among treatments (P>0.05. In experiment II, reactive oxygen species in oocytes were elevated in treatments with low oxygen tension which was independent of oocyte density (P<0.05. Additionally, ROS levels in IVM medium were higher in treatments with high oocyte density by volume of medium, which was independent of oxygen tension (P<0.05. In Experiment III, the fertilization and penetration rates were higher in the treatment with 20% oxygen tension and high oocyte density (P<0.05. Furthermore, a high incidence of polyspermy was observed in groups with high oxygen tension and low oocyte

  2. The effect of follicular fluid hormones on oocyte recovery after ovarian stimulation: FSH level predicts oocyte recovery

    Directory of Open Access Journals (Sweden)

    Rinaudo Paolo F


    Full Text Available Abstract Background Ovarian stimulation for assisted reproductive technology (ART overcomes the physiologic process to develop a single dominant follicle. However, following stimulation, egg recovery rates are not 100%. The objective of this study is to determine if the follicular fluid hormonal environment is associated with oocyte recovery. Methods This is a prospective study involving patients undergoing ART by standard ovarian stimulation protocols at an urban academic medical center. A total of 143 follicular fluid aspirates were collected from 80 patients. Concentrations of FSH, hCG, estradiol, progesterone, testosterone and prolactin were determined. A multivariable regression analysis was used to investigate the relationship between the follicular fluid hormones and oocyte recovery. Results Intrafollicular FSH was significantly associated with oocyte recovery after adjustment for hCG (Adjusted odds ratio (AOR = 1.21, 95%CI 1.03–1.42. The hCG concentration alone, in the range tested, did not impact the odds of oocyte recovery (AOR = 0.99, 95%CI 0.93–1.07. Estradiol was significantly associated with oocyte recovery (AOR = 0.98, 95% CI 0.96–0.99. After adjustment for progesterone, the strength of association between FSH and oocyte recovery increased (AOR = 1.84, 95%CI 1.45–2.34. Conclusion The relationship between FSH and oocyte recovery is significant and appears to work through mechanisms independent of the sex hormones. FSH may be important for the physiologic event of separation of the cumulus-oocyte complex from the follicle wall, thereby influencing oocyte recovery. Current methods for inducing the final stages of oocyte maturation, with hCG administration alone, may not be optimal. Modifications of treatment protocols utilizing additional FSH may enhance oocyte recovery.

  3. Cryotop and development of vitrified immature bovine oocytes

    Directory of Open Access Journals (Sweden)

    H Hajarian


    Full Text Available The effectiveness of different cryodevices (open-pulled straw (OPS, electron microscopy grid (EMG, and Cryotop was evaluated for vitrification of immature bovine oocytes. Polar body, metaphase II stage (MII, survivability, and subsequent developmental rates were determined. Only oocytes with four or five layers of cumulus cells were used. Oocytes were equilibrated in two vitrification solutions - 1: 10% DMSO + 10% ethylene glycol (EG for 30-45sec and 2: 20% DMSO + 20% EG +0.5M sucrose for 25sec -, mounted on one of the cryodevices and directly plunged into liquid nitrogen for 10 days. Immature vitrified oocytes using Cryotop showed the highest rates of polar body extrusion (PB and nuclear maturity (MII; 41 and 58% respectively. Vitrified oocytes using OPS and EMG showed 26 and 32%; and 35 and 46% of PB and MII rates, respectively. The highest survivability resulted from Cryotop and EMG groups and no significant difference was found between them. Vitrified oocytes using Cryotop had the highest cleavage and blastocyst rates. All of the mean rates for vitrified immature oocytes were significantly lower than that of control group (P<0.05. The results of this study showed the superiority of Cryotop device for vitrification of immature bovine oocytes

  4. (LH) on in vitro maturation of Egyptian buffalo oocytes

    African Journals Online (AJOL)

    Microsoft Corporation


    Mar 8, 2012 ... immature bovine oocytes. Gamete Res. 24:197-204. Deshmukh SP, Pawshe CH, Ingawale MV, Deshmukh SG (2010). In vitro maturation of buffalo immature oocyte after Vitrification with combination of Ethylene Glycol and Dimethyl Sulfoxide. Proceedings. 9th World Buffalo Congress, pp. 911-921. Duncan ...

  5. Nuclear maturation of immature bovine oocytes after vitrification ...

    African Journals Online (AJOL)



    Mar 21, 2011 ... compared for cryopreservation of immature bovine oocytes. After warming, COCs were cultured in vitro for 24 h. The polar body (PB+) and metaphase-II (MII) stage rates differed significantly among treatment groups. Oocytes vitrified using cryotop solution and device showed higher percentages of PB+ (36 ...

  6. Effect of collection techniques on cumulus oocyte complexes (COCs ...

    African Journals Online (AJOL)

    The experiment was undertaken to study the effect of collection techniques on cumulus oocyte complexes (COCs) recovery, in vitro maturation (IVM) and in vitro fertilization (IVF) of goat oocytes. COCs were collected by three techniques viz. puncture, slicing and aspiration of goat ovaries obtained at slaughterhouse.

  7. Nuclear maturation of immature bovine oocytes after vitrification ...

    African Journals Online (AJOL)

    In the second experiment, effectiveness of both vitrification methods was compared for cryopreservation of immature bovine oocytes. After warming, COCs were cultured in vitro for 24 h. The polar body (PB+) and metaphase-II (MII) stage rates differed significantly among treatment groups. Oocytes vitrified using cryotop ...

  8. Human oocyte chromosome analysis: complicated cases and major ...

    Indian Academy of Sciences (India)

    Human oocytes that remained unfertilized in programmes of assisted reproduction have been analysed cytogenetically for more than 20 years to assess the incidence of aneuploidy in female gametes. However, the results obtained so far are not indisputable as a consequence of difficulties in evaluating oocyte chromosome ...

  9. Perinatal outcomes in 375 children born after oocyte donation

    DEFF Research Database (Denmark)

    Malchau, Sara S; Loft, Anne; Larsen, Elisabeth C


    To describe perinatal outcomes in children born after oocyte donation (OD) compared with in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and spontaneous conception (SC).......To describe perinatal outcomes in children born after oocyte donation (OD) compared with in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and spontaneous conception (SC)....

  10. Endoplasmic reticulum protein 29 (ERp29, a protein related to sperm maturation is involved in sperm-oocyte fusion in mouse

    Directory of Open Access Journals (Sweden)

    Zhu Yemin


    Full Text Available Abstract Background Sperm-oocyte fusion is a critical step in fertilization, which requires a series of proteins from both spermatozoa and oocyte to mediate membrane adhesion and subsequent fusion. A rat spermatozoa membrane protein is endoplasmic reticulum protein 29 (ERp29, which significantly increases on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda as the sperm undergo epididymal maturation. Moreover, ERp29 facilitates viral infection via mediating membrane penetration. We determined if in addition to promoting sperm maturation ERp29 may also play a role in facilitating gamete fusion during the fertilization process. Methods Laser scanning confocal microscopy (LSCM and Western blot analysis were employed to probe for ERp29 protein in BALB/c mouse epididymal and acrosome-reacted spermatozoa. We prepared rabbit polyclonal antibodies against mouse recombinant ERp29 (rERp29 to characterize: 1 fertilization rate (FR; 2 fertilization index (FI; 3 sperm motility and 4 acrosome reaction (AR. Results Confocal microscopy indicated that ERp29 was partially localized at the sperm head of the epididymal caput as well as over the whole head and part of the principal piece of the tail region from the epididymal cauda. However, when the acrosome reacted, ERp29 remained in the equatorial and post-acrosomal regions of the sperm head, which is the initial site of sperm-oocyte membrane fusion. Such localization changes were confirmed based on the results of Western blot analysis. Furthermore, the antibodies against mouse rERp29 inhibited the spermatozoa from penetrating into the zona pellucida (ZP-free oocytes. The functional blocking antibodies reduced both mouse sperm-oocyte FR and FI at concentrations of 100 and 200 micro g/ml compared with pre-immunized rabbit IgG or with anti-mouse recombinant bactericidal/permeability-increasing protein (BPI, a sperm surface protein unrelated to sperm-oocyte fusion antibodies

  11. Ambient ionisation mass spectrometry for lipid profiling and structural analysis of mammalian oocytes, preimplantation embryos and stem cells. (United States)

    Ferreira, Christina R; Jarmusch, Alan K; Pirro, Valentina; Alfaro, Clint M; González-Serrano, Andres F; Niemann, Heiner; Wheeler, Matthew B; Rabel, Rathnaweera A C; Hallett, Judy E; Houser, Rebecca; Kaufman, Annemarie; Cooks, R Graham


    Lipids play fundamental roles in mammalian embryo preimplantation development and cell fate. Triacylglycerol accumulates in oocytes and blastomeres as lipid droplets, phospholipids influence membrane functional properties, and essential fatty acid metabolism is important for maintaining the stemness of cells cultured in vitro. The growing impact that lipids have in the field of developmental biology makes analytical approaches to analyse structural information of great interest. This paper describes the concept and presents the results of lipid profiling by mass spectrometry (MS) of oocytes and preimplantation embryos, with special focus on ambient ionisation. Based on our previous experience with oocytes and embryos, we aim to convey that ambient MS is also valuable for stem cell differentiation analysis. Ambient ionisation MS allows the detection of a wide range of lipid classes (e.g. free fatty acids, cholesterol esters, phospholipids) in single oocytes, embryos and cell pellets, which are informative of in vitro culture impact, developmental and differentiation stages. Background on MS principles, the importance of underused MS scan modes for structural analysis of lipids, and statistical approaches used for data analysis are covered. We envisage that MS alone or in combination with other techniques will have a profound impact on the understanding of lipid metabolism, particularly in early embryo development and cell differentiation research.

  12. Adaptation of intestinal enzymes to seasonal and dietary changes in a hibernator: the European hamster (Cricetus cricetus). (United States)

    Galluser, M; Raul, F; Canguilhem, B


    Effects of diet, hibernation and seasonal variations on hydrolase activities were determined in mucosa and purified brush border membranes of the small intestine of European hamsters. Wild hamsters captured in April and fed for several weeks with an equilibrated laboratory chow (20% protein, 50% carbohydrates) exhibited a rise in disaccharidase activities (sucrase, isomaltase, lactase) but no changes in aminopeptidase N activity. During deep hibernation, in contrast to sucrase and isomaltase activities which showed only minor changes, lactase activity was significantly enhanced along the jejunoileum, and aminopeptidase N activity was maximum in the ileum. After a short period (48 h) of wakefulness and feeding following 10 days of starvation during the hibernation period, the activities of the disaccharidases and of aminopeptidase N returned to values measured in active animals. In contrast to the nutritional state, which has an important impact on the activities of intestinal enzymes, season has little effect on the intestine of the active animal under a controlled environment. The pattern of enzyme activities which occurs along the small intestine in the hibernating animal may be a prerequisite for optimum digestion during the short phases of waking during the hibernation period of the European hamster.

  13. First attempts to cryopreserve red abalone (Haliotis rufescens oocytes

    Directory of Open Access Journals (Sweden)

    Ramírez Torrez, A.


    Full Text Available Overall, few advances in the cryopreservation of complex cells such as oocytes, embryo or tissue have been registered and in less quantity have been reported for aquatic species. Abalone has high economic interest worldwide and the conservation of abalone germplasm may help to enhance its culture and develop repopulation programs. In this work, we reported the cytotoxic effect of two concentration of trehalose (0.2 and 0.4 M on red abalone oocytes incubated for 10, 15 and 20 min. Also, we reported the cryopreservation of red abalone oocytes using a 3-steps cryopreservation protocol and 5 thawing protocols. Significant differences on cytotoxic effect were found (p<0.01. However, none of the cryoprotectant was optimum to cryopreserve red abalone oocyte. In conclusion, it is necessary to find an appropriate method to dehydrate or make the cryoprotectant penetrate on the abalone oocyte before proceeding to cryopreservation.

  14. α-Linolenic Acid-Enriched Diet Prevents Myocardial Damage and Expands Longevity in Cardiomyopathic Hamsters (United States)

    Fiaccavento, Roberta; Carotenuto, Felicia; Minieri, Marilena; Masuelli, Laura; Vecchini, Alba; Bei, Roberto; Modesti, Andrea; Binaglia, Luciano; Fusco, Angelo; Bertoli, Aldo; Forte, Giancarlo; Carosella, Luciana; Di Nardo, Paolo


    Randomized clinical trials have demonstrated that the increased intake of ω-3 polyunsaturated fatty acids significantly reduces the risk of ischemic cardiovascular disease, but no investigations have been performed in hereditary cardiomyopathies with diffusely damaged myocardium. In the present study, δ-sarcoglycan-null cardiomyopathic hamsters were fed from weaning to death with an α-linolenic acid (ALA)-enriched versus standard diet. Results demonstrated a great accumulation of ALA and eicosapentaenoic acid and an increased eicosapentaenoic/arachidonic acid ratio in cardiomyopathic hamster hearts, correlating with the preservation of myocardial structure and function. In fact, ALA administration preserved plasmalemma and mitochondrial membrane integrity, thus maintaining proper cell/extracellular matrix contacts and signaling, as well as a normal gene expression profile (myosin heavy chain isoforms, atrial natriuretic peptide, transforming growth factor-β1) and a limited extension of fibrotic areas within ALA-fed cardiomyopathic hearts. Consequently, hemodynamic indexes were safeguarded, and more than 60% of ALA-fed animals were still alive (mean survival time, 293 ± 141.8 days) when all those fed with standard diet were deceased (mean survival time, 175.9 ± 56 days). Therefore, the clinically evident beneficial effects of ω-3 polyunsaturated fatty acids are mainly related to preservation of myocardium structure and function and the attenuation of myocardial fibrosis. PMID:17148657

  15. Reduced aflatoxicosis in livers of hamsters fed a manganese sulfate supplement. (United States)

    Hastings, C E; Llewellyn, G C


    Male, weanling Syrian hamsters (Mesocricetus auratus) were given (for 10 weeks) diets supplemented with manganese sulfate, aflatoxin, or a combination of both. All animals were killed and a histopathologic evaluation was performed on each liver to assess the influence of a manganese-supplemented diet on aflatoxicosis. Serum cholesterol and liver glycogen levels were also analyzed to further study the interaction of manganese and aflatoxin. Characteristic aflatoxin-induced precancerous histopathologic changes were observed in animals receiving the toxin. These changes included bile duct cell hyperplasia, enlarged nuclei, nuclear inclusions, and megala-hepatocytes. The dietary manganese addition to aflatoxin animals caused a slight reduction in the bile duct cell hyperplasia and significantly reduced the enlarged nuclei and nuclear inclusions. The latter indicates that the manganese may be influencing membrane chemistry. Animals receiving aflatoxin alone showed significantly increased serum cholesterol and liver glycogen. The cholesterol levels were significantly increased over the aflatoxin-induced levels when manganese was given in combination with the aflatoxin. The manganese lowered the increased liver glycogen levels caused by the aflatoxin. Dietary manganese shows some potential in suppressing several, but not all, of the aspects of developing aflatoxicosis in the hamster. The specific mode and site of action of manganese requires additional study.

  16. In Vitro Growth and Maturation of Vitrified-Warmed Bovine Oocytes Collected from Early Antral Follicles (United States)

    HIRAO, Yuji; SOMFAI, Tamás; NARUSE, Kenji


    Abstract. Cryopreservation of growing oocytes enriches the choice of timing and location of artificial embryo production. However, completion of oocyte growth after warming is crucial when using such cryopreserved oocytes. Our research objective was to develop a sequential system that incorporates cryopreservation of growing bovine oocytes and their subsequent in vitro growth. Oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 µm were vitrified-warmed and then cultured for 14 days. The percentage of surviving oocytes following cryopreservation and 14-day culture was approximately 80%. More than half of the surviving oocytes were capable of maturing to metaphase II after in vitro maturation; the rate was comparable to that of control oocytes grown in vitro without cryopreservation. Taken together, the combined protocols for vitrification-warming of growing oocytes and subsequent in vitro growth can produce oocytes capable of undergoing meiotic maturation. PMID:24126072

  17. Male-specific submaxillary gland protein, a lipocalin allergen of the golden hamster, differs from the lipocalin allergens of Siberian and Roborovski dwarf hamsters. (United States)

    Hilger, Christiane; Dubey, Ved P; Lentz, Delphine; Davril, Caroline; Revets, Dominique; Muller, Claude P; Diederich, Claire; De La Barrière, Hélène; Codreanu-Morel, Françoise; Morisset, Martine; Lehners, Christiane; De, Prabir K; Hentges, François


    An increasing number of asthma cases upon exposure to hamsters and anaphylactic reactions following hamster bites are being reported, but the allergens responsible are still poorly characterized. In the Golden hamster, male-specific submaxillary gland protein (MSP), a lipocalin expressed in a sex- and tissue-specific manner in the submaxillary and lacrimal glands, is secreted in the saliva, tears and urine. The purpose of this study was to determine if MSP is an allergen, to identify IgE-reactive proteins of different hamster species and to analyse potential cross-reactivities. Fur extracts were prepared from four hamster species. Hamster-allergic patients were selected based on a history of positive IgE-test to hamster epithelium. The IgE-reactivity of patients' sera was investigated by means of immunoblot and ELISA. IgE-reactive proteins in fur extracts and the submaxillary gland were identified using anti-MSP antibodies, Edman sequencing or mass spectrometry. MSP was purified from Golden hamster and recombinant MSP was expressed in E. coli. Four patients had IgE-antibodies against 20.5-kDa and 24-kDa proteins of Golden hamster fur extract, which were identified as MSP. IgE-reactive MSP-like proteins were detected in European hamster fur extract. Three patient sera showed IgE-reactive bands at 17-21 kDa in Siberian and Roborovski hamster fur extracts. These proteins were identified as two closely related lipocalins. Immunoblot inhibition experiments showed that they are cross-reactive and are different from MSP. MSP lipocalin of the Golden hamster was identified as an allergen, and it is different from the cross-reactive lipocalin allergens of Siberian and Roborovski hamsters. Our findings highlight the need for specific tools for the in vitro and in vivo diagnosis of allergy to different hamster species. © 2015 S. Karger AG, Basel.

  18. The total pregnancy potential per oocyte aspiration after assisted reproduction-in how many cycles are biologically competent oocytes available? (United States)

    Lemmen, J G; Rodríguez, N M; Andreasen, L D; Loft, A; Ziebe, S


    While stimulation of women prior to assisted reproduction is associated with increased success rates, the total biological pregnancy potential per stimulation cycle is rarely assessed. Retrospective sequential cohort study of the cumulative live birth rate in 1148 first IVF/ICSI-cycles and 5-year follow up of frozen embryo replacement (FER) cycles were used. Oocyte number, number of embryos transferred, and cryopreserved/thawed and transferred embryos in a FER cycle were registered for all patients. Children per oocyte and per transferred embryo and percentage of cycles with births were calculated. We obtained 9529 oocytes. Embryos (2507) were transferred in either fresh or FER cycles, resulting in 422 births and 474 live born children. Median age of the women was 32.5 years (range 20-41.5 years). In total, 34.3 % of all cycles ended with a live birth while in 65.7 % of the cycles, no oocytes were capable of developing into a child. The average number of oocytes needed per live born child after transfer of fresh and thawed embryos was 20 as only 5.0 % of oocytes aspirated in the first IVF/ICSI cycle had the competence to develop into a child. In our setting, overall 5.0 % of the oocytes in a first cycle were biologically competent and in around 2/3 of all cycles, none of the oocytes had the potential to result in the birth of a child.

  19. Copulatory and agonistic behavior in Syrian hamsters following social defeat. (United States)

    Jeffress, Elizabeth C; Huhman, Kim L


    Syrian hamsters are highly aggressive animals that reliably defend their home territory. After social defeat, however, hamsters no longer defend their home cage but instead display submissive and defensive behavior toward an intruder, a response that we have termed conditioned defeat. Plasma testosterone is significantly reduced in Syrian hamsters following repeated defeat suggesting that social defeat might also impair copulatory behavior. The present study aimed to determine whether copulatory behavior in male Syrian hamsters is suppressed following repeated social defeats and additionally whether exposure to a hormone-primed stimulus female after social defeat reduces the behavioral response to defeat. Hamsters were paired with an aggressive opponent for one or nine defeats using a resident-intruder model, while controls were placed into the empty cage of a resident aggressor. On the day after the last treatment, half of the hamsters were paired with a receptive female for 10 min. There were no significant differences in the copulatory behavior of defeated versus non-defeated hamsters, and the opportunity to copulate had no effect on subsequent conditioned defeat testing, as defeated animals displayed significantly more submissive behavior than did non-defeated animals. The current data suggest that conditioned defeat is not necessarily a maladaptive response to social stress, at least in terms of reproductive behavior, but may instead represent a viable behavioral strategy adopted by losing animals following social defeat. Further, these data indicate that conditioned defeat is relatively persistent and stable, as the opportunity to copulate does not reduce the subsequent display of submissive behavior. © 2013 Wiley Periodicals, Inc.

  20. Dietary fiber improves lipid homeostasis and modulates adipocytokines in hamsters. (United States)

    Hung, Shao-Ching; Bartley, Glenn; Young, Scott Andrew; Albers, David Robert; Dielman, Demetrius Richard; Anderson, William Henry Kerr; Yokoyama, Wallace


    The hypocholesterolemic and hypoglycemic effects of various natural and semisynthetic dietary fibers have been studied for their potential use in the prevention and improvement of metabolic syndrome. Of these dietary fibers, hydroxypropyl methylcellulose (HPMC) has been shown to lower plasma cholesterol and reduce weight gain. However, the underlying mechanisms are not known. In the present study, we examined associations between plasma adipocytokine levels and both lipid metabolism and insulin sensitivity after HPMC intake in golden Syrian hamsters. In addition, endogenous adiponectin from hamster plasma was purified and characterized. Hamsters were treated with HPMC (2% and 4% in a high-fat diet) or 2% or 4% microcrystalline cellulose (MCC; control diet) for 8 weeks. Plasma glucose, insulin, lipids, adiponectin, leptin, and hepatic lipid levels were assessed using standard techniques. After 8 weeks of feeding, plasma total cholesterol and triglyceride levels in hamsters fed the 4% HPMC-supplemented diet were significantly lower than in hamsters fed the control diet. Moreover, a significant increase in adiponectin levels and a decrease in leptin levels were observed in hamsters fed the 4% HPMC-supplemented diet. Hamster adiponectin was found to be comprised of 244 amino acid residues with an apparent molecular weight of 30 kDa, consistent with the adiponectin reported in other species. Reductions in plasma cholesterol and triglyceride levels were correlated with a decrease in plasma leptin and an increase in adiponectin. These results suggest that adipocytokines are regulated by HPMC and may play a pivotal role in the hypocholesterolemic effect. © 2009 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Blackwell Publishing Asia Pty Ltd.

  1. A reaction-diffusion model of CO2 influx into an oocyte. (United States)

    Somersalo, Erkki; Occhipinti, Rossana; Boron, Walter F; Calvetti, Daniela


    We have developed and implemented a novel mathematical model for simulating transients in surface pH (pH(S)) and intracellular pH (pH(i)) caused by the influx of carbon dioxide (CO(2)) into a Xenopus oocyte. These transients are important tools for studying gas channels. We assume that the oocyte is a sphere surrounded by a thin layer of unstirred fluid, the extracellular unconvected fluid (EUF), which is in turn surrounded by the well-stirred bulk extracellular fluid (BECF) that represents an infinite reservoir for all solutes. Here, we assume that the oocyte plasma membrane is permeable only to CO(2). In both the EUF and intracellular space, solute concentrations can change because of diffusion and reactions. The reactions are the slow equilibration of the CO(2) hydration-dehydration reactions and competing equilibria among carbonic acid (H(2)CO(3))/bicarbonate (HCO(3)(-)) and a multitude of non-CO(2)/HCO(3)(-) buffers. Mathematically, the model is described by a coupled system of reaction-diffusion equations that-assuming spherical radial symmetry-we solved using the method of lines with appropriate stiff solvers. In agreement with experimental data [Musa-Aziz et al. 2009, PNAS 106 5406-5411], the model predicts that exposing the cell to extracellular 1.5% CO(2)/10 mM HCO(3)(-) (pH 7.50) causes pH(i) to fall and pH(S) to rise rapidly to a peak and then decay. Moreover, the model provides insights into the competition between diffusion and reaction processes when we change the width of the EUF, membrane permeability to CO(2), native extra- and intracellular carbonic anhydrase-like activities, the non-CO(2)/HCO(3)(-) (intrinsic) intracellular buffering power, or mobility of intrinsic intracellular buffers. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Frog oocytes to unveil the structure and supramolecular organization of human transport proteins.

    Directory of Open Access Journals (Sweden)

    Marc J Bergeron

    Full Text Available Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM and single particle analysis (SPA of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4 and the aquaporin-1 (AQP1 water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12 family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

  3. Efficient Gene Targeting in Golden Syrian Hamsters by the CRISPR/Cas9 System


    Zhiqiang Fan; Wei Li; Sang R Lee; Qinggang Meng; Bi Shi; Bunch, Thomas D.; Kenneth L White; Il-Keun Kong; Zhongde Wang


    The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)--three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, ...

  4. Role of caloric homeostasis and reward in alcohol intake in Syrian golden hamsters


    Gulick, Danielle; Green, Alan I.


    The Syrian golden hamster drinks alcohol readily, but only achieves moderate blood alcohol levels, and does not go through withdrawal from alcohol. Because the hamster is a model of caloric homeostasis, both caloric content and reward value may contribute to the hamster’s alcohol consumption. The current study examines alcohol consumption in the hamster when a caloric or non-caloric sweet solution is concurrently available and caloric intake in the hamster before, during, and after exposure t...

  5. The Syrian hamster model of hantavirus pulmonary syndrome (United States)

    Safronetz, David; Ebihara, Hideki; Feldmann, Heinz; Hooper, Jay W.


    Hantavirus pulmonary syndrome (HPS) is a relatively rare, but frequently fatal disease associated with New World hantaviruses, most commonly Sin Nombre and Andes viruses in North and South America, respectively. It is characterized by fever and the sudden, rapid onset of severe respiratory distress and cardiogenic shock, which can be fatal in up to 50% of cases. Currently there are no approved antiviral therapies or vaccines for the treatment or prevention of HPS. A major obstacle in the development of effective medical countermeasures against highly pathogenic agents like the hantaviruses is recapitulating the human disease as closely as possible in an appropriate and reliable animal model. To date, the only animal model that resembles HPS in humans is the Syrian hamster model. Following infection with Andes virus, hamsters develop HPS-like disease which faithfully mimics the human condition with respect to incubation period and pathophysiology of disease. Perhaps most importantly, the sudden and rapid onset of severe respiratory distress observed in humans also occurs in hamsters. The last several years has seen an increase in studies utilizing the Andes virus hamster model which have provided unique insight into HPS pathogenesis as well as potential therapeutic and vaccine strategies to treat and prevent HPS. The purpose of this article is to review the current understanding of HPS disease progression in Syrian hamsters and discuss the suitability of utilizing this model to evaluate potential medical countermeasures against HPS. PMID:22705798

  6. Social context modulates food hoarding in Syrian hamsters

    Directory of Open Access Journals (Sweden)

    Bibiana Montoya


    Full Text Available The effect of the presence of a con-specific in the temporal organization of food hoarding was studied in two varieties of Syrian hamster (Mesocricetus auratus: golden and long-haired. Four male hamsters of each variety were used. Their foraging behavior was observed during four individual and four shared trials in which animals were not competing for the same food source or territory. During individual trials, long-haired hamsters consumed food items directly from the food source, transporting and hoarding only remaining pieces. During shared trials, the long-haired variety hoarded food items before consumption, and increased the duration of hoarding trips, food handling in the storage, and cache size. Golden hamsters maintained the same temporal organization of hoarding behavior (i.e., hoarding food items before consumption throughout both individual and shared trials. However, the golden variety increased handling time at the food source and decreased the duration of hoarding trips, the latency of hoarding and storing size throughout the shared trials. In Syrian hamsters, the presence of a con-specific may signal high probability of food source depletion suggesting that social pressures over food availability might facilitate hoarding behavior. Further studies are required to evaluate cost-benefit balance of food hoarding and the role of cache pilferage in this species.

  7. Regulation of lipid metabolism by obeticholic acid in hyperlipidemic hamsters. (United States)

    Dong, Bin; Young, Mark; Liu, Xueqing; Singh, Amar Bahadur; Liu, Jingwen


    The farnesoid X receptor (FXR) plays critical roles in plasma cholesterol metabolism, in particular HDL-cholesterol (HDL-C) homeostasis. Obeticholic acid (OCA) is a FXR agonist being developed for treating various chronic liver diseases. Previous studies reported inconsistent effects of OCA on regulating plasma cholesterol levels in different animal models and in different patient populations. The mechanisms underlying its divergent effects have not yet been thoroughly investigated. The scavenger receptor class B type I (SR-BI) is a FXR-modulated gene and the major receptor for HDL-C. We investigated the effects of OCA on hepatic SR-BI expression and correlated such effects with plasma HDL-C levels and hepatic cholesterol efflux in hyperlipidemic hamsters. We demonstrated that OCA induced a time-dependent reduction in serum HDL-C levels after 14 days of treatment, which was accompanied by a significant reduction of liver cholesterol content and increases in fecal cholesterol in OCA-treated hamsters. Importantly, hepatic SR-BI mRNA and protein levels in hamsters were increased to 1.9- and 1.8-fold of control by OCA treatment. Further investigations in normolipidemic hamsters did not reveal OCA-induced changes in serum HDL-C levels or hepatic SR-BI expression. We conclude that OCA reduces plasma HDL-C levels and promotes transhepatic cholesterol efflux in hyperlipidemic hamsters via a mechanism involving upregulation of hepatic SR-BI.

  8. Hamster and Murine Models of Severe Destructive Lyme Arthritis

    Directory of Open Access Journals (Sweden)

    Erik Munson


    Full Text Available Arthritis is a frequent complication of infection in humans with Borrelia burgdorferi. Weeks to months following the onset of Lyme borreliosis, a histopathological reaction characteristic of synovitis including bone, joint, muscle, or tendon pain may occur. A subpopulation of patients may progress to a chronic, debilitating arthritis months to years after infection which has been classified as severe destructive Lyme arthritis. This arthritis involves focal bone erosion and destruction of articular cartilage. Hamsters and mice are animal models that have been utilized to study articular manifestations of Lyme borreliosis. Infection of immunocompetent LSH hamsters or C3H mice results in a transient synovitis. However, severe destructive Lyme arthritis can be induced by infecting irradiated hamsters or mice and immunocompetent Borrelia-vaccinated hamsters, mice, and interferon-gamma- (IFN-γ- deficient mice with viable B. burgdorferi. The hamster model of severe destructive Lyme arthritis facilitates easy assessment of Lyme borreliosis vaccine preparations for deleterious effects while murine models of severe destructive Lyme arthritis allow for investigation of mechanisms of immunopathology.

  9. Pineal melatonin synthesis in Syrian hamsters: A summary (United States)

    Rollag, M. D.


    During the past decade there has been ample documentation of the proposition that the pineal gland mediates photoperiodic influences upon reproductive behavior of hamsters. It is commonly hypothesized that the pineal gland expresses its activity by transformation of photoperiodic information into an hormonal output, that hormone being melatonin. If this hypothesis is correct, there must be some essential diffrence in melatonin's output when hamsters are exposed to different photoperiodic environments. The experiments summarized in this communication analyze pineal melatonin contents in Syrian hamsters maintained in a variety of photoperiodic conditions during different physiological states. The results demonstrate that adult hamsters have a daily surge in pineal melatonin content throughout their lifetime when exposed to simulated annual photoperiodic cycles. There is some fluctuation in the amount of pineal melatonin produced during different physiological states and photoperiodic environments, but these fluctuations seem small when compared to those normally found for other regulatory hormones. When hamsters are exposed to different photoperiodic regimens, the daily melatonin surge maintains a relatively constant phase relationship with respect to the onset of daily activity. There is a concomitant change in its phase relationship with respect to light-dark transitions.

  10. Hamster and Murine Models of Severe Destructive Lyme Arthritis (United States)

    Munson, Erik; Nardelli, Dean T.; Du Chateau, Brian K.; Callister, Steven M.; Schell, Ronald F.


    Arthritis is a frequent complication of infection in humans with Borrelia burgdorferi. Weeks to months following the onset of Lyme borreliosis, a histopathological reaction characteristic of synovitis including bone, joint, muscle, or tendon pain may occur. A subpopulation of patients may progress to a chronic, debilitating arthritis months to years after infection which has been classified as severe destructive Lyme arthritis. This arthritis involves focal bone erosion and destruction of articular cartilage. Hamsters and mice are animal models that have been utilized to study articular manifestations of Lyme borreliosis. Infection of immunocompetent LSH hamsters or C3H mice results in a transient synovitis. However, severe destructive Lyme arthritis can be induced by infecting irradiated hamsters or mice and immunocompetent Borrelia-vaccinated hamsters, mice, and interferon-gamma- (IFN-γ-) deficient mice with viable B. burgdorferi. The hamster model of severe destructive Lyme arthritis facilitates easy assessment of Lyme borreliosis vaccine preparations for deleterious effects while murine models of severe destructive Lyme arthritis allow for investigation of mechanisms of immunopathology. PMID:22461836

  11. Dormancy and activation of human oocytes from primordial and primary follicles: molecular clues to oocyte regulation

    DEFF Research Database (Denmark)

    Ernst, Emil Hagen; Grøndahl, Marie Louise; Grund, Simon


    as putative mediators of oocyte dormancy and activation. WHAT IS KNOWN ALREADY: Cellular signaling pathways including PI3K/AKT and AKT/mTOR as well as TGF-β and IGF signaling are known to regulate the primordial-to-primary transition in mammalian follicle development. STUDY DESIGN, SIZE, DURATION: We...... isolated by Laser Capture Microdissection, and submitted to the HiSeq Illumina platform. Data mapping, quality control, filtering and expression analysis were performed using Tophat (2.0.4), Cufflinks (2.0.2), BWA (0.6.2) and software R. Modeling of complex biological systems was performed using the IPA......® software. Finally, qPCR and immunohistochemistry were employed to explore expression and localization of selected genes and products in human ovarian tissue. MAIN RESULTS AND THE ROLE OF CHANCE: We found 223 and 268 genes down-regulated and up-regulated, respectively, in the oocytes during the human...

