The Effect of Lysophosphatidic Acid during In Vitro Maturation of Bovine Oocytes: Embryonic Development and mRNA Abundances of Genes Involved in Apoptosis and Oocyte Competence

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Dorota Boruszewska

2014-01-01

Full Text Available In the present study we examined whether LPA can be synthesized and act during in vitro maturation of bovine cumulus oocyte complexes (COCs. We found transcription of genes coding for enzymes of LPA synthesis pathway (ATX and PLA2 and of LPA receptors (LPAR 1–4 in bovine oocytes and cumulus cells, following in vitro maturation. COCs were matured in vitro in presence or absence of LPA (10−5 M for 24 h. Supplementation of maturation medium with LPA increased mRNA abundance of FST and GDF9 in oocytes and decreased mRNA abundance of CTSs in cumulus cells. Additionally, oocytes stimulated with LPA had higher transcription levels of BCL2 and lower transcription levels of BAX resulting in the significantly lower BAX/BCL2 ratio. Blastocyst rates on day 7 were similar in the control and the LPA-stimulated COCs. Our study demonstrates for the first time that bovine COCs are a potential source and target of LPA action. We postulate that LPA exerts an autocrine and/or paracrine signaling, through several LPARs, between the oocyte and cumulus cells. LPA supplementation of maturation medium improves COC quality, and although this was not translated into an enhanced in vitro development until the blastocyst stage, improved oocyte competence may be relevant for subsequent in vivo survival.

Transcriptome dynamics and molecular cross-talk between bovine oocyte and its companion cumulus cells

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Looft C

2011-01-01

Full Text Available Abstract Background The bi-directional communication between the oocyte and its companion cumulus cells (CCs is crucial for development and functions of both cell types. Transcripts that are exclusively expressed either in oocytes or CCs and molecular mechanisms affected due to removal of the communication axis between the two cell types is not investigated at a larger scale. The main objectives of this study were: 1. To identify transcripts exclusively expressed either in oocyte or CCs and 2. To identify those which are differentially expressed when the oocyte is cultured with or without its companion CCs and vice versa. Results We analyzed transcriptome profile of different oocyte and CC samples using Affymetrix GeneChip Bovine Genome array containing 23000 transcripts. Out of 13162 genes detected in germinal vesicle (GV oocytes and their companion CCs, 1516 and 2727 are exclusively expressed in oocytes and CCs, respectively, while 8919 are expressed in both. Similarly, of 13602 genes detected in metaphase II (MII oocytes and CCs, 1423 and 3100 are exclusively expressed in oocytes and CCs, respectively, while 9079 are expressed in both. A total of 265 transcripts are differentially expressed between oocytes cultured with (OO + CCs and without (OO - CCs CCs, of which 217 and 48 are over expressed in the former and the later groups, respectively. Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs + OO or without (CCs - OO their enclosed oocytes. Of these, 320 and 246 are over expressed in CCs + OO and CCs - OO, respectively. While oocyte specific transcripts include those involved in transcription (IRF6, POU5F1, MYF5, MED18, translation (EIF2AK1, EIF4ENIF1 and CCs specific ones include those involved in carbohydrate metabolism (HYAL1, PFKL, PYGL, MPI, protein metabolic processes (IHH, APOA1, PLOD1, steroid biosynthetic process (APOA1, CYP11A1, HSD3B1, HSD3B7. Similarly, while transcripts over expressed in OO + CCs

CHEMERIN (RARRES2) decreases in vitro granulosa cell steroidogenesis and blocks oocyte meiotic progression in bovine species.

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Reverchon, Maxime; Bertoldo, Michael J; Ramé, Christelle; Froment, Pascal; Dupont, Joëlle

2014-05-01

CHEMERIN, or RARRES2, is a new adipokine that is involved in the regulation of adipogenesis, energy metabolism, and inflammation. Recent data suggest that it also plays a role in reproductive function in rats and humans. Here we studied the expression of CHEMERIN and its three receptors (CMKLR1, GPR1, and CCRL2) in the bovine ovary and investigated the in vitro effects of this hormone on granulosa cell steroidogenesis and oocyte maturation. By RT-PCR, immunoblotting, and immunohistochemistry, we found CHEMERIN, CMKLR1, GPR1, and CCRL2 in various ovarian cells, including granulosa and theca cells, corpus luteum, and oocytes. In cultured bovine granulosa cells, INSULIN, IGF1, and two insulin sensitizers-metformin and rosiglitazone-increased rarres2 mRNA expression whereas they decreased cmklr1, gpr1, and cclr2 mRNA expression. Furthermore, TNF alpha and ADIPONECTIN significantly increased rarres2 and cmklr1 expression, respectively. In cultured bovine granulosa cells, human recombinant CHEMERIN (hRec, 200 ng/ml) reduced production of both progesterone and estradiol, cholesterol content, STAR abundance, CYP19A1 and HMGCR proteins, and the phosphorylation levels of MAPK3/MAPK1 in the presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). All of these effects were abolished by using an anti-CMKLR1 antibody. In bovine cumulus-oocyte complexes, the addition of hRec (200 ng/ml) in the maturation medium arrested most oocytes at the germinal vesicle stage, and this was associated with a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. Thus, in cultured bovine granulosa cells, hRec decreases steroidogenesis, cholesterol synthesis, and MAPK3/1 phosphorylation, probably through CMKLR1. Moreover, in cumulus-oocyte complexes, it blocked meiotic progression at the germinal vesicle stage and inhibited MAPK3/1 phosphorylation in both the oocytes and cumulus cells during in vitro maturation. © 2014 by the Society for the Study of Reproduction, Inc.

Relationship between growth hormone concentrations in bovine oocytes and follicular fluid and oocyte developmental competence

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S Modina

2009-08-01

Full Text Available In the last few years, several works suggest that Growth Hormone (GH is involved in follicular development and oocyte maturation. These actions may reflect endocrine roles of pituitary GH and also account for local autocrine or paracrine activities of GH produced in reproductive tissue. This study was aimed to verify whether the developmental competence of bovine female gametes might be related to ovarian GH.We evaluated the localisation and distribution of GH in the cumulus oocytes complexes (COCs and the concentration of GH in the oocytes and in the follicular fluids (FF from ovaries classified on the basis of the follicles number. Oocytes retrieved from ovaries with more than 10 follicles of 2 to 5 mm in diameter (High ovaries, Hi show higher rate of maturation and blastocyst formation than those retrieved from ovaries with less than 10 follicles (Low ovaries, Lo. At the same time we measured Estrogen (E2 and Progesterone (P4 concentrations in FF, to relate oocytes quality, GH concentration and follicle health. GH localization in COCs and oocytes was performed by indirect immunofluorescence and its concentration within the ooplasm was evaluated by microspectrophotometer analysis. GH, E2 and P4 concentrations in FF were measured by an Enzyme Linked ImmunoSorbent assay (ELISA.We observed a positive, diffuse signal at cytoplasmic level in most of the cumulus cells, with no differences between COCs collected from Hi and Lo ovaries. On the contrary, GH level was significantly higher in the oocytes collected from Lo ovaries than in those recovered from Hi ovaries. Finally we found that also GH level in the FF was inversely related to the oocytes developmental capability. We suggest that the increase of GH in the oocytes and in the FF derived from Lo ovaries might be interpreted as attempt of the follicular environment to improve ovarian activity and in turn oocytes developmental competence in a autocrineparacrine manner. Moreover, E2, and P4 levels

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

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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.

Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes.

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Bilodeau-Goeseels, Sylvie

2011-01-01

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures. Copyright © 2011 Wiley Periodicals, Inc.

PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

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

2016-08-02

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.

Vitrification of bovine matured oocytes and blastocysts in a paper container.

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Paul, Ashit Kumar; Liang, Yuanyuan; Srirattana, Kanokwan; Nagai, Takashi; Parnpai, Rangsun

2018-02-01

In the present study, we aimed to determine the applicability of a paper container for the vitrification of in vitro matured (IVM) bovine oocytes. In experiment 1, IVM oocytes were exposed to vitrification solution (20% dimethylsulfoxide (DMSO), 20% ethylene glycol (EG), and 5 mol/L sucrose), using a two-step method, for 30 s; loaded onto either a paper container or Cryotop; and stored in liquid nitrogen. No significant difference (P container and Cryotop. In experiment 2, IVM oocytes were exposed to either a two- or three-step vitrification solution. The three-step vitrification solution was not significantly different from the two-step solution in terms of oocyte survival, cleavage and blastocyst rates. In experiment 3, in vitro produced blastocysts were graded according to the manual of the International Embryo Transfer Society (grades 1 and 2) and vitrified using the two- and three-step methods. For grade 2 blastocysts, the three-step method showed significantly higher (P < 0.05) survival and hatched blastocyst rates than the two-step method, whereas for grade 1 blastocysts, no significant difference was observed. In conclusion, the paper device and three-step technique are suitable for oocytes and embryo vitrification. © 2017 Japanese Society of Animal Science.

Bovine cumulus-oocyte disconnection in vitro

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Maddox-Hyttel, Poul

1987-01-01

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...

Successful vitrification of bovine immature oocyte using liquid helium instead of liquid nitrogen as cryogenic liquid.

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Yu, Xue-Li; Xu, Ya-Kun; Wu, Hua; Guo, Xian-Fei; Li, Xiao-Xia; Han, Wen-Xia; Li, Ying-Hua

2016-04-01

The objectives of this study were to compare the effectiveness of liquid helium (LHe) and liquid nitrogen (LN2) as cryogenic liquid for vitrification of bovine immature oocytes with open-pulled straw (OPS) system and determine the optimal cryoprotectant concentration of LHe vitrification. Cumulus oocyte complexes were divided into three groups, namely, untreated group (control), LN2 vitrified with OPS group, and LHe vitrified with OPS group. Oocyte survival was assessed by morphology, nuclear maturation, and developmental capability. Results indicated that the rates of normal morphology, maturation, cleavage, and blastocyst (89.3%, 52.8%, 42.7%, and 10.1%, respectively) in the LHe-vitrified group were all higher than those (79.3%, 43.4%, 34.1%, and 4.7%) in the LN2-vitrified group (P 0.05). The maturation rate of the EDS35 group (65.0%) was higher than those of the EDS30 (51.3%), EDS40 (50.1%), EDS45 (52.1%), and EDS50 groups (36.9%; P liquid for vitrification of bovine immature oocytes, and it is more efficient than LN2-vitrified oocytes in terms of blastocyst production. EDS35 was the optimal cryoprotectant agent combination for LHe vitrification in this study. Copyright © 2016 Elsevier Inc. All rights reserved.

Naturally occurring mastitis disrupts developmental competence of bovine oocytes.

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Roth, Z; Dvir, A; Kalo, D; Lavon, Y; Krifucks, O; Wolfenson, D; Leitner, G

2013-10-01

We examined the effects of naturally occurring mastitis on bovine oocyte developmental competence in vitro. Specifically, we investigated the effects of intramammary infection on the ovarian pool of oocytes (i.e., follicle-enclosed oocytes) and their ability to undergo in vitro maturation, fertilization, and further development to the blastocyst stage. Culled Holstein cows (n=50) from 9 commercial dairy farms in Israel were allotted to 3 groups according to somatic cell count (SCC) records of the last 3 monthly milk tests as well as of quarter samples collected before slaughter: (1) low SCC (n=7), (2) medium SCC (n=16), or (3) high SCC (n=27). Means of SCC values differed among low-, medium-, and high-SCC groups: 148,000, 311,000 and 1,813,000 cell/mL milk, respectively. Milk yield and days in milk did not differ among the 3 groups. Bacterial isolates included coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, or no bacteria found. Ovaries were collected at the abattoir and brought to the laboratory. Cumulus oocyte complexes were recovered separately from each cow and subjected individually to in vitro maturation and fertilization, followed by 8d in culture. The number of aspirated oocytes did not differ among groups, with a range of 17 to 21 oocytes per cow. The proportion of oocytes that cleaved into 2- to 4-cell-stage embryos (86.1 ± 3.4%) did not differ among groups. In contrast, mean percentages of embryos developed to the blastocyst stage on d 7 and 8 after fertilization were less in both medium- and-high SCC groups than in the low-SCC group (5.6 ± 2.3 and 4.1 ± 1.8 vs. 18.1 ± 4.6%, respectively). Additional analysis indicated that cleavage and blastocyst-formation rates did not differ among the bacterial types in the low-, medium-, and high-SCC groups. These are the first results to demonstrate that naturally occurring mastitis disrupts the developmental competence of the ovarian pool of oocytes, (i.e., oocytes at the

Production of bovine hand-made cloned embryos by zygote-oocyte cytoplasmic hemi-complementation.