  12. Social oocyte cryopreservation: a portrayal of Brazilian women. (United States)

    Santo, Elisangela V Espirito; Dieamant, Felipe; Petersen, Claudia G; Mauri, Ana L; Vagnini, Laura D; Renzi, Adriana; Zamara, Camila; Oliveira, João Batista A; Baruffi, Ricardo L R; Franco, José G


    This study aimed to determine what Brazilian childless women of reproductive age think about oocyte cryopreservation to postpone pregnancy and their reasons for performing or not performing this procedure. Women of reproductive age were randomly selected from the general population using different e-mail lists and were invited to participate in the study by completing an online web survey regarding social oocyte cryopreservation. The survey was also distributed through social media to women of reproductive age. Although most of the responders had a partner (86.9%) and had already planned the pregnancy of their first child (69.6%), 85.4% (379) considered the potential of social oocyte freezing to improve their chances of giving birth later in life. Those that had already planned pregnancy were two times more likely to intend to freeze their oocytes (p=0.03). The most important barrier for not undergoing oocyte cryopreservation was cost. The women who indicated that they could not currently undergo the procedure now because of cost were two times (p=0.03) more likely to intend to cryopreserve their oocytes than women who thought that they would not need to delay pregnancy. Brazilian women who think that they are not ready to have a family are discovering the option of oocyte cryopreservation. Most participants considered safeguarding their reproductive potential. Making the procedure more accessible could give women the opportunity to make proactive decisions about the future of their fertility.

  13. [The cytogenetics of human oocytes: 40 years of progress]. (United States)

    Pellestor, F; Andréo, B; Anahory, T; Déchaud, H; Hédon, B; Hamamah, S


    Chromosomal abnormalities account for the majority of pre- and post- implantation embryo wastage in humans. Most of these abnormalities result from maternal meiotic errors, which preferentially occur during the first meiotic division. Consequently, the cytogenetic analysis of human oocytes has then been considered as a highly valuable source of data for the investigation of both the occurrence and the origin of chromosomal abnormalities in human. During the last 4 decades, the cytogenetic analysis of human oocytes has never stopped progressing, according to the advents of new technologies. Both karyotyping and molecular cytogenetic studies have been reported to date, providing a large body of data on the incidence and the distribution of chromosomal abnormalities in human female gametes. However, these studies display a great variability in results, which may be essentially attributable to the limitations of these techniques when applied to human oocytes. The most relevant analysis have led to the estimate that 15-20% of human oocytes present chromosome abnormalities, and they have emphasized the implication of both whole chromosome non-disjunction and chromatid separation in the occurrence of aneuploidy in human oocytes. The effect of advanced maternal age on the incidence of aneuploidy in human oocytes has also been clearly evidenced by recent reports based on large sample of oocytes or polar bodies.

  14. Oocytes transport across the oviduct of Murrah and Nelore cows

    Directory of Open Access Journals (Sweden)

    P.S. Baruselli


    Full Text Available In order to verify the causes of the low embryo recovery rate in superovulated buffaloes, the effect of species and of estradiol-17β (E2 treatment were evaluated on oocyte transport across the oviduct in Murrah and Nelore. The females were synchronised with progesterone plus estradiol benzoate followed by an injection of PGF2α and eCG. The ovulation was induced with GnRH and 48hs after the animals were slaughtered and the oviducts removed. The oviducts were washed with HBSS and oocytes of both species were inserted into infundibulum portion. The oviducts were put in a dish with HBSS with or without E2 and incubated for 24 h. The oviduct were then flushed with DPBS, in order to recover and count the oocytes. Data were analyzed by ANOVA. There was no effect of interaction. The total number of oocytes and the recovery rate were higher for Nelore than Murrah (P<0.05 oviducts. There was no effect of treatment on these variables. The number of oocytes from buffaloes and bovine recovered was similar. These results indicate that oocytes transport across the oviduct of Murrah or Nelore does not depend on the oocyte species and is not influenced by E2.

  15. A quantitative assessment of follicle size on oocyte developmental competence (United States)

    Rosen, Mitchell P.; Shen, Shehua; Dobson, Anthony T.; Rinaudo, Paolo F.; McCulloch, Charles E.; Cedars, Marcelle I.


    Objective To quantitatively assess the impact of follicle size on oocyte maturation, fertilization, and embryo quality. Design Prospective study. Setting Academic medical center. Patient(s) Couples undergoing ovarian stimulation and in vitro fertilization (IVF). Intervention(s) A total of 235 cycles were monitored prospectively, and 2934 oocytes were collected from five groups of follicle size. Repeated measures multivariate analyses were used to compare the smaller follicle sizes with the lead follicle. Main Outcome Measure(s) Oocyte maturation, fertilization, and embryo quality. Result(s) Compared with the lead follicular group (>18 mm), the odds of a mature oocyte from a 16 to 18 mm size follicle were 37% and declined progressively with each size. The odds of fertilization of oocytes from follicles 16 to 18 mm in size was 28% less than the lead group and decreased with each size. The rate of polyspermy with conventional insemination was increased for the smaller follicular groups (adjusted odds ratio =2.37). Follicle size did not predict embryo cell number, but embryos from smaller follicles had a statistically significantly higher fragmentation compared with the lead group. Conclusion(s) The lead follicular group was most likely to have a mature oocyte that was capable of fertilization and best suited for development into a high-quality embryo. The smaller follicles were capable of producing metaphase II oocytes that could fertilize, but at rates approaching only 60% that of the lead follicular group. PMID:18249377

  16. Nucleologenesis and nucleolotransfer in mammalian oocytes: A review

    Directory of Open Access Journals (Sweden)

    Michal Benc


    Full Text Available An effort to improve development potential of early embryos is one of the main goals of biotechnology in the area of reproductive biology with application in veterinary or human medicine. Recent observations of the function of nucleolus or rather its forms before, during and after the fertilisation or parthenogenetic activation show the key role(s of nucleolus in the processes of early genome activation. The nucleolus is a subnuclear structure (organelle mainly involved in regulation of transcription and translation. This organelle has been characterized in detail by immunofluorescence, cell transfection and proteomics. This data was, however, mostly obtained in nucleoli of differentiated eukaryotic cells. Much less is known about the nucleolar structural changes and related functional processes in growing and fully grown mammalian oocytes, zygotes and early cleavage stage embryos, especially in the context of embryonic genome activation. It has been shown, that nucleoli in mammalian oocytes and early embryos have several forms and functions, which vary during the oocyte growth and embryonic development. Certain functions have not been fully described or explained, yet. The method of enucleolation, which allows to remove nucleoli from the oocytes or to exchange nucleoli between oocytes or zygotes, together with their proteomic and structural analyses brought new information about functions of nucleoli in oocytes and early cleavage-stage embryos and allowed to explain some new key roles of nucleoli during oocyte maturation and early embryonic development.

  17. Filter-Aided Sample Preparation (FASP) for Improved Proteome Analysis of Recombinant Chinese Hamster Ovary Cells. (United States)

    Coleman, Orla; Henry, Michael; Clynes, Martin; Meleady, Paula


    Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell line for biopharmaceutical production because of their ability to correctly fold and posttranslationally modify recombinant proteins that are compatible with human use. Proteomics, along with other 'omic platforms, are being used to understand the biology of CHO cells with the ultimate aim of enhancing CHO cell factories for more efficient production of biopharmaceuticals. In this chapter, we will describe an efficient protocol called Filter Aided Sample Preparation (FASP) for the extraction of proteins from CHO cells for proteomic studies. FASP uses a common ultrafiltration device whereby the membrane pores are small enough to allow contaminating detergents to pass through, while proteins are too large and are retained and concentrated in the filter unit. This method of sample preparation and protein digestion is universally applicable and can be easily employed in any proteomics facilities as standard everyday laboratory reagents and equipment are used.

  18. Potential targets of transforming growth factor-beta1 during inhibition of oocyte maturation in zebrafish

    Directory of Open Access Journals (Sweden)

    Clelland Eric


    Full Text Available Abstract Background TGF-beta is a multifunctional growth factor involved in regulating a variety of cellular activities. Unlike mammals, the function of TGF-beta in the reproduction of lower vertebrates, such as fish, is not clear. Recently, we showed that TGF-beta1 inhibits gonadotropin- and 17alpha, 20beta-dihydroxyprogesterone (DHP-induced maturation in zebrafish. The aim of the present study was to investigate the mechanisms underlying this action. Method To determine if the effect of TGF-beta1 on oocyte maturation involves transcription and/or translation, ovarian follicles were pre-treated with actinomycin D, a blocker of transcription, and cyclohexamide, an inhibitor of translation, and incubated with hCG or DHP, either alone or in combination with TGF-beta1 and oocyte maturation scored. To determine the effect of TGF-beta1 on mRNA levels of several key effectors of oocyte maturation, three sets of experiments were performed. First, follicles were treated with control medium or TGF-beta1 for 2, 6, 12, and 24 h. Second, follicles were treated with different concentrations of TGF-beta1 (0 to 10 ng/ml for 18 h. Third, follicles were incubated with hCG in the absence or presence of TGF-beta1 for 18 h. At the end of each experiment, total RNA was extracted and reverse transcribed. PCR using primers specific for 20beta-hydroxysteroid dehydrogenase (20beta-HSD which is involved in DHP production, follicle stimulating hormone receptor (FSHR, luteinizing hormone receptor (LHR, the two forms of membrane progestin receptor: mPR-alpha and mPR-beta, as well as GAPDH (control, were performed. Results Treatment with actinomycin D, a blocker of transcription, reduced the inhibitory effect of TGF-beta1 on DHP-induced oocyte maturation, indicating that the inhibitory action of TGF-beta1 is in part due to regulation of gene transcription. Treatment with TGF-beta1 caused a dose and time-dependent decrease in mRNA levels of 20beta-HSD, LHR and mPR-beta in

  19. Role of cumulus cells during vitrification and fertilization of mature bovine oocytes

    NARCIS (Netherlands)

    Ortiz-Escribano, N.; Smits, K.; Piepers, S.; Abbeel, Van den E.; Woelders, H.; Soom, Van A.


    This study was designed to determine the role of cumulus cells during vitrification of bovine oocytes. Mature cumulus-oocyte complexes (COCs) with many layers of cumulus cells, corona radiata oocytes (CRs), with a few layers of cumulus cells, and denuded oocytes (DOs) without cumulus cells were

  20. Gene expression profiles of single human mature oocytes in relation to age

    DEFF Research Database (Denmark)

    Grøndahl, M L; Andersen, Claus Yding; Bogstad, J


    The development competence of human oocytes declines with increasing age. The objective of this study was to investigate the effect of age on gene expression profile in mature human oocytes.......The development competence of human oocytes declines with increasing age. The objective of this study was to investigate the effect of age on gene expression profile in mature human oocytes....

  1. 9 CFR 3.36 - Primary enclosures used to transport live guinea pigs and hamsters. (United States)


    ... live guinea pigs and hamsters. 3.36 Section 3.36 Animals and Animal Products ANIMAL AND PLANT HEALTH..., Care, Treatment, and Transportation of Guinea Pigs and Hamsters Transportation Standards § 3.36 Primary enclosures used to transport live guinea pigs and hamsters. No person subject to the Animal Welfare...

  2. MicroRNA Expression during Bovine Oocyte Maturation and Fertilization

    Directory of Open Access Journals (Sweden)

    Graham C. Gilchrist


    Full Text Available Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV oocytes, metaphase II (MII oocytes, and presumptive zygotes (PZ. Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR. Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05. To determine whether changes in specific primary miRNA (pri-miRNA transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo.

  3. Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Desrosiers, R.R.; Romanik, E.A.; O' Connor, C.M. (Worcester Foundation for Experimental Biology, Shrewsbury, MA (USA))


    The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-(methyl-3H)methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-(methyl-3H)methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly.

  4. Melatonin-induced increase of lipid droplets accumulation and in vitro maturation in porcine oocytes is mediated by mitochondrial quiescence. (United States)

    He, Bin; Yin, Chao; Gong, Yabin; Liu, Jie; Guo, Huiduo; Zhao, Ruqian


    Melatonin, the major pineal secretory product, has a significant impact on the female reproductive system. Recently, the beneficial effects of melatonin on mammalian oocyte maturation and embryonic development have drawn increased attention. However, the exact underlying mechanisms remain to be fully elucidated. This study demonstrates that supplementing melatonin to in vitro maturation (IVM) medium enhances IVM rate, lipid droplets (LDs) accumulation as well as triglyceride content in porcine oocytes. Decrease of mitochondrial membrane potential, mitochondrial respiratory chain complex IV activity as well as mitochondrial reactive oxygen species (mROS) content indicated that melatonin induced a decrease of mitochondrial activity. The copy number of mitochondrial DNA (mtDNA) which encodes essential subunits of oxidative phosphorylation (OXPHOS), was not affected by melatonin. However, the expression of mtDNA-encoded genes was significantly down-regulated after melatonin treatment. The DNA methyltransferase DNMT1, which regulates methylation and expression of mtDNA, was increased and translocated into the mitochondria in melatonin-treated oocytes. The inhibitory effect of melatonin on the expression of mtDNA was significantly prevented by simultaneous addition of DNMT1 inhibitor, which suggests that melatonin regulates the transcription of mtDNA through up-regulation of DNMT1 and mtDNA methylation. Increase of triglyceride contents after inhibition of OXPHOS indicated that mitochondrial quiescence is crucial for LDs accumulation in oocytes. Taken together, our results suggest that melatonin-induced reduction in mROS production and increase in IVM, and LDs accumulation in porcine oocytes is mediated by mitochondrial quiescence. © 2017 Wiley Periodicals, Inc.

  5. Oocytes Polar Body Detection for Automatic Enucleation

    Directory of Open Access Journals (Sweden)

    Di Chen


    Full Text Available Enucleation is a crucial step in cloning. In order to achieve automatic blind enucleation, we should detect the polar body of the oocyte automatically. The conventional polar body detection approaches have low success rate or low efficiency. We propose a polar body detection method based on machine learning in this paper. On one hand, the improved Histogram of Oriented Gradient (HOG algorithm is employed to extract features of polar body images, which will increase success rate. On the other hand, a position prediction method is put forward to narrow the search range of polar body, which will improve efficiency. Experiment results show that the success rate is 96% for various types of polar bodies. Furthermore, the method is applied to an enucleation experiment and improves the degree of automatic enucleation.

  6. Rapamycin Rescues the Poor Developmental Capacity of Aged Porcine Oocytes

    Directory of Open Access Journals (Sweden)

    Seung Eun Lee


    Full Text Available Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 μM rapamycin/24 h, 47.52±5.68 or control oocytes (44 h IVM; 42.14±4.40 significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68 (p<0.05. Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK, and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1 compared with untreated, 24 h-aged IVM oocytes (p<0.05. Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS activity and DNA fragmentation (p<0.05, and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05 and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1, anti-apoptosis (BCL2L1 and BIRC5; p<0.05, and development (NANOG and SOX2; p<0.05 genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3 compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

  7. Effect of bismuth subsalicylate on Clostridium difficile colitis in hamsters. (United States)

    Chang, T W; Dong, M Y; Gorbach, S L


    The therapeutic effect of bismuth subsalicylate (BSS, Pepto-Bismol) in Clostridium difficile colitis was studied in golden Syrian hamsters. C. difficile was fed to the hamsters by orogastric intubation 2-3 days after their arrival. Clindamycin (1.5 mg per animal) was given intraperitoneally 4 days later. Twenty-four hours after challenge with clindamycin, animals were given BSS at dosages of 5, 10, and 15 mg twice daily for 5 days by orogastric intubation. Controls included untreated animals and those given 5 mg of vancomycin once daily by intubation. Delay in the time of death was observed in all BSS-treated animals and was statistically significant on days 4-6 in those receiving 15 mg twice daily. Vancomycin produced a greater delay in death than did BSS. Our study suggests that BSS at a dosage of 15 mg twice daily has some therapeutic effect on C. difficile colitis in hamsters.

  8. Effect of oocyte selection, estradiol and antioxidant treatment on in vitro maturation of oocytes collected from prepubertal Boer goats

    Directory of Open Access Journals (Sweden)

    George W. Smith


    Full Text Available Development of improved procedures for in vitro maturation of oocytes collected from prepubertal goats has applications for in vitro embryo production and accompanying strategies for genetic improvement. The objective of described studies was to determine the effects of oocyte grade, in vitro maturation time, antioxidant supplementation and concentrations of estradiol in the maturation medium on in vitro maturation of oocytes harvested from 1-6 mm follicles present on the ovaries (obtained from an abattoir of 1-6 month-old prepubertal Boer goats. Rates of progression to metaphase II were greater for grade 1 oocytes (>3 compact layers of cumulus cells and evenly granulated cytoplasm than grade 2 oocytes (in vitro maturation in the presence of high concentrations of estradiol (10 and 100 mg/mL on progression to metaphase II was observed, and no effect was observed in response to 1 mg/mL estradiol treatment as compared with control. Results suggest that oocyte selection and beta-mercaptoethanol supplementation can positively influence progression to metaphase II of oocytes harvested from ovaries of prepubertal goats, whereas high concentrations of estradiol are inhibitory to in vitro maturation.

  9. Foodborne transmission of nipah virus in Syrian hamsters. (United States)

    de Wit, Emmie; Prescott, Joseph; Falzarano, Darryl; Bushmaker, Trenton; Scott, Dana; Feldmann, Heinz; Munster, Vincent J


    Since 2001, outbreaks of Nipah virus have occurred almost every year in Bangladesh with high case-fatality rates. Epidemiological data suggest that in Bangladesh, Nipah virus is transmitted from the natural reservoir, fruit bats, to humans via consumption of date palm sap contaminated by bats, with subsequent human-to-human transmission. To experimentally investigate this epidemiological association between drinking of date palm sap and human cases of Nipah virus infection, we determined the viability of Nipah virus (strain Bangladesh/200401066) in artificial palm sap. At 22°C virus titers remained stable for at least 7 days, thus potentially allowing food-borne transmission. Next, we modeled food-borne Nipah virus infection by supplying Syrian hamsters with artificial palm sap containing Nipah virus. Drinking of 5×10⁸ TCID₅₀ of Nipah virus resulted in neurological disease in 5 out of 8 hamsters, indicating that food-borne transmission of Nipah virus can indeed occur. In comparison, intranasal (i.n.) inoculation with the same dose of Nipah virus resulted in lethal respiratory disease in all animals. In animals infected with Nipah virus via drinking, virus was detected in respiratory tissues rather than in the intestinal tract. Using fluorescently labeled Nipah virus particles, we showed that during drinking, a substantial amount of virus is deposited in the lungs, explaining the replication of Nipah virus in the respiratory tract of these hamsters. Besides the ability of Nipah virus to infect hamsters via the drinking route, Syrian hamsters infected via that route transmitted the virus through direct contact with naïve hamsters in 2 out of 24 transmission pairs. Although these findings do not directly prove that date palm sap contaminated with Nipah virus by bats is the origin of Nipah virus outbreaks in Bangladesh, they provide the first experimental support for this hypothesis. Understanding the Nipah virus transmission cycle is essential for preventing

  10. Diet-induced dyslipidemia impairs reverse cholesterol transport in hamsters. (United States)

    Tréguier, Morgan; Briand, François; Boubacar, Adamou; André, Agnès; Magot, Thierry; Nguyen, Patrick; Krempf, Michel; Sulpice, Thierry; Ouguerram, Khadija


    Reverse cholesterol transport (RCT) is an anti-atherogenic process by which cholesterol is effluxed from peripheral tissues by high-density lipoprotein (HDL) and returned to the liver for excretion into the bile and faeces. Dyslipidemia is thought to impair RCT through higher triglyceride-rich lipoprotein (TRL), low HDL-cholesterol and higher activity of cholesteryl ester transfer protein (CETP), which transfers cholesteryl esters from HDL to TRL for further hepatic uptake. As CETP pathway would represent a major route in human RCT, we therefore investigated whether diet-induced dyslipidemia impairs RCT in hamster, a CETP-expressing species. Golden Syrian hamsters were fed a chow or chow+0·3% cholesterol diet over 4 weeks. Biochemical parameters and in vivo VLDL-triglycerides secretion (Triton WR-1339 injection) were then measured. In vitro macrophage cholesterol efflux was measured, and in vivo macrophage-to-faeces RCT was also assessed after an intraperitoneal injection of (3) H-cholesterol-labelled hamster primary macrophages. Cholesterol-enriched diet increased plasma total cholesterol (144%), triglycerides (101%), VLDL-triglycerides secretion (175%), CETP activity (44%) and reduced HDL-cholesterol/total cholesterol ratio by 20% (P diet significantly increased hepatic total cholesterol and triglycerides by 459 and 118% and increased aortic total cholesterol content by 304%. In vitro cholesterol efflux from macrophages to plasma was significantly reduced by 25% with plasma from cholesterol-fed hamsters. In vivo RCT experiments showed a significant 75% reduction of macrophage-derived cholesterol faecal excretion in cholesterol-fed hamsters. Overall, these data demonstrate that diet-induced dyslipidemia severely impairs in vivo RCT in hamsters. © 2011 The Authors. European Journal of Clinical Investigation © 2011 Stichting European Society for Clinical Investigation Journal Foundation.

  11. The thalamic intergeniculate leaflet modulates photoperiod responsiveness in Siberian hamsters. (United States)

    Freeman, David A; Dhandapani, Krishnan M; Goldman, Bruce D


    Siberian hamsters are seasonal breeders that use changes in day length to synchronize their reproductive effort with those times of the year most favorable for successful reproduction. The ability of Siberian hamsters to measure and respond to changes in day length depends upon accurate photoentrainment of the circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. Two pathways have been characterized through which entraining stimuli reach the SCN: the retinohypothalamic tract (RHT), which transmits light information from the retinae, and the geniculohypothalamic tract (GHT) from the intergeniculate leaflet of the thalamus (IGL), which is involved in transmitting both photic and nonphotic cues. Ablating the IGL/GHT results in only modest alterations in entrainment to static day lengths and fails to interfere with seasonal responses induced by transfer from static long day to static short day lengths. Because several studies suggest that the IGL may be involved in tracking the time of dusk and dawn, we sought to determine whether an intact IGL is necessary for hamsters to respond to a simulated natural photoperiod (SNP) in which the time of dusk and dawn gradually changes in a pattern approximating the rate of change in day length that occurs during autumn at the latitude this species inhabits in nature. The results indicate that neurochemical lesions of the IGL alter both the pattern of circadian entrainment and photoperiodic responsiveness of Siberian hamsters to an SNP. Both intact and IGL-lesioned hamsters exhibited testicular regression in shortening day lengths, but only IGL-intact hamsters exhibited seasonal pelage molt.

  12. Morphological evaluation of canine oocytes recovered in different phases of the estrous cycle


    Sánchez R., Alfonso; Escuela de Medicina Veterinaria, Universidad Santo Tomás, Viña del Mar; Ahumada C., Carolina; Escuela de Medicina Veterinaria, Universidad Santo Tomás, Viña del Mar


    The aim of the study was to obtain and morphologically evaluate canine oocytes from females at different phases of the estrous cycle and to compare the proportions of oocytes potentially suitable for in vitro maturation (IVM). Ovaries of 24 bitches, 6 for each phase of the estrous cycle, were sliced to recover the oocytes. These were morphologically classified into fit and unfit oocytes for IVM. The largest proportion of oocytes fit for IVM (86.6%) was recovered from females in proestrus and ...

  13. The Hamster Buccal Pouch Model of Oral Carcinogenesis. (United States)

    Nagini, Siddavaram; Kowshik, Jaganathan


    The hamster buccal pouch (HBP) carcinogenesis model is one of the most well-characterized animal tumor models used as a prelude to investigate multistage oral carcinogenesis and to assess the efficacy of chemointervention. Hamster buccal pouch carcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) show extensive similarities to human oral squamous cell carcinomas. The HBP model offers a number of advantages including a simple and predictable tumor induction procedure, easy accessibility for examination and follow-up of lesions, and reproducibility. This model can be used to test both chemopreventive and chemotherapeutic agents.

  14. Genotoxic activity of nitrilotriacetic acid in Chinese hamster cells. (United States)

    Modesti, D; Tanzarella, C; Degrassi, F


    Nitrilotriacetic acid (NTA), a chelating agent, was tested for its ability to induce chromosomal damage in Chinese hamster cells. The chemical was shown to exert a weak genotoxic activity increasing the frequency of micronuclei after prolonged treatments. The analysis of kinetochore containing-micronuclei showed that NTA prevailingly induces chromosomal aberrations as compared to chromosome loss in hamster cells. Furthermore, immunostaining with an alpha-tubulin antibody showed clear alterations in the interphase microtubule network of cells treated for 24 h with 3 mM NTA. The microtubule effects of the chemical may be partly responsible for its cytotoxic effects.

  15. Effect of PCMBS on CO2 permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant. (United States)

    Cooper, G J; Boron, W F


    A recent study on Xenopus oocytes [N. L. Nakhoul, M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 (Cell Physiol. 43): C543-548, 1998] injected with carbonic anhydrase showed that expressing aquaporin 1 (AQP1) increases by approximately 40% the rate at which exposing the cell to CO2 causes intracellular pH to fall. This observation is consistent with several interpretations. Overexpressing AQP1 might increase apparent CO2 permeability by 1) allowing CO2 to pass through AQP1, 2) stimulating injected carbonic anhydrase, 3) enhancing the CO2 solubility of the membrane's lipid, or 4) increasing the expression of a native "gas channel." The purpose of the present study was to distinguish among these possibilities. We found that expressing the H2O channel AQP1 in Xenopus oocytes increases the CO2 permeability of oocytes in an expression-dependent fashion, whereas expressing the K+ channel ROMK1 has no effect. The mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS), which inhibits the H2O movement through AQP1, also blocks the AQP1-dependent increase in CO2 permeability. The mercury-insensitive C189S mutant of AQP1 increases the CO2 permeability of the oocyte to the same extent as does the wild-type channel. However, the C189S-dependent increase in CO2 permeability is unaffected by treatment with PCMBS. These data rule out options 2-4 listed above. Thus our results suggest that CO2 passes through the pore of AQP1 and are the first data to demonstrate that a gas can enter a cell by a means other than diffusing through the membrane lipid.

  16. Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence

    Directory of Open Access Journals (Sweden)

    Yokoi Hayato


    Full Text Available Abstract Background Large volumes of lymph can be collected from the eye-sacs of bubble-eye goldfish. We attempted to induce vitellogenin (Vtg in the eye-sac lymph of bubble-eye goldfish and develop a method for visualizing Vtg incorporation by zebrafish oocytes using FITC-labeling. Methods Estrogen efficiently induced Vtg in the eye-sac lymph of goldfish. After FITC-labeled Vtg was prepared, it was injected into mature female zebrafish. Results Incorporation of FITC-labeled Vtg by zebrafish oocytes was detected in in vivo and in vitro experiments. The embryos obtained from zebrafish females injected with FITC-labeled Vtg emitted FITC fluorescence from the yolk sac and developed normally. Conclusion This method for achieving Vtg incorporation by zebrafish oocytes could be useful in experiments related to the development and endocrinology of zebrafish oocytes.

  17. Early effect of boron neutron capture therapy mediated by boronophenylalanine (BPA-BNCT) on mast cells in premalignant tissue and tumors of the hamster cheek pouch. (United States)

    Aromando, Romina F; Trivillin, Verónica A; Heber, Elisa M; Pozzi, Emiliano; Schwint, Amanda E; Itoiz, María E


    Mast cell (MC) activation in the hamster cheek pouch cancerization model is associated with the increase in tumor cell proliferation, mediated in turn by tryptase, a protease released from mast cell granules after activation. Tryptase induces tumor cell proliferation through the activation of PAR-2 (protease activated receptor-2) on the plasma membrane of carcinoma cells. The therapeutic success of boron neutron capture therapy mediated by boronophenylalanine (BPA-BNCT) in tumor control in the hamster cheek pouch oral cancer model has been previously reported by our laboratory. Early effects of BPA-BNCT on tumors of the hamster cheek pouch include a reduction in DNA-synthesis with the concomitant decrease in the proliferation of malignant cells. The aim of the present study was to investigate the early histological changes in mast cells after BPA-BNCT in tumors and premalignant tissue of the hamster cheek pouch. Tumor-bearing pouches were treated with BPA-BNCT or beam only (neutron irradiation without prior administration of the boron compound) and sacrificed 1day after treatment. The samples were fixed in Carnoy fixative and stained with alcian blue-safranin to identify all the populations of mast cells. Total, active and inactive mast cells (MC) were counted in the connective tissue and the adventitious tissue underlying the pouch wall and at the base of the tumors in pouches treated with BPA-BNCT, in keeping with a previously described technique. BPA-BNCT induced a marked reduction in the total number of mast cells in the pouch (pBNCT and beam only elicited a qualitative change in the secretion modality of the granule content. Although further studies are needed to evaluate the subcellular effect of BNCT on mast cell granule secretion, the reduction in cell proliferation induced by BPA-BNCT would be partially due to the decrease in total mast cells in the hamster check pouch. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  18. Oocyte surface in four teleost fish species postspawning and fertilization


    Rizzo,Elizete; Moura,Thais F.C.; Sato,Yoshimi; Bazzoli,Nilo


    Cytological and cytochemical studies were carried out to investigate the surface characteristics of oocytes of four teleost species from the São Francisco river. The fishes were submitted to hypophysation at the Três Marias Hybrobiology and Fishculture Station, Minas Gerais, Brazil, in January 1996. Postspawning, oocytes of the curimatãs Prochilodus affinis, Prochilodus marggravii and dourado Salminus brasiliensis were surrounded by a thick, three-layered zona pellucida with radial striae. Th...

  19. [Lipids in the sperm plasma membrane and their role in fertilization]. (United States)

    Niu, Dong-Mei; Wang, Jun-Jun


    Sexual reproduction is marked by the fusion of the sperm cell with the oocyte during fertilization to produce the diploid zygote, in which the lipids in the sperm plasma membrane play an important role. Due to the loss of most cell organelles and DNA transcription, spermatozoa lack protein expression and vesicular transport. However, the lipids of the sperm plasma membrane undergo complicated dynamic changes, which may facilitate the capacitation, binding with zona pellucida, acrosome reaction and fusion of the sperm cell with the oocyte. This paper summarizes the progress in the studies of the lipids in the sperm plasma membrane, their composition, structure, peroxidation, metabolism and role in fertilization.

  20. Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system.

    Directory of Open Access Journals (Sweden)

    Zhiqiang Fan

    Full Text Available The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs--three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C--and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.

  1. Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system. (United States)

    Fan, Zhiqiang; Li, Wei; Lee, Sang R; Meng, Qinggang; Shi, Bi; Bunch, Thomas D; White, Kenneth L; Kong, Il-Keun; Wang, Zhongde


    The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)--three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C--and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.

  2. Cloning of a hamster anti-mouse CD79B antibody sequences and identification of a new hamster immunoglobulin lambda constant IGLC gene region. (United States)

    Haggart, Ryan; Perera, Jason; Huang, Haochu


    Anti-CD79 antibodies have been effective at targeting B cell lymphoma cells and depleting B cells in animal models. In order to engineer recombinant antibodies with additional effector functions in mice, we cloned and sequenced the full-length cDNAs of the heavy and light chain of a hamster anti-mouse CD79B antibody. Although hamster antibodies represent a unique source of monoclonal antibodies against mouse, rat, and human antigens, sequence information of hamster immunoglobulins (IG) is sparse. Here, we report a new hamster (Cricetulus migratorius) IG lambda constant (IGLC) gene region that is most homologous to mouse IGLC2 and IGLC3.

  3. The Role of Mitochondria from Mature Oocyte to Viable Blastocyst

    Directory of Open Access Journals (Sweden)

    Scott Chappel


    Full Text Available The oocyte requires a vast supply of energy after fertilization to support critical events such as spindle formation, chromatid separation, and cell division. Until blastocyst implantation, the developing zygote is dependent on the existing pool of mitochondria. That pool size within each cell decreases with each cell division. Mitochondria obtained from oocytes of women of advanced reproductive age harbor DNA deletions and nucleotide variations that impair function. The combination of lower number and increased frequency of mutations and deletions may result in inadequate mitochondrial activity necessary for continued embryo development and cause pregnancy failure. Previous reports suggested that mitochondrial activity within oocytes may be supplemented by donor cytoplasmic transfer at the time of intracytoplasmic sperm injection (ICSI. Those reports showed success; however, safety concerns arose due to the potential of two distinct populations of mitochondrial genomes in the offspring. Mitochondrial augmentation of oocytes is now reconsidered in light of our current understanding of mitochondrial function and the publication of a number of animal studies. With a better understanding of the role of this organelle in oocytes immediately after fertilization, blastocyst and offspring, mitochondrial augmentation may be reconsidered as a method to improve oocyte quality.