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Mezzalira, Joana Claudia; Ohlweiler, Lain Uriel; da Costa Gerger, Renato Pereira; Casali, Renata; Vieira, Fabiano Koerich; Ambrósio, Carlos Eduardo; Miglino, Maria Angélica; Rodrigues, José Luiz; Mezzalira, Alceu; Bertolini, Marcelo

2011-02-01

The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; hand-made cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

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

Effect of antifreeze glycoprotein 8 supplementation during vitrification on the developmental competence of bovine oocytes.

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Liang, Shuang; Yuan, Bao; Kwon, Jeong-Woo; Ahn, Mija; Cui, Xiang-Shun; Bang, Jeong Kyu; Kim, Nam-Hyung

2016-07-15

The purpose of this study was to investigate the effect of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the survival, fertilization, and embryonic development of bovine oocytes and the underlying molecular mechanism(s). Survival, fertilization, early embryonic development, apoptosis, DNA double-strand breaks, reactive oxygen species levels, meiotic cytoskeleton assembly, chromosome alignment, and energy status of mitochondria were measured in the present experiments. Compared with that in the nonsupplemented group; survival, monospermy, blastocyst formation rates, and blastomere counts were significantly higher in the AFGP8-supplemented animals. Oocytes of the latter group also presented fewer double-strand breaks and lower cathepsin B and caspase activities. Rates of normal spindle organization and chromosome alignment, actin filament impairment, and mitochondrial distribution were significantly higher in the AFGP8-supplemented group. In addition, intracellular reactive oxygen species levels significantly decreased in the AFGP8-supplemented groups, maintaining a higher ΔΨm than that in the nonsupplemented group. Taken together, these results indicated that supplementation with AFGP8 during vitrification has a protective effect on bovine oocytes against chilling injury. Copyright © 2016 Elsevier Inc. All rights reserved.

Mitochondrial Patterns in Bovine Oocytes with Different Meiotic Competence Related to Their in vitro Maturation

Czech Academy of Sciences Publication Activity Database

Jeseta, M.; Čtvrtlíková Knitlová, D.; Hanzalová, K.; Hulínská, P.; Hanuláková, S.; Milakovič, I.; Němcová, Lucie; Kaňka, Jiří; Machatková, M.

2014-01-01

Roč. 49, č. 3 (2014), s. 469-475 ISSN 0936-6768 R&D Projects: GA MZe QI91A018 Institutional support: RVO:67985904 Keywords : bovine oocytes Subject RIV: GI - Animal Husbandry ; Breeding Impact factor: 1.515, year: 2014

Role of Fas-Mediated Apoptosis and Follicle-Stimulating Hormone on the Developmental Capacity of Bovine Cumulus Oocyte Complexes in Vitro

NARCIS (Netherlands)

Pomar, F.J.; Roelen, B.A.J.; Slot, K.A.; Tol, van H.T.A.; Colenbrander, B.; Teerds, K.J.

2004-01-01

Follicular atresia is believed to be largely regulated by apoptosis. To further understand how apoptosis can affect cumulus cells and oocytes we have evaluated the incidence and regulation of apoptosis affecting bovine cumulus oocyte complexes in vitro. Expression of components of the Fas signaling

Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification.

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

2015-01-01

Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage? The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods. The use of more 'steps' of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time. Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study. Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem-Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification. The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less

Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development.

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Morató, Roser; Izquierdo, Dolors; Paramio, Maria Teresa; Mogas, Teresa

2008-10-01

Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.

Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes.

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Giulia Rusciano

Full Text Available Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs. Our results suggest that vitrification induces a transformation of the protein secondary structure from the α-helices to the β-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our

Effects of green tea polyphenols, insulin-like growth factor I and glucose on developmental competence of bovine oocytes

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Zhengguang Wang

2012-12-01

Full Text Available The present study examined the effects of green tea polyphenols (GTP, insulin-like growth factor-I (IGF-I and glucose on oocyte in vitro maturation, subsequent embryo development and blastocyst quality in bovine. Cumulus-oocyte complexes (COC were aspirated from the ovaries and cultured in synthetic oviduct fluid supplemented with MEM amino acids (SOFaa media supplemented with one of the following supplements: GTP (0, 10, 15 and 20 µM, IGF-I (0, 50, 100 and 150 ng/mL or glucose (0, 1.5, 5.6 and 20 mM for 24 h. The results showed that oocytes cultured in media supplemented with 15 µM GTP, 100 ng/mL IGF-I and 5.6 mM glucose, in separate experiments, have higher cleavage and blastocyst rates compared with oocytes cultured in media without or with other concentration of GTP, IGF-I and glucose. Then these three substances with the concentration above were added together into SOFaa media and constituted a modified medium (Modified SOFaa. The COC were cultured in control SOFaa media and modified SOFaa media, respectively. The results showed that modified SOFaa media increased the intracellular glutathione concentration of matured oocytes, blastocyst rates and total cell numbers and cell numbers of inner cell mass per blastocyst compared with the control. Supplementing of GTP, IGF-I and glucose synchronously to maturation media can increase the intracellular GSH concentration of oocytes after in vitro maturation, and improve the embryo development and blastocyst quality in bovine.

Oocyte mitochondrial deletions and heteroplasmy in a bovine model of ageing and ovarian stimulation.

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Hammond, Elizabeth R; Green, Mark P; Shelling, Andrew N; Berg, Martin C; Peek, John C; Cree, Lynsey M

2016-04-01

Maternal ageing and ovarian stimulation result in the accumulation of mitochondrial DNA (mtDNA) deletions and heteroplasmy in individual oocytes from a novel bovine model for human assisted reproductive technology (ART). The levels of mtDNA deletions detected in oocytes increased with ovarian ageing. Low levels of mtDNA heteroplasmy were apparent across oocytes and no relationship was identified with respect to ovarian ageing or ovarian stimulation. Oocyte quality decreases with ovarian ageing and it is postulated that the mtDNA may have a role in this decline. The impact of ovarian stimulation on oocyte quality is poorly understood. Human studies investigating these effects are often limited by the use of low quality oocytes and embryos, variation in age and ovarian stimulation regimens within the patients studied, as well as genetic and environmental variability. Further, no study has investigated mtDNA heteroplasmy in individual oocytes using next-generation sequencing (NGS), and little is known about whether the oocyte accumulates heteroplasmic mtDNA mutations following ageing or ovarian stimulation. A novel bovine model for the effect of stimulation and age in human ART was undertaken using cows generated by somatic cell nuclear transfer (SCNT) from one founder, to produce a homogeneous population with reduced genetic and environmental variability. Oocytes and somatic tissues were collected from young (3 years of age; n = 4 females) and old (10 years of age; n = 5 females) cow clones following multiple natural ovarian cycles, as well as oocytes following multiple mild (FSH only) and standard (based on human a long GnRH agonist protocol) ovarian stimulation cycles. In addition, oocytes were recovered in a natural cycle from naturally conceived cows aged 4-13.5 years (n = 10) to provide a heterogeneous cohort for mtDNA deletion studies. The presence or absence of mtDNA deletions were investigated using long-range PCR in individual oocytes (n = 62). To determine

Structural Changes in Cattle Immature Oocytes Subjected to Slow Freezing and Vitrification

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H. Wahid*, M. Thein1, E.A. El-Hafez2, M.O. Abas3, K. Mohd Azam4, O. Fauziah5, Y. Rosnina and H. Hajarian

2012-05-01

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.

The role of the PI3K-Akt signaling pathway in the developmental competence of bovine oocytes.

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Gabriella Mamede Andrade

Full Text Available The ovarian follicle encloses oocytes in a microenvironment throughout their growth and acquisition of competence. Evidence suggests a dynamic interplay among follicular cells and oocytes, since they are constantly exchanging "messages". We dissected bovine ovarian follicles and recovered follicular cells (FCs-granulosa and cumulus cells and cumulus-oocyte complexes (COCs to investigate whether the PI3K-Akt signaling pathway impacted oocyte quality. Following follicle rupture, COCs were individually selected for in vitro cultures to track the follicular cells based on oocyte competence to reach the blastocyst stage after parthenogenetic activation. Levels of PI3K-Akt signaling pathway components in FCs correlated with oocyte competence. This pathway is upregulated in FCs from follicles with high-quality oocytes that are able to reach the blastocyst stage, as indicated by decreased levels of PTEN and increased levels of the PTEN regulators bta-miR-494 and bta-miR-20a. Using PI3K-Akt responsive genes, we showed decreased FOXO3a levels and BAX levels in lower quality groups, indicating changes in cell cycle progression, oxidative response and apoptosis. Based on these results, the measurement of levels of PI3K-Akt pathway components in FCs from ovarian follicles carrying oocytes with distinct developmental competences is a useful tool to identify putative molecular pathways involved in the acquisition of oocyte competence.

Influence of commercially available follicle stimulating hormone on the in vitro maturation of bovine oocytes

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Maria Valéria de Oliveira Santos

2017-06-01

Full Text Available The work aimed (Experiment I to compare commercial representations of porcine follicle stimulating hormone (FSH, Pluset® vs. Folltropin® in concentration (10 ?g/mL and time (24 h standard (more used in protocols of in vitro maturation, IVM; (Experiment II to evaluate the best incubation time (6 h vs. 16 h vs. 24 h and, (Experiment III analyze varying concentrations (1.0 ?g/mL vs. 2.5 ?g/mL vs. 10.0 ?g/mL of representations of FSH on the IVM of bovine oocytes. Thus, oocytes were recovered and submitted to IVM under appropriate conditions. After the IVM, oocytes were evaluated for expansion of cumulus cells (CCs, presence of the first polar body (1PB and metaphase plate (MII. All the data were analyzed by the Fisher exact test (P 0.05 was observed in maturation rates of the oocytes incubated with FSH Pluset® or Folltropin®, assessed by expansion of CCs (97.6% vs. 94.3%, presence of 1PB (76.6% vs. 69.4% and MII (70.0% vs. 68.6%. In Experiment II, when the incubation time with FSH was evaluated, both Pluset® as Folltropin® showed lower rate of expansion of CCs when they were present only in the first 6 h of IVM. As for presence of 1PB, differences were observed in relation to Pluset® while Folltropin® showed similar results in all incubation times. Regarding the MII, no difference was observed between the incubation times with FSH Pluset® and Folltropin®. In Experiment III, no difference was observed in the expansion of CCs, presence of 1PB and MII for concentrations evaluated FSH Pluset® and Folltropin®. Therefore, the FSH Pluset® and Folltropin® have the same efficiency in IVM of bovine oocytes. Regarding the incubation time, it is recommended to maintain FSH (Pluset® or Folltropin® throughout the period of IVM, since there was no difference in the results of presence of MII. Furthermore, the concentration of 1.0 ?g/mL of FSH Pluset® and Folltropin® is as effective as 10 ?g/mL and can therefore be used for IVM of oocytes.

The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

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Lee, So-Rim; Xu, Yong-Nan; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

2015-11-01

Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation. © 2015 Wiley Periodicals, Inc.

Extension of the culture period for the in vitro growth of bovine oocytes in the presence of bone morphogenetic protein-4 increases oocyte diameter, but impairs subsequent developmental competence.

Science.gov (United States)

Yang, Yinghua; Kanno, Chihiro; Sakaguchi, Kenichiro; Yanagawa, Yojiro; Katagiri, Seiji; Nagano, Masashi

2017-11-01

Bone morphogenetic protein-4 (BMP-4) inhibits luteinization of granulosa cells during in vitro growth (IVG) culture of bovine oocytes; however, oocytes derived from a 12 day IVG were less competent for development than in vivo-grown oocytes. We herein investigated whether an extended IVG culture with BMP-4 improves oocyte growth and development to blastocysts after in vitro fertilization. Oocyte-granulosa cell complexes (OGCs) were cultured for 14 or 16 days with BMP-4 (10 ng/mL), while a 12 day culture with BMP-4 served as the in vitro control. OGC viability was maintained for the 16 day culture with BMP-4 (83.2%), but was significantly lower without BMP-4 (58.9%) than the control (83.0%). Prolong-cultured oocytes at 16 days had statistically greater diameter (114.6 μm) than the control (111.7 μm). IVG oocytes with BMP-4 for the 16 day culture had a similar nuclear maturation rate to the control (approximately 67%); however, blastocyst rates in BMP-4 treated oocytes of 14 (1.8%) and 16 day (0%) IVG were statistically lower than that of 12 day IVG (9.0%). In conclusion, BMP-4 maintained OGC viability and promoted oocyte growth in a prolonged culture, but impaired the developmental competence of oocytes. Prolonged culture may not be an appropriate strategy for enhancing the developmental competence of IVG oocytes. © 2017 Japanese Society of Animal Science.

The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

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Sirini, Matias A; Anchordoquy, Juan Mateo; Anchordoquy, Juan Patricio; Pascua, Ana M; Nikoloff, Noelia; Carranza, Ana; Relling, Alejandro E; Furnus, Cecilia C

2017-10-01

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

A frozen-thawed in vitro-matured bovine oocyte derived calf with normal growth and fertility.