  4. Resveratrol protects mouse oocytes from methylglyoxal-induced oxidative damage.

    Directory of Open Access Journals (Sweden)

    Yu Liu

    Full Text Available Methylglyoxal, a reactive dicarbonyl compound, is mainly formed from glycolysis. Methylglyoxal can lead to the dysfunction of mitochondria, the depletion of cellular anti-oxidation enzymes and the formation of advanced glycation ends. Previous studies showed that the accumulation of methylglyoxal and advanced glycation ends can impair the oocyte maturation and reduce the oocyte quality in aged and diabetic females. In this study, we showed that resveratrol, a kind of phytoalexin found in the skin of grapes, red wine and other botanical extracts, can alleviate the adverse effects caused by methylglyoxal, such as inhibition of oocyte maturation and disruption of spindle assembly. Besides, methylglyoxal-treated oocytes displayed more DNA double strands breaks and this can also be decreased by treatment of resveratrol. Further investigation of these processes revealed that methylglyoxal may affect the oocyte quality by resulting in excessive reactive oxygen species production, aberrant mitochondrial distribution and high level lipid peroxidation, and resveratrol can block these cytotoxic changes. Collectively, our results showed that resveratrol can protect the oocytes from methylglyoxal-induced cytotoxicity and this was mainly through the correction of the abnormity of cellular reactive oxygen species metabolism.

  5. Frequency of Aneuploidy Related to Age in Porcine Oocytes (United States)

    Musilova, Petra; Pavlok, Antonin; Kubelka, Michal; Motlik, Jan; Rubes, Jiri; Anger, Martin


    It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed. PMID:21556143

  6. Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa. (United States)

    van Tol, H T; van Eijk, M J; Mummery, C L; van den Hurk, R; Bevers, M M


    Cumulus oocyte complexes (COCs) and cumulus oocyte complexes connected to a piece of the membrane granulosa (COCGs) were isolated from bovine antral follicles with a diameter of 2 to 8 mm. After culture of COCGs without gonadotrophic hormones for 22 hr approximately 50% of the oocytes were still in the germinal vesicle (GV) stage. Histology of the COCGs showed that the pieces of the membrana granulosa were free of thecal cells and parts of the basal membrane. This indicates that the membrana granulosa solely inhibits the progression of meiosis. To investigate the effect of gonadotropins on the resumption of meiosis of oocytes from small and medium sized antral follicles, COCs and COCGs were cultured with or without rec-hFSH or hCG. Addition of 0.05 IU rec-hFSH to the culture medium of COCGs resulted in germinal vesicle breakdown in 97.8% of the oocytes compared to 46% in the control group, and an increase of the diameter of the COCs (479 microns vs. 240 microns in the control group). Addition of 0.05 IU hCG to the culture medium had no effect on nuclear maturation (47.2% GV vs. 48.5% GV in the control group) nor on cumulus expansion (246 microns vs. 240 microns in the control group). RT-PCR on cDNA of the follicular wall, cumulus cells, granulosa cells, COCs, and oocytes revealed that mRNA for FSH receptor was present in all cell types except oocytes. mRNA of the LH receptor was detected exclusively in thecal cells. Nucleotide sequence analysis and alignment of the cloned PCR products showed the presence of two isoforms of the FSH receptor mRNA and two isoforms of the LH receptor mRNA. It is concluded that, in vitro, resumption of meiosis of oocytes, originating from small and medium sized antral follicles and meiotically arrested by the membrana granulosa, is triggered by FSH and not by LH. This is supported by the fact that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of these follicles.

  7. Total number of oocytes and zygotes are predictive of live birth pregnancy in fresh donor oocyte in vitro fertilization cycles. (United States)

    Hariton, Eduardo; Kim, Keewan; Mumford, Sunni L; Palmor, Marissa; Bortoletto, Pietro; Cardozo, Eden R; Karmon, Anatte E; Sabatini, Mary E; Styer, Aaron K


    To evaluate the association of oocyte donor-recipient characteristics, oocyte donor response, and live birth pregnancy rate following fresh donor oocyte IVF-ET. Retrospective cohort study. Academic reproductive medicine practice. Two hundred thirty-seven consecutive fresh donor oocyte IVF-ET cycles from January 1, 2007 to December 31, 2013 at the Massachusetts General Hospital Fertility Center. None. Live birth rate per cycle initiated. The mean (±SD) age of oocyte donors and recipients was 27.0 ± 3.7 and 41.4 ± 4.6 years, respectively. Oocyte donor demographic/reproductive characteristics, ovarian reserve testing, and peak serum E2 during ovarian stimulation were similar among cycles which did and did not result in live birth, respectively. Overall implantation, clinical pregnancy, and live birth pregnancy rates per cycle initiated were 40.5%, 60.8%, and 54.9%, respectively. The greatest probability of live birth was observed in cycles with >10 oocytes retrieved, mature oocytes, oocytes with normal fertilization (zygote-two pronuclear stage), and cleaved embryos. The number of oocytes (total and mature), zygotes, and cleaved embryos are associated with live birth following donor oocyte IVF cycles. These findings suggest that specific peri-fertilization factors may be predictive of pregnancy outcomes following donor oocyte IVF cycles. Copyright © 2017 American Society for Reproductive Medicine. All rights reserved.

  8. Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes. (United States)

    Mertl, Manuela; Daniel, Hannelore; Kottra, Gabor


    The adaptation of the capacity of the intestinal peptide transporter PEPT1 to varying substrate concentrations may be important with respect to its role in providing bulk quantities of amino acids for growth, development, and other nutritional needs. In the present study, we describe a novel phenomenon of the regulation of PEPT1 in the Xenopus oocyte system. Using electrophysiological and immunofluorescence methods, we demonstrate that a prolonged substrate exposure of rabbit PEPT1 (rPEPT1) caused a retrieval of transporters from the membrane. Capacitance as a measure of membrane surface area was increased in parallel with the increase in rPEPT1-mediated transport currents with a slope of approximately 5% of basal surface per 100 nA. Exposure of oocytes to the model peptide Gly-l-Gln for 2 h resulted in a decrease in maximal transport currents with no change of membrane capacitance. However, exposure to substrate for 5 h decreased transport currents but also, in parallel, surface area by endocytotic removal of transporter proteins from the surface. The reduction of the surface expression of rPEPT1 was confirmed by presteady-state current measurements and immunofluorescent labeling of rPEPT1. A similar simultaneous decrease of current and surface area was also observed when endocytosis was stimulated by the activation of PKC. Cytochalasin D inhibited all changes evoked by either dipeptide or PKC stimulation, whereas the PKC-selective inhibitor bisindolylmaleimide only affected PKC-stimulated endocytotic processes but not substrate-dependent retrieval of rPEPT1. Coexpression experiments with human Na(+)-glucose transporter 1 (hSGLT1) revealed that substrate exposure selectively affected PEPT1 but not the activity of hSGLT1.

  9. Oocyte Donation Pregnancies- Non-Disclosure of Oocyte Recipient Status to Obstetric Care Providers and Perinatal Outcomes.

    LENUS (Irish Health Repository)


    Oocyte donation pregnancies- non-disclosure of oocyte recipient (OR) status to obstetric care providers and perinatal outcomes.Many studies report a higher rate of pregnancy-induced hypertension (PIH) and severe pre-eclampsia (PET) in OR pregnancies. The objective is to determine the rates of non-disclosure of OR pregnancy to obstetric care providers and also the rates of perinatal complications.

  10. Oocyte-specific deletion of N-WASP does not affect oocyte polarity, but causes failure of meiosis II completion. (United States)

    Wang, Zhen-Bo; Ma, Xue-Shan; Hu, Meng-Wen; Jiang, Zong-Zhe; Meng, Tie-Gang; Dong, Ming-Zhe; Fan, Li-Hua; Ouyang, Ying-Chun; Snapper, Scott B; Schatten, Heide; Sun, Qing-Yuan


    There is an unexplored physiological role of N-WASP (neural Wiskott-Aldrich syndrome protein) in oocyte maturation that prevents completion of second meiosis. In mice, N-WASP deletion did not affect oocyte polarity and asymmetric meiotic division in first meiosis, but did impair midbody formation and second meiosis completion. N-WASP regulates actin dynamics and participates in various cell activities through the RHO-GTPase-Arp2/3 (actin-related protein 2/3 complex) pathway, and specifically the Cdc42 (cell division cycle 42)-N-WASP-Arp2/3 pathway. Differences in the functions of Cdc42 have been obtained from in vitro compared to in vivo studies. By conditional knockout of N-WASP in mouse oocytes, we analyzed its in vivo functions by employing a variety of different methods including oocyte culture, immunofluorescent staining and live oocyte imaging. Each experiment was repeated at least three times, and data were analyzed by paired-samples t-test. Oocyte-specific deletion of N-WASP did not affect the process of oocyte maturation including spindle formation, spindle migration, polarity establishment and maintenance, and homologous chromosome or sister chromatid segregation, but caused failure of cytokinesis completion during second meiosis (P meiosis completion and failures in this process that affect oocyte quality. None. This work was supported by the National Basic Research Program of China (No. 2012CB944404) and the National Natural Science Foundation of China (Nos 30930065, 31371451, 31272260 and 31530049). There are no potential conflicts of interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:

  11. Cystolithiasis in a Syrian hamster: a different outcome | Petrini ...

    African Journals Online (AJOL)

    A 14-month-old intact male Syrian hamster was admitted for lethargy and hematuria. A total body radiographic image and abdominal ultrasonography showed the presence of a vesical calculus. During cystotomy, a sterile urine sample was obtained and sent to the diagnostic laboratory along with the urolith for analysis.

  12. Characterisation of monoclonal antibodies specific for hamster leukocyte differentiation molecules. (United States)

    Rees, Jennifer; Haig, David; Mack, Victoria; Davis, William C


    Flow cytometry was used to identify mAbs that recognize conserved epitopes on hamster leukocyte differentiation molecules (hLDM) and also to characterize mAbs developed against hLDM. Initial screening of mAbs developed against LDMs in other species yielded mAbs specific for the major histocompatibility (MHC) II molecule, CD4 and CD18. Screening of sets of mAbs developed against hLDM yielded 22 new mAbs, including additional mAbs to MHC II molecules and mAbs that recognize LDMs expressed on all leukocytes, granulocytes, all lymphocytes, all T cells, a subset of T cells, or on all B cells. Based on comparison of the pattern of expression of LDMs expressed on all hamster leukocytes with the patterns of expression of known LDMs in other species, as detected by flow cytometry (FC), four mAbs are predicted to recognize CD11a, CD44, and CD45. Cross comparison of mAbs specific for a subset of hamster T cells with a cross reactive mAb known to recognize CD4 in mice and one recognising CD8 revealed they recognize CD4. The characterization of these mAbs expands opportunities to use hamsters as an additional model species to investigate the mechanisms of immunopathogenesis of infectious diseases. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Humanizing recombinant glycoproteins from Chinese hamster ovary cells

    DEFF Research Database (Denmark)

    Hansen, Anders Holmgaard; Amann, Thomas; Kol, Stefan

    hamster ovary (CHO) cells are making a very heterogeneous mixture of NGlycans. We speculate that the CHO pattern of N-Glycans would affect half-life and/or efficacy of the glycoprotein in the bloodstream making it unsuitable for human intravenous use, whereas our humanized version would be identical...

  14. Biodynamics of cholesterol and bile acids in the lithiasic hamster. (United States)

    Khallou, J; Riottot, M; Parquet, M; Verneau, C; Lutton, C


    By using the isotopic equilibrium method in the young male Syrian hamster, the rates of cholesterol turnover processes, i.e. dietary cholesterol absorption, cholesterol synthesis, cholesterol excretion in the faeces and urine and cholesterol transformation into bile acids, were determined in the hamster receiving a control (C) or a lithogenic diet (L) for 7 weeks. At the end of this period the gall bladder of all animals in group L contained cholesterol gallstones. The coefficient of dietary cholesterol absorption was reduced by 26%, cholesterol synthesis and cholesterol faecal excretion were twofold higher in group L than in group C. Bile acid content in the small intestine was diminished in group L, but bile acid composition was similar in the two groups. The increase in cholesterogenesis in lithiasic animals essentially took place in the liver. Bile acid biosynthesis did not significantly differ in the two groups, but represented only 35% of total cholesterol input (dietary absorption + internal secretion) in group L v. 52% in group C. Thus, in the lithiasic hamster, hepatic synthesis of cholesterol and bile acids are not coupled. The molar percentage of cholesterol in bile was twofold higher in group L than in group C but those of bile acids and of phospholipids were not modified. In the lithiasic hamster the specific activity of biliary cholesterol was similar to that in plasma and liver. Consequently, biliary cholesterol does not derive directly from cholesterol newly synthesized in the liver but from hepatic cholesterol rapidly exchangeable with plasma cholesterol.

  15. Enhanced longevity in tau mutant Syrian hamsters, Mesocricetus auratus

    NARCIS (Netherlands)

    Oklejewicz, Malgorzata; Daan, Serge

    The single-gene mutation tau in the Syrian hamster shortens the circadian period by about 20% in the homozygous mutant and simultaneously increases the mass-specific metabolic rate by about 20%. Both effects might be expected to lead to a change in longevity. To test such expectations, the life span

  16. Het effect van hamsterbeheer op de overwintering bij hamsters

    NARCIS (Netherlands)

    Beek, van der M.; Ligtenberg, H.; Haye, la M.J.J.


    De afgelopen decennia is het aantal hamsters (Cricetus cricetus) in Nederland sterk afgenomen. Door onderzoek te verrichten naar de levenscyclus en naar de effecten van agrarisch beheer is getracht de afname te verklaren en oplossingen aan te dragen. In voorjaar 2006 is een verkennend onderzoek

  17. Early Postnatal Development of the South African Hamster ...

    African Journals Online (AJOL)

    The South African Hamster Mystromys albicaudatus has been bred in the laboratory of the Medical Ecology Centre since 1941. It is of interest taxonomically in that it is the sole representative left in Africa of the subfamily Cricetinae (Davis 1962). It has been used in Medical Research on poliomyelitis, benign histoplasmosis, ...

  18. Bioavailability and disposition of solanine in rats and hamsters

    NARCIS (Netherlands)

    Groen K; Pereboom-de Fauw DPKH; Besamusca P; Beekhof PK; Speijers GJA; Derks HJGM


    The toxicokinetics of [3H]-alpha-solanine after oral (po) and intravenous (iv) administration in rats and hamsters were studied, in order to decide which is the most appropriate model in risk assessment studies. The iv dose was 54 mug/kg; the oral dose was 170 mug/kg. After iv administration, the

  19. Rudimentary coronary artery in Syrian hamsters (Mesocricetus auratus). (United States)

    Durán, A C; Arqué, J M; Fernández, B; Fernández, M C; Fernández-Gallego, T; Rodríguez, C; Sans-Coma, V


    Congenital underdevelopment of one or more main branches of the coronary arteries has been reported in man, but not in non-human mammals. In man, this defective coronary artery arrangement may cause myocardial ischaemia and even sudden death. The main goal of this study was to describe the coronary artery distribution patterns associated with the presence of a markedly underdeveloped (rudimentary) coronary artery in Syrian hamsters. Moreover, an attempt was made to explain the morphogenesis of these patterns, according to current knowledge on coronary artery development. Eleven affected hamsters belonging to a laboratory inbred family were examined by means of internal casts of the heart, great arterial trunks and coronary arteries. The aortic valve was tricuspid (normal) in seven hamsters and bicuspid in the other four. A rudimentary coronary artery arose from the right side of the aortic valve in four specimens, from the left side of the aortic valve in a further three, and from the dorsal aortic sinus in the remaining four. In all cases, a second, well-developed coronary artery provided for all the coronary blood flow. Except for the existence of a rudimentary coronary artery, the present anomalous coronary artery distribution patterns are similar to coronary artery patterns reported in Syrian hamsters, dogs and humans in association with a solitary coronary ostium in aorta. We suggest that an unusual prolonged time interval in the development of the embryonic coronary stems might be a key factor in the formation of coronary arteries displaying significantly dissimilar developmental degrees.

  20. Development of Taenia pisiformis in golden hamster (Mesocricetus auratus

    Directory of Open Access Journals (Sweden)

    Maravilla Pablo


    Full Text Available Abstract The life cycle of Taenia pisiformis includes canines as definitive hosts and rabbits as intermediate hosts. Golden hamster (Mesocricetus auratus is a rodent that has been successfully used as experimental model of Taenia solium taeniosis. In the present study we describe the course of T. pisiformis infection in experimentally infected golden hamsters. Ten females, treated with methyl-prednisolone acetate were infected with three T. pisiformis cysticerci each one excised from one rabbit. Proglottids released in faeces and adults recovered during necropsy showed that all animals were infected. Eggs obtained from the hamsters' tapeworms, were assessed for viability using trypan blue or propidium iodide stains. Afterwards, some rabbits were inoculated with eggs, necropsy was performed after seven weeks and viable cysticerci were obtained. Our results demonstrate that the experimental model of adult Taenia pisiformis in golden hamster can replace the use of canines in order to study this parasite and to provide eggs and adult tapeworms to be used in different types of experiments.

  1. A Simplified Method for Three-Dimensional (3-D) Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice (United States)

    Higuchi, Carolyn M.; Maeda, Yuuki; Horiuchi, Toshitaka; Yamazaki, Yukiko


    In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART). Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture) for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture). We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-), A (Membrane/activin+), M (Matrigel/activin-), and M+A (Matrigel/activin+). We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A). Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM) and in vitro fertilization (IVF). Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A) than with those grown in membrane culture (C, A). In particular, activin A treatment further improved 3-D culture (M+A) success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian culture

  2. A Simplified Method for Three-Dimensional (3-D Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice.

    Directory of Open Access Journals (Sweden)

    Carolyn M Higuchi

    Full Text Available In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART. Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture. We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-, A (Membrane/activin+, M (Matrigel/activin-, and M+A (Matrigel/activin+. We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A. Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM and in vitro fertilization (IVF. Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A than with those grown in membrane culture (C, A. In particular, activin A treatment further improved 3-D culture (M+A success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian

  3. Cell volume and membrane stretch independently control K+ channel activity

    DEFF Research Database (Denmark)

    Bomholtz, Sofia Hammami; Willumsen, Niels J; Olsen, Hervør L


    . To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (approximately 50 microm(2) macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current...... increases with increasing negative hydrostatic pressure (suction) applied to the pipette. Thus, at a pipette pressure of -5.0 +/- 0.1 mmHg the increase amounted to 381 +/- 146% (mean +/- S.E.M., n = 6, P KCNQ1 channel, the current...... was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude...

  4. Oocyte development and fecundity type of the Brazilian Snapper Lutjanus alexandrei Moura & Lindeman, 2007 (Perciformes: Lutjanidae). (United States)

    Fernandes, C A F; Oliveira, P G V; Oliveira, C H B; Hazin, F H V; Travassos, P


    Lutjanid species exhibit multiple spawning behaviour during an extended spawning season in warm months, asynchronous oocyte development and indeterminate fecundity. Although early studies have contributed to knowledge of the reproductive cycle of many species within the group, they have not considered aspects about the number of cortical alveoli oocyte stage throughout maturity phases along spawning season. The latter aspect is also considered very important to confirm indeterminate fecundity hypothesis. In the present study, were analyzed 154 Brazilian snapper Lutjanus alexandrei female gonads obtained from artisanal fisheries in Pernambuco State (Brazil) between October 2010 and March 2011. Were measured oocyte size frequency distribution for maturity phases (developing, spawning capable and actively spawning), and oocyte development stage (unyolked oocytes, cortical alveoli, primary, secondary and tertiary vitellogenic oocytes and hydrated oocytes), and also the oocyte stage frequency during spawning season. The frequency of cortical alveoli oocyte stage was constantly found in the spawning period (>37%), showing slight variation throughout maturity phases. The absence of gap in the oocyte size frequency distribution between primary and secondary oocyte growth stages during spawning season is a strong indicator of continuous oocyte recruitment from reserve stocks. In addition, co-occurrence of tertiary vitellogenic oocytes, hydrated oocytes, post-ovulatory follicles and yellow-brown bodies in the histological sections of ovaries reinforce indeterminate fecundity hypothesis.

  5. Vitrification of mouse MII oocytes: Developmental competency using paclitaxel. (United States)

    Fesahat, Farzaneh; Faramarzi, Azita; Khoradmehr, Arezoo; Omidi, Marjan; Anbari, Fatemeh; Khalili, Mohammad Ali


    Oocyte cryopreservation provides an important alternative for fertility preservation for women who will be treated with cytotoxic drugs. However, it can cause spindle disorganization of microtubules, putting the zygote at risk for aneuploidy. Paclitaxel is known to stabilize the microtubules that constitute the spindle. The aim of this study was to investigate the suitable concentration of paclitaxel for adding to the vitrification media to improve the developmental potential of post-thawed mature oocytes to blastocyst formation in mice. A total of 300 MII oocytes were retrieved from superovulated mice, and were divided into three groups of control, Experimental I, and Experimental II. Oocytes in Experimental I and Experimental II were cryopreserved in the presence of 0.5μM or 1μM of paclitaxel in vitrification media, respectively. After thawing, all oocytes were incubated in G-IVF medium for 1 hour. From each group,12 oocytes were selected for viability evaluation by Hoechst/propidium iodide nuclear staining. Standard in vitro fertilization was performed on the rest of the oocytes and embryo development was followed to the blastocyst stage. Fertilization rate was not significantly different between the three groups. However, the cleavage rate (55%) in Experimental II group was significantly lower compared to Experimental I (88%) and control groups (83%). There was a detectable difference between the three groups at the blastocyst rate (Experimental I and control groups, p = 0.004; Experimental II vs. control and Experimental I, p < 0.001). The highest rates of parthenogenesis and arrest were in Experimental II (16% and 21%, respectively) compared with control (6% and 5%, respectively) and Experimental I (5% and 3%, respectively). There was also a significant decrease in viability rate of oocytes in Experimental II compared to the other groups. A high concentration of paclitaxel, an anticancer drug, interrupted the mouse oocyte competency when supplemented to

  6. Seasonal aspects of sleep in the Djungarian hamster

    Directory of Open Access Journals (Sweden)

    Deboer Tom


    Full Text Available Abstract Background Changes in photoperiod and ambient temperature trigger seasonal adaptations in the physiology and behaviour of many species, including the Djungarian hamster. Exposure of the hamsters to a short photoperiod and low ambient temperature leads to a reduction of the polyphasic distribution of sleep and waking over the light and dark period. In contrast, a long photoperiod enhances the daily sleep-wake amplitude leading to a decline of slow-wave activity in NREM sleep within the light period. It is unknown whether these changes can be attributed specifically to photoperiod and/or ambient temperature, or whether endogenous components are contributing factors. The influence of endogenous factors was investigated by recording sleep in Djungarian hamsters invariably maintained at a low ambient temperature and fully adapted to a short photoperiod. The second recording was performed when they had returned to summer physiology, despite the maintenance of the 'winter' conditions. Results Clear winter-summer differences were seen in sleep distribution, while total sleep time was unchanged. A significantly higher light-dark cycle modulation in NREM sleep, REM sleep and waking was observed in hamsters in the summer physiological state compared to those in the winter state. Moreover, only in summer, REM sleep episodes were longer and waking bouts were shorter during the light period compared to the dark period. EEG power in the slow-wave range (0.75–4.0 Hz in both NREM sleep and REM sleep was higher in animals in the summer physiological state than in those in the 'winter' state. In winter SWA in NREM sleep was evenly distributed over the 24 h, while in summer it decreased during the light period and increased during the dark period. Conclusion Endogenous changes in the organism underlie the differences in sleep-wake redistribution we have observed previously in hamsters recorded in a short and long photoperiod.

  7. Seasonal aspects of sleep in the Djungarian hamster. (United States)

    Palchykova, Svitlana; Deboer, Tom; Tobler, Irene


    Changes in photoperiod and ambient temperature trigger seasonal adaptations in the physiology and behaviour of many species, including the Djungarian hamster. Exposure of the hamsters to a short photoperiod and low ambient temperature leads to a reduction of the polyphasic distribution of sleep and waking over the light and dark period. In contrast, a long photoperiod enhances the daily sleep-wake amplitude leading to a decline of slow-wave activity in NREM sleep within the light period. It is unknown whether these changes can be attributed specifically to photoperiod and/or ambient temperature, or whether endogenous components are contributing factors. The influence of endogenous factors was investigated by recording sleep in Djungarian hamsters invariably maintained at a low ambient temperature and fully adapted to a short photoperiod. The second recording was performed when they had returned to summer physiology, despite the maintenance of the 'winter' conditions. Clear winter-summer differences were seen in sleep distribution, while total sleep time was unchanged. A significantly higher light-dark cycle modulation in NREM sleep, REM sleep and waking was observed in hamsters in the summer physiological state compared to those in the winter state. Moreover, only in summer, REM sleep episodes were longer and waking bouts were shorter during the light period compared to the dark period. EEG power in the slow-wave range (0.75-4.0 Hz) in both NREM sleep and REM sleep was higher in animals in the summer physiological state than in those in the 'winter' state. In winter SWA in NREM sleep was evenly distributed over the 24 h, while in summer it decreased during the light period and increased during the dark period. Endogenous changes in the organism underlie the differences in sleep-wake redistribution we have observed previously in hamsters recorded in a short and long photoperiod.

  8. Distinct mechanisms eliminate mother and daughter centrioles in meiosis of starfish oocytes. (United States)

    Borrego-Pinto, Joana; Somogyi, Kálmán; Karreman, Matthia A; König, Julia; Müller-Reichert, Thomas; Bettencourt-Dias, Mónica; Gönczy, Pierre; Schwab, Yannick; Lénárt, Péter


    Centriole elimination is an essential process that occurs in female meiosis of metazoa to reset centriole number in the zygote at fertilization. How centrioles are eliminated remains poorly understood. Here we visualize the entire elimination process live in starfish oocytes. Using specific fluorescent markers, we demonstrate that the two older, mother centrioles are selectively removed from the oocyte by extrusion into polar bodies. We show that this requires specific positioning of the second meiotic spindle, achieved by dynein-driven transport, and anchorage of the mother centriole to the plasma membrane via mother-specific appendages. In contrast, the single daughter centriole remaining in the egg is eliminated before the first embryonic cleavage. We demonstrate that these distinct elimination mechanisms are necessary because if mother centrioles are artificially retained, they cannot be inactivated, resulting in multipolar zygotic spindles. Thus, our findings reveal a dual mechanism to eliminate centrioles: mothers are physically removed, whereas daughters are eliminated in the cytoplasm, preparing the egg for fertilization. © 2016 Borrego-Pinto et al.

  9. mRNA from NCB-20 cells encodes the N-methyl-D-aspartate/phencyclidine receptor: a Xenopus oocyte expression study. (United States)

    Lerma, J; Kushner, L; Spray, D C; Bennett, M V; Zukin, R S


    The mouse neuroblastoma--Chinese hamster brain hybrid cell line NCB-20 is the only clonal cell line in which binding studies indicate the presence of phencyclidine (PCP) receptors. We report here that Xenopus oocytes injected with NCB-20 cell poly(A)+ RNA express N-methyl-D-aspartate (NMDA)-activated channels and that these channels include the PCP receptor site. In injected oocytes, NMDA application evoked a partially desensitizing inward current that was potentiated by glycine, blocked by the competitive antagonist D-2-amino-5-phosphonovaleric acid, blocked by Mg2+ and by Zn2+, and blocked in a use-dependent manner by the PCP receptor ligands PCP and MK-801. There was little or no response to kainate or quisqualate (agonists of the other excitatory amino acid receptors), to gamma-aminobutyric acid (an inhibitory transmitter), or to glycine (an inhibitory transmitter as well as an allosteric potentiator of NMDA channels). Thus, NMDA/PCP receptors expressed from NCB-20 cell mRNA exhibit properties similar to those of the neuronal receptors. The absence of expression of other excitatory amino acid receptors in this system makes it particularly useful for study of NMDA-evoked responses without interference from responses mediated by other receptors. Moreover, NCB-20 mRNA may be an appropriate starting material for cloning the cDNA(s) encoding the NMDA/PCP-receptor complex. PMID:2537982

  10. Membrane dynamics

    DEFF Research Database (Denmark)

    Bendix, Pól Martin


    Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...... for the lateral organization of membranes as wells as for physical properties like bending, permeability and elasticity...

  11. Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation, anti-polyspermy defense, and assisted reproduction. (United States)

    Sutovsky, Peter; Manandhar, Gaurishankar; Wu, Alex; Oko, Richard


    Perinuclear theca (PT) is the cytoskeletal coat of mammalian sperm nucleus that is removed from the sperm head at fertilization. PT harbors the sperm borne, oocyte-activating factor (SOAF), a yet-to-be-characterized substance responsible for triggering the signaling cascade of oocyte activation, thought to be dependent on intra-oocyte calcium release. The present article reviews the current knowledge on the biogenesis and molecular composition of sperm PT. Possible functions of sperm PT during natural and assisted fertilization, and in the initiation of embryonic development are discussed. Furthermore, evidence is provided that SOAF is transferred from the sperm PT to oocyte cytoplasm through the internalization and rapid solubilization of the post-acrosomal PT. It is shown that during natural fertilization the sperm PT dissolves in the oocyte cytoplasm concomitantly with sperm nuclear decondensation and the initiation of pronuclear development. SOAF activity is preserved in the differentially extracted sperm heads only if the integrity of PT is maintained. After intracytoplasmic sperm injection (ICSI), activation occurs only in those oocytes in which the injected spermatozoon displays complete or partial dissolution of PT. In the latter case, the residual PT of the sub-acrosomal and/or post-acrosomal sperm region may persist on the apical surface of the sperm nucleus/male pronucleus and may cause a delay or arrest of zygotic development. We propose that the sperm PT harbors SOAF in the post-acrosomal sheath, as this is the first part of the sperm cytosol to enter the oocyte cytoplasm and its disassembly appears sufficient to initiate the early events of oocyte activation. Dissolution of the sub-acrosomal part of the PT, on the other hand, appears necessary to insure complete DNA decondensation in the internalized sperm nucleus and initiate DNA synthesis of both pronuclei. The release of the SOAF from the sperm head into oocyte cytoplasm at fertilization ultimately

  12. Successful pregnancy and delivery after ICSI with artificial oocyte activation by calcium ionophore in in-vitro matured oocytes: a case report. (United States)

    Kim, Jun-Woo; Yang, Seong-Ho; Yoon, San-Hyun; Kim, Sang-Don; Jung, Jae-Hoon; Lim, Jin-Ho


    The achievement of a successful pregnancy and delivery after oocyte activation with calcium ionophore is reported in a couple having low fertilization rates after intracytoplasmic sperm injection (ICSI) of in-vitro matured oocytes. A couple, in which the wife had polycystic ovary syndrome and the husband had moderate oligoteratozoospermia, showed a low fertilization rate in a previous in-vitro maturation cycle (2/11 [18.2%]). The most likely cause of complete fertilization failure or low fertilization rates is failure of oocyte activation. Therefore, artificial oocyte activation by calcium ionophore was combined with ICSI to achieve viable fertilized oocytes. Oocytes were stimulated with calcium ionophore for 30 min after ICSI. The fertilization rate of oocytes activated with calcium ionophore (13/15 [86.7%] and 7/9 [77.8%]) was higher than that of the non-activated oocytes. In the latest cycle, three embryos derived from the activated oocytes were transferred into the uterus on day 3. Subsequently, two gestational sacs were identified on ultrasound. The patient delivered dizygotic twins (girl 2260 g and boy 2760 g) at 35 weeks and 6 days gestation by caesarean section. This result suggests that calcium ionophore could be useful for oocyte fertilization in couples with low fertilization rates after ICSI of in-vitro matured oocytes. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  13. WNT4-like protein is a cortical granule component in mouse oocytes and functions in regulating preimplantation embryogenesis. (United States)

    Liu, Min; Yang, Huei-Ting


    Mammalian cortical granules (CG) are membrane-bound organelles located in the cortex of the unfertilized oocytes. Upon fertilization, CG undergo exocytosis to function in blocking polyspermy. While cortical granules are important in fertilization, their exact biochemical composition and reproductive function have not been fully defined. In the present study, a 66 kDa wingless-type MMTV integration site family, member 4 (WNT4)-like protein, with mouse CG origin was identified. Oocytes that were double labeled with lectin Lens culinaris agglutinin (LCA) and WNT4 antibody showed colocalization of the WNT4 molecules and cortical granules. The disappearance of WNT4 molecules in the artificially activated oocytes that were devoid of cortical granules confirmed their granule origin. Following fertilization, WNT4 remained associated with zygotes and blastomeres of 2-cell and 8-cell embryos; however the amount of protein present was reduced more than 2-fold as embryos developed. Prior to implantation, WNT4 appeared to be detectable only in the trophoblast cells. Our functional study revealed that WNT4 molecules were involved in regulating zygotic cleavage and early embryogenesis. To our knowledge, this is the first study demonstrating mammalian cortical granules contain signaling molecules that are involved in the regulation of the first phase of embryonic development.