Science.gov (United States)

Otoi, T; Yamamoto, K; Koyama, N; Tachikawa, S; Suzuki, T

1996-08-01

The growth and fertility of a female calf obtained from a frozen-thawed bovine oocyte was assessed. The birth weight of the calf was lower than the mean birth weight of calves from in vitro fertilized embryos (IVF-controls) and calves obtained by artificial insemination (AI-controls). The growth rate of the calf up to 6 months was slower than that of the IVF-controls, but similar to that of the AI-controls. When the calf developed into a heifer (200 kg), she was inseminated with frozen semen and 280 days later delivered a male calf. The chromosoms of this cow were normal. These findings suggest that the growth and fertility of the calf derived from the frozen oocyte are normal.

MicroRNA-212 post-transcriptionally regulates oocyte-specific basic-helix-loop-helix transcription factor, factor in the germline alpha (FIGLA, during bovine early embryogenesis.

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Swamy K Tripurani

Full Text Available Factor in the germline alpha (FIGLA is an oocyte-specific basic helix-loop-helix transcription factor essential for primordial follicle formation and expression of many genes required for folliculogenesis, fertilization and early embryonic survival. Here we report the characterization of bovine FIGLA gene and its regulation during early embryogenesis. Bovine FIGLA mRNA expression is restricted to gonads and is detected in fetal ovaries harvested as early as 90 days of gestation. FIGLA mRNA and protein are abundant in germinal vesicle and metaphase II stage oocytes, as well as in embryos from pronuclear to eight-cell stage but barely detectable at morula and blastocyst stages, suggesting that FIGLA might be a maternal effect gene. Recent studies in zebrafish and mice have highlighted the importance of non-coding small RNAs (microRNAs as key regulatory molecules targeting maternal mRNAs for degradation during embryonic development. We hypothesized that FIGLA, as a maternal transcript, is regulated by microRNAs during early embryogenesis. Computational predictions identified a potential microRNA recognition element (MRE for miR-212 in the 3' UTR of the bovine FIGLA mRNA. Bovine miR-212 is expressed in oocytes and tends to increase in four-cell and eight-cell stage embryos followed by a decline at morula and blastocyst stages. Transient transfection and reporter assays revealed that miR-212 represses the expression of FIGLA in a MRE dependent manner. In addition, ectopic expression of miR-212 mimic in bovine early embryos dramatically reduced the expression of FIGLA protein. Collectively, our results demonstrate that FIGLA is temporally regulated during bovine early embryogenesis and miR-212 is an important negative regulator of FIGLA during the maternal to zygotic transition in bovine embryos.

Effect of supplementation of green tea polyphenols on the developmental competence of bovine oocytes in vitro

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Z.G. Wang

2007-08-01

Full Text Available The objective of the present study was to examine the effect of green tea polyphenols (GTPs supplementation during in vitro maturation, in vitro fertilization, and in vitro culture on the developmental competence of bovine oocytes. Cumulus-oocyte complexes aspirated from the ovaries were matured in vitro (38.5ºC for 24 h and fertilized (38.5ºC for 15-18 h and embryos were cultured (38.5ºC for 192 h in a defined conditioned medium with or without GTPs supplementation. The GTPs used in the present study contained 99% catechin derivatives, with the major components being 50% (--epigallocatechin gallate, 22% (--epicatechin gallate, 18% (--epigallocatechin, and 10% (--epicatechin. Four replicate trials were done for each type of experiment. GTPs supplementation (15 µM of the maturation medium led to a significant increase in the rate of blastocyst formation (34.0 vs 21.4%, P < 0.05. However, the rate of blastocyst formation was not improved when higher GTPs concentrations (20 or 25 µM were added to the in vitro maturation medium. During in vitro fertilization, supplementation with higher GTPs concentrations (20 or 25 µM significantly reduced the rate of blastocyst formation (P < 0.05. Supplementation of the culture medium with 15 µM GTPs improved the rate of blastocyst formation, while higher GTPs concentrations (25 µM significantly reduced embryo development (P < 0.05. In conclusion, these results demonstrate that supplementation with GTPs at low concentration (15 µM during in vitro maturation and in vitro culture improved the developmental competence of bovine oocytes.

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence.

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Md Mahmodul Hasan Sohel

Full Text Available Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down in exosomes and 30 miRNAs differentially expressed (21 up and 9 down in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.

Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation.

Science.gov (United States)

Labas, Valérie; Teixeira-Gomes, Ana-Paula; Bouguereau, Laura; Gargaros, Audrey; Spina, Lucie; Marestaing, Aurélie; Uzbekova, Svetlana

2018-03-20

Intact cell MALDI-TOF mass spectrometry (ICM-MS) was adapted to bovine follicular cells from individual ovarian follicles to obtain the protein/peptide signatures (top-down workflow using high resolution MS/MS (TD HR-MS) was performed on the protein extracts from oocytes, CC and GC. The TD HR-MS proteomic approach allowed for: (1) identification of 386 peptide/proteoforms encoded by 194 genes; and (2) characterisation of proteolysis products likely resulting from the action of kallikreins and caspases. In total, 136 peaks observed by ICM-MS were annotated by TD HR-MS (ProteomeXchange PXD004892). Among these, 16 markers of maturation were identified, including IGF2 binding protein 3 and hemoglobin B in the oocyte, thymosins beta-4 and beta-10, histone H2B and ubiquitin in CC. The combination of ICM-MS and TD HR-MS proved to be a suitable strategy to identify non-invasive markers of oocyte quality using limited biological samples. Intact cell MALDI-TOF mass spectrometry on single oocytes and their surrounding cumulus cells, coupled to an optimised top-down HR-MS proteomic approach on ovarian follicular cells, was used to identify specific markers of oocyte meiotic maturation represented by whole low molecular weight proteins or products of degradation by specific proteases. Copyright © 2017 Elsevier B.V. All rights reserved.

mRNA levels of imprinted genes in bovine in vivo oocytes, embryos and cross species comparisons in humans, mice and pigs

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Twenty-six confirmed imprinted genes in the bovine were quantified in in vivo produced oocytes and embryos. Eighteen were detectable and their transcriptional abundance were categorized into five patterns: largely decreased (MEST and PLAGL1); first decreased and then increased (CDKN1C and IGF2R); p...

Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation.

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Kenji Ezoe

Full Text Available Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK or 3-isobutyl-1-methylxanthine (IBMX to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.

The copper transporter (SLC31A1/CTR1) is expressed in bovine spermatozoa and oocytes: Copper in IVF medium improves sperm quality.

Science.gov (United States)

Anchordoquy, J P; Anchordoquy, J M; Pascua, A M; Nikoloff, N; Peral-García, P; Furnus, C C

2017-07-15

Adequate dietary intake of copper (Cu) is required for normal reproductive performance in cattle. The objective of this study was to investigate the pregnancy rates from cattle with deficient, marginal and adequate Cu plasma concentration at the beginning of artificial insemination protocol. Moreover, we determined Cu concentrations present in bovine oviductal fluid (OF), and the effects of Cu on fertilizing ability of bovine spermatozoa. Also, the presence of Cu transporter, SLC31A1 (also known as CTR1), in spermatozoa and in vitro matured oocyte were investigated. We found no differences in pregnancy rates among animals with adequate, marginal, and deficient Cu concentrations measured in plasma at the beginning of fixed-time artificial insemination (FTAI) protocol. Copper concentrations in OF were 38.3 ± 2.17 μg/dL (mean ± SEM) regardless of cupremia levels. The addition of 40 μg/dL Cu to IVF medium enhanced total and progressive motility, sperm viability, functional sperm membrane integrity (HOST), sperm-zona binding, and pronuclear formation. On the other hand, the presence of Cu in IVF medium did not modify acrosome integrity and cleavage rates after IVF, but impaired blastocyst rates. Cu transporter SLC31A1 was detected in bovine spermatozoa in the apical segment of acrosome, and in the oocyte matured in vitro. In conclusion, the results obtained in the present study determined that cupremia levels at the beginning of FTAI protocol did not influence the pregnancy rates at 60 d after insemination. The presence of CTR1 in bovine mature oocyte and spermatozoa, as well as the beneficial effect of Cu on sperm quality would suggest an important role of this mineral during the fertilization process. Copyright © 2017 Elsevier Inc. All rights reserved.

Characteristics of bovine oocytes with different meiotic competence in terms of their mitochondrial status and expression of nuclear-encoded factors

Czech Academy of Sciences Publication Activity Database

Machatková, M.; Jeseta, M.; Hulínská, P.; Knitlová, D.; Němcová, Lucie; Kaňka, Jiří

2012-01-01

Roč. 47, č. 5 (2012), s. 806-814 ISSN 0936-6768 R&D Projects: GA MZe QI91A018 Grant - others:GA ČR(CZ) GD523/08/H064 Program:GD Institutional research plan: CEZ:AV0Z50450515 Keywords : bovine oocytes * mitochondrial status * nuclear-encoded factors Subject RIV: GI - Animal Husbandry ; Breeding Impact factor: 1.392, year: 2012

Follicle and oocyte growth in early postnatal calves: cytochemical, autoradiographical and electron microscopical studies

International Nuclear Information System (INIS)

Mhawi, A.J.; Kaňka, J.; Motlík, J.

1991-01-01

The initiation of oocyte and follicle growth was studied in 1- and 3-d-old calf ovaries using cytochemical, autoradiographical and electron microscopical approaches. Attention was only paid to unilaminar ovarian follicles that were classified into 3 categories: unilaminar flattened (UF), unilaminar flatto-cuboidal (UFC) and unilaminar cuboidal (UC) ovarian follicles when the oocyte was surrounded by 1 layer of flattened, a mixture of flattened and cuboidal and entirely cuboidal follicle cells, respectively. Our findings suggested that oocytes within each of these follicle categories were in different developmental stages. Furthermore, electron microscopic observations revealed that early after birth, oocyte nuclei characteristic of diplotene configuration (aggregation of the nuclear chromatin into moderately electron-dense small patches and fibrillo-granular texture of the nucleolus) were encountered in 41% of the UF follicles. The rest of the UF as well as all of the UFC and UC follicles were found to contain dictyate oocytes in which the chromatin was highly decondensed and the nucleolus differentiated into fibrillar, fibrillo-granular and granular components. The present results also indicated that the complete transition of the surrounding follicle cells from flattened to cuboidal shape and the morphological changes of the oocyte endoplasmic reticulum and mitochondria were 2 complementary events essential for initiation of oocyte growth

Retinol improves bovine embryonic development in vitro

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Edwards J Lannett

2004-12-01

Full Text Available Abstract Retinoids are recognized as important regulators of vertebrate development, cell differentiation, and tissue function. Previous studies, performed both in vivo and in vitro, indicate that retinoids influence several reproductive events, including follicular development, oocyte maturation and early embryonic development. The present study evaluated in vitro effects of retinol addition to media containing maturing bovine oocytes and developing embryos in both a low oxygen atmosphere (7% and under atmospheric oxygen conditions (20%. In the first experiment, abbatoir collected bovine oocytes were matured in the presence or absence of varying concentrations of retinol. After a 22–24 hour maturation period the oocytes were fertilized, denuded 18 hours later and cultured in a modified synthetic oviductal fluid (mSOF in a humidified atmosphere at 38.5 degrees C, 5% CO2, 7% O2 and 88% N2. Cleavage rates did not differ among control and retinol-treated oocytes in all three experiments. Addition of 5 micromolar retinol to the maturation medium (IVM tended (p

Late effects of 60Co γ radiation on the bovine oocyte as reflected by oocyte survival, follicular development, and reproductive performance

International Nuclear Information System (INIS)

Erickson, B.H.; Reynolds, R.A.; Murphree, R.L.

1976-01-01

Long-term effects of ionizing radiation on the oocyte of a long-lived mammal have not been determined. Therefore, 280 15- to 18-month-old grade Herefords were partitioned among five groups and irradiated with either 0, 200, 300, 400, or 300 + 300 (55 days between fractions) R of 60 Co γ radiation at exposure rates of 0.69 or 0.78 R/min. Dose to the ovaries was 37 +- 3 percent of the dose measured in air. Germ cell effects were assessed in the survivors through (1) counts of germ cells and follicles in serially sectioned ovaries, (2) incidence of ovarian abnormalities, and (3) reproductive performance. Neither germ cell nor follicular counts were significantly affected by irradiation (P greater than 0.05). Incidence of ovarian abnormalities was not altered nor was reproductive performance or quality of offspring. It was concluded, therefore, that a near lethal postpuberally applied dose of γ radiation would not affect the reproductive capacity of the bovine female

Heat stress effects on the cumulus cells surrounding the bovine oocyte during maturation: altered matrix metallopeptidase 9 and progesterone production.