  14. Ovarian ageing: the role of mitochondria in oocytes and follicles. (United States)

    May-Panloup, Pascale; Boucret, Lisa; Chao de la Barca, Juan-Manuel; Desquiret-Dumas, Valérie; Ferré-L'Hotellier, Véronique; Morinière, Catherine; Descamps, Philippe; Procaccio, Vincent; Reynier, Pascal


    There is a great inter-individual variability of ovarian ageing, and almost 20% of patients consulting for infertility show signs of premature ovarian ageing. This feature, taken together with delayed childbearing in modern society, leads to the emergence of age-related ovarian dysfunction concomitantly with the desire for pregnancy. Assisted reproductive technology is frequently inefficacious in cases of ovarian ageing, thus raising the economic, medical and societal costs of the procedures. Ovarian ageing is characterized by quantitative and qualitative alteration of the ovarian oocyte reserve. Mitochondria play a central role in follicular atresia and could be the main target of the ooplasmic factors determining oocyte quality adversely affected by ageing. Indeed, the oocyte is the richest cell of the body in mitochondria and depends largely on these organelles to acquire competence for fertilization and early embryonic development. Moreover, the oocyte ensures the uniparental transmission and stability of the mitochondrial genome across the generations. This review focuses on the role played by mitochondria in ovarian ageing and on the possible consequences over the generations. PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning mitochondria and ovarian ageing, in animal and human species. Searches were performed using keywords belonging to three groups: 'mitochondria' or 'mitochondrial DNA'; 'ovarian reserve', 'oocyte', 'ovary' or 'cumulus cells'; and 'ageing' or 'ovarian ageing'. These keywords were combined with other search phrases relevant to the topic. References from these articles were used to obtain additional articles. There is a close relationship, in mammalian models and humans, between mitochondria and the decline of oocyte quality with ageing. Qualitatively, ageing-related mitochondrial (mt) DNA instability, which leads to the accumulation of mtDNA mutations in the oocyte, plays a key role in

  15. Mural granulosa cell gene expression associated with oocyte developmental competence

    Directory of Open Access Journals (Sweden)

    Jiang Jin-Yi


    Full Text Available Abstract Background Ovarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an in vivo immature rat model, gene expression profile in granulosa cells, which may be linked to the developmental competence of the oocyte. We hypothesized that expression of specific genes in granulosa cells may be correlated with the developmental competence of the oocyte. Methods Immature rats were injected with eCG and 24 h thereafter with anti-eCG antibody to induce follicular atresia or with pre-immune serum to stimulate follicle development. A high percentage (30-50%, normal developmental competence, NDC of oocytes from eCG/pre-immune serum group developed to term after embryo transfer compared to those from eCG/anti-eCG (0%, poor developmental competence, PDC. Gene expression profiles of mural granulosa cells from the above oocyte-collected follicles were assessed by Affymetrix rat whole genome array. Results The result showed that twelve genes were up-regulated, while one gene was down-regulated more than 1.5 folds in the NDC group compared with those in the PDC group. Gene ontology classification showed that the up-regulated genes included lysyl oxidase (Lox and nerve growth factor receptor associated protein 1 (Ngfrap1, which are important in the regulation of protein-lysine 6-oxidase activity, and in apoptosis induction, respectively. The down-regulated genes included glycoprotein-4-beta galactosyltransferase 2 (Ggbt2, which is involved in the regulation of extracellular matrix organization and biogenesis. Conclusions The data in the present study demonstrate a close association between specific gene expression in mural granulosa cells and

  16. Effect of kisspeptin on in vitro maturation of sheep oocytes

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    Priyanka Byri


    Full Text Available Aim: The aim of this study was to investigate the effect of kisspeptin (KP on in vitro maturation (IVM of sheep oocytes aspirated from the ovaries collected from slaughterhouse. Materials and Methods: Two different experiments were conducted to investigate the effect of KP (5, 10 and 15 μg/ml alone (experiment 1 or in combination with follicle-stimulating hormone (FSH, luteinizing hormone (LH, and Estradiol (E2 (experiment 2 on IVM of sheep oocytes. Tissue culture medium 199 supplemented with Gentamicin was used as control medium. Good quality oocytes were randomly allocated into different IVM media and cultured at 38.5°C in 5% CO2 under humidified atmosphere for 24 h. The oocytes were evaluated for their cumulus cell expansion (CCE and extrusion of the 1st polar body (PB at the end of maturation. Results: The proportion of oocytes showing CCE and extrusion of PB was highest when the oocytes were matured in the medium supplemented with 10 μg/ml of KP. In experiment 2, oocytes were matured in 12 different maturation media (G1-G12: G1: Control, G2: KP alone, G3: FSH, G4: FSH+KP, G5: LH, G6: LH+KP, G7: E2, G8: E2+KP, G9: FSH+LH+E2, G10: FSH+LH+E2+KP, G11: FSH+LH+E2+fetal bovine serum (FBS, G12: FSH+LH+E2+FBS+KP. The proportion of oocytes showing cumulus expansion and PB extrusion was highest (98.33±1.05 and 89.17±2.38 when they were matured in FSH+LH+E2+FBS+KP (G12 and was significantly higher than other groups. The proportion of CCE and extrusion of PB was significantly increased when KP was supplemented to FSH and E2, but no effect was observed with LH. The maturation rates were significantly increased when FSH, LH, and E2 (G9 containing media were additionally supplemented with KP (G10. Conclusion: This study demonstrated that the addition of KP (10 μg/ml to the FSH, LH, and E2 supplemented media would enhance the sheep oocyte maturation in vitro.

  17. Time-Lapse Dynamics of the Mouse Oocyte Chromatin Organisation during Meiotic Resumption

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    Martina Belli


    Full Text Available In the mammalian oocyte, distinct patterns of centromeres and pericentromeric heterochromatin localisation correlate with the gamete’s developmental competence. Mouse antral oocytes display two main types of chromatin organisation: SN oocytes, with a ring of Hoechst-positive chromatin surrounding the nucleolus, and NSN oocytes lacking this ring. When matured to MII and fertilised, only SN oocytes develop beyond the 2-cell, and reach full term. To give detailed information on the dynamics of the SN or NSN chromatin during meiosis resumption, we performed a 9 hr time-lapse observation. The main significant differences recorded are: (1 reduction of the nuclear area only in SN oocytes; (2 ~17 min delay of GVBD in NSN oocytes; (3 chromatin condensation, after GVBD, in SN oocytes; (4 formation of 4-5 CHCs in SN oocytes; (5 increase of the perivitelline space, ~57 min later in NSN oocytes; (6 formation of a rosette-like disposition of CHCs, ~84 min later in SN oocytes; (7 appearance of the MI plate ~40 min later in NSN oocytes. Overall, we described a pathway of transition from the GV to the MII stage that is punctuated of discrete recordable events showing their specificity and occurring with different time kinetics in the two types of oocytes.

  18. PTK2b function during fertilization of the mouse oocyte. (United States)

    Luo, Jinping; McGinnis, Lynda K; Carlton, Carol; Beggs, Hilary E; Kinsey, William H


    Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development. Published by Elsevier Inc.

  19. Highly efficient vitrification method for cryopreservation of human oocytes. (United States)

    Kuwayama, Masashige; Vajta, Gábor; Kato, Osamu; Leibo, Stanley P


    Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.

  20. Kif4 Is Essential for Mouse Oocyte Meiosis. (United States)

    Camlin, Nicole J; McLaughlin, Eileen A; Holt, Janet E


    Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age.

  1. The Effects of Progesterone on Oocyte Maturation and Embryo Development

    Directory of Open Access Journals (Sweden)

    Saeed Zavareh


    Full Text Available Oocyte maturation and embryo development are controlled by intra-ovarian factors suchas steroid hormones. Progesterone (P4 exists in the follicular fluid that contributes tonormal mammalian ovarian function and has several critical functions during embryodevelopment and implantation, including endometrial receptivity, embryonic survivalduring gestation and transformation of the endometrial stromal cells to decidual cells.It is well known that the physiological effects of P4 during the pre-implantation stages ofsome mammal’s embryos are mediated by P4 receptors and their gene expression is determined.The effects of P4 on oocytes and embryo development have been assessed bysome investigations, with contradictory results. P4, a dominant steroid in follicular fluidat approximately 18 hours after the luteinizing hormone (LH surge may have a criticalrole in maturation of oocytes at the germinal stage. However, it has been shown that differentconcentrations of P4 could not improve in vitro maturation rates of germinal vesicles(GV in cumulus oocyte complexes (COCs and cumulus denuded oocytes (CDOs.Culture media supplemented with P4 significantly improved mouse embryo development.In addition, an in vivo experimental design has shown high blastocyst survival andimplantation rates in P4-treated mice.In this review we explain some of the findings that pertain to the effects of P4 onoocyte maturation and embryo development both in vitro and in vivo.

  2. Coexpression of MT1 and RORalpha1 melatonin receptors in the Syrian hamster Harderian gland. (United States)

    Tomás-Zapico, Cristina; Antonio Boga, José; Caballero, Beatriz; Vega-Naredo, Ignacio; Sierra, Verónica; Alvarez-García, Oscar; Tolivia, Delio; Josefa Rodríguez-Colunga, María; Coto-Montes, Ana


    Melatonin acts through several specific receptors, including membrane receptors (MT(1) and MT(2)) and members of the RZR/ROR nuclear receptors family, which have been identified in a large variety of mammalian and nonmammalian cells types. Both membrane and nuclear melatonin receptors have been partially characterized in Harderian gland of the Syrian hamster. Nevertheless, the identities of these receptors were unknown until this study, where the coexistence of MT(1) and RORalpha(1) in this gland was determined by nested RT-PCR followed by amplicon sequencing and Western-blot. Furthermore, the cellular localization of both receptors was determined by immunohistochemistry. Thus, MT(1) receptor was localized exclusively at the basal side of the cell acini, supporting the hypothesis that this receptor is activated by the pineal-synthesized melatonin. On the contrary, although a RORalpha(1)-immunoreactivity was observed in nuclei of epithelial cells of both sexes, an extranuclear specific staining, which was more frequently among those cells of males, was also seen. The implication of this possible nuclear exclusion of RORalpha(1) on the role of this indoleamine against oxidative stress is discussed.

  3. Sensibility of the hamster (Cricetus auratus to the Treponema pertenue

    Directory of Open Access Journals (Sweden)

    F. Nery-Guimarães


    Full Text Available In two experiments, 8 Hamsters inoculated with material from yaws lesions (Treponema pertenue, developed skin lesions considered specific by their clinical and histopathological aspects and by the presence of treponemae. These lesions appeared on the scrotumm, testicle, prepuce, anus, tail, muzzle, back and hinders paws (palm surface. In the internal organs no treponemae were found in direct examinations and inoculation of brain, spleen and lymph node. The incubation period was of 35 days for the testicle, 55 days for the scrotum and 107 days for peritoneal cavity inoculation. Positive sub-inoculations were obtained. The serum reactions (Qasserman's and Kahn's were negative in all 5 tested Hamsters. Out of 4 normal females matched to infected males two developed nasal lesions resulting from direct contact. Apparently the genital lesions hindered copulation. Hamsters are very well suited for an experimental study of yaws.Em 2 experiências, 8 Hamsters inoculados com material direto de lesões boubáticas (Treponema pertenue, desenvolveram lesões cutâneas consideradas específicas, pelo aspecto clínico e histopatológico e pela presentça de treponemas. Essas lesões se manifestaram no escrôto, testículo, prepúcio, anus, cauda, focinho, dorso e patas posteriores (face palmar. Nos órgãos internos não foram vistos treponemas ao exame direto e, uma vez, por inoculação de cérebro, baço e gânglio linfático. O período incubativo foi de 35 dias pela via testicular, 55 dias pela via escrotal e 107 dias pela via peritonial. Foram obtidas sub-inoculações positivas para Hamsters normais. As experiências continuam. De 4 fêmeas normais, acasaladas com 4 hamsters infectados apenas 2 mostraram lesões positivas resultantes de contágio direto. Aparentemente, não houve copulação e, se esta ocorreu, não determinou fecundação.

  4. Reception and transduction of the serotonin signal responsible for meiosis reinitiation in oocytes of the Japanese clam Ruditapes philippinarum. (United States)

    Gobet, I; Durocher, Y; Leclerc, C; Moreau, M; Guerrier, P


    Prophase-arrested oocytes of Ruditapes philippinarum are triggered to undergo germinal vesicle breakdown under the influence of the neurohormone serotonin (5HT) and then arrest in metaphase 1. Our data show that these oocytes possess a single class of original 5HT receptors. Their binding parameters have been determined on semipurified membrane preparations incubated with [3H]5HT. No significant differences were observed when comparing 5HT-competent and -incompetent batches as well as prophase- or metaphase-arrested oocytes. Specific experiments including incubation with mastoparan or mas 7, GTP iontophoresis, and IP3 quantification strongly suggest that these receptors must be coupled with G-proteins to be functional. Peak change in IP3 mass occurs at 3 min and is likely to trigger the 5HT-dependent Ca2+ transient that begins at this time. In metaphase-arrested oocytes, binding of 5HT to its receptors no longer produces a Ca2+ surger. This is likely to result from a negative retrocontrol loop which would involve kinase C and exert its effect upstream of the Ca2+ surge. Indeed, the phorbol ester PMA proved able to reduce the Ca2+ response and to block 5HT action when applied during the first 3 min corresponding to the hormone-dependent period. Such an inhibition was reversed in the presence of 5 microM of the C kinase inhibitor GF109203X and could be bypassed by ionophore, ammonia, and thapsigargin, which trigger a receptor-independent Ca2+ surge.

  5. Heat and cold acclimation in helium-cold hypothermia in the hamster. (United States)

    Musacchia, X. J.


    A study was made of the effects of acclimation of hamsters to high (34-35 C) and low (4-5 C) temperatures for periods up to 6 weeks on the induction of hypothermia in hamsters. Hypothermia was achieved by exposing hamsters to a helox mixture of 80% helium and 20% oxygen at 0 C. Hypothermic induction was most rapid (2-3 hr) in heat-acclimated hamsters and slowest (6-12 hr) in cold-acclimated hamsters. The induction period was intermediate (5-8 hr) in room temperature nonacclimated animals (controls). Survival time in hypothermia was relatable to previous temperature acclimations. The hypothesis that thermogenesis in cold-acclimated hamsters would accentuate resistance to induction of hypothermia was substantiated.

  6. Hamsters' (Mesocricetus auratus) memory in a radial maze analog: the role of spatial versus olfactory cues. (United States)

    Tonneau, François; Cabrera, Felipe; Corujo, Alejandro


    The golden hamster's (Mesocricetus auratus) performance on radial maze tasks has not been studied a lot. Here we report the results of a spatial memory task that involved eight food stations equidistant from the center of a circular platform. Each of six male hamsters depleted the food stations along successive choices. After each choice and a 5-s retention delay, the hamster was brought back to the center of the platform for the next choice opportunity. When only one baited station was left, the platform was rotated to evaluate whether olfactory traces guided hamsters' choices. Results showed that despite the retention delay hamsters performed above chance in searching for food. The choice distributions observed during the rotation probes were consistent with spatial memory and could be explained without assuming guidance by olfactory cues. The radial maze analog we devised could be useful in furthering the study of spatial memory in hamsters.

  7. Obstetric and neonatal outcome after oocyte donation in 106 women with Turner syndrome

    DEFF Research Database (Denmark)

    Hagman, Anna; Loft, Anne; Wennerholm, Ulla-Britt


    What are the obstetric and neonatal outcomes of deliveries after oocyte donation (OD) in women with Turner syndrome (TS)?......What are the obstetric and neonatal outcomes of deliveries after oocyte donation (OD) in women with Turner syndrome (TS)?...

  8. Human oocyte vitrification: the permeability of metaphase II oocytes to water and ethylene glycol and the appliance toward vitrification. (United States)

    Mullen, Steven F; Li, Mei; Li, Yuan; Chen, Zi-Jiang; Critser, John K


    To determine the permeability of human metaphase II oocytes to ethylene glycol and water in the presence of ethylene glycol, and to use this information to develop a method to vitrify human oocytes. An incomplete randomized block design. A university-affiliated assisted reproductive center. Women undergoing assisted reproduction in the Center for Reproductive Medicine at Shandong University. Oocytes were exposed to 1.0 molar ethylene glycol in a single step and photographed during subsequent volume excursions. A two-parameter model was employed to estimate the permeability to water and ethylene glycol. Water permeability ranged from 0.15 to 1.17 microm/(min.atm), and ethylene glycol permeability ranged from 1.5 to 30 microm/min between 7 degrees C at 36 degrees C. The activation energies for water and ethylene glycol permeability were 14.42 Kcal/mol and 21.20 Kcal/mol, respectively. Despite the lower permeability of human metaphase II oocytes to ethylene glycol compared with previously published values for propylene glycol and dimethylsulfoxide, methods to add and remove human oocytes with a vitrifiable concentration of ethylene glycol can be designed that prevent excessive osmotic stress and minimize exposure to high concentrations of this compound.

  9. Cryopreservation of embryos and oocytes in human assisted reproduction. (United States)

    Konc, János; Kanyó, Katalin; Kriston, Rita; Somoskői, Bence; Cseh, Sándor


    Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification) of human embryos and oocytes are summarized.

  10. Cryopreservation of Mammalian Oocyte for Conservation of Animal Genetics

    Directory of Open Access Journals (Sweden)

    Jennifer R. Prentice


    Full Text Available The preservation of the female portion of livestock genetics has become an international priority; however, in situ conservation strategies are extremely expensive. Therefore, efforts are increasingly focusing on the development of a reliable cryopreservation method for oocytes, in order to establish ova banks. Slow freezing, a common method for cryopreservation of oocytes, causes osmotic shock (solution effect and intracellular ice crystallization leading to cell damage. Vitrification is an alternative method for cryopreservation in which cells are exposed to a higher concentration of cryoprotectants and frozen with an ultra rapid freezing velocity, resulting in an ice crystal free, solid glass-like structure. Presently, vitrification is a popular method for cryopreservation of embryos. However, vitrification of oocytes is still challenging due to their complex structure and sensitivity to chilling.

  11. Developmental history of the mammalian oocyte: insight from mouse mutations. (United States)

    Rawls, A; McGaughey, R W; Wilson-Rawls, J


    Growth and differentiation of the mammalian oocyte is regulated with the coordinate development of the granulosa cells. The complex signaling pathways that regulate the growth and development of mammalian oocytes are beginning to be elucidated through the use of gene targeting. These technologies have provided new insight into the roles of specific genes during the development of the germ cells and gonads, as well as post-pubertal development of oocytes. In many cases, these studies have resulted in a new understanding of the function of certain genes, in others they have provided new genes and pathways to be studied in mammalian reproductive biology. Ultimately, these studies will shed light on human genetic disease and infertility.

  12. A non-exchangeable fluorescent phospholipid analog as a membrane traffic marker of the endocytic pathway

    NARCIS (Netherlands)

    Willem, J; ter Beest, M; Scherphof, G; Hoekstra, D


    The fluorescent phospholipid analog N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE) was inserted into the plasma membrane of Baby hamster kidney cells at low temperature (2 degrees C). The mobility characteristics of the analog--as revealed by fluorescence photobleaching

  13. Experimental treatment with sodium stibogluconate of hamsters infected with Leishmania (Leishmania) chagasi and Leishmania (Leishmania) amazonensis


    Elizabeth M. de Figueiredo; Silva,Jaime Costa e; Brazil,Reginaldo P


    The present paper reports the experimental treatment of hamsters infected with Leishmania chagasi and Leishmania amazonensis with sodium stibogluconate (20mg/kg/day x 20 days). Only with L. chagasi did the treatment result in the complete elimination of parasites from the spleen. However, no parasitological cure was achieved in hamsters infected with L. amazonensis.O presente trabalho é um relato do tratamento experimental de hamsters infectado com Leishmania chagasi e Leishmania amazonensis ...

  14. Cell-Mediated Cytotoxicity Toward Measles Virus-Infected Target Cells in Randomly Bred Syrian Hamsters


    Cremer, Natalie E.; O'Keefe, Beatrice; Hagens, Shirley J.; Diggs, Janice


    Cell-mediated cytotoxicity (CMC) toward measles virus-infected cells was studied by a 51Cr release assay with spleen cells from hamsters inoculated with measles virus (strain Lec) or control antigen and with spleen cells from normal hamsters. Spleen cells from measles virus-inoculated hamsters showed greater CMC toward infected than toward noninfected target cells (designated specific CMC). Specific CMC was maximal 7 days after virus inoculation and was declining by 9 to 10 days. Effector cel...

  15. Clinical definition paper on in vitro maturation of human oocytes. (United States)

    Dahan, Michael H; Tan, Seang Lin; Chung, Jintae; Son, Weon-Young


    In vitro maturation (IVM) of human oocytes is a reproductive technique which has been practiced for 25 years and is gaining popularity. However, the techniques used for IVM differ substantially across clinics and they result in extremely variable pregnancy rates, partially due to some of these differences in protocols. Such differences include the use in some cycles of hCG triggering prior to oocyte retrieval and the use of a few days of gonadotrophin treatment to support moderate follicle growth. Other important factors are patient selection (including those with polycystic ovaries or decreased ovarian reserve), the number of embryos transferred and cleavage-stage embryo or blastocyst transfer. There are also substantial differences of opinion among clinicians regarding IVM and what it implies. Due to the large variation in protocols, a decision was made to write this paper in an attempt to introduce uniformity when comparing treatments and outcomes of IVM. A clinical definition of IVM was developed: The retrieval of oocytes from small and intermediate sized follicles in an ovary before the largest follicle has surpassed 13 mm in mean diameter. The use of short gonadotrophin stimulation should be acknowledged. However, it should be stated that metaphase II oocytes also have the potential to be collected at that time in the cycles associated with either hCG or GnRH agonist priming. Many feel this is not IVM because some mature oocytes are retrieved, therefore, we recommend renaming this procedure either natural cycle IVF or modified natural cycle IVF (if gonadotrophin stimulation is given) with early triggering, combined with IVM The percentage as well as the absolute number of mature oocytes at retrieval should be indicated. The use of these titles will allow transparency when comparing results of IVM cycles. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For

  16. Leukotriene inhibition in hamster periodontitis. A histochemical and morphometric study

    Directory of Open Access Journals (Sweden)

    B. Baroukh


    Full Text Available The effects of leukotriene (LT inhibition on gingival and adjacent bone compartments were assessed by using phenidone (100 mg/kg/d and ketoconazole (50 mg/kg/d given for 4 weeks to periodontitis-affected hamsters. In the gingiva the two agents significantly decreased PMNL recruitment and migration and increased the vascular lumen. At the bone level, they reduced significantly preosteoclast and osteoclast numbers but did not affect osteoclast activity. Phenidone had no action on periodontitis induced inhibition of bone formation; in contrast ketoconazole enhanced formation. As both phenidone and ketoconazole are unspecific LT inhibitors it cannot be ascertained that the effects observed were actually due to LT inhibition. However, phenidone and ketoconazole induced changes different from indomethacin used in previous studies to inhibit the cyclooxygenase pathway. These discrepancies suggest that LT inhibition occurred in the present study and that they participate in gingival inflammation and osteoclastic destruction during hamster periodontitis.

  17. Spontaneous fibrosarcoma in a Djungarian hamster (phodopus sungorus). (United States)

    Kondo, Hirotaka; Onuma, Mamoru; Ito, Hidetoshi; Shibuya, Hisashi; Sato, Tsuneo


    A 1.5-y-old female Djungarian hamster (Phodopus sungorus) presented with a large subcutaneous mass surrounding the right shoulder. Radiography revealed dislocation of the right humeral articulation and osteolytic lesions of the right scapula. Histologically, the mass was composed of spindle to stellate cells arranged in fascicles interwoven with delicate collagen fibers, and neoplastic cells infiltrated the bone, skeletal muscle, and subcutaneous tissues. Neoplastic cells stained intensely positive for vimentin and negative for S100 protein, neurofilament, and desmin. A minority of neoplastic cells (10% to 20%) stained moderately for smooth muscle actin. The mass was diagnosed as a fibrosarcoma. Although fibrosarcomas are relatively common in dogs and cats, this is the first report of fibrosarcoma in a domestic Djungarian hamster.

  18. Cystolithiasis in a Syrian hamster: a different outcome

    Directory of Open Access Journals (Sweden)

    D. Petrini


    Full Text Available A 14-month-old intact male Syrian hamster was admitted for lethargy and hematuria. A total body radiographic image and abdominal ultrasonography showed the presence of a vesical calculus. During cystotomy, a sterile urine sample was obtained and sent to the diagnostic laboratory along with the urolith for analysis. Urine culture was found negative for bacterial growth, and the urolith was identified as a calcium-oxalate stone. Diet supplementation with palmitoylethanolamide, glucosamine and hesperidin was adopted the day after discharge. One year follow up revealed no presence of vesical calculi. Although this is the report of a single clinical case, this outcome differs from the results reported in the literature characterized by recurrences after few months. Considering the positive outcome and the beneficial properties of palmitoylethanolamide, glucosamine, and hesperidin, these nutritional elements in Syrian hamsters, are recommended to reduce recurrence after surgical treatment of urolithiasis.

  19. Cytotoxic effects of permethrin in oocytes of Rhipicephalus sanguineus (Acari: Ixodidae) fully engorged females: I. Direct or indirect action of the acaricide in germ cells? (United States)

    Roma, Gislaine Cristina; Furquim, Karim Christina Scopinho; Bechara, Gervásio Henrique; Camargo Mathias, Maria Izabel


    Given the wide use of synthetic chemicals to control ticks, this study evaluated the effects of the permethrin pyrethroid on oocytes of Rhipicephalus sanguineus fully engorged females in order to examine whether this compound, in addition to the proven neurotoxic effect, also acts directly on germ cells. The results revealed that permethrin effectively inhibits and/or interrupts the reproductive process of R. sanguineus. Exposed oocytes exhibited prominent structural changes such as altered shape of cells and germ vesicle (oocyte nucleus), cytoplasmic vacuolation, and decrease of yolk granules. The composition of the latter, however, was not altered. These findings confirm those already reported by Roma et al. (Food Chem Toxicol 48:825-830, 2010) demonstrating that permethrin acts on germ cells of R. sanguineus via direct absorption from the hemolymph by pedicel cells, or by the oocyte plasmic membrane. On the other hand, these results contradict studies reporting that acaricides act exclusively on the nervous systems of ticks and that all the changes in other organs are a result from the indirect action of these chemical compounds, because blocking of the nervous system would compromise the normal metabolism of other organs (dependent on sensory information).

  20. Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability


    Díez, Carmen; Duque, Paloma; Gómez, Enrique; Hidalgo, C.O. (Carlos); Tamargo, Carolina; Rodríguez, Aida; Fernández, Lina; Varga, Santiago; Fernández, Alba; Facal, Nieves; Carbajo, Maite


    The nuclear stage at which oocytes are cryopreserved influences further development ability and cryopreservation affects ultrastructure of both cumulus cells and the oocyte. In this work, we analyze the effects of vitrification at different nuclear and cytoplasmic maturation stages on the oocyte ultrastructure and developmental ability. Culture in TCM199 + PVA with roscovitine 25 M during 24 h led to meiotic arrest (MA) in cumulus-oocyte complexes (COCs), while permissive in vitro...

  1. An improved vitrification protocol for equine immature oocytes, resulting in a first live foal. (United States)

    Ortiz-Escribano, N; Bogado Pascottini, O; Woelders, H; Vandenberghe, L; De Schauwer, C; Govaere, J; Van den Abbeel, E; Vullers, T; Ververs, C; Roels, K; Van De Velde, M; Van Soom, A; Smits, K


    The success rate for vitrification of immature equine oocytes is low. Although vitrified-warmed oocytes are able to mature, further embryonic development appears to be compromised. The aim of this study was to compare two vitrification protocols, and to examine the effect of the number of layers of cumulus cells surrounding the oocyte during vitrification of immature equine oocytes. Experimental in vitro and in vivo trials. Immature equine oocytes were vitrified after a short exposure to high concentrations of cryoprotective agents (CPAs), or a long exposure to lower concentrations of CPAs. In Experiment 1, the maturation of oocytes surrounded by multiple layers of cumulus cells (CC oocytes) and oocytes surrounded by only corona radiata (CR oocytes) was investigated. In Experiment 2, spindle configuration was determined for CR oocytes vitrified using the two vitrification protocols. In Experiment 3, further embryonic development was studied after fertilisation and culture. Embryo transfer was performed in a standard manner. Similar nuclear maturation rates were observed for CR oocytes vitrified using the long exposure and nonvitrified controls. Furthermore, a lower maturation rate was obtained for CC oocytes vitrified with the short exposure compared to control CR oocytes (P = 0.001). Both vitrification protocols resulted in significantly higher rates of aberrant spindle configuration than the control groups (Pfoal. The relatively low number of equine oocytes and embryo transfer procedures performed. For vitrification of immature equine oocytes, the use of 1) CR oocytes, 2) a high concentration of CPAs, and 3) a short exposure time may be key factors for maintaining developmental competence. © 2017 EVJ Ltd.

  2. Influence of co-culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus-enclosed porcine oocytes in a defined system. (United States)

    Appeltant, Ruth; Somfai, Tamás; Kikuchi, Kazuhiro; Maes, Dominiek; Van Soom, Ann


    Co-culture of cumulus-oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte-secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β-mercaptoethanol. Cumulus-oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co-culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus-enclosed porcine oocytes in a defined system. © 2015 Japanese Society of Animal Science.

  3. Photoperiodic Influences on Ultradian Rhythms of Male Siberian Hamsters (United States)

    Prendergast, Brian J.; Zucker, Irving


    Seasonal changes in mammalian physiology and behavior are proximately controlled by the annual variation in day length. Long summer and short winter day lengths markedly alter the amplitude of endogenous circadian rhythms and may affect ultradian oscillations, but the threshold photoperiods for inducing these changes are not known. We assessed the effects of short and intermediate day lengths and changes in reproductive physiology on circadian and ultradian rhythms of locomotor activity in Siberian hamsters. Males were maintained in a long photoperiod from birth (15 h light/day; 15 L) and transferred in adulthood to 1 of 7 experimental photoperiods ranging from 14 L to 9 L. Decreases in circadian rhythm (CR) robustness, mesor and amplitude were evident in photoperiods ≤14 L, as were delays in the timing of CR acrophase and expansion of nocturnal activity duration. Nocturnal ultradian rhythms (URs) were comparably prevalent in all day lengths, but 15 L markedly inhibited the expression of light-phase URs. The period (τ’), amplitude and complexity of URs increased in day lengths ≤13 L. Among hamsters that failed to undergo gonadal regression in short day lengths (nonresponders), τ’ of the dark-phase UR was longer than in photoresponsive hamsters; in 13 L the incidence and amplitude of light-phase URs were greater in hamsters that did not undergo testicular regression. Day lengths as long as 14 L were sufficient to trigger changes in the waveform of CRs without affecting UR waveform. The transition from a long- to a short-day ultradian phenotype occurred for most UR components at day lengths of 12 L–13 L, thereby establishing different thresholds for CR and UR responses to day length. At the UR-threshold photoperiod of 13 L, differences in gonadal status were largely without effect on most UR parameters. PMID:22848579

  4. Circadian arrhythmia dysregulates emotional behaviors in aged Siberian hamsters (United States)

    Prendergast, Brian J.; Onishi, Kenneth G.; Patel, Priyesh N.; Stevenson, Tyler J.


    Emotional behaviors are influenced by the circadian timing system. Circadian disruptions are associated with depressive-like symptoms in clinical and preclinical populations. Circadian rhythm robustness declines markedly with aging and may contribute to susceptibility to emotional dysregulation in aged individuals. The present experiments used a model of chronic circadian arrhythmia generated noninvasively, via a series of circadian-disruptive light treatments, to investigate interactions between circadian desynchrony and aging on depressive- and anxiety-like behaviors, and on limbic neuroinflammatory gene expression that has been linked with emotionality. We also examined whether a social manipulation (group housing) would attenuate effects of arrhythmia on emotionality. In aged (14-18 months of age) male Siberian hamsters, circadian arrhythmia increased behavioral despair and decreased social motivation, but decreased exploratory anxiety. These effects were not evident in younger (5-9 months of age) hamsters. Social housing (3-5 hamsters/cage) abolished the effects of circadian arrhythmia on emotionality. Circadian arrhythmia alone was without effect on hippocampal or cortical interleukin-1β (IL-1β) and indoleamine 2,3-dioxygenase (Ido) mRNA expression in aged hamsters, but social housing decreased hippocampal IL-1β and Ido mRNAs. The data demonstrate that circadian disruption can negatively impact affective state, and that this effect is pronounced in older individuals. Although clear associations between circadian arrhythmia and constitutive limbic proinflammatory activity were not evident, the present data suggest that social housing markedly inhibits constitutive hippocampal IL-1β and Ido activity, which may contribute to the ameliorating effects of social housing on a number of emotional behaviors. PMID:24333374

  5. Spontaneous Fibrosarcoma in a Djungarian Hamster (Phodopus sungorus)


    Kondo, Hirotaka; ONUMA, Mamoru; Ito, Hidetoshi; Shibuya, Hisashi; Sato, Tsuneo


    A 1.5-y-old female Djungarian hamster (Phodopus sungorus) presented with a large subcutaneous mass surrounding the right shoulder. Radiography revealed dislocation of the right humeral articulation and osteolytic lesions of the right scapula. Histologically, the mass was composed of spindle to stellate cells arranged in fascicles interwoven with delicate collagen fibers, and neoplastic cells infiltrated the bone, skeletal muscle, and subcutaneous tissues. Neoplastic cells stained intensely po...

  6. Clozapine chronically suppresses alcohol drinking in Syrian golden hamsters (United States)

    Chau, David T.; Gulick, Danielle; Xie, Haiyi; Dawson, Ree; Green, Alan I.


    Alcohol use disorder is common in patients with schizophrenia and is associated with poor clinical outcomes. Preliminary reports from our group and others suggest that the atypical antipsychotic clozapine may decrease alcohol use in these patients. We have previously shown that clozapine suppresses alcohol consumption for 9 days in Syrian golden hamsters. Here, we assessed the effects of clozapine on alcohol consumption in hamsters over a 27-day period, using a continuous access, 2-bottle (15% alcohol vs. water) protocol. Clozapine (4, 8, or 12 mg/kg/day, injected subcutaneously [s.c.]) dose-dependently suppressed alcohol drinking, while increasing food and water intake, and there was no tolerance within individual groups to this effect of clozapine over time. In a separate experiment, the effects of clozapine on sucrose and water drinking and food intake over a 9-day period were assessed. Clozapine (4, 8, or 12 mg/kg/day s.c.) failed to suppress sucrose (0.09 M), food, or water consumption at any time-point tested. In a related study, assessment of blood alcohol levels in hamsters indicated that blood alcohol levels were maintained within a narrow and moderate range (7–13 mg/dL), levels noted by others to produce physiologic effects in rodents. The ability of clozapine to suppress alcohol drinking in the hamster over an extended period of time without suppressing sucrose, water, or food consumption is consistent with preliminary reports indicating that clozapine limits frequent alcohol use, even producing abstinence in many patients with schizophrenia. PMID:19895824

  7. Pharmaceutical Options for Triggering of Final Oocyte Maturation in ART

    DEFF Research Database (Denmark)

    Castillo, Juan Carlos; Humaidan, Peter; Bernabéu, Rafael


    Since the pioneering days of in vitro fertilization, hCG has been the gold standard to induce final follicular maturation. We herein reviewed different pharmaceutical options for triggering of final oocyte maturation in ART. The new upcoming agent seems to be GnRHa with its potential advantages...... over hCG trigger. GnRHa triggering elicits a surge of gonadotropins resembling the natural midcycle surge of gonadotropins, without the prolonged action of hCG, resulting in the retrieval of more mature oocytes and a significant reduction in or elimination of OHSS as compared to hCG triggering...

  8. Antibody inhibition of protein activity in starfish oocytes. (United States)

    Okumura, Eiichi; Hara, Masatoshi; Kishimoto, Takeo


    Antibodies are widely utilized in cell and molecule biology for immunoblots, immunostaining, immunoprecipitation, immunoaffinity purification, and immunoassay. Some antibodies can be used for in vivo inhibition experiments. These antibodies bind to their target molecules and neutralize their functions, providing functional information in the study of their biological role. Here, we describe our methods for obtaining inhibitory antibodies against desired proteins. We then describe in the starfish oocyte system how to inhibit a target protein, even in the nucleus, by injection of antibody into the cytoplasm, and how to evaluate antibody inhibition of cell cycle regulators in small numbers of oocytes.

  9. Artificial intelligence techniques for embryo and oocyte classification. (United States)

    Manna, Claudio; Nanni, Loris; Lumini, Alessandra; Pappalardo, Sebastiana


    One of the most relevant aspects in assisted reproduction technology is the possibility of characterizing and identifying the most viable oocytes or embryos. In most cases, embryologists select them by visual examination and their evaluation is totally subjective. Recently, due to the rapid growth in the capacity to extract texture descriptors from a given image, a growing interest has been shown in the use of artificial intelligence methods for embryo or oocyte scoring/selection in IVF programmes. This work concentrates the efforts on the possible prediction of the quality of embryos and oocytes in order to improve the performance of assisted reproduction technology, starting from their images. The artificial intelligence system proposed in this work is based on a set of Levenberg-Marquardt neural networks trained using textural descriptors (the local binary patterns). The proposed system was tested on two data sets of 269 oocytes and 269 corresponding embryos from 104 women and compared with other machine learning methods already proposed in the past for similar classification problems. Although the results are only preliminary, they show an interesting classification performance. This technique may be of particular interest in those countries where legislation restricts embryo selection. One of the most relevant aspects in assisted reproduction technology is the possibility of characterizing and identifying the most viable oocytes or embryos. In most cases, embryologists select them by visual examination and their evaluation is totally subjective. Recently, due to the rapid growth in our capacity to extract texture descriptors from a given image, a growing interest has been shown in the use of artificial intelligence methods for embryo or oocyte scoring/selection in IVF programmes. In this work, we concentrate our efforts on the possible prediction of the quality of embryos and oocytes in order to improve the performance of assisted reproduction technology

  10. Prevalence of intestinal parasites in hamsters and rabbits in some pet shops of Turkey. (United States)

    Sürsal, Neslihan; Gökpinar, Sami; Yildiz, Kader


    In this study, we aimed to determine the parasite species carried by hamsters and rabbits purchased from some commercial pet shops in Turkey. For this purpose, the fecal samples of clinically healthy Syrian hamsters, dwarf hamsters, and crossbred rabbits were collected from 22 pet shops randomly selected in Ankara and Kirikkale provinces, located in Central Anatolia Region of Turkey. The fecal samples were examined with centrifuge flotation technique using saturated salt solution. Parasitic infection rate was 57.5% in dwarf hamsters, 54.9% in Syrian hamsters, and 56.3% in crossbreed rabbits. Trichurid eggs were the most prevalent parasite in the feces of Syrians hamsters (28.1%). The other parasites of Syrian hamsters were as follows: Eimeria spp. oocysts (15.4%) and the eggs of H. nana (11.2%), Syphacia spp. (11%). and Aspiculuris spp. (5.6 %). Only trichurid eggs were observed in the fecal samples of dwarf hamsters (51.5%). Oocysts of Eimeria spp. (52.7%) and the eggs of P. ambiguus (3.6%) were detected in the feces of rabbits. Within the scope of this study, the detection of H. nana eggs, a zoonotic parasite, in the feces of Syrian hamster was quite remarkable for public health.

  11. The pathology of experimental Schistosoma curassoni infections in mice and hamsters. (United States)

    Vercruysse, J; Fransen, J; Southgate, V R; Rollinson, D


    The histopathology of experimental Schistosoma curassoni infections in white mice and hamsters was studied. In mice, hepatic lesions were severe with characteristic extensive perilobular fibrosis and large perilobular granulomas throughout the parenchyma. Only a few granulomas were detected in the lung, small intestine, and rectum of mice. In hamsters, lesions in the liver were limited. Few granulomas were found but the giant cell reaction was pronounced. Lesions in the lung and small intestine were minimal. Many subserosal and submucosal epithelioid cell granulomas were in the colon and rectum of hamsters. Parasites were not detected in the bladder of either mice or hamsters.

  12. Female-biased anorexia and anxiety in the Syrian hamster. (United States)

    Shannonhouse, John L; Fong, Li An; Clossen, Bryan L; Hairgrove, Ross E; York, Daniel C; Walker, Benjamin B; Hercules, Gregory W; Mertesdorf, Lauren M; Patel, Margi; Morgan, Caurnel


    Anorexia and anxiety cause significant mortality and disability with female biases and frequent comorbidity after puberty, but the scarcity of suitable animal models impedes understanding of their biological underpinnings. It is reported here that in adult or weanling Syrian hamsters, relative to social housing (SH), social separation (SS) induced anorexia characterized as hypophagia, weight loss, reduced adiposity, and hypermetabolism. Following anorexia, SS increased reluctance to feed, and thigmotaxis, in anxiogenic environments. Importantly, anorexia and anxiety were induced post-puberty with female biases. SS also reduced hypothalamic corticotrophin-releasing factor mRNA and serum corticosteroid levels assessed by RT-PCR and RIA, respectively. Consistent with the view that sex differences in adrenal suppression contributed to female biases in anorexia and anxiety by disinhibiting neuroimmune activity, SS elevated hypothalamic interleukin-6 and toll-like receptor 4 mRNA levels. Although corticosteroids were highest during SH, they were within the physiological range and associated with juvenile-like growth of white adipose, bone, and skeletal muscle. These results suggest that hamsters exhibit plasticity in bioenergetic and emotional phenotypes across puberty without an increase in stress responsiveness. Thus, social separation of hamsters provides a model of sex differences in anorexia and anxiety during adulthood and their pathogeneses during adolescence. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Learned magnetic compass orientation by the Siberian hamster, Phodopus sungorus

    Energy Technology Data Exchange (ETDEWEB)

    Deutschlander, Mark E.; Freake, Michael J.; Borland, Christopher; Phillips, John B.; Madden, R C.; Anderson, Larry E.; Wilson, B W.


    Magnetic orientation has been demonstrated in Siberian hamsters, Phodopus sungorus. The behavior, using a nest building assay, shows a directional preference in nest position and appears in this animal to be a learned behavior. Hamsters were housed prior to testing in rectangular cages aligned along perpendicular axes. When subsequently tested in a radially-symmetrical arena, the hamsters positioned their nests in a bimodal distribution that coincided with the magnetic direction of the long-axis of the holding cages. In addition, results are presented that illustrate some of the factors that can influence behavioral responses to the magnetic field. In particular for P. sungorus, holding conditions prior to testing and the presence of non-magnetic cues may influence the strength and expression of magnetic orientation. Failure to consider these and other factors may help to explain why previous attempts to demonstrate magnetic orientation in a number of rodent species have failed or, when positive results have been obtained, have been difficult to replicate in other laboratories.

  14. Melanin content of hamster tissues, human tissues, and various melanomas

    Energy Technology Data Exchange (ETDEWEB)

    Watts, K.P.; Fairchild, R.G.; Slatkin, D.N.; Greenberg, D.; Packer, S.; Atkins, H.L.; Hannon, S.J.


    Melanin content (percentage by weight) was determined in both pigmented and nonpigmented tissues of Syrian golden hamsters bearing Greene melanoma. Melanin content was also measured in various other melanoma models (B-16 in C57 mice, Harding-Passey in BALB/c mice, and KHDD in C3H mice) and in nine human melanomas, as well as in selected normal tissues. The purpose was to evaluate the possible efficacy of chlorpromazine, which is known to bind to melanin, as a vehicle for boron transport in neutron capture therapy. Successful therapy would depend upon selective uptake and absolute concentration of borated compounds in tumors; these parameters will in turn depend upon melanin concentration in melanomas and nonpigmented ''background'' tissues. Hamster whole eyes, hamster melanomas, and other well-pigmented animal melanomas were found to contain 0.3 to 0.8% melanin by weight, whereas human melanomas varied from 0.1 to 0.9% (average, 0.35%). Other tissues, with the exception of skin, were lower in content by a factor of greater than or equal to30. Melanin pigment was extracted from tissues, and the melanin content was determined spectrophotometrically. Measurements were found to be sensitive to the presence of other proteins. Previous procedures for isolating and quantifying melanin often neglected the importance of removing proteins and other interfering nonmelanic substances.

  15. Isotretinoin-induced craniofacial malformations in humans and hamsters. (United States)

    Willhite, C C; Hill, R M; Irving, D W


    Oral administration of 40-80 mg/d of isotretinoin (13-cis-retinoic acid, Ro 4-3780, Accutane) during the first month of human pregnancy can induce severe congenital malformation. The human Accutane dysmorphic syndrome includes rudimentary external ears, absent or imperforate auditory canals, a triangular microcephalic skull, cleft palate, depressed midface, and anomalies of the brain, jaw, and heart. Children who suffer from this syndrome have large occiputs with narrowing of the frontal bone. Microphthalmia is reported in two cases; notations are made about the orbits in three cases; and the fact that infants could not follow with their eyes is noted in three cases. A decrease in muscle tone is noted in six, cleft palate is present in four, and limb reduction defects are described in two. The cardiac malformations usually include overriding aorta, interrupted or hypoplastic aortic arch, and septation defects of atria and ventricles. There are at least two cases of abnormal origin of the subclavian arteries. Oral isotretinoin in the pregnant hamster also induces similar congenital malformations. A human case of isotretinoin-induced dysmorphia is presented and compared with other affected infants and affected hamsters. The metabolic fate and pharmacokinetic parameters of isotretinoin in humans and rodents are discussed in relation to the teratogenic response. The results suggest that humans are approximately 16 times more sensitive to the teratogenic effects of oral isotretinoin than are hamsters.

  16. The signaling pathways by which the Fas/FasL system accelerates oocyte aging. (United States)

    Zhu, Jiang; Lin, Fei-Hu; Zhang, Jie; Lin, Juan; Li, Hong; Li, You-Wei; Tan, Xiu-Wen; Tan, Jing-He


    In spite of great efforts, the mechanisms for postovulatory oocyte aging are not fully understood. Although our previous work showed that the FasL/Fas signaling facilitated oocyte aging, the intra-oocyte signaling pathways are unknown. Furthermore, the mechanisms by which oxidative stress facilitates oocyte aging and the causal relationship between Ca2+ rises and caspase-3 activation and between the cell cycle and apoptosis during oocyte aging need detailed investigations. Our aim was to address these issues by studying the intra-oocyte signaling pathways for Fas/FasL to accelerate oocyte aging. The results indicated that sFasL released by cumulus cells activated Fas on the oocyte by increasing reactive oxygen species via activating NADPH oxidase. The activated Fas triggered Ca2+ release from the endoplasmic reticulum by activating phospholipase C-γ pathway and cytochrome c pathway. The cytoplasmic Ca2+ rises activated calcium/calmodulin-dependent protein kinase II (CaMKII) and caspase-3. While activated CaMKII increased oocyte susceptibility to activation by inactivating maturation-promoting factor (MPF) through cyclin B degradation, the activated caspase-3 facilitated further Ca2+releasing that activates more caspase-3 leading to oocyte fragmentation. Furthermore, caspase-3 activation and fragmentation were prevented in oocytes with a high MPF activity, suggesting that an oocyte must be in interphase to undergo apoptosis.

  17. Role of growth hormone and growth hormone receptor in oocyte maturation

    NARCIS (Netherlands)

    Bevers, MM; Izadyar, F


    Near the completion of growth, mammalian oocytes acquire the competence to resume and complete meiosis. In vivo the preovulatory LH surge triggers the resumption of meiosis in the oocyte contained in preovulatory follicles. When immature oocytes and the surrounding cumulus cells are released from

  18. Influence of antral follicle size on oocyte characteristics and embryo development in the bovine

    DEFF Research Database (Denmark)

    Lequarre, Anne Sophie; Vigneron, Céline; Ribaucour, Fabrice


    The developmental competence of bovine oocytes isolated from antral follicles of different sizes was assessed in three European laboratories (Belgium, UCL; Denmark, DIAS; France, INRA). Using the same protocol for in vitro production of embryos, the oocytes isolated from follicles with a diameter...... ≥6 mm always gave a higher blastocyst rate than oocytes from follicles...

  19. Effect of stage of follicular growth during superovulation on developmental competence of bovine oocytes

    DEFF Research Database (Denmark)

    Humblot, P; Holm, P; Lonergan, P


    The final steps of oocyte capacitation and maturation are critical for embryonic development but detailed information is scarce on how the oocyte is affected during this period. In this study, 2033 oocytes were collected from 106 superovulated cattle at four different time points before ovulation...

  20. Nerve Growth Factor Increases mRNA Levels for the Prion Protein and the β -amyloid Protein Precursor in Developing Hamster Brain (United States)

    Mobley, William C.; Neve, Rachael L.; Prusiner, Stanley B.; McKinley, Michael P.


    Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

  1. Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence. (United States)

    Canosa, S; Adriaenssens, T; Coucke, W; Dalmasso, P; Revelli, A; Benedetto, C; Smitz, J


    Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. Single oocytes and their corresponding CC were analysed. Oocytes were examined using PLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P ZP area and thickness in mature oocytes than in immature oocytes (all P ZP retardance was

  2. An omega-3 fatty acid-enriched diet prevents skeletal muscle lesions in a hamster model of dystrophy. (United States)

    Fiaccavento, Roberta; Carotenuto, Felicia; Vecchini, Alba; Binaglia, Luciano; Forte, Giancarlo; Capucci, Enrico; Maccari, Anna Maria; Minieri, Marilena; Di Nardo, Paolo


    Currently, despite well-known mutational causes, a universal treatment for neuromuscular disorders is still lacking, and current therapeutic efforts are mainly restricted to symptomatic treatments. In the present study, δ-sarcoglycan-null dystrophic hamsters were fed a diet enriched in flaxseed-derived ω3 α-linolenic fatty acid from weaning until death. α-linolenic fatty acid precluded the dystrophic degeneration of muscle morphology and function. In fact, in dystrophic animals fed flaxseed-derived α-linolenic fatty acid, the histological appearance of the muscular tissue was improved, the proliferation of interstitial cells was decreased, and the myogenic differentiation originated new myocytes to repair the injured muscle. In addition, muscle myofibers were larger and cell membrane integrity was preserved, as witnessed by the correct localization of α-, β-, and γ-sarcoglycans and α-dystroglycan. Furthermore, the cytoplasmic accumulation of both β-catenin and caveolin-3 was abolished in dystrophic hamster muscle fed α-linolenic fatty acid versus control animals fed standard diet, while α-myosin heavy chain was expressed at nearly physiological levels. These findings, obtained by dietary intervention only, introduce a novel concept that provides evidence that the modulation of the plasmalemma lipid profile could represent an efficacious strategy to ameliorate human muscular dystrophy.

  3. The fertilization ability and developmental competence of bovine oocytes grown in vitro. (United States)

    Makita, Miho; Ueda, Mayuko; Miyano, Takashi


    In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4-0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts.

  4. The fertilization ability and developmental competence of bovine oocytes grown in vitro (United States)

    MAKITA, Miho; UEDA, Mayuko; MIYANO, Takashi


    In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4−0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts. PMID:27151093

  5. Vitrification of oocytes from endangered Mexican gray wolves (Canis lupus baileyi). (United States)

    Boutelle, S; Lenahan, K; Krisher, R; Bauman, K L; Asa, C S; Silber, S


    Careful genetic management, including cryopreservation of genetic material, is central to conservation of the endangered Mexican gray wolf. We tested a technique, previously used to vitrify human and domestic animal oocytes, on oocytes from domestic dogs as a model and from the endangered Mexican wolf. This method provided a way to conserve oocytes from genetically valuable older female Mexican wolves as an alternative to embryos for preserving female genes. Oocytes were aspirated from ovaries of 36 female dogs in December and March (0 to 65 oocytes per female) and from six female wolves (4 to 73 per female) during their physiologic breeding season, or following stimulation with the GnRH agonist deslorelin. Oocytes from dogs were pooled; half were immediately tested for viability and the remainder vitrified, then warmed and tested for viability. All oocytes were vitrified by being moved through media of increasing cryoprotectant concentration, placed on Cryotops, and plunged into liquid nitrogen. There was no difference in viability (propidium iodide staining) between fresh and vitrified, warmed dog oocytes (65.7 and 61.0%, respectively, P = 0.27). Oocyte viability after warming was similarly assessed in a subset of wolves (4 to 15 oocytes from each of three females; total 29 oocytes). Of these, 57.1% of the post-thaw intact oocytes were viable, which was 41.4% of all oocytes warmed. These were the first oocytes from a canid or an endangered species demonstrated to have maintained viability after vitrification and warming. Furthermore, our results demonstrated that vitrification of oocytes with the Cryotop technique was an option for preserving female gametes from Mexican wolves for future use in captive breeding programs, although in vitro embryo production techniques must first be developed in canids for this technique to be used. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Andes Virus M Genome Segment is Not Sufficient to Confer the Virulence Associated With Andres Virus in Syrian Hamsters

    National Research Council Canada - National Science Library

    McElroy, A


    ...) in North and South America, respectively. Although ANDV causes a lethal HPS-like disease in hamsters, SNV, and all other HPS-associated hantaviruses that have been tested, cause asymptomatic infections of laboratory animals, including hamsters...

  7. Genetic spatial structure of European common hamsters (Cricetus cricetus) - a result of repeated range expansion and demographic bottlenecks

    NARCIS (Netherlands)

    Neumann, K.; Michaux, R.; Maak, S.; Jansman, H.A.H.; Kayser, A.; Mundt, G.; Gattermann, R.


    The spatial genetic structure of common hamsters (Cricetus cricetus) was investigated using three partial mitochondrial (mt) genes and 11 nuclear microsatellite loci. All marker systems revealed significant population differentiation across Europe. Hamsters in central and western Europe belong

  8. The presence of opioidergic pinealocytes in the pineal gland of the European hamster (Cricetus cricetus): an immunocytochemical study

    DEFF Research Database (Denmark)

    Coto-Montes, A.; Masson-Pévet, M.; Pévet, P.


    Neurobiologi, pineal gland, leu-enkephalin, Met-enkephalin, synaptic contacts, paracrine regulation, European hamster, cricetus cricetus (rodents)......Neurobiologi, pineal gland, leu-enkephalin, Met-enkephalin, synaptic contacts, paracrine regulation, European hamster, cricetus cricetus (rodents)...

  9. Sequencing, annotation and analysis of the Syrian hamster (Mesocricetus auratus) transcriptome. (United States)

    Tchitchek, Nicolas; Safronetz, David; Rasmussen, Angela L; Martens, Craig; Virtaneva, Kimmo; Porcella, Stephen F; Feldmann, Heinz; Ebihara, Hideki; Katze, Michael G


    The Syrian hamster (golden hamster, Mesocricetus auratus) is gaining importance as a new experimental animal model for multiple pathogens, including emerging zoonotic diseases such as Ebola. Nevertheless there are currently no publicly available transcriptome reference sequences or genome for this species. A cDNA library derived from mRNA and snRNA isolated and pooled from the brains, lungs, spleens, kidneys, livers, and hearts of three adult female Syrian hamsters was sequenced. Sequence reads were assembled into 62,482 contigs and 111,796 reads remained unassembled (singletons). This combined contig/singleton dataset, designated as the Syrian hamster transcriptome, represents a total of 60,117,204 nucleotides. Our Mesocricetus auratus Syrian hamster transcriptome mapped to 11,648 mouse transcripts representing 9,562 distinct genes, and mapped to a similar number of transcripts and genes in the rat. We identified 214 quasi-complete transcripts based on mouse annotations. Canonical pathways involved in a broad spectrum of fundamental biological processes were significantly represented in the library. The Syrian hamster transcriptome was aligned to the current release of the Chinese hamster ovary (CHO) cell transcriptome and genome to improve the genomic annotation of this species. Finally, our Syrian hamster transcriptome was aligned against 14 other rodents, primate and laurasiatheria species to gain insights about the genetic relatedness and placement of this species. This Syrian hamster transcriptome dataset significantly improves our knowledge of the Syrian hamster's transcriptome, especially towards its future use in infectious disease research. Moreover, this library is an important resource for the wider scientific community to help improve genome annotation of the Syrian hamster and other closely related species. Furthermore, these data provide the basis for development of expression microarrays that can be used in functional genomics studies.

  10. Ecdysteroids and oocyte development in the black fly Simulium vittatum

    Directory of Open Access Journals (Sweden)

    Hagedorn Henry H


    Full Text Available Abstract Background Oocyte development was studied in the autogenous black fly, Simulium vittatum (Diptera, Nematocera, a vector of Onchocerca volvulus, the causative agent of onchocerciasis. Results Oocyte growth was nearly linear between adult eclosion and was complete by 72 hours at 21°C. The oocyte became opaque at 14 hours after eclosion indicating the initiation of protein yolk deposition. The accumulation of vitellogenin was measured using SDS-PAGE. The density of the yolk protein bands at about 200 and 65 kDa increased during the first and second days after eclosion. The amount of protein in the 200 kDa band of vitellogenin, determined using densitometry, rapidly increased between 12 and 25 hours after eclosion. Ecdysteroid levels were measured using a competitive ELISA. Ecdysteroid levels increased rapidly and subsequently declined during the first day after eclosion. Conclusion These data show a correlation between the appearance of vitellogenin in the oocyte, and the rise in ecdysteroids. A possible relationship to molting of the nematode, Onchocerca volvulus, is discussed.

  11. Protein Tyrosine Kinase Signaling During Oocyte Maturation and Fertilization (United States)

    McGinnis, Lynda K.; Carroll, David J.; Kinsey, William H.


    The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre-positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC-family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK-mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC-family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans. PMID:21681843

  12. Finite element model to study calcium distribution in oocytes ...

    African Journals Online (AJOL)

    A program has been developed in MATLAB 7.10 for the entire problem and executed to obtain numerical results. The numerical results have been used to study the effect of buffers, RyR and VGCC on calcium distribution in oocyte. The results indicate that buffers can significantly decrease the calcium concentration and ...

  13. Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Peaucellier, G.; Andersen, A.C.; Kinsey, W.H. (Univ. of Miami School of Medicine, FL (USA))


    We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.

  14. Aurelia aurita (Cnidaria oocytes' contact plate structure and development.

    Directory of Open Access Journals (Sweden)

    Leonid S Adonin

    Full Text Available One of the A. aurita medusa main mesoglea polypeptides, mesoglein, has been described previously. Mesoglein belongs to ZP-domain protein family and therefore we focused on A.aurita oogenesis. Antibodies against mesoglein (AB RA47 stain the plate in the place where germinal epithelium contacts oocyte on the paraffin sections. According to its position, we named the structure found the "contact plate". Our main instrument was AB against mesoglein. ZP-domain occupies about half of the whole amino acid sequence of the mesoglein. Immunoblot after SDS-PAGE and AU-PAGE reveals two charged and high M(r bands among the female gonad germinal epithelium polypeptides. One of the gonads' polypeptides M(r corresponds to that of mesogleal cells, the other ones' M(r is higher. The morphological description of contact plate formation is the subject of the current work. Two types of AB RA47 positive granules were observed during progressive oogenesis stages. Granules form the contact plate in mature oocyte. Contact plate of A.aurita oocyte marks its animal pole and resembles Zona Pellucida by the following features: (1 it attracts spermatozoids; (2 the material of the contact plate is synthesized by oocyte and stored in granules; (3 these granules and the contact plate itself contain ZP domain protein(s; (4 contact plate is an extracellular structure made up of fiber bundles similar to those of conventional Zona Pellucida.

  15. Gene expression and maturation evaluation of sheep oocytes ...

    African Journals Online (AJOL)

    I. A. H. Barakat


    Dec 21, 2017 ... media of sheep oocytes resulted in an improvement of embryo development to blastocyst stage. The molecular modifications of COCs during maturation include accumulation of mRNA and proteins, which originate from long stages of development during oogenesis and folliculogenesis (Kempisty et al.,.

  16. High hydrostatic pressure treatment of porcine oocytes induces parthenogenetic activation

    DEFF Research Database (Denmark)

    Lin, Lin; Pribenszky, Csaba; Molnár, Miklós


    An innovative technique called high hydrostatic pressure (HHP) treatment has recently been reported to improve the cryosurvival of gametes and embryos in certain mammalian species, including the mouse, pig, and cattle. In the present study the parthenogenetic activation (PA) of pig oocytes caused...

  17. characteristics of mature oocytes from four species of marine teleosts

    African Journals Online (AJOL)


    Aug 9, 1988 ... fish induced to complete gametogenesis and spawn by means of hormone injections (Kuo, Nash & Shehadeh. 1974; Zhitenev, Kalinin & Abayev 1974). Whether these oocytes are of comparable quality to those produced in nattire has not been established. The present article describes mature translucent.

  18. Pelvic abscess complicating transvaginal oocyte retrieval: A case ...

    African Journals Online (AJOL)

    Pelvic abscess complicating transvaginal oocyte retrieval for in vitro fertilization is uncommon. Difficulties and/or delays in diagnosis, attributable to the rarity of the pathology, are associated with complications that lead to severe maternal and perinatality morbidity and mortality. In this report, we present a 37 year old ...

  19. A simplified approach for oocyte enucleation in mammalian cloning. (United States)

    Iuso, Domenico; Czernik, Marta; Zacchini, Federica; Ptak, Grazyna; Loi, Pasqualino


    Despite its success in almost all farm and laboratory animals, somatic cell nuclear transfer (SCNT) is still a low-efficiency technique. In this investigation, we determined the impact of each enucleation step on oocyte viability (assessed by parthenogenetic activation): Hoechst (HO) staining, cytochalasin B, ultraviolet (UV) exposure, and demecolcine. Our data showed that of all the factors analyzed, UV exposure impaired oocyte development (cleavage, 59% for untreated oocytes vs. 8% UV exposed; blastocyst stage, 32% untreated vs. 0% UV exposed). A minor toxicity was detected following demecolcine treatment (cleavage, 62%; blastocyst stage, 13%). Next, we compared HO/UV (canonical) and demecolcine-assisted enucleation (DAE), with a straight removal of metaphase chromosomes without any chemical or physical aid (straight enucleation). DAE improved the preimplantation development of sheep cloned embryos compared to HO/UV enucleation (cleavage, 38% vs. 19%; blastocysts, 17% vs. 4%), yet straight enucleation resulted in the highest cleavage and blastocysts rates (61% and 30%, respectively). We concluded that: (1) UV exposure harms sheep oocyte and embryo development; (2) DAE may represent an alternative approach, especially for unskilled operators; and (3) straight enucleation remains, in our estimation, the most reliable and least harmful protocol for SCNT.

  20. Characteristics of failed fertilized oocytes in patients with severe obesity

    Directory of Open Access Journals (Sweden)

    E A Pigarova


    Full Text Available Реферат по статье: Machtinger R, Combelles CM, Missmer SA, Correia KF, Fox JH, Racowsky C. The association between severe obesity and characteristics of failed fertilized oocytes. Hum Reprod. 2012 Nov;27(11:3198-207.

  1. Successful cryopreservation of buffalo ovaries using in situ oocyte cryopreservation

    Directory of Open Access Journals (Sweden)

    Saber Mohammed Abd-Allah


    Full Text Available To improve the efficiency and efficacy of cryopreservation of ovaries, we developed a new method termed in situ oocyte (ISO cryopreservation. ISO cryopreservation is a multistep procedure that involves aspiration of follicular fluid and then perfusion of antral follicles and diffusion of whole buffalo ovaries with cryoprotectant agent (CPA, rapid cooling, storage, thawing and, finally, dilution and removal of the CPA with return to physiological environment. Our study compared ISO cryo ovaries with cryo-diffused ovaries. We systematically examined the effects of ISO cryo and diffuse cryo on ovaries by morphological examination and with viability tests. The percentages of morphologically normal and viable follicular oocytes from ISO cryo were significantly higher than those that resulted from the cryo-diffused method (p<0.01. The quality of follicular oocytes from ISO cryo ovaries appeared better than that achieved from cryo-diffused ovaries. In conclusion, this study shows that ISO cryo is highly efficient for cryopreservation of oocytes and ovarian tissue.

  2. In vitro maturation of sheep oocytes in different concentrations of ...

    African Journals Online (AJOL)

    The aim of the study was to determine the optimum concentration of the mare serum (MS) for sheep in vitro oocyte maturation. Sheep ovaries were collected from a local abattoir and transported within 1 h to the laboratory in a warm saline solution (30 – 35oC), supplemented with 100 IU penicillin G and 100 g streptomycin ...

  3. Effect of melatonin on in vitro maturation of bovine oocytes

    African Journals Online (AJOL)



    Apr 26, 2010 ... To investigate the effect of different concentrations of melatonin on bovine oocytes in vitro maturation, varying concentrations of melatonin .... solution (30 - 35°C) containing 100 IU/ml penicillin and 100 g/ml streptomycin (15140-122 .... The adverse effect will compromise fertility potential (Peter and Adashi, ...

  4. GGPP-Mediated Protein Geranylgeranylation in Oocyte Is Essential for the Establishment of Oocyte-Granulosa Cell Communication and Primary-Secondary Follicle Transition in Mouse Ovary.

    Directory of Open Access Journals (Sweden)

    Chen Jiang


    Full Text Available Folliculogenesis is a progressive and highly regulated process, which is essential to provide ova for later reproductive life, requires the bidirectional communication between the oocyte and granulosa cells. This physical connection-mediated communication conveys not only the signals from the oocyte to granulosa cells that regulate their proliferation but also metabolites from the granulosa cells to the oocyte for biosynthesis. However, the underlying mechanism of establishing this communication is largely unknown. Here, we report that oocyte geranylgeranyl diphosphate (GGPP, a metabolic intermediate involved in protein geranylgeranylation, is required to establish the oocyte-granulosa cell communication. GGPP and geranylgeranyl diphosphate synthase (Ggpps levels in oocytes increased during early follicular development. The selective depletion of GGPP in mouse oocytes impaired the proliferation of granulosa cells, primary-secondary follicle transition and female fertility. Mechanistically, GGPP depletion inhibited Rho GTPase geranylgeranylation and its GTPase activity, which was responsible for the accumulation of cell junction proteins in the oocyte cytoplasm and the failure to maintain physical connection between oocyte and granulosa cells. GGPP ablation also blocked Rab27a geranylgeranylation, which might account for the impaired secretion of oocyte materials such as Gdf9. Moreover, GGPP administration restored the defects in oocyte-granulosa cell contact, granulosa cell proliferation and primary-secondary follicle transition in Ggpps depletion mice. Our study provides the evidence that GGPP-mediated protein geranylgeranylation contributes to the establishment of oocyte-granulosa cell communication and then regulates the primary-secondary follicle transition, a key phase of folliculogenesis essential for female reproductive function.