Science.gov (United States)

Rispoli, L A; Payton, R R; Gondro, C; Saxton, A M; Nagle, K A; Jenkins, B W; Schrick, F N; Edwards, J L

2013-08-01

When the effects of heat stress are detrimental during maturation, cumulus cells are intimately associated with the oocyte. To determine the extent to which heat stress affects these cells, in this study, transcriptome profiles of the cumulus that surrounded control and heat-stressed oocytes (41 °C during the first 12 h only and then shifted back to 38.5 °C) during in vitro maturation (IVM) were compared using Affymetrix bovine microarrays. The comparison of cumulus-derived profiles revealed a number of transcripts whose levels were increased (n=11) or decreased (n=13) ≥ twofold after heat stress exposure (P1.7-fold decrease in the protein levels of latent matrix metallopeptidase 9 (proMMP9). Heat-induced reductions in transcript levels were noted at 6 h IVM with reductions in proMMP9 protein levels at 18 h IVM (P=0.0002). Independent of temperature, proMMP9 levels at 24 h IVM were positively correlated with the development rate of blastocysts (R²=0.36; P=0.002). The production of progesterone increased during maturation; heat-induced increases were evident by 12 h IVM (P=0.002). Both MMP9 and progesterone are associated with the developmental competence of the oocyte; thus, it seems plausible for some of the negative consequences of heat stress on the cumulus-oocyte complex to be mediated through heat-induced perturbations occurring in the surrounding cumulus.

Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.

Science.gov (United States)

Li, H J; Sutton-McDowall, M L; Wang, X; Sugimura, S; Thompson, J G; Gilchrist, R B

2016-04-01

Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P gap junctions between

Ascorbic acid effects on in vitro maturation of mouse oocyte with or ...

African Journals Online (AJOL)

STORAGESEVER

2009-10-19

Oct 19, 2009 ... tomycin (Sigma, S-9137), 6 mg/ml penicillin (Biochrom, A321-42) and 5% fetal bovine serum (FBS) (Hyclone, SH 30070.03). The follicles were punctured using a 28-gauge needle. Immature cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were collected and randomly transferred to ...

In vitro production of bovine embryos

DEFF Research Database (Denmark)

Stroebech, L.; Mazzoni, Gianluca; Pedersen, Hanne Skovsgaard

2015-01-01

be improved, and aspects related to the oocyte donor, oocyte maturation and the recipients are addressed in the following. Also, some of the future aspects of genomic selection and systems biology are addressed with particular focus on the Brazilian-Danish collaboration in the so-called GIFT-project.......In vitro production (IVP) of bovine embryos has become a widespread technology implemented in cattle breeding and production. The implementation of genomic selection and systems biology adds great dimensions to the impact of bovine IVP. The physical procedures included in the IVP process can still...

Effect of jasplakinolide on the in vitro maturation of bovine oocytes ...

African Journals Online (AJOL)

Jasplakinolide (JAS), a cytotoxic natural product, induces actin polymerization and increases microfilament assembly. The knowledge about the effect of JAS on oocyte meiosis in mammals is limited. The present study was to investigate the effect of JAS on the events of oocyte meiosis such as spindle configuration, ...

Effect of stage of follicular growth during superovulation on developmental competence of bovine oocytes

DEFF Research Database (Denmark)

Humblot, P; Holm, Peter; Lonergan, P

2005-01-01

. Follicular characteristics were measured and oocyte quality was assessed by morphology, mRNA expression of eight marker genes or developmental ability after in vitro/in vivo maturation and subsequent in vitro fertilization and culture. Approaching ovulation, expected increases in follicular size and cumulus...... expansion suggested progression of oocyte maturation. No differences were found in the expression patterns of analyzed genes, except for heat-shock-protein (Hsp) that was lower in in vivo matured oocytes collected shortly before ovulation. Oocytes collected at this time also had higher developmental ability...... measured as blastocyst rates (57.6 after in vitro production while no differences were found between oocytes recovered earlier at the first three time points (39.3-41.5. We conclude that oocytes recovered late in the preovulatory period are more developmentally competent than oocytes recovered at the pre...

Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species.

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Lagutina, Irina; Zakhartchenko, Valeri; Fulka, Helena; Colleoni, Silvia; Wolf, Eckhard; Fulka, Josef; Lazzari, Giovanna; Galli, Cesare

2011-04-01

The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

On-chip enucleation of an oocyte by untethered microrobots

International Nuclear Information System (INIS)

Ichikawa, Akihiko; Sakuma, Shinya; Sugita, Masakuni; Shoda, Tatsuro; Tamakoshi, Takahiro; Arai, Fumihito; Akagi, Satoshi

2014-01-01

We propose a novel on-chip enucleation of an oocyte with zona pellucida by using a combination of untethered microrobots. To achieve enucleation within the closed space of a microfluidic chip, two microrobots, a microknife and a microgripper were integrated into the microfluidic chip. These microrobots were actuated by an external magnetic force produced by permanent magnets placed on the robotic stage. The tip of the microknife was designed by considering the biological geometric feature of an oocyte, i.e. the oocyte has a polar body in maturation stage II. Moreover, the microknife was fabricated by using grayscale lithography, which allows fabrication of three-dimensional microstructures. The microgripper has a gripping function that is independent of the driving mechanism. On-chip enucleation was demonstrated, and the enucleated oocytes are spherical, indicating that the cell membrane of the oocytes remained intact. To confirm successful enucleation using this method, we investigated the viability of oocytes after enucleation. The results show that the production rate, i.e. the ratio between the number of oocytes that reach the blastocyst stage and the number of bovine oocytes after nucleus transfer, is 100%. The technique will contribute to complex cell manipulation such as cell surgery in lab-on-a-chip devices. (paper)

Insulin during in vitro oocyte maturation has an impact on development, mitochondria, and cytoskeleton in bovine day 8 blastocysts.