  5. Chromosome Cohesion Established by Rec8-Cohesin in Fetal Oocytes Is Maintained without Detectable Turnover in Oocytes Arrested for Months in Mice. (United States)

    Burkhardt, Sabrina; Borsos, Máté; Szydlowska, Anna; Godwin, Jonathan; Williams, Suzannah A; Cohen, Paula E; Hirota, Takayuki; Saitou, Mitinori; Tachibana-Konwalski, Kikuë


    Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis [1]. Rec8-containing cohesin, bound to Smc3/Smc1α or Smc3/Smc1β, maintains bivalent cohesion in mammalian meiosis [2-6]. In females, meiotic DNA replication and recombination occur in fetal oocytes. After birth, oocytes arrest at the prolonged dictyate stage until recruited to grow into mature oocytes that divide at ovulation. How cohesion is maintained in arrested oocytes remains a pivotal question relevant to maternal age-related aneuploidy. Hypothetically, cohesin turnover regenerates cohesion in oocytes. Evidence for post-replicative cohesion establishment mechanism exists, in yeast and invertebrates [7, 8]. In mouse fetal oocytes, cohesin loading factor Nipbl/Scc2 localizes to chromosome axes during recombination [9, 10]. Alternatively, cohesion is maintained without turnover. Consistent with this, cohesion maintenance does not require Smc1β transcription, but unlike Rec8, Smc1β is not required for establishing bivalent cohesion [11, 12]. Rec8 maintains cohesion without turnover during weeks of oocyte growth [3]. Whether the same applies to months or decades of arrest is unknown. Here, we test whether Rec8 activated in arrested mouse oocytes builds cohesion revealed by TEV cleavage and live-cell imaging. Rec8 establishes cohesion when activated during DNA replication in fetal oocytes using tamoxifen-inducible Cre. In contrast, no new cohesion is detected when Rec8 is activated in arrested oocytes by tamoxifen despite cohesin synthesis. We conclude that cohesion established in fetal oocytes is maintained for months without detectable turnover in dictyate-arrested oocytes. This implies that women's fertility depends on the longevity of cohesin proteins that established cohesion in utero. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes. (United States)

    Jørgensen, Morten Egevang; Nour-Eldin, Hussam Hassan; Halkier, Barbara Ann


    Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.

  7. Membrane tension and membrane fusion


    Kozlov, Michael M.; Chernomordik, Leonid V.


    Diverse cell biological processes that involve shaping and remodeling of cell membranes are regulated by membrane lateral tension. Here we focus on the role of tension in driving membrane fusion. We discuss the physics of membrane tension, forces that can generate the tension in plasma membrane of a cell, and the hypothesis that tension powers expansion of membrane fusion pores in late stages of cell-to-cell and exocytotic fusion. We propose that fusion pore expansion can require unusually la...

  8. Development of the competence of bovine oocytes to release cortical granules and block polyspermy after meiotic maturation. (United States)

    Wang, W; Hosoe, M; Li, R; Shioya, Y


    Bovine immature oocytes do not have the ability to block polyspermic penetration. The present study was conducted to determine whether this is correlated to cortical granule (CG) distribution and the competence of oocytes to release CG upon sperm penetration, and whether the ability of bovine oocytes to release CG develops during in vitro maturation. Fluorescein isothiocyanate-conjugated Lens culinaris agglutinin was used for detecting CG in immature and mature oocytes before and after sperm penetration and electric stimulation. The labeled oocytes were examined with laser confocal and fluorescent microscopes. The results show that CG exist as clusters in all immature oocytes. The CG were not released from immature oocytes exposed to electric pulse or penetrated by spermatozoa, resulting in 94% of oocytes being polyspermic. When immature oocytes were cultured for 22 h in vitro, 81% extruded the first polar body and reached metaphase II. In mature oocytes, 25% of oocytes showed CG clusters, 42% and 33% of oocytes showed partial and complete CG dispersion, respectively. When mature oocytes were inseminated in vitro, only 15% of oocytes were polyspermic. Cortical granule exocytosis occurred in 97% of oocytes after sperm penetration and 84% of oocytes released all of the CG 18 h after insemination. Electric pulse induced all of the mature oocytes to release CG but only 55% released all of their CG 18 h post stimulation. These results indicate that polyspermy in immature bovine oocytes is the result of the complete failure of the oocyte to release CG after sperm penetration. Bovine oocytes became competent to release CG by sperm penetration and electric stimulation after meiotic maturation. These results provide evidence that CG exocytosis plays an important role(s) in the establishment of the block to polyspermy in bovine oocytes.

  9. Oocyte development in cattle: physiological and genetic aspects

    Directory of Open Access Journals (Sweden)

    Jack H. Britt


    Full Text Available Oocytes in cattle are formed during embryogenesis and develop within individual follicles in the cortex of the ovary. Dormant primordial follicles become active and undergo progressive development at regular intervals commencing during the late fetal stage and continuing throughout adulthood. Once activated, follicles and oocytes in a cohort either grow to maturation and ovulation or undergo atresia, ultimately depleting the ovaries of germ cells. It takes an estimated 100 days for a follicle and its oocyte to reach the mature ovulatory stage. Within an individual, number of follicles in one ovary is similar to number in the other ovary; however, there are large differences among individuals in total number of follicles present in both ovaries. In follicles that are recruited into the preovulatory pool, the fluid filled antrum enlarges and growth can be monitored by ultrasonography. Preovulatory follicles grow in waves rather than in a continuous stream, and number of follicles detected in a wave is related positively to number of microscopic follicles populating the ovarian cortex. Number of follicles in a wave is highly repeatable and the estimated heritability of number of follicles in heifers is approximately 0.35. Monitoring preovulatory follicle numbers by ultrasonography can be used to identify females that produce more embryos following superovulation or more natural twin births. Number of follicles in a wave can be influenced by energy balance and exogenous hormones, and quality of oocytes within those follicles can be influenced environmental factors, hormones and vitamins. The potential exists to increase genetic progress through repeatedly harvesting oocytes from preovulatory follicles of genetically superior females.

  10. Aurora B regulates spindle bipolarity in meiosis in vertebrate oocytes. (United States)

    Shao, Hua; Ma, Chunqi; Zhang, Xuan; Li, Ruizhen; Miller, Ann L; Bement, William M; Liu, X Johné


    Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine(122) is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity.

  11. Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification. (United States)

    Apparicio, M; Ruggeri, E; Luvoni, G C


    The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n = 72) using a vitrification kit for bovine embryo or slow frozen (n = 69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48 h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n = 92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p vitrification compared to 60.9% of those submitted to slow freezing procedure (p bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48 h of culture. © 2012 Blackwell Verlag GmbH.

  12. Calcium transients during early development in single starfish (Asterias forbesi) oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Eisen, A.; Reynolds, G.T.


    Maturation and fertilization of the starfish oocyte are putative calcium-dependent events. The authors have investigated the spatial distribution and temporal dynamics of this calcium dependence in single oocytes of Asterias forbesi. They used the calcium photoprotein, aequorin, in conjunction with a microscope-photomultiplier and microscope-image intensifier. Surprisingly, in contrast to earlier work with Marasthenias glacialis, there is no detectable increase in intracellular-free calcium in the oocyte of A. forbesi in response to the maturation hormone 1-methyl adenine. During fertilization of the same, matured, A. forbesi oocyte there is a large increase in intracellular-free calcium. The calcium concentration increases to approx.1 at the point of insemination and the region of elevated free calcium expands across the oocyte in approx.20 s (17-19/sup 0/C). After the entire oocyte reaches an elevated concentration of free calcium, the concentration decreases uniformly throughout the oocyte over the next several minutes.

  13. Utility of ultrasound stimulation for activation of pig oocytes matured in vitro. (United States)

    Sato, Keisuke; Yoshida, Mitsutoshi; Miyoshi, Kazuchika


    The present study was carried out to examine the development of pig oocytes after exposing to ultrasound under various conditions. When oocytes were exposed to ultrasound in the sorbitol medium, the blastocyst formation rate was significantly (P ultrasound in the sorbitol medium (P ultrasound with 10% duty cycle was significantly (P ultrasound with 50% duty cycle. The blastocyst formation rate of oocytes exposed to ultrasound for 30 sec was significantly (P ultrasound. The pronuclear formation and second polar body extrusion rates of oocytes exposed to ultrasound did not differ from eclectically activated oocytes. Although there was no significant difference in the blastocyst formation rates between different activation methods, the mean number of cells in the blastocysts developed from oocytes activated by exposing to ultrasound was significantly (P ultrasound stimulation can induce the nuclear activation and parthenogenetic development of pig oocytes matured in vitro.

  14. Effect of deuterium on the circadian period and metabolism in wild-type and tau mutant Syrian hamsters

    NARCIS (Netherlands)

    Oklejewicz, M; Hut, RA; Daan, S


    Homozygous tau mutant Syrian hamsters (tau-/-) have a free-running circadian period (tau) around 20 h and a proportionally higher metabolic rate compared with wild-type hamsters (tau+/+) with a period of circa 24 h. In this study, we applied deuterium oxide (D2O) to hamsters to test whether

  15. Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts from In Vitro Maturation of Bovine Oocytes

    Directory of Open Access Journals (Sweden)

    Jolanta Opiela


    Full Text Available The objective of the present study was to evaluate the effect of hyaluronan (HA during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC, and obtained blastocysts. COCs were matured in vitro in control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001 was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01. Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higher Bax mRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

  16. A Comparison of Hamster Anesthetics and Their Effect on Mosquito Blood Feeding (United States)

    Hamsters or mice are often anesthetized when they are used as the hosts for insect feeding experiments. An experiment was done to determine if there was a difference in mosquito blood feeding success when fed on hamsters anesthetized using two commonly used protocols. The number of blood-fed females...

  17. Lack of negative effects on Syrian hamsters and Mongolian gerbils housed in the same secondary enclosure. (United States)

    Pritchett-Corning, Kathleen R; Gaskill, Brianna N


    In cases where different species might be housed in the same room or secondary enclosure, the Guide for the Care and Use of Laboratory Animals recommends that the animals should be behaviorally compatible and have the same health status. Syrian hamsters and Mongolian gerbils, both desert-dwelling rodents, appear to be reasonable candidates for such a combination. This study was undertaken to evaluate whether housing hamsters and gerbils in the same secondary enclosure is an acceptable practice. Weanling and breeding-age hamsters and gerbils were housed in open-topped cages in an isolator for 5 mo; the isolator also contained with nude and haired mice, which acted as sentinels. Cages housing hamsters and gerbils were rotated between species, and dirty bedding was exchanged between species in an effort to transmit microorganisms. In addition, sentinel mice housed in the isolator were supplied with dirty bedding from both hamsters and gerbils. Neither species showed clinical signs of illness, the health status of neither the hamsters nor the gerbils changed significantly, and the sentinel mice acquired only 2 infectious organisms, a Helicobacter species and Staphylococcus aureus. Both hamsters and gerbils bred successfully when housed together in the same isolator, and no infanticide or mortality was seen. Breeding performance did not differ between isolator breeding and barrier breeding. This study supports the housing of hamsters and gerbils in the same secondary enclosure.

  18. Syrian Hamsters as a Small Animal Model for Emerging Infectious Diseases: Advances in Immunologic Methods. (United States)

    Warner, Bryce M; Safronetz, David; Kobinger, Gary P


    The use of small animal models for the study of infectious disease is critical for understanding disease progression and for developing prophylactic and therapeutic treatment options. For many diseases, Syrian golden hamsters have emerged as an ideal animal model due to their low cost, small size, ease of handling, and ability to accurately reflect disease progression in humans. Despite the increasing use and popularity of hamsters, there remains a lack of available reagents for studying hamster immune responses. Without suitable reagents for assessing immune responses, researchers are left to examine clinical signs and disease pathology. This becomes an issue for the development of vaccine and treatment options where characterizing the type of immune response generated is critical for understanding protection from disease. Despite the relative lack of reagents for use in hamsters, significant advances have been made recently with several hamster specific immunologic methods being developed. Here we discuss the progress of this development, with focus on classical methods used as well as more recent molecular methods. We outline what methods are currently available for use in hamsters and what is readily used as well as what limitations still exist and future perspectives of reagent and assay development for hamsters. This will provide valuable information to researchers who are deciding whether to use hamsters as an animal model.

  19. Diet affects resting, but not basal metabolic rate of normothermic Siberian hamsters acclimated to winter. (United States)

    Gutowski, Jakub P; Wojciechowski, Michał S; Jefimow, Małgorzata


    We examined the effect of different dietary supplements on seasonal changes in body mass (m(b)), metabolic rate (MR) and nonshivering thermogenesis (NST) capacity in normothermic Siberian hamsters housed under semi-natural conditions. Once a week standard hamster food was supplemented with either sunflower and flax seeds, rich in polyunsaturated fatty acids (FA), or mealworms, rich in saturated and monounsaturated FA. We found that neither of these dietary supplements affected the hamsters' normal winter decrease in m(b) and fat content nor their basal MR or NST capacity. NST capacity of summer-acclimated hamsters was lower than that of winter-acclimated ones. The composition of total body fat reflected the fat composition of the dietary supplements. Resting MR below the lower critical temperature of the hamsters, and their total serum cholesterol concentration were lower in hamsters fed a diet supplemented with mealworms than in hamsters fed a diet supplemented with seeds. These results indicate that in mealworm-fed hamsters energy expenditure in the cold is lower than in animals eating a seed-supplemented diet, and that the degree of FA unsaturation of diet affects energetics of heterotherms, not only during torpor, but also during normothermy. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Functional evidence for alternative ANG II-forming pathways in hamster cardiovascular system

    NARCIS (Netherlands)

    Nishimura, H; Buikema, H; Baltatu, O; Ganten, D; Urata, H


    Like human chymase, hamster chymase is an ANG II-forming enzyme, but pathophysiological roles of chymase are still unknown. We determined the functional conversion of ANG I and [Pro(11), D-Ala(12)]ANG I, a chymase-selective substrate, to ANG II in the hamster cardiovascular system. ANG I and

  1. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin


    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  2. Winter hibernation and UCHL1-p34cdc2 association in toad oocyte maturation competence.

    Directory of Open Access Journals (Sweden)

    Zhichao Kuang

    Full Text Available Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1 is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34(cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13(suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34(cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte's dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34(cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.

  3. Composition and significance of detergent resistant membranes in mouse spermatozoa. (United States)

    Nixon, Brett; Bielanowicz, Amanda; McLaughlin, Eileen A; Tanphaichitr, Nongnuj; Ensslin, Michael A; Aitken, R John


    Mammalian spermatozoa acquire the ability to fertilize an oocyte as they ascend the female reproductive tract. This process is characterized by a complex cascade of biophysical and biochemical changes collectively know as "capacitation." The attainment of a capacitated state is accompanied by a dramatic reorganization of the surface architecture to render spermatozoa competent to recognize the oocyte and initiate fertilization. Emerging evidence indicates that this process is facilitated by molecular chaperone-mediated assembly of a multimeric receptor complex on the sperm surface. However, the mechanisms responsible for gathering key recognition molecules within this putative complex have yet to be defined. In this study, we provide the first evidence that chaperones partition into detergent resistant membrane fractions (DRMs) within capacitated mouse spermatozoa and co-localize in membrane microdomains enriched with the lipid raft marker, G(M1) ganglioside. During capacitation, these microdomains coalesce within the apical region of the sperm head, a location compatible with a role in sperm-zona pellucida interaction. Significantly, DRMs isolated from spermatozoa possessed the ability to selectively bind to the zona pellucida of unfertilized, but not fertilized, mouse oocytes. A comprehensive proteomic analysis of the DRM fractions identified a total of 100 proteins, a number of which have previously been implicated in sperm-oocyte interaction. Collectively, these data provide compelling evidence that mouse spermatozoa possess membrane microdomains that provide a platform for the assembly of key recognition molecules on the sperm surface and thus present an important mechanistic insight into the fundamental cell biological process of sperm-oocyte interaction. (c) 2008 Wiley-Liss, Inc.

  4. Spontaneous nonneoplastic lesions in control Syrian hamsters in three 24-month long-term carcinogenicity studies. (United States)

    McInnes, Elizabeth F; Ernst, Heinrich; Germann, Paul-Georg


    Information about the incidence of spontaneously occurring, nonneoplastic background findings in Syrian hamsters is essential if Syrian hamsters are to be used for toxicity studies. Male and female Syrian hamsters of the strain Han:AURA from the Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM) breeding colony were maintained as control animals for carcinogenicity studies and were examined for the presence of nonneoplastic background findings either when they died or when the study was terminated. The nonneoplastic background lesions observed at an incidence of >50% (high), >25% (moderate), and >10% (low) in either male or female animals or in both sexes in one or more long-term studies are detailed. The results are compared to previous published reports of nonneoplastic, spontaneous background lesions in Syrian hamsters. Background information about the incidence of background lesions in Syrian hamsters on short- and long-term studies is useful to both toxicologists and toxicological pathologists. © 2014 by The Author(s).

  5. Mechanisms by which a lack of germinal vesicle (GV) material causes oocyte meiotic defects: a study using oocytes manipulated to replace GV with primary spermatocyte nuclei. (United States)

    Zhang, Jie; Cui, Wei; Li, Qing; Wang, Tian-Yang; Sui, Hong-Shu; Wang, Jun-Zuo; Luo, Ming-Jiu; Tan, Jing-He


    Oocytes with germinal vesicles (GVs) replaced with somatic nuclei exhibit meiotic abnormalities. Although this suggests an exclusive role for GV material in meiosis, mechanisms by which a lack of GV material causes meiotic defects are unknown. Knowledge of these mechanisms will help us to understand meiotic control, nuclear-cytoplasmic interactions, and cellular reprogramming. This study showed that although oocytes with prometaphase I chromosomes replaced with primary spermatocyte nuclei (PSN) did not, oocytes with GV replaced with PSN (PSG oocytes) did display meiotic defects. Among the defects, insufficient chromosome condensation with chromosome bridges was associated with spindle abnormalities. Abnormal spindle migration, cortical nonpolarization, and the aberrant spindle caused randomly positioning of cleavage furrows, leading to large first polar bodies (PB1) and unequal allocation of chromosomes and mitogen-activated protein kinases (MAPK) between oocyte and PB1. Spindle assembly checkpoint was activated but did not stop the incorrect division. The unequal MAPK allocation resulted in differences in pronuclear formation and PB1 degeneration; oocytes receiving more MAPK were more capable of forming pronuclear rudiments, whereas PB1 receiving more MAPK degenerated sooner than those that received less. Because none of the PSG oocytes or the enucleated GV oocytes injected with sperm heads showed cortical polarization in spite of chromosome localization close to the oolemma and because the PSG oocytes receiving more MAPK could form only pronuclear rudiments and not normal pronuclei, we suggest that the GV material plays essential roles in polarization and pronuclear formation on top of those played by chromosomes or MAPK. In conclusion, using PSG oocytes as models, this study has revealed the primary pathways by which a lack of GV material cause meiotic defects, laying a foundation for future research on the role of GV material in oocyte meiotic control.

  6. Entamoeba histolytica: liver invasion and abscess production by intraperitoneal inoculation of trophozoites in hamsters, Mesocricetus auratus. (United States)

    Shibayama, M; Campos-Rodríguez, R; Ramírez-Rosales, A; Flores-Romo, L; Espinosa-Cantellano, M; Martínez-Palomo, A; Tsutsumi, V


    Intraperitoneal inoculation of axenically cultured Entamoeba histolytica trophozoites constitutes an easy to perform, highly reproducible procedure for inducing amebic liver abscesses in hamsters. Efficiency in abscess production (95% of infected animals after 1 week) was similar to data reported using direct intrahepatic or intraportal inoculation. The morphological sequence of infection shows that amebas in the peritoneal cavity initially produce a large exudate constituted mainly of acute inflammatory cells. These cells form a rim of polymorphonuclear leukocytes surrounding the amebas, which adhere to the trophozoite and can sometimes be observed polarized to one end of the parasite, suggesting capping of surface receptors. Early stages are also characterized by the production of distant inflammatory reactions in the hepatic portal spaces. At 6 h postintraperitoneal inoculation, larger foci of inflammatory reactions surrounding amebas are developed in the peritoneum, extending to and damaging the liver surface membranes as well as the serosa of other internal organs. Thereafter, tissue damage progresses deeper into the liver parenchyma, and a few days later, coalescing granulomas and large necrotic areas are observed in the liver tissue. Based on the present morphological time-sequence study, we suggest that inflammatory cells associated with E. histolytica trophozoites play an important role in commencing the damage of liver sheaths and producing the subsequent parenchymal lesions. The simplicity and reliability of this model are important factors to consider when large numbers of experimentally induced amebic liver abscesses are needed.

  7. Syrian hamsters (Mesocricetus auratus) with simultaneous intestinal Giardia sp., Spironucleus sp., and trichomonad infections. (United States)

    Sheppard, Barbara J; Stockdale Walden, Heather D; Kondo, Hirotaka


    A commercial facility producing hamsters with a history of infection by dwarf tapeworm (Hymenolepis nana) submitted 15 animals for necropsy and postmortem parasitological and microscopic examination. No tapeworms were detected grossly or microscopically. Fecal examination including gastrointestinal mucosal smears demonstrated mixed intestinal bacteria and low numbers of Giardia sp. Histologic examination of small intestine demonstrated filling of the small intestinal crypts by large numbers of 7-9 µm × 3 µm, rod to crescent or teardrop-shaped flagellates consistent with Spironucleus sp. These organisms had two 1-µm, basophilic, oval nuclei and multiple superficial flagella-like structures. Much larger 10-15 µm × 8-10 µm, oval to pear-shaped organisms were also present in lower numbers and usually located with the crypts. These larger flagellates had multiple flagella and a basophilic rod-shaped nucleus. The larger flagellates included Giardia sp., which had an intimate interface with the surface of the mucosal epithelium, bilaterally symmetry, and binucleation. Lower numbers of trichomonads were also present and were distinguished by an undulating surface membrane and a single nucleus. The mucosa was hyperplastic and moderately inflamed. Although the tapeworm infection was resolved, diagnosis of multiple intestinal flagellates by fecal examination is complicated by the varying sensitivity and diagnostic accuracy of different types of fecal analysis for different flagellate types. Key differences in the morphology and location of the different types of flagellates as observed by histology of intestinal tissues provide important additional diagnostic information to distinguish trichomonads, Spironucleus sp., and Giardia sp.

  8. Ultrastructure of Giardia duodenalis trophozoites group from hamster: some relevant aspects

    Directory of Open Access Journals (Sweden)

    Maria Inês L. Sogayar


    Full Text Available Throphozoites of Giardia duodenalis group obtained from fragments or scratched of hamster's mucosa were examined by transission electron microscopy. The fine structure of the trophozoites are presented and comapred with those described for other animals. Some of the trophozoites present the cytoplasm full of glycogen, rough endoplasmic reticulum-like structures and homogeneous inclusions not enclosed by membranes, recognized as lipid drops, which had not been observed in Giardia from other animals. The adhesive disk is composed of a layer of microtubules, from which fibrous ribbons extend into the cytoplasm; these ribbons are linked by layer of crossbridge filaments that shows an intermediary dense band, described for the first time in this paper. The authors regard this band as the result of the cross-bridge filaments slinding in the medium region between adjacent fibrous ribbons, and suggest a contractile activity for them. The role of the adhesive disk on the trophozoite mechanism of attachment to host mucosa is also discussed.

  9. The hamster cheek pouch model for field cancerization studies. (United States)

    Monti-Hughes, Andrea; Aromando, Romina F; Pérez, Miguel A; Schwint, Amanda E; Itoiz, Maria E


    External carcinogens, such as tobacco and alcohol, induce molecular changes in large areas of oral mucosa, which increase the risk of malignant transformation. This condition, known as 'field cancerization', can be detected in biopsy specimens using histochemical techniques, even before histological alterations are identified. The efficacy of these histochemical techniques as biomarkers of early cancerization must be demonstrated in appropriate models. The hamster cheek pouch oral cancer model, universally employed in biological studies and in studies for the prevention and treatment of oral cancer, is also an excellent model of field cancerization. The carcinogen is applied in solution to the surface of the mucosa and induces alterations that recapitulate the stages of cancerization in human oral mucosa. We have demonstrated that the following can be used for the early detection of cancerized tissue: silver staining of nucleolar organizer regions; the Feulgen reaction to stain DNA followed by ploidy analysis; immunohistochemical analysis of fibroblast growth factor-2, immunohistochemical labeling of proliferating cells to demonstrate an increase of epithelial cell proliferation in the absence of inflammation; and changes in markers of angiogenesis (i.e. those indicating vascular endothelial growth factor activity, endothelial cell proliferation and vascular density). The hamster cheek pouch model of oral cancer was also proposed and validated by our group for boron neutron capture therapy studies for the treatment of oral cancer. Clinical trials of this novel treatment modality have been performed and are underway for certain tumor types and localizations. Having demonstrated the efficacy of boron neutron capture therapy to control tumors in the hamster cheek pouch oral cancer model, we adapted the model for the long-term study of field cancerized tissue. We demonstrated the inhibitory effect of boron neutron capture therapy on tumor development in field

  10. Spermatozoa as a transport system of large unilamellar lipid vesicles into the oocyte. (United States)

    Geerts, N; McGrath, J; Stronk, J N; Vanderlick, T K; Huszar, G


    In addition to their role as man-made membranes, vesicles continue to be investigated as carriers for drug delivery. While most research focuses on their injectable properties, here a new delivery strategy is proposed. It is shown that spermatozoa can transport vesicles of variable composition. For human spermatozoa, the vesicles started to show binding after 20 mol% of the nonbinding vesicle backbone lipids were substituted with positive, negative, cerebroside or ganglioside lipids. Vesicle binding is a dynamic process with constant 'on' and 'off' binding. The physiological and motility attributes of the spermatozoa are not affected by the attached vesicles. Sperm swimming characteristics changed only marginally. Also, the activation status of the acrosomal membrane, tested with the fluorescent probe Pisum sativum agglutinin, was not affected by vesicle binding. Moreover, the hyaluronic acid-binding test showed that viable, fully developed spermatozoa will attach and remain bound to hyaluronic acid-coated slides regardless of vesicle binding. Therefore a new 'hybrid' delivery system was created with human spermatozoa, and tested with a mouse IVF system. Large unilamellar vesicles physisorbed to mouse spermatozoa can not only penetrate the mouse oocytes in these proof-of-principle experiments, but also deliver the cargo placed within the vesicles. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  11. Estudios inmunologicos en hamsters (Cricetus auratus) infectados con Schistosoma mansoni


    Eduardo Monge; Paulo M Z Coelho; Tavares, Carlos A. P.


    Los resultados de este trabajo muestran que el hamster (Cricetus auratus) puede ser utilizado como un modelo experimental para estudios inmunológicos en la infección por Schistosoma mansoni. Los datos obtenidos, relativos a inmunidad concomitante, producción de anticuerpo letal e inmunosupresión se asemejan a los conseguidos en otros modelos experimentales ya establecidos. Estas observaciones indican que el hámster, además de ser un hospedero satisfactorio para el mantenimiento del parásito e...

  12. Imaging visceral leishmaniasis in real time with golden hamster model: Monitoring the parasite burden and hamster transcripts to further characterize the immunological responses of the host. (United States)

    Rouault, Eline; Lecoeur, Hervé; Meriem, Asma Ben; Minoprio, Paola; Goyard, Sophie; Lang, Thierry


    Characterizing the clinical, immunological and parasitological features associated with visceral leishmaniasis is complex. It involves recording in real time and integrating quantitative multi-parametric data sets from parasite infected host tissues. Although several models have been used, hamsters are considered the bona fide experimental model for Leishmania donovani studies. To study visceral leishmaniasis in hamsters we generated virulent transgenic L. donovani that stably express a reporter luciferase protein. Two complementary methodologies were combined to follow the infectious process: in vivo imaging using luciferase-expressing Leishmania and real time RT-PCR to quantify both Leishmania and host transcripts. This approach allows us: i) to assess the clinical outcome of visceral leishmaniasis by individual monitoring of hamster weight, ii) to follow the parasite load in several organs by real time analysis of the bioluminescence in vivo and through real time quantitative PCR analysis of amastigote parasite transcript abundance ex vivo, iii) to evaluate the immunological responses triggered by the infection by quantifying hamster transcripts on the same samples and iv) to limit the number of hamsters selected for further analysis. The overall data highlight a correlation between the transcriptional cytokine signatures of hamster affected tissues and the amastigote burden fluctuations, thus providing new insights into the immunopathological process driven by L. donovani in the tissues of mammalian hosts. Finally, they suggest organ-specific immune responses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Expression of E-cadherin and N-cadherin in perinatal hamster ovary: possible involvement in primordial follicle formation and regulation by follicle-stimulating hormone. (United States)

    Wang, Cheng; Roy, Shyamal K


    We examined the expression and hormonal regulation of E-cadherin (CDH1) and N-cadherin (CDH2) with respect to primordial follicle formation. Hamster Cdh1 and Cdh2 cDNA and amino acid sequences were more than 90% similar to those of the mouse, rat, and human. Although CDH1 expression remained exclusively in the oocytes during neonatal ovary development, CDH2 expression shifted from the oocytes to granulosa cells of primordial follicles on postnatal day (P)8. Subsequently, strong CDH2 expression was restricted to granulosa cells of growing follicles. Cdh2 mRNA levels in the ovary decreased from embryonic d 13 through P10 with a transient increase on P7, which was the day before the appearance of primordial follicles. Cdh1 mRNA levels decreased from embryonic d 13 through P3 and then showed a transient increase on P8, coinciding with the formation of primordial follicles. CDH1 and CDH2 expression were consistent with that of mRNA. Neutralization of FSH in utero impaired primordial follicle formation with an associated decrease in Cdh2 mRNA and CDH2, but an increase in Cdh1 mRNA and CDH1 expression. The altered expression was reversed by equine chorionic gonadotropin treatment on P1. Whereas a CDH2 antibody significantly reduced the formation of primordial and primary follicles in vitro, a CDH1 antibody had the opposite effect. This is the first evidence to suggest that primordial follicle formation requires a differential spatiotemporal expression and action of CDH1 and CDH2. Further, FSH regulation of primordial follicle formation may involve the action of CDH1 and CDH2.

  14. Localization of calcium in differentiating odontoblasts and ameloblasts before and during early dentinogenesis and amelogenesis in hamster tooth germs. (United States)

    Lyaruu, D M; Bronckers, A L; Burger, E H; Wöltgens, J H


    Potassium pyroantimonate-osmium tetroxide cytochemistry has been used to study the distribution of ionic calcium in hamster tooth germs during cell differentiation and during early dentinogenesis and amelogenesis. Before the onset of mineralization, pyroantimonate (PA) reaction product was found in the nucleus of differentiating preameloblasts and preodontoblasts. In the predentin, it was preferentially located along striated collagen fibrils, lying perpendicular to the basal lamina. At the onset of mineralization, a pronounced increase of PA reaction product was evident in the predentin and on the plasma membrane and in mitochondria of both preodontoblasts and preameloblasts opposite the mineralizing mantle dentin. During early enamel mineralization, PA reaction product was present in the "growing" crystal ends, while in the secretory ameloblasts, most of the PA reaction product was localized on the cytoplasmic side of the apical plasma membranes and in mitochondria. When Tomes' processes developed, PA reaction product, both cytoplasmic and membrane bound, was low or absent deep in the processes, but gradually increased toward the apical terminal web. A corresponding gradient of PA reaction product was observed on the opposing enamel crystallites. From this study we conclude that both preodontoblasts and preameloblasts seem to be involved in calcium acquisition necessary for the early stages of mantle dentin mineralization. Tomes' processes seem to regulate the entry of calcium into the enamel mineralization front.

  15. Raman micro-spectroscopy can be used to investigate the developmental stage of the mouse oocyte.

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    Bryony Davidson

    Full Text Available In recent years, the uptake of assisted reproductive techniques such as in vitro fertilisation has risen exponentially. However, there is much that is still not fully understood about the biochemical modifications that take place during the development and maturation of the oocyte. As such, it is essential to further the understanding of how oocyte manipulation during these procedures ultimately affects its developmental potential; yet, there are few methods currently available which are capable of providing a quantitative measure of oocyte quality. Raman spectroscopy enables investigation of the global biochemical profile of intact cells without the need for labelling. Here, Raman spectra were acquired from the ooplasm of mouse oocytes at various stages of development, from late pre-antral follicles, collected after in vitro maturation within their ovarian follicles and from unstimulated and stimulated ovulatory cycles. Using a combination of univariate and multivariate statistical methods, it was found that ooplasm lipid content could be used to discriminate between different stages of oocyte development. Furthermore, the spectral profiles of mature oocytes revealed that oocytes which have developed in vitro are protein-deficient when compared to in vivo grown oocytes. Finally, the ratio of two Raman peak intensities, namely 1605∶1447 cm⁻¹, used as a proxy for the protein-to-lipid ratio of the ooplasm, was shown to be indicative of the oocyte's quality. Together, results indicate that Raman spectroscopy may present an alternative analytical tool for investigating the biochemistry of oocyte developmental stage and quality.

  16. Release of sICAM-1 in oocytes and in vitro fertilized human embryos.

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    Monica Borgatti

    Full Text Available During the last years, several studies have reported the significant relationship between the production of soluble HLA-G molecules (sHLA-G by 48-72 hours early embryos and an increased implantation rate in IVF protocols. As consequence, the detection of HLA-G modulation was suggested as a marker to identify the best embryos to be transferred. On the opposite, no suitable markers are available for the oocyte selection.The major finding of the present paper is that the release of ICAM-1 might be predictive of oocyte maturation. The results obtained are confirmed using three independent methodologies, such as ELISA, Bio-Plex assay and Western blotting. The sICAM-1 release is very high in immature oocytes, decrease in mature oocytes and become even lower in in vitro fertilized embryos. No significant differences were observed in the levels of sICAM-1 release between immature oocytes with different morphological characteristics. On the contrary, when the mature oocytes were subdivided accordingly to morphological criteria, the mean sICAM-I levels in grade 1 oocytes were significantly decreased when compared to grade 2 and 3 oocytes.The reduction of the number of fertilized oocytes and transferred embryos represents the main target of assisted reproductive medicine. We propose sICAM-1 as a biochemical marker for oocyte maturation and grading, with a possible interesting rebound in assisted reproduction techniques.

  17. In Vitro Maturation and Embryo Development to blastocyst Mouse Germinal Vesicle Oocytes after Vitrification

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    M Nikseresht


    Full Text Available Abstract Background & aim: Vitrification is a simple and ultra rapid technique for the conservation of fertility. Improving pregnancy rate associate with the use of cryopreserved oocytes would be an important advanced in human assisted reproductive technology (ART. The purpose of this study was to evaluate survival, oocytes maturation and embryo development to the blastocyst stage after vitrification of oocytes germinal vesicle-stage and multi stage Methods: In the present experimental study, germinal vesicle oocytes with or without cumulus cells were transferred to vitrification solution containing 30% (v/v ethylene glycol, 18% (w/v Ficoll-70, and 0.3 M sucrose, either by single step or in a step-wise way. After vitrification and storage in liquid nitrogen, the oocytes were thawed and washed twice in culture medium TCM119, and then subjected to in vitro maturation, fertilization, and culture. Data analysis was performed by using One-way variance and Tukey tests. Results: Oocytes survival, metaphase 2 stage oocyte maturation, fertilization and embryo formed blastocyst in vitrification methods multistage were significantly higher than the single step procedure (P<0/05 Conclusion: The Germinal vesicle stage oocytes vitrified with cumulus cells and stepwise procedure had positive effect on the survival, maturation and developmental rate on blastocyst compared to oocytes without cumulus cell and single step procedure. Key words: Germinal Vesicle Oocyte, Blastocyst, Vitrification, Ethylene glycol

  18. Use of Rat Estrus Serum for in Vitro Maturation of Bovine Oocytes

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    AR Rafati


    Full Text Available Introduction: Superovulation produces complications in some patients, so invitro maturation of oocytes is used to decrease or eliminate these complications and improve IVF. Moreover, IVM is used for different aspects of reproductive researches. Slaughterhouse ovaries are the main source of oocytes for IVM and IVF studies. Different media has been introduced and experimented for in vitro maturation of oocytes. Animal's serum at estrus stage contains different hormones and proteins which are essential for oocyte maturation. The aim of this study was to compare three culture media for in vitro maturation (IVM of bovine oocytes; 1(controlTCM-199, 2HCG and follicular fluid (FF and 3 antibiotic. Methods: Rat estrus serum (RSS or fetal bovine serum (FBS was added to control medium. Total of 1789 compact cumulus oocyte complexes (COCs were aspirated from ovaries of slaughtered animals. Oocytes were randomly cultured in mentioned media and incubated in 38.5◦c, 5% CO2 and 95% humidity for 24 hours. The maturation of oocytes was judged according to cumulus cell expansion or randomly orcein stained oocytes and observation of polar bodies. Results: The results showed that maturation rate was significantly higher in second and third group (90.2%, 78.7% as compared to the control group (p<0.001. There was no significant difference between second and third groups (90.2 % vs. 86.6%. Conclusion: RSS is as effective as FBS for IVM of bovine oocytes and can be used as an alternative.

  19. Developmental potential of prepubertal mouse oocytes is compromised due mainly to their impaired synthesis of glutathione.

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    Guang-Zhong Jiao

    Full Text Available Although oocytes from prepubertal animals are found less competent than oocytes from adults, the underlying mechanisms are poorly understood. Using the mouse oocyte model, this paper has tested the hypothesis that the developmental potential of prepubertal oocytes is compromised due mainly to their impaired potential for glutathione synthesis. Oocytes from prepubertal and adult mice, primed with or without eCG, were matured in vitro and assessed for glutathione synthesis potential, oxidative stress, Ca(2+ reserves, fertilization and in vitro development potential. In unprimed mice, abilities for glutathione synthesis, activation, male pronuclear formation, blastocyst formation, cortical granule migration and polyspermic block were all compromised significantly in prepubertal compared to adult oocytes. Cysteamine and cystine supplementation to maturation medium significantly promoted oocyte glutathione synthesis and blastocyst development but difference due to maternal age remained. Whereas reactive oxygen species (ROS levels increased, Ca(2+ storage decreased significantly in prepubertal oocytes. Levels of both catalytic and modifier subunits of the γ-glutamylcysteine ligase were significantly lower in prepubertal than in adult oocytes. Maternal eCG priming improved all the parameters and eliminated the age difference. Together, the results have confirmed our hypothesis by showing that prepubertal oocytes have a decreased ability to synthesize glutathione leading to an impaired potential to reduce ROS and to form male pronuclei and blastocysts. The resulting oxidative stress decreases the intracellular Ca(2+ store resulting in impaired activation at fertilization, and damages the microfilament network, which affects cortical granule redistribution leading to polyspermy.

  20. Vitrification of human immature oocytes before and after in vitro maturation: a review. (United States)

    Khalili, Mohammad Ali; Shahedi, Abbas; Ashourzadeh, Sareh; Nottola, Stefania Annarita; Macchiarelli, Guido; Palmerini, Maria Grazia


    The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.

  1. Protein deubiquitination during oocyte maturation influences sperm function during fertilisation, antipolyspermy defense and embryo development. (United States)

    Yi, Young-Joo; Sutovsky, Miriam; Song, Won-Hee; Sutovsky, Peter


    Ubiquitination is a covalent post-translational modification of proteins by the chaperone protein ubiquitin. Upon docking to the 26S proteasome, ubiquitin is released from the substrate protein by deubiquitinating enzymes (DUBs). We hypothesised that specific inhibitors of two closely related oocyte DUBs, namely inhibitors of the ubiquitin C-terminal hydrolases (UCH) UCHL1 (L1 inhibitor) and UCHL3 (L3 inhibitor), would alter porcine oocyte maturation and influence sperm function and embryo development. Aberrant cortical granule (CG) migration and meiotic spindle defects were observed in oocytes matured with the L1 or L3 inhibitor. Embryo development was delayed or blocked in oocytes matured with the general DUB inhibitor PR-619. Aggresomes, the cellular stress-inducible aggregates of ubiquitinated proteins, formed in oocytes matured with L1 inhibitor or PR-619, a likely consequence of impaired protein turnover. Proteomic analysis identified the major vault protein (MVP) as the most prominent protein accumulated in oocytes matured with PR-619, suggesting that the inhibition of deubiquitination altered the turnover of MVP. The mitophagy/autophagy of sperm-contributed mitochondria inside the fertilised oocytes was hindered by DUB inhibitors. It is concluded that DUB inhibitors alter porcine oocyte maturation, fertilisation and preimplantation embryo development. By regulating the turnover of oocyte proteins and mono-ubiquitin regeneration, the DUBs may promote the acquisition of developmental competence during oocyte maturation.

  2. Oocyte development and fecundity type of the skipjack, Katsuwonus pelamis, in the Western Indian Ocean (United States)

    Grande, Maitane; Murua, Hilario; Zudaire, Iker; Korta, Maria


    The study aims to define the oogenesis pattern of the skipjack (Katsuwonus pelamis) of the Western Indian Ocean basin in terms of oocyte growth and recruitment style. The main objective is to define the type of fecundity regulation (i.e. determinate or indeterminate) based on four lines of evidence: (a) oocyte size-frequency distribution; (b) seasonal variation of the relative number and percentage of oocyte stages, (c) diameter of the advanced vitellogenic oocytes in females in the spawning capable phase; and (d) incidence of atresia throughout the spawning season. The samples were collected from 2009 to 2010 in the Western Indian Ocean, and 673 ovaries were classified in the different reproductive phases using histological staging. Moreover, the oocyte size distribution of 93 mature individuals was described by the newly implemented image analysis method. This species showed a broad oocyte size frequency distribution with no gap formation between the primary and secondary oocyte growth stages. There was no seasonal variation in the percentage of oocyte stages in ovaries in the spawning capable phase, and the diameter of those oocytes at the most advanced vitellogenic stage was approximately constant during the sampling period. These facts provide evidence of continuous oocyte recruitment into the standing stock of developing oocytes. Moreover, when reaching the end of the active reproductive period (i.e. February and March) the prevalence of atresia increased. This is a mechanism adopted by fishes of the indeterminate fecundity type to reabsorb the surplus oocyte production. Based on the findings, we state that the skipjack in the Western Indian Ocean shows asynchronous oocyte growth and an indeterminate fecundity type.

  3. In vitro fertilization of porcine oocytes is affected by spermatic coincubation time

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    Guilherme Oberlender

    Full Text Available Abstract: The aim was to study the effects of different gamete coincubation times on porcine in vitro fertilization (IVF, and to verify whether efficiency could be improved by reducing oocyte exposure time to spermatozoa during IVF. In groups of 50, a total of 508 immature cumulus-oocyte complexes (COCs were matured in NCSU-37 medium. The COCs were cultured for 44 hours and then inseminated with in natura semen (2,000 spermatozoa/oocyte. The sperm and oocytes were coincubated according to the following treatments (T: T1 = oocytes exposed to spermatozoa for one hour (173 oocytes, T2 = oocytes exposed to spermatozoa for two hours (170 oocytes, and T3 = oocytes exposed to spermatozoa for three hours (165 oocytes. After these coincubation periods, the oocytes were washed in fertilization medium (TALP medium to remove spermatozoa not bound to the zona pellucida and cultured in another similar medium (containing no sperm. Eighteen to twenty hours after fertilization, the putative zygotes were stained in Hoechst-33342 to evaluate the IVF results. The penetration rate was higher (P0.05 between oocytes exposed to spermatozoa for one (T1 and three hours (T3. However, optimum (P=0.048 results were obtained after two hours of coincubation, when the rate of fertilization performance was 50.16±8.52%. The number of penetrated sperm per oocyte, as well as male pronucleus formation, did not differ (P>0.05 between the treatments evaluated. Under these assay conditions, especially in relation to the sperm concentration used, gamete coincubation for a period of two hours appears to be optimal for monospermy and fertilization performance. Thus, it is the optimal time period for obtaining a large number of pig embryos capable of normal development.

  4. Multiple requirements of PLK1 during mouse oocyte maturation.

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    Petr Solc

    Full Text Available Polo-like kinase 1 (PLK1 orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1's functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC. Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.

  5. Anastral spindle assembly and γ-tubulin in Drosophila oocytes

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    Hallen Mark A


    Full Text Available Abstract Background Anastral spindles assemble by a mechanism that involves microtubule nucleation and growth from chromatin. It is still uncertain whether γ-tubulin, a microtubule nucleator essential for mitotic spindle assembly and maintenance, plays a role. Not only is the requirement for γ-tubulin to form anastral Drosophila oocyte meiosis I spindles controversial, but its presence in oocyte meiosis I spindles has not been demonstrated and is uncertain. Results We show, for the first time, using a bright GFP fusion protein and live imaging, that the Drosophila maternally-expressed γTub37C is present at low levels in oocyte meiosis I spindles. Despite this, we find that formation of bipolar meiosis I spindles does not require functional γTub37C, extending previous findings by others. Fluorescence photobleaching assays show rapid recovery of γTub37C in the meiosis I spindle, similar to the cytoplasm, indicating weak binding by γTub37C to spindles, and fits of a new, potentially more accurate model for fluorescence recovery yield kinetic parameters consistent with transient, diffusional binding. Conclusions The FRAP results, together with its mutant effects late in meiosis I, indicate that γTub37C may perform a role subsequent to metaphase I, rather than nucleating microtubules for meiosis I spindle formation. Weak binding to the meiosis I spindle could stabilize pre-existing microtubules or position γ-tubulin for function during meiosis II spindle assembly, which follows rapidly upon oocyte activation and completion of the meiosis I division.

  6. Effect of guaianolides in the meiosis reinitiation of amphibian oocytes. (United States)

    Zapata-Martínez, J; Sánchez-Toranzo, G; Chaín, F; Catalán, C A N; Bühler, M I


    Sesquiterpene lactones (STLs) are a large and structurally diverse group of plant metabolites generally found in the Asteraceae family. STLs exhibit a wide spectrum of biological activities and it is generally accepted that their major mechanism of action is the alkylation of the thiol groups of biological molecules. The guaianolides is one of various groups of STLs. Anti-tumour and anti-migraine effects, an allergenic agent, an inhibitor of smooth muscle cells and of meristematic cell proliferation are only a few of the most commonly reported activities of STLs. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under stimulus with progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. There are previous records of the inhibitory effect of dehydroleucodin (DhL), a guaianolide lactone, on the progression of meiosis. It has been also shown that DhL and its 11,13-dihydroderivative (2H-DhL; a mixture of epimers at C-11) act as blockers of the resumption of meiosis in fully grown ovarian oocytes from the amphibian Rhinella arenarum (formerly classified as Bufo arenarum). The aim of this study was to analyze the effect of four closely related guaianolides, i.e., DhL, achillin, desacetoxymatricarin and estafietin as possible inhibitors of meiosis in oocytes of amphibians in vitro and discuss some structure-activity relationships. It was found that the inhibitory effect on meiosis resumption is greater when the lactone has two potentially reactive centres, either a α,β-α',β'-diunsaturated cyclopentanone moiety or an epoxide group plus an exo-methylene-γ-lactone function.

  7. Social forces can impact the circadian clocks of cohabiting hamsters (United States)

    Paul, Matthew J.; Indic, Premananda; Schwartz, William J.


    A number of field and laboratory studies have shown that the social environment influences daily rhythms in numerous species. However, underlying mechanisms, including the circadian system's role, are not known. Obstacles to this research have been the inability to track and objectively analyse rhythms of individual animals housed together. Here, we employed temperature dataloggers to track individual body temperature rhythms of pairs of cohabiting male Syrian hamsters (Mesocricetus auratus) in constant darkness and applied a continuous wavelet transform to determine the phase of rhythm onset before, during, and after cohabitation. Cohabitation altered the predicted trajectory of rhythm onsets in 34% of individuals, representing 58% of pairs, compared to 12% of hamsters single-housed as ‘virtual pair’ controls. Deviation from the predicted trajectory was by a change in circadian period (τ), which tended to be asymmetric—affecting one individual of the pair in nine of 11 affected pairs—with hints that dominance might play a role. These data implicate a change in the speed of the circadian clock as one mechanism whereby social factors can alter daily rhythms. Miniature dataloggers coupled with wavelet analyses should provide powerful tools for future studies investigating the principles and mechanisms mediating social influences on daily timing. PMID:24500164

  8. Anti-hypercholesterolemic effect of Saccharomyces boulardii in the hamster. (United States)

    Girard, Philippe; Pansart, Yannick; Verleye, Marc


    Hypercholesterolemia is a major risk factor for coronary artery disease and probiotics have been suggested as tools to manage elevated cholesterol levels. The present study investigated the ability of the biotherapeutic agent Saccharomyces boulardii (Sb-Biocodex) to reduce the hypercholesterolemia induced by a 0.1% cholesterol-enriched diet in the hamster. In a first experiment, chronic oral treatment with S. boulardii at 12 × 10(10) CFU/kg (3 g/kg) twice a day was started from the beginning of the cholesterol diet and continued for 14 days ('preventive protocol'). In the second experiment, S. boulardii was given 14 days after the beginning of the cholesterol diet when hypercholesterolemia had developed and continued for an additional 14 days ('curative protocol'). In the preventive protocol, administration of the yeast significantly reduced hypercholesterolemia (14%) induced by the cholesterol-enriched diet compared to the group receiving only the cholesterol diet. In the curative protocol, S. boulardii significantly reduced hypercholesterolemia (12%) induced by the cholesterol-enriched diet, too. Moreover, the yeast significantly decreased the serum triglyceride increase by 39%. S. boulardii possesses anti-hypercholesterolemic properties in the hamster worthy of further evaluation in clinical studies. © 2014 S. Karger AG, Basel.

  9. Serum lysophospholipid levels are altered in dyslipidemic hamsters. (United States)

    Suárez-García, Susana; Caimari, Antoni; Del Bas, Josep Maria; Suárez, Manuel; Arola, Lluís


    Dyslipidemias are common disorders that predispose individuals to severe diseases. It is known that healthy living habits can prevent dyslipidemias if they are diagnosed properly. Therefore, biomarkers that assist in diagnosis are essential. The aim of this study was to identify biomarkers of dyslipidemia progression, which in turn disclose its etiology. These findings will pave the way for examinations of the regulatory mechanisms involved in dyslipidemias. Hamsters were fed either a normal-fat diet (NFD) or a high-fat diet. Some of the NFD-fed animals were further treated with the hyperlipidemic agent Poloxamer 407. Non-targeted metabolomics was used to investigate progressive changes in unknown serum metabolites. The hepatic expression of putative biomarker-related genes was also analyzed. The serum levels of lysophospholipids (Lyso-PLs) and their related enzymes lecithin-cholesterol acyltransferase (LCAT), secreted phospholipase A2 (sPLA2) and paraoxonase-1 were altered in dyslipidemic hamsters. Lysophosphatidylcholine levels were increased in diet-induced dyslipidemic groups, whereas lysophosphatidylethanolamine levels increased in response to the chemical treatment. The liver was significantly involved in regulating the levels of these molecules, based on the modified expression of endothelial lipase (Lipg), sPLA2 (Pla2g2a) and acyltransferases (Lcat and Lpcat3). We concluded that Lyso-PL evaluation could aid in the comprehensive diagnosis and management of lipid disorders.

  10. Differentiation of hamster liver oval cell following Clonorchis sinensis infection. (United States)

    Yoon, B I; Jung, S Y; Hur, K; Lee, J H; Joo, K H; Lee, Y S; Kim, D Y


    Oval cells which appear in the liver after hepatic injuries are suspected to be progenitor cells for both hepatocytes and bile duct cells. Oval cell isolated from the livers of the hamsters treated with diethylnitrosamine and 2-acetylaminofluorene and infected with Clonorchis sinensis (CS). cultured for 2 weeks and evaluated for differentiation and plasticity by electron microscopy and immunohistochemistry. In the CS-uninfected group, glycogen granules and peroxisomes were noted in the cells that were cultured for 2 weeks. Starting at 1 week postculture, immunoreactivity of the cells to cytokeratin 19 markedly decreased but that to albumin and alpha-fetoprotein gradually increased. This means that oval cells isolated from hamsters that were not infected with CS differentiated toward hepatocyte lineage. However, in the CS-infected group, cultured cells contained numerous rough endoplasmic reticulum and showed immunoreactivity that was generally in reverse to that of CS-uninfected group, meaning that cells isolated following CS infection were primed by CS and differentiated toward bile duct cell lineage. The results of this study suggested that oval cells are indeed bipolar progenitor cells for hepatocytes and bile duct cells and can differentiate toward either lineage depending upon the priming factor.

  11. Expression of thioredoxin during progression of hamster and human cholangiocarcinoma. (United States)

    Yoon, Byung-Il; Kim, Yeong-Hun; Yi, Jung-Yeon; Kang, Min-Soo; Jang, Ja-June; Joo, Kyoung-Hwan; Kim, Yongbaek; McHugh Law, J; Kim, Dae-Yong


    Thioredoxin (Trx) is a multifunctional redox protein that has growth-promoting and anti-apoptotic effects on cells and protects cells from endogenous and exogenous free radicals. Recently, altered expression of Trx has been reported in various cancers. In the present study, we investigated altered expression of Trx at the precancerous and carcinogenic phases during cholangiocarcinogenesis in a hamster cholangiocarcinoma (ChC) model, using semiquantitative immunohistochemical and Western blot analyses. Moreover, to determine if the results correlated well with those in human ChCs, we carried out a comparative immunohistochemical study for Trx in tissue-arrayed human ChCs with different grades of tumor cell differentiation. Trx was found highly expressed in the cytoplasm of dysplastic bile ducts with highly abnormal growth patterns and ChCs irrespective of tumor type or tumor cell differentiation. Overexpression of Trx at the precancerous and carcinogenic phases was further supported by significant elevation of Trx protein in Western blotting. The results from the hamster ChCs were in good agreement with those from human ChCs. Our results strongly suggested that the redox regulatory function of Trx plays an important role in bile duct cell transformation and tumor progression during cholangiocarcinogenesis.

  12. Developmental competence of ovine oocyte following vitrification: effect of oocyte developmental stage, cumulus cells, cytoskeleton stabiliser, FBS concentration, and equilibration time. (United States)

    Shirazi, Abolfazl; Taheri, Fatemeh; Nazari, Hassan; Norbakhsh-Nia, Maryam; Ahmadi, Ebrahim; Heidari, Banafsheh


    The aim of the present study was to examine the effects of fetal bovine serum (FBS) concentration, equilibration time, and oocyte pre-treatment with cytochalasin B (CCB) on subsequent development of vitrified-warmed ovine immature (GVCOCs) and matured (MII) oocytes with (MIICOCs) or without cumulus cells (MIIDOs). In Experiment 1, the effects of FBS concentrations (10 and 20%) during the vitrification-warming procedure were examined. Survival rates after warming were not different between GVCOCs, MIICOCs and MIIDOs oocytes. After in vitro fertilization, rate of cleaved embryos in MIICOCs group at the presence of 20%FBS was higher than MIIDOs and GVCOCs groups. In Experiment 2, the effects of equilibration times (5, 7, and 10 min) were examined. There was no difference in survival rate of vitrified-warmed oocytes equilibrated at different times. Although, the rate of cleavage in MIICOCs and MIIDOs oocytes equilibrated for 10 and 7 min, respectively, was higher than 5 min equilibrated MIIDOs and 7 and 10 min equilibrated GVCOCs oocytes. In Experiment 3, the effects of oocyte pre-treatment with CCB were examined. Despite the insignificant difference in survival rate of vitrified-warmed ovine immature and matured oocytes, the rates of cleavage in CCB pretreated groups were significantly lower than untreated groups. Moreover, the blastocysts were only derived from those cumulus enclosed vitrified-warmed germinal vesicle (GV) and MII oocytes that had been exposed to 10% FBS in the absence of CCB. In conclusion, the presence of cumulus cells, 10% FBS, and the omission of CCB were beneficial for post-warming development of vitrified ovine oocytes.

  13. Prematuration with cyclic adenosine monophosphate modulators alters cumulus cell and oocyte metabolism and enhances developmental competence of in vitro-matured mouse oocytes. (United States)

    Zeng, Hai-Tao; Richani, Dulama; Sutton-McDowall, Melanie L; Ren, Zi; Smitz, Johan E J; Stokes, Yvonne; Gilchrist, Robert B; Thompson, Jeremy G


    Oocyte in vitro maturation (IVM) is an important assisted reproductive technology and research tool. The adoption of IVM into routine clinical practice has been hindered by its significantly lower success rates compared to conventional in vitro fertilization. Cyclic AMP (cAMP) modulation and follicle-stimulating hormone (FSH), independently, have long been known to improve IVM oocyte developmental competence. This study comprehensively examined the effects of FSH and cAMP/cGMP modulation, alone and in combination, on IVM oocyte metabolism and developmental outcomes. Mouse cumulus-oocyte complexes (COCs) were subjected to a 1 h prematuration phase ± the cAMP modulator forskolin and cAMP/cGMP modulator 3-isobutyl-1-methylxanthine followed by IVM ± FSH. Prematuration with these cyclic nucleotide modulators or IVM with FSH significantly improved oocyte developmental competence and reduced spindle abnormalities compared to spontaneous IVM (no treatment); however, these two treatments in combination endowed even greater developmental competence (improved subsequent blastocyst rates and quality; P < 0.05), albeit blastocyst yield and quality remained significantly lower than that of oocytes matured in vivo. A significant additive effect of combined IVM treatments was evident as increased COC lactate production and oxygen consumption and enhanced oocyte oxidative metabolism, ATP production, ATP:ADP ratio, and glutathione levels (P < 0.05). Nevertheless, IVM increased reactive oxygen species production, particularly as a consequence of FSH addition, relative to in vivo matured oocytes. In conclusion, improvements in the embryo yield following IVM is associated with increased COC oxygen consumption and oocyte oxidative metabolism, but these remain metabolically and developmentally less competent relative to in vivo derived oocytes. © 2014 by the Society for the Study of Reproduction, Inc.

  14. A cdk1 gradient guides surface contraction waves in oocytes. (United States)

    Bischof, Johanna; Brand, Christoph A; Somogyi, Kálmán; Májer, Imre; Thome, Sarah; Mori, Masashi; Schwarz, Ulrich S; Lénárt, Péter


    Surface contraction waves (SCWs) in oocytes and embryos lead to large-scale shape changes coupled to cell cycle transitions and are spatially coordinated with the cell axis. Here, we show that SCWs in the starfish oocyte are generated by a traveling band of myosin II-driven cortical contractility. At the front of the band, contractility is activated by removal of cdk1 inhibition of the RhoA/RhoA kinase/myosin II signaling module, while at the rear, contractility is switched off by negative feedback originating downstream of RhoA kinase. The SCW's directionality and speed are controlled by a spatiotemporal gradient of cdk1-cyclinB. This gradient is formed by the release of cdk1-cyclinB from the asymmetrically located nucleus, and progressive degradation of cyclinB. By combining quantitative imaging, biochemical and mechanical perturbations with mathematical modeling, we demonstrate that the SCWs result from the spatiotemporal integration of two conserved regulatory modules, cdk1-cyclinB for cell cycle regulation and RhoA/Rok/NMYII for actomyosin contractility.Surface contraction waves (SCWs) are prominent shape changes coupled to cell cycle transitions in oocytes. Here the authors show that SCWs are patterned by the spatiotemporal integration of two conserved modules, cdk1-cyclinB for cell cycle regulation and RhoA/Rok/NMYII for actomyosin contractility.

  15. Ca2+ Homeostasis Regulates Xenopus Oocyte Maturation1 (United States)

    Sun, Lu; Hodeify, Rawad; Haun, Shirley; Charlesworth, Amanda; MacNicol, Angus M.; Ponnappan, Subramaniam; Ponnappan, Usha; Prigent, Claude; Machaca, Khaled


    In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte’s competence to undergo maturation in preparation for fertilization and embryonic development. PMID:18094360

  16. Storage time does not modify the gene expression profile of cryopreserved human metaphase II oocytes. (United States)

    Stigliani, Sara; Moretti, Stefano; Anserini, Paola; Casciano, Ida; Venturini, Pier Luigi; Scaruffi, Paola


    Does storage time have any impact on the transcriptome of slowly frozen cryopreserved human metaphase II (MII) oocytes? The length of cryostorage has no effect on the gene expression profile of human MII oocytes. Oocyte cryopreservation is a widely used technique in IVF for storage of surplus oocytes, as well as for fertility preservation (i.e. women undergoing gonadotoxic therapies) and oocyte donation programs. Although cryopreservation has negative impacts on oocyte physiology and it is associated with decrease of transcripts, no experimental data about the effect of storage time on the oocyte molecular profile are available to date. This study included 27 women, ≤38 years aged, without any ovarian pathology, undergoing IVF treatment. Surplus MII oocytes were donated after written informed consent. A total of 31 non-cryopreserved oocytes and 68 surviving slow-frozen/rapid-thawed oocytes (32 oocytes cryostored for 3 years and 36 cryostored for 6 years) were analyzed. Pools of ≈10 oocytes for each group were prepared. Total RNA was extracted from each pool, amplified, labeled and hybridized on oligonucleotide microarrays. Analyses were performed by R software using the limma package. Comparison of gene expression profiles between surviving thawed oocytes after 3 and 6 years of storage in liquid nitrogen found no differently expressed genes. The expression profiles of cryopreserved MII oocytes significantly differed from those of non-cryopreserved oocytes in 107 probe sets corresponding to 73 down-regulated and 29 up-regulated unique transcripts. Gene Ontology analysis by DAVID bioinformatics resource disclosed that cryopreservation deregulates genes involved in oocyte function and early embryo development, such as chromosome organization, RNA splicing and processing, cell cycle, cellular response to DNA damage and to stress, DNA repair, calcium ion binding, malate dehydrogenase activity and mitochondrial activity. Among the probes significantly up-regulated in

  17. A lethal disease model for New World hantaviruses using immunosuppressed Syrian hamsters. (United States)

    Vergote, Valentijn; Laenen, Lies; Vanmechelen, Bert; Van Ranst, Marc; Verbeken, Erik; Hooper, Jay W; Maes, Piet


    Hantavirus, the hemorrhagic causative agent of two clinical diseases, is found worldwide with variation in severity, incidence and mortality. The most lethal hantaviruses are found on the American continent where the most prevalent viruses like Andes virus and Sin Nombre virus are known to cause hantavirus pulmonary syndrome. New World hantavirus infection of immunocompetent hamsters results in an asymptomatic infection except for Andes virus and Maporal virus; the only hantaviruses causing a lethal disease in immunocompetent Syrian hamsters mimicking hantavirus pulmonary syndrome in humans. Hamsters, immunosuppressed with dexamethasone and cyclophosphamide, were infected intramuscularly with different New World hantavirus strains (Bayou virus, Black Creek Canal virus, Caño Delgadito virus, Choclo virus, Laguna Negra virus, and Maporal virus). In the present study, we show that immunosuppression of hamsters followed by infection with a New World hantavirus results in an acute disease that precisely mimics both hantavirus disease in humans and Andes virus infection of hamsters. Infected hamsters showed specific clinical signs of disease and moreover, histological analysis of lung tissue showed signs of pulmonary edema and inflammation within alveolar septa. In this study, we were able to infect immunosuppressed hamsters with different New World hantaviruses reaching a lethal outcome with signs of disease mimicking human disease.

  18. Effect of naltrexone on food intake and body weight in Syrian hamsters depends on metabolic status. (United States)

    Jones, Juli E; Corp, Eric S


    Opioids are a family of neuropeptides involved in the control of food intake and regulation of body weight. In general, nonselective opioid antagonists have inhibited food intake in a variety of paradigms in rodent species. Syrian hamsters may be an exception to the general findings. In a previous report, we showed that systemic administration of an opioid antagonist, naltrexone, for 2 days increased body weight in female Syrian hamsters. To confirm the extent of these finding we designed the present experiment testing the effect of a chronic 6-day infusion of naltrexone on food intake, water intake, and body weight in freely feeding male hamsters. In addition, we examined the effect of acute administration of naltrexone on food intake in both ad-libitum-fed and food-deprived hamsters. We found that chronic systemic administration of naltrexone caused a significant increase in food intake and body weight. Second, acute administration of naltrexone decreased food intake after a 48-h fast but had no effect in ad-libitum-fed hamsters. Water consumption was not altered in any experimental paradigm. Our results suggest that opioid circuits in Syrian hamsters may function tonically to suppress food intake and body weight when Syrian hamsters are in positive energy balance. Paradoxically, opioids may enhance food intake after a sustained fast.


    Directory of Open Access Journals (Sweden)

    A. Mohammadi-Rousheh


    Full Text Available Rate of in vitro maturation of oocytes is one of the challenges of assisted reproductive techniques. In this study we investigated the effects of supplementation of cysteamine on the rate of in vitro maturation of oocytes in two different media. Germinal vesicle oocytes were collected from mouse ovary and cultured in two media (TCM199 and MEME with 0, 50, 100, 200, 500 µM/ml cysteamine. Number of germinal vesicle breakdowns and metaphase II oocytes were recorded. The results showed that the rate of in vitro maturation in 100 µM/ml cysteamine was significantly higher compared to control (P < 0.05. Evaluation of two media in this study showed that TCM199 improved the rate of in vitro maturation and oocyte maturation better than MEME; however, this difference was not statistically significant. These findings indicate that TCM199 as compared to MEME was better in rate of in vitro maturation of oocytes.

  20. Conservation of RNA polymerase during maturation of the Rana pipiens oocyte

    Energy Technology Data Exchange (ETDEWEB)

    Hollinger, T.G.; Smith, L.D.


    DNA-dependent RNA polymerase was extracted from oocytes of the frog, Rana pipiens. The bulk of the enzyme activity was present in the germinal vesicle and the amounts of each major form of such activity did not significantly change during oocyte maturation. Therefore, either nuclear polymerase activity is conserved after breakdown of the oocyte nucleus during maturation or, alternatively, de novo synthesis of the enzymes must occur during oocyte maturation concomitant with degradation. We have measured rates of protein synthesis in oocytes and determined a maximum rate of synthesis for RNA polymerases. Our kinetic studies show that no more than 20, 10, and 5 percent of RNA polymerases type I, IIa, and IIb, respectively, could be synthesized during steroid-induced oocyte maturation. These results thus show that the bulk of RNA polymerase accumulates in the germinal vesicle during oogenesis, is dispersed into the cytoplasm during maturation, and, since only limited synthesis seems to be occurring, the polymerase is available during embryogenesis.

  1. Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes. (United States)

    Tsuji, Ai; Nakamura, Toshinobu; Shibata, Katsumi


    Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.

  2. Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Grunnet, M; Angelo, K


    and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual-function...... vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels....... will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells...

  3. Modification of the pathogenicity of Schistosoma mattheei for sheep by passage of the parasite in hamsters. (United States)

    Taylor, M G; James, E R; Nelson, G S; Bickle, Q; Dunne, D W; Dobinson, A R; Dargie, J D; Berry, C I; Hussein, M F


    Border Leicester X Suffolk sheep infected with a strain of S. mattheei maintained in hamsters do not develop the same pathological changes as Romney Marsh sheep infected with the same strain of parasite before hamster passage. To determine the cause of this reduced pathogenicity, five Romney Marsh sheep were each infected with 10 000 cercariae of the hamster-passaged parasite and five with 10 000 cercariae of a S. mattheei strain from Onderstepoort, South Africa, passaged exclusively through sheep. Striking pathological and parasitological differences were found between the two strains. Infection with the "sheep" strain was lethal, whereas infection with the "hamster" strain produced little evidence of clinical disease. By 13 weeks post-infection the mean body weight of the sheep infected with the sheep strain had declined by 15% compared with both the uninfected controls and the sheep infected with the hamster strain, and the mean PCV was lowered to 20% in the sheep strain infected animals. Egg production began at seven weeks with the sheep strain, faecal counts rising to more than 300 e.p.g., whereas only two of the sheep infected with the hamster strain passed eggs in the faeces (at nine weeks) and the maximum egg count was 50 e.p.g. Twice as many adult worms of the sheep strain were recovered, and, although the number of eggs found in the tissues "per worm pair" was not significantly different, overall egg production was higher for the sheep strain; also more of the sheep strain eggs were deposited in the intestines. Similar parasite differences were seen in a supplementary study in mice and it seemed that "attenuation" of the parasite had occurred, presumably due to its maintenance in hamsters. Histopathological observations and faecal egg counts both indicated an inability of hamster strain eggs to penetrate the intestinal lumen; this was probably important in reducing the pathogenicity of the hamster strain.

  4. Determination of elements in blood of golden hamster by NAA; Determinacao de elementos em sangue de hamster dourado usando AAN

    Energy Technology Data Exchange (ETDEWEB)

    Aguiar, Rodrigo Oliveira de


    In the present study Neutron Activation Analysis technique has been used to determine, simultaneously, some element concentrations of clinical relevance in whole blood samples of golden hamster. The normal range for Br, Ca, Cl K, Mg, Na and S considering 2 {sigma} (Two Standard deviations) was 0.011 0.047 gL{sup -1} (Br); 0.11 0.35 gL{sup -1} (Ca); 2.11 3.75 gL{sup -1} (Cl); 1.35 2.79 gL{sup -1} (K), 0.026 0.090 gL{sup -1} (Mg), 1.03 2.51 gL{sup -1} (Na) e 0.97 2.01 gL{sup -1} (S). The knowledge of these limits became possible to perform clinical investigation in this animal model using whole blood. The comparison with the results from human being whole blood estimation (Hamster and human) became possible to check the similarities or physiologic differences, an important data for animal experimentation. (author)

  5. Omega 3 Fatty Acids Promote Macrophage Reverse Cholesterol Transport in Hamster Fed High Fat Diet


    Fatima Kasbi Chadli; Hassane Nazih; Michel Krempf; Patrick Nguyen; Khadija Ouguerram


    The aim of this study was to investigate macrophage reverse cholesterol transport (RCT) in hamster, a CETP-expressing species, fed omega 3 fatty acids (ω3PUFA) supplemented high fat diet (HFD). Three groups of hamsters (n = 6/group) were studied for 20 weeks: 1) control diet: Control, 2) HFD group: HF and 3) HFD group supplemented with ω3PUFA (EPA and DHA): HFω3. In vivo macrophage-to-feces RCT was assessed after an intraperitoneal injection of (3)H-cholesterol-labelled hamster primary macrop...

  6. Infestivity of Demodex canis to hamster skin engrafted onto SCID mice. (United States)

    Tani, Kenji; Une, Satoshi; Hasegawa, Atsuhiko; Adachi, Makoto; Kanda, Naoko; Watanabe, Shin-ichi; Nakaichi, Munekazu; Taura, Yasuho


    We demonstrated that Demodex canis was transferred to skin xenografts of a dog and a hamster onto severe combined immunodeficiency mice. After the transfer of mites, the number of eggs, larvae, nymphs and adult mites per gram of canine and hamster xenografts increased, whereas no live mites were detected on murine allograft. These results indicate that D. canis proliferates in hair follicles of dog and hamster skins but not in murine allograft. Therefore, D. canis may have host preference but not strict host-specificity.

  7. Structural changes of in vitro matured buffalo and bovine oocytes following cryopreservation


    Marina De Blasi; Evelina Mariotti; Salvatore Velotto; Marcello Rubessa; Serena Di Francesco; Bianca Gasparrini


    The aim of this work was to evaluate chromatin and spindle organization of buffalo and bovine in vitro matured oocytes after vitrification/warming by Cryotop and after their exposure to cryoprotectants (CP). In vitro matured oocytes were vitrified/warmed and exposed to the vitrification/warming solutions containing ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose as CP. Two hours after warming, oocytes were fixed and immunostained for microtubules and nuclei and examined by fluores...

  8. Cryopreservation of bovine oocytes: is cryoloop vitrification the future to preserving the female gamete?


    Mavrides, Andreas; Morroll, David


    International audience; The cryoloop is a technique where a thin nylon loop is used to suspend a film of cryoprotectant containing the oocytes and directly immersing them in liquid nitrogen. 508 bovine oocytes were collected, of these 351 were cryopreserved by slow freezing using standard straws or a new vitrification method using our self-constructed cryoloops and the remainder were controls. After thawing, the oocytes were inseminated by ICSI or standard IVF. The cryoloop vitrification meth...

  9. Ultrastructure and Development of Vitrified/Warmed Bovine Oocytes Matured with 9-cis Retinoic Acid


    Rodríguez, Aida; Gómez, Enrique; Antolín, Isaac; Duque, Paloma; Hidalgo, C.O. (Carlos); Alonso, Cristina; Tamargo, Carolina; Fernández, Lina; Carbajo, Maite; Facal, Nieves; Caamaño, J.N. (José); Díez, Carmen


    In this work we analyze the effects of vitrification on the ultrastructure and developmental ability of bovine oocytes matured in the presence of 9-cis-retinoic acid (RA). Bovine cumulus oocyte complexes (COCs) were matured in a basic medium containing 10% fetal calf serum (FCS), 9-cis-RA or polyvinyl alcohol (PVA). Mature oocytes were vitrified using the Open Pulled Straw method with minor modifications. A group of fresh and vitrified/warmed COCs was fixed for ultrastructural analysis, wh...

  10. Vitrification affects nuclear maturation and gene expression of immature human oocytes

    Directory of Open Access Journals (Sweden)

    Abbas Shahedi


    Full Text Available Background: Vitrification of oocytes is a fast-freezing technique, which may affect the quality of the human oocyte, and consequently affects the embryo development, pregnancy and birth. The aim of the current study was to investigate the consequence of in-vitro vitrification on maturation status of immature human oocytes, additionally, expression levels of stress, and apoptosis related genes. Materials and Methods: The total of 213 human immature oocytes which routinely discarded from assisted reproduction clinics were collected and divided into two groups including: (I fresh germinal vesicle (GV oocytes (n=106 (matured in-vitro  (fIVM , and  (II GV oocytes (n=107 that initially vitrified, then matured in  in-vitro (vIVM. After 36 hours of incubation, the oocytes were evaluated for nuclear maturation and expression level of DNA methyltransferase (DNMT1, stress related genes (Sod1 and Hsp70, and apoptotic related genes (Bax and Bcl-2 by quantitative Real-Time PCR. Results: Oocyte maturation rates were reduced in vIVM compared to fIVM oocytes (P=0.001. The expression of stress (Sod1 and Hsp70, and apoptotic-related genes (Bax and Bcl-2 in vIVM were significantly higher compared to the fIVM group. Additionally, pro-apoptotic gene up-regulated 4.3 times more than anti-apoptotic gene in vIVM oocyte. However, DNMT1 gene expression was reduced in vIVM oocyte (P = 0.047. Conclusions: The low survival rate of vitrified In-vitro matured GV oocytes could definitely be explained by the alterations of their gene expression profile. 

  11. Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse. (United States)

    Clarke, H J; Masui, Y


    Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.

  12. Proteomics-based systems biology modeling of bovine germinal vesicle stage oocyte and cumulus cell interaction.

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    Divyaswetha Peddinti

    Full Text Available BACKGROUND: Oocytes are the female gametes which establish the program of life after fertilization. Interactions between oocyte and the surrounding cumulus cells at germinal vesicle (GV stage are considered essential for proper maturation or 'programming' of oocytes, which is crucial for normal fertilization and embryonic development. However, despite its importance, little is known about the molecular events and pathways involved in this bidirectional communication. METHODOLOGY/PRINCIPAL FINDINGS: We used differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT on bovine GV oocyte and cumulus cells and identified 811 and 1247 proteins in GV oocyte and cumulus cells, respectively; 371 proteins were significantly differentially expressed between each cell type. Systems biology modeling, which included Gene Ontology (GO and canonical genetic pathway analysis, showed that cumulus cells have higher expression of proteins involved in cell communication, generation of precursor metabolites and energy, as well as transport than GV oocytes. Our data also suggests a hypothesis that oocytes may depend on the presence of cumulus cells to generate specific cellular signals to coordinate their growth and maturation. CONCLUSIONS/SIGNIFICANCE: Systems biology modeling of bovine oocytes and cumulus cells in the context of GO and protein interaction networks identified the signaling pathways associated with the proteins involved in cell-to-cell signaling biological process that may have implications in oocyte competence and maturation. This first comprehensive systems biology modeling of bovine oocytes and cumulus cell proteomes not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level.

  13. Effect of jasplakinolide on the in vitro maturation of bovine oocytes ...

    African Journals Online (AJOL)

    The results showed that (1) JAS affected oocyte maturation and haploid composition of matured oocytes in a dose-dependent manner. The maturation rates were 70.3, 53.1, 39.7 and 20.6% after culturing oocytes in JAS at 100, 200, 300 and 400 nM for 24 h, respectively, which were significantly lower than the control group ...

  14. Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa. (United States)

    Bedford, S J; Kurokawa, M; Hinrichs, K; Fissore, R A


    In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain in horse oocytes the [Ca(2+)](i)-releasing activity and activating capacity of equine sperm extracts corresponding to the activity present in a single stallion spermatozoon; and (iii) to determine whether equine oocytes respond with [Ca(2+)](i) transients and activation when fertilized using the intracytoplasmic sperm injection (ICSI) procedure. The results of this study indicate that equine sperm extracts are able to induce [Ca(2+)](i) oscillations, activation and embryo development in mouse oocytes. Furthermore, in horse oocytes, injection of sperm extracts induced persistent [Ca(2+)](i) oscillations that lasted for >60 min and initiated oocyte activation. Nevertheless, injection of a single stallion spermatozoon did not consistently initiate [Ca(2+)](i) oscillations in horse oocytes. It is concluded that stallion sperm extracts can efficiently induce [Ca(2+)](i) responses and parthenogenesis in horse oocytes, and can be used to elucidate the signalling mechanism of fertilization in horses. Conversely, the inconsistent [Ca(2+)](i) responses obtained with sperm injection in horse oocytes may explain, at least in part, the low developmental success obtained using ICSI in large animal species.

  15. Characterization of the IGF2 Imprinted Gene Methylation Status in Bovine Oocytes during Folliculogenesis.

    Directory of Open Access Journals (Sweden)

    Anelise dos Santos Mendonça

    Full Text Available DNA methylation reprogramming occurs during mammalian gametogenesis and embryogenesis. Sex-specific DNA methylation patterns at specific CpG islands controlling imprinted genes are acquired during this window of development. Characterization of the DNA methylation dynamics of imprinted genes acquired by oocytes during folliculogenesis is essential for understanding the physiological and genetic aspects of female gametogenesis and to determine the parameters for oocyte competence. This knowledge can be used to improve in vitro embryo production (IVP, specifically because oocyte competence is one of the most important aspects determining the success of IVP. Imprinted genes, such as IGF2, play important roles in embryo development, placentation and fetal growth. The aim of this study was to characterize the DNA methylation profile of the CpG island located in IGF2 exon 10 in oocytes during bovine folliculogenesis. The methylation percentages in oocytes from primordial follicles, final secondary follicles, small antral follicles, large antral follicles, MII oocytes and spermatozoa were 73.74 ± 2.88%, 58.70 ± 7.46%, 56.00 ± 5.58%, 65.77 ± 5.10%, 56.35 ± 7.45% and 96.04 ± 0.78%, respectively. Oocytes from primordial follicles showed fewer hypomethylated alleles (15.5% than MII oocytes (34.6% (p = 0.039; spermatozoa showed only hypermethylated alleles. Moreover, MII oocytes were less methylated than spermatozoa (p<0.001. Our results showed that the methylation pattern of this region behaves differently between mature oocytes and spermatozoa. However, while this region has a classical imprinted pattern in spermatozoa that is fully methylated, it was variable in mature oocytes, showing hypermethylated and hypomethylated alleles. Furthermore, our results suggest that this CpG island may have received precocious reprogramming, considering that the hypermethylated pattern was already found in growing oocytes from primordial follicles. These results

  16. Vitrification of human germinal vesicle oocytes; before or after in vitro maturation?

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    Evangelia Kasapi


    Full Text Available Background The use of immature oocytes derived from stimulated cycles could be of great importance, particularly for urgent fertility preservation cases. The current study aimed to determine whether in vitro maturation (IVM was more successful before or after vitrification of these oocytes. Materials and Methods This prospective study was performed in a private in vitro fertilization (IVF center. We collected 318 germinal vesicle (GV oocytes from 104 stimulated oocyte donation cycles. Oocytes were divided into two groups according to whether vitrification was applied at the GV stage (group 1 or in vitro matured to the metaphase II (MII stage and then vitrified (group 2. In the control group (group 3, oocytes were in vitro matured without vitrification. In all three groups, we assessed survival rate after warming, maturation rate, and MII-spindle/chromosome configurations. The chi-square test was used to compare rates between the three groups. Statistical significance was defined at P<0.05 and we used Bonferroni criterion to assess statistical significance regarding the various pairs of groups. The Statistical Package for the Social Sciences version 17.0 was used to perform statistical analysis. Results There was no significant difference in the survival rate after vitrification and warming of GV (93.5% and MII oocytes (90.8%. A significantly higher maturation rate occurred when IVM was performed before vitrification (82.9% compared to after vitrification (51%. There was no significant difference in the incidence of normal spindle/ chromosome configurations among warmed oocytes matured in vitro before (50.0% or after (41.2% vitrification. However, a higher incidence of normal spindle/chromosome configurations existed in the in vitro matured oocytes which were not subjected to vitrification (fresh oocytes, 77.9%. Conclusion In stimulated cycles, vitrification of in vitro matured MII oocytes rather than GV oocytes seems to be more efficient. This

  17. Parthenogenic blastocysts derived from cumulus-free in vitro matured human oocytes.

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    Sohyun L McElroy

    Full Text Available BACKGROUND: Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. METHODOLOGY/PRINCIPAL FINDING: Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin, a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. CONCLUSIONS/SIGNIFICANCE: Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear

  18. Oocyte maturation, embryo development and gene expression following two different methods of bovine cumulus-oocyte complexes vitrification. (United States)

    Azari, Mehdi; Kafi, Mojtaba; Ebrahimi, Bita; Fatehi, Roya; Jamalzadeh, Mahboobeh


    To examine the maturational competence, embryo development and expression of genes involved in oocyte maturation and cumulus expansion (GDF9, BMP15, HAS2, TNFAIP6, FGF17 and FSHr) following two standard methods of bovine COCs vitrification. Bovine cumulus-oocyte complexes (COCs) were aspirated from slaughtered ovaries and then distributed into three groups: non-vitrified COCs (control), vitrification 1 group (V1); vitrification was performed by 15% ethylene glycol (EG) and 15% DMSO in holding media (TCM-199 with 20% FCS); and vitrification 2 group (V2); vitrification was performed by 40% EG in holding media. After vitrification, COCs were warmed in two steps and cultured and then evaluated for nuclear maturation, embryo development and gene expressions. The mean (±SD) percentages of nuclear maturation and blastocyst/cleaved were higher in control group (79.5 ± 8.0 and 31.0 ± 5.1%) than the V1 (34.8 ± 9.1 and 4.4 ± 5.1%) and V2 (47.8 ± 11.7 and 7.1 ± 5.8%) groups (P vitrification groups (P vitrification procedure and conditions. Using EG alone for vitrification of bovine immature COCs, resulted in higher expression of GDF9, BMP15 and production of more in vitro matured and cleaved oocytes.

  19. Corona cell RNA sequencing from individual oocytes revealed transcripts and pathways linked to euploid oocyte competence and live birth. (United States)

    Parks, Jason C; Patton, Alyssa L; McCallie, Blair R; Griffin, Darren K; Schoolcraft, William B; Katz-Jaffe, Mandy G


    Corona cells surround the oocyte and maintain a close relationship through transzonal processes and gap junctions, and may be used to assess oocyte competence. In this study, the corona cell transcriptome of individual cumulus oocyte complexes (COCs) was investigated. Isolated corona cells were collected from COCs that developed into euploid blastocysts and were transferred in a subsequent frozen embryo transfer. Ten corona cell samples underwent RNA-sequencing to generate unique gene expression profiles. Live birth was compared with negative implantation after the transfer of a euploid blastocyst using bioinformatics and statistical analysis. Individual corona cell samples produced a mean of 21.2 million sequence reads, and 307 differentially expressed transcrpits (P corona cell transcriptome was successfully generated using RNA-sequencing. Key genes and signalling pathways were identified in association with implantation outcome after the transfer of a euploid blastocyst in a frozen embryo transfer. These data could provide novel biomarkers for the non-invasive assessment of embryo viability. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  20. Inhibitory effects of tramadol on nicotinic acetylcholine receptors in adrenal chromaffin cells and in Xenopus oocytes expressing α7 receptors (United States)

    Shiraishi, Munehiro; Minami, Kouichiro; Uezono, Yasuhito; Yanagihara, Nobuyuki; Shigematsu, Akio; Shibuya, Izumi


    Tramadol has been used clinically as an analgesic; however, the mechanism of its analgesic effects is still unknown.We used bovine adrenal chromaffin cells to investigate effects of tramadol on catecholamine secretion, nicotine-induced cytosolic Ca2+ concentration ([Ca2+]i) increases and membrane current changes. We also investigated effects of tramadol on α7 nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes.Tramadol concentration-dependently suppressed carbachol-induced catecholamine secretion to 60% and 27% of the control at the concentration of 10 and 100 μM, respectively, whereas it had little effect on veratridine- or high K+-induced catecholamine secretion.Tramadol also suppressed nicotine-induced ([Ca2+]i) increases in a concentration-dependent manner. Tramadol inhibited nicotine-induced inward currents, and the inhibition was unaffected by the opioid receptor antagonist naloxone.Tramadol inhibited nicotinic currents carried by α7 receptors expressed in Xenopus oocytes.Tramadol inhibited both α-bungarotoxin-sensitive and -insensitive nicotinic currents in bovine adrenal chromaffin cells.In conclusion, tramadol inhibits catecholamine secretion partly by inhibiting nicotinic AChR functions in a naloxone-insensitive manner and α7 receptors are one of those inhibited by tramadol. PMID:12010769

  1. Short-day response in Djungarian hamsters of different circadian phenotypes. (United States)

    Schöttner, Konrad; Schmidt, Maren; Hering, Anke; Schatz, Juliane; Weinert, Dietmar


    In Djungarian hamsters (Phodopus sungorus) bred at the authors' institute, a certain number of animals show activity patterns incompatible with proper entrainment of their endogenous circadian pacemaker to the environmental light-dark (LD) cycle. Even though the activity-offset in these animals is stably coupled to "light-on," activity-onset is increasingly delayed, leading to a compression of the activity time (α). If α falls below a critical value, the circadian rhythm in these so called delayed activity-onset (DAO) hamsters starts to free-run and finally breaks down. Animals then show an arrhythmic activity pattern (AR hamsters). Previous studies revealed the mechanisms of photic entrainment have deteriorated (DAO) or the suprachiasmatic nucleus (SCN) does not generate a rhythmic signal (AR). The aim of the present study was to investigate the consequences that these deteriorations have upon photoperiodic time measurement. Animals were bred and kept under standardized housing conditions with food and water ad libitum and a 14L/10D (long day, LD) regimen. Locomotor activity was recorded continuously using passive infrared motion detectors. Body mass, testes size, and fur coloration were measured weekly or biweekly to further quantify the photoperiodic reaction. In a first experiment, adult male wild-type (WT), DAO, and AR hamsters were transferred initially to a 16L/8D cycle. After 3-4 wks, the light period was shortened symmetrically by 8 h. After 14 wks, none of the DAO and AR hamsters, and only 1 of 8 WT hamsters showed short-day (SD) traits. Therefore, in a second experiment, hamsters were transferred to SD conditions (8L/16D cycle) for 8 wks directly from standard LD conditions. In 6 of 7 WT hamsters, activity time expanded, body mass and testes size decreased, and fur coloration changed from summer to winter pelage. In contrast, none of the DAO and AR hamsters displayed an SD response. In a third experiment, DAO and AR hamsters were kept in constant

  2. Membrane Biophysics

    CERN Document Server

    Ashrafuzzaman, Mohammad


    Physics, mathematics and chemistry all play a vital role in understanding the true nature and functioning of biological membranes, key elements of living processes. Besides simple spectroscopic observations and electrical measurements of membranes we address in this book the phenomena of coexistence and independent existence of different membrane components using various theoretical approaches. This treatment will be helpful for readers who want to understand biological processes by applying both simple observations and fundamental scientific analysis. It provides a deep understanding of the causes and effects of processes inside membranes, and will thus eventually open new doors for high-level pharmaceutical approaches towards fighting membrane- and cell-related diseases.

  3. Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP

    DEFF Research Database (Denmark)

    Hou, Y -p; Liu, Ying; Dai, Y -p


    The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1 h, followed by parthenogenetic activation...... evaluating other activation protocols with 9% ethanol, calcium ionophore A23187, or ionomycin alone, or in combination with DMAP or cycloheximide (CHX). In conclusion, the oocyte activation protocol affected developmental capacity of vitrified bovine oocytes; 9% ethanol (5 min) followed by 6-DMAP (4 h...

  4. Structural Changes in Cattle Immature Oocytes Subjected to Slow Freezing and Vitrification

    Directory of Open Access Journals (Sweden)

    H. Wahid*, M. Thein1, E.A. El-Hafez2, M.O. Abas3, K. Mohd Azam4, O. Fauziah5, Y. Rosnina and H. Hajarian


    Full Text Available This study was conducted to evaluate the effect of different cryopreservation methods (slow-freezing and vitrification on structural changes of bovine immature oocytes. Bovine ovaries were collected from local abattoirs. Cumulus-oocyte-complexes (COCs were retrieved using aspiration method from 2-6 mm follicles. In Experiment 1, selected oocytes were randomly divided into 4 treatment groups namely freezing solution-exposed, frozen-thawed, vitrification solution-exposed and vitrified-thawed and then oocytes abnormalities were examined under a stereomicroscope. In Experiment 2, oocytes were randomly allocated to the same grouping as experiment 1 plus control group. Following freezing or vitrification, all oocytes were fixed in glutaraldehyde and processed for transmission electron microscopy. In experiment 1, there was a higher incidence of abnormalities in the frozen-thawed and vitrified-warmed oocytes compared to those in freezing solution and vitrification solution-exposed groups (P<0.05. In experiment 2, there were marked alterations in the perivitelline space, microvilli and vesicles of frozen-thawed and vitrified-warmed oocytes characterized by loss of elasticity and integrity of cytoplasmic processes and microvilli following cooling and warming. In conclusion, ethylene glycol-based freezing and vitrification solutions are suitable choices for cryopreservation of immature oocytes and most organelles are able to retain their normal morphology following cryopreservation and thawing processes.

  5. A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep. (United States)

    Mullen, S F; Fahy, G M


    Vitrification as a means of cryopreservation has become a standard approach for oocytes from livestock. This paradigm shift occurred primarily as a result of the demonstration in 1996 that bovine oocytes are extremely susceptible to chilling injury. Since that early work, numerous devices have been used as supports for oocytes during so-called "ultra-rapid cooling", and occasionally, trials involving the deposition of small volumes of media containing oocytes directly into liquid nitrogen to facilitate cooling have been reported. Results reporting blastocyst development exceeding 10% are common, but variability remains high, and a standard method for bovine oocytes remains to be established. Oocytes from pigs are particularly difficult to cryopreserve, even with the use of ultrarapid cooling approaches. Few reports have demonstrated blastocyst development exceeding 5%. The application of hydrostatic pressure before vitrification appears to impart stress tolerance to porcine oocytes, as the results of some treatments have shown development to blastocysts at proportions >10%. Work on sheep oocyte vitrification is relatively new, and a few articles have reported blastocyst development at 10% or more. Messenger RNA levels are reportedly altered in sheep oocytes as a result of vitrification, and damage to the cytoskeleton is common across species. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Advances in Collection, Transport and Maturation of Equine Oocytes for Assisted Reproductive Techniques. (United States)

    Carnevale, Elaine M


    Assisted reproductive techniques that are based on oocyte manipulations have gained acceptance in the equine industry. Methods to collect and handle immature or maturing oocytes have been developed, and systems to ship oocytes now allow for collection in one location and intracytoplasmic sperm injection (ICSI) in another. Subsequently, ICSI-produced embryos can be transferred onsite, shipped to another location, or cryopreserved. Methods for the collection, identification, culture, maturation, and shipment of equine oocytes are reviewed, with an emphasis on procedures from laboratories providing clinical services with documented success. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Individual luteolysis pattern after GnRH-agonist trigger for final oocyte maturation.

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    Barbara Lawrenz

    Full Text Available Final oocyte maturation using GnRH-agonist trigger in a GnRH-antagonist protocol is increasingly common, as ovarian hyperstimulation syndrome is almost completely avoided. However, this approach might lead to reduced pregnancy rates due to severe luteolysis. This proof of concept study evaluated the extend of luteolysis by measuring progesterone levels 48 hours after oocyte retrieval in 51 patients, who received GnRH-agonist trigger for final oocyte maturation in a GnRH-antagonist protocol due to the risk of ovarian hyperstimulation syndrome. It was shown, that luteolysis after GnRHa-trigger differs greatly among patients, with progesterone levels ranging from 13.0 ng/ml to ≥ 60.0 ng/ml, 48 hours after oocyte retrieval. Significant positive correlations could be demonstrated between progesterone levels and the number of ovarian stimulation and suppression days (p = 0.006 and p = 0.002 respectively, the total amount of medication used for ovarian suppression (p = 0.015, the level of progesterone on the day of final oocyte maturation (p = 0.008 and the number of retrieved oocytes (p = 0.019. Therefore it was concluded, that luteolysis after GnRH-agonist trigger is patient-specific and also luteal phase support requires individualization. Longer stimulation duration as well as a higher level of progesterone on the day of final oocyte maturation and more retrieved oocytes will result in higher levels of progesterone 48 hours after oocyte retrieval.

  8. Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns.

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    Guang-Jian Jiang

    Full Text Available It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.

  9. Oocyte batch development and enumeration in the European anchovy (Engraulis encrasicolus

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    Full Text Available An alternative method to the traditional hydrated oocyte (HO method has been evaluated for the Sicilian anchovy, Engraulis encrasicolus. The method is based on the processing of ovarian whole mount images and the identification of the spawning batch in oocyte size frequency distributions and shows the advantage that it can be applied to various oocyte stages rather than strictly to the HO stage. Despite the peculiar elliptical shape of anchovy oocytes, this image analysis technique was fully successful since the yolked stage appeared to perform equally to the HO stage for anchovy batch fecundity measurements.

  10. Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro (United States)

    Liu, Shuzhen; Jiang, Ligang; Zhong, Tao; Kong, Shuhui; Zheng, Rongbin; Kong, Fengyun; Zhang, Cong; Zhang, Lei; An, Liguo


    Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus–oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus–oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro. PMID:26275143

  11. Identification of some unknown transcripts from SSH cDNA library of buffalo follicular oocytes. (United States)

    Rajput, S K; Kumar, P; Roy, B; Verma, A; Pandey, H P; Singh, D; De, S; Datta, T K


    A buffalo oocyte-specific subtracted cDNA library was constructed to identify exclusively or preferentially oocyte-expressed genes. The library represented an enriched population of transcripts obtained from oocytes of diverse ovarian follicular origin and at different stages of in vitro maturation. A total of 1173 high-quality sequences of oocyte-specific genes were clustered into 645 unique sequences, out of which 65.76% were represented as singlets and 34.26% as contig expressed sequence tags (ESTs; clusters). Analysis of sequences revealed that 498 of these sequences were identified as a known sequence in mammalian species including buffalo, 103 as uncharacterized ESTs and 44 unknown sequences including 1 novel EST, so far not reported in any species. Gene ontology annotation classified these sequences into functional categories of cellular events and biological processes associated with oocyte competence. Expression status of the isolated unknown ESTs confirmed that many of these are expressed in oocytes exclusively and in others preferentially, some in excess of 80-fold greater in comparison with a variety of somatic tissues. The isolated novel EST was detected to be expressed exclusively in oocytes and testicular cells only. To our knowledge, this is the first report giving a detailed transcriptome account of oocyte-expressed genes in buffalo. This study will provide important information on the physiological control of oocyte development, as well as many questions yet to be addressed on the reproductive process of buffalo.

  12. The role of phosphatidylinositol signaling pathway in regulating serotonin-induced oocyte maturation in Mercenaria mercenaria (United States)

    Wang, Qing; Zhang, Tao


    Serotonin (5-HT) has been found to stimulate meiotic maturation of oocytes in many molluscs. During maturation, several signaling pathways are involved, especially the phosphatidylinositol and cAMP pathways. In order to examine the possible role of the phosphatidylinositol signaling pathway in regulating oocyte maturation in Mercenaria mercenaria, the effects of the activator/inhibitor of phospholipase (PLC) and protein kinase C (PKC) on serotonin-induced maturation were studied. Results show that high-concentrations of neomycin (inhibitor of PLC) blocked oocyte maturation, while 9, 10-dimethyl-1, 2-benzanthracene (DMBA, activator of PLC) promoted oocyte maturation in the presence of serotonin. It was also found that in the presence of serotonin, phorbol 12-myristate 13-acetate (PMA, activator of PKC) inhibited oocyte maturation, while sphingosine (inhibitor of PKC) stimulated oocyte maturation. These results indicate that serotonin-induced oocyte maturation requires the activation of the phosphatidylinositol pathway. Decrease of PLC inhibited serotonin-induced oocyte maturation, whereas a decrease of PKC stimulated the maturation. Thus, our study indicates that serotonin promotes maturation of M. mercenaria oocytes through PLC stimulated increase in calcium ion concentration via inositol-1, 4, 5-trisphosphate (IP3) but not PKC.

  13. The Relationship Between Transcript Expression Levels of Nuclear Encoded (TFAM, NRF1 and Mitochondrial Encoded (MT-CO1 Genes in Single Human Oocytes During Oocyte Maturation

    Directory of Open Access Journals (Sweden)

    Ghaffari Novin M.


    Full Text Available In some cases of infertility in women, human oocytes fail to mature when they reach the metaphase II (MII stage. Mitochondria plays an important role in oocyte maturation. A large number of mitochondrial DNA (mtDNA, copied in oocytes, is essential for providing adenosine triphosphate (ATP during oocyte maturation. The purpose of this study was to identify the relationship between transcript expression levels of the mitochondrial encoded gene (MT-CO1 and two nuclear encoded genes, nuclear respiratory factor 1 (NRF1 and mitochondrial transcription factor A (TFAM in various stages of human oocyte maturation. Nine consenting patients, age 21-35 years old, with male factors were selected for ovarian stimulation and intracytoplasmic sperm injection (ICSI procedures. mRNA levels of mitochondrial- related genes were performed by singlecell TaqMan® quantitative real-time polymerase chain reaction (qRT-PCR. There was no significant relationship between the relative expression levels in germinal vesicle (GV stage oocytes (p = 0.62. On the contrary, a significant relationship was seen between the relative expression levels of TFAM and NRF1 and the MT-CO1 genes at the stages of metaphase I (MI and MII (p = 0.03 and p = 0.002. A relationship exists between the transcript expression levels of TFAM and NRF1, and MT-CO1 genes in various stages of human oocyte maturation.

  14. Digital multiplexed mRNA analysis of functionally important genes in single human oocytes and correlation of changes in transcript levels with oocyte protein expression☆ (United States)

    Riris, Solon; Webster, Philippa; Homer, Hayden


    Objective To investigate functionally important transcripts in