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Laskowski, Denise; Båge, Renée; Humblot, Patrice; Andersson, Göran; Sirard, Marc-André; Sjunnesson, Ylva

2017-10-01

Insulin is a key metabolic hormone that controls energy homeostasis in the body, including playing a specific role in regulating reproductive functions. Conditions associated with hyperinsulinemia can lower developmental rates in bovine in vitro embryo production and are linked to decreased fertility in humans, as in cases of obesity or type 2 diabetes. Embryo quality is important for fertility outcome and it can be assessed by choosing scoring standards for various characteristics, such as developmental stage, quality grade, cell number, mitochondrial pattern or actin cytoskeleton structure. Changes in the embryo's gene expression can reflect environmental impacts during maturation and may explain morphological differences. Together with morphological evaluation, this could enable better assessment and possibly prediction of the developmental potential of the embryo. The aim of this study was to use a bovine model to identify potential gene signatures of insulin-induced changes in the embryo by combining gene expression data and confocal microscopy evaluation. Bovine embryos were derived from oocytes matured in two different insulin concentrations (10 µg mL - 1 and 0.1 µg mL - 1 ), then stained to distinguish f-Actin, DNA and active mitochondria. The total cell number of the embryo, quality of the actin cytoskeleton and mitochondrial distribution were assessed and compared to an insulin-free control group. A microarray-based transcriptome analysis was used to investigate key genes involved in cell structure, mitochondrial function and cell division. Our results indicate that insulin supplementation during oocyte maturation leads to lower blastocyst rates and a different phenotype, characterised by an increased cell number and different actin and mitochondrial distribution patterns. These changes were reflected by an up-regulation of genes involved in cell division (MAP2K2; DHCR7), cell structure (LMNA; VIM; TUBB2B; TUBB3; TUBB4B) and mitochondrial activation

Bovine Lhx8, a Germ Cell-Specific Nuclear Factor, Interacts with Figla.

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Liyuan Fu

Full Text Available LIM homeobox 8 (Lhx8 is a germ cell-specific transcription factor essential for the development of oocytes during early oogenesis. In mice, Lhx8 deficiency causes postnatal oocyte loss and affects the expression of many oocyte-specific genes. The aims of this study were to characterize the bovine Lhx8 gene, determine its mRNA expression during oocyte development and early embryogenesis, and evaluate its interactions with other oocyte-specific transcription factors. The bovine Lhx8 gene encodes a protein of 377 amino acids. A splice variant of Lhx8 (Lhx8_v1 was also identified. The predicted bovine Lhx8 protein contains two LIM domains and one homeobox domain. However, one of the LIM domains in Lhx8_v1 is incomplete due to deletion of 83 amino acids near the N terminus. Both Lhx8 and Lhx8_v1 transcripts were only detected in the gonads but none of the somatic tissues examined. The expression of Lhx8 and Lhx8_v1 appears to be restricted to oocytes as none of the transcripts was detectable in granulosa or theca cells. The maternal Lhx8 transcript is abundant in GV and MII stage oocytes as well as in early embryos but disappear by morula stage. A nuclear localization signal that is required for the import of Lhx8 into nucleus was identified, and Lhx8 is predominantly localized in the nucleus when ectopically expressed in mammalian cells. Finally, a novel interaction between Lhx8 and Figla, another transcription factor essential for oogenesis, was detected. The results provide new information for studying the mechanisms of action for Lhx8 in oocyte development and early embryogenesis.

The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation.

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Kátia R L Schwarz

Full Text Available This study aimed to determine the influence of cyclic guanosine 3'5'-monophosphate (cGMP and cGMP-dependent kinase (PKG during in vitro maturation (IVM on lipolysis-related parameters in bovine cumulus-oocyte complexes (COCs, and on embryo development and cryosurvival. COCs were matured with cGMP/PKG modulators and assessed for metaphase II rates (MII, cGMP levels, lipid content in oocytes (OO, transcript abundance for genes involved in lipolysis (ATGL and lipid droplets (PLIN2 in cumulus cells (CC and OO, and presence of phosphorylated (active hormone sensitive lipase (HSLser563 in OO. Embryo development, lipid contents and survival to vitrification were also assessed. Phosphodiesterase 5 inhibition (PDE5; cGMP-hydrolyzing enzyme with 10-5M sildenafil (SDF during 24 h IVM increased cGMP in COCs (56.9 vs 9.5 fMol/COC in untreated controls, p<0.05 and did not affect on maturation rate (84.3±6.4% MII. Fetal calf serum (FCS in IVM medium decreased cGMP in COCs compared to bovine serum albumin (BSA + SDF (19.6 vs 66.5 fMol/COC, respectively, p<0.05. FCS increased lipid content in OO (40.1 FI, p<0.05 compared to BSA (34.6 FI, while SDF decreased (29.8 and 29.6 FI, with BSA or FCS, respectively p<0.05. PKG inhibitor (KT5823 reversed this effect (38.9 FI, p<0.05. ATGL and PLIN2 transcripts were detected in CC and OO, but were affected by cGMP and PKG only in CC. HSLser563 was detected in OO matured with or without modulators. Reduced lipid content in embryos were observed only when SDF was added during IVM and IVC (27.6 FI compared to its use in either or none of the culture periods (34.2 FI, p<0.05. Survival to vitrification was unaffected by SDF. In conclusion, cGMP and PKG are involved in lipolysis in OO and possibly in CC and embryos; serum negatively affects this pathway, contributing to lipid accumulation, and cGMP modulation may reduce lipid contents in oocytes and embryos, but without improving embryo cryotolerance.

Vitrificação de ovócitos imaturos de bovinos utilizando etilenoglicol associado à trehalose e polivinilpirrolidona Vitrification of immature bovine oocytes, by the ethylene glycol associated with trehalose and polivinylpyrrolidone

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M.R. Souza

2003-10-01

Full Text Available Avaliaram-se os efeitos da vitrificação de ovócitos imaturos de bovinos utilizando o etilenoglicol (EG associado à trehalose e à polivinilpirrolidona (PVP. Utilizaram-se ovócitos provenientes de ovários de vacas abatidas em matadouro, distribuídos aleatoriamente em três tratamentos (T. TI - ovócitos não desnudados e não congelados, TII - ovócitos vitrificados com cumulus oophorus e TIII - ovócitos desnudados vitrificados. A percentagem de ovócitos recuperados e ovócitos com morfologia normal após a vitrificação foi diferente entre TII e TIII (92,2 e 72,6%; 79,0 e 63,6%, respectivamente. Os ovócitos normais foram cultivados à 38,5ºC em atmosfera de 5% de CO2 por 24 horas. Após o cultivo, os ovócitos foram fecundados e os embriões cultivados in vitro por sete dias. Foram encontradas diferenças entre tratamentos quanto às taxas de maturação nuclear, fecundação e clivagem (83,9, 70,0 e 44,0%; 17,5, 23,7 e 5,1%; 0,0, 0,0 e 0,0% para os tratamentos I, II e III, respectivamente. Apenas no TI foram obtidas mórulas e blastocistos (21,4%. Os procedimentos de vitrificação, segundo os protocolos utilizados, não são indicados para a criopreservação de ovócitos imaturos de bovinos.This study aimed to evaluate the effects of vitrification procedure of immature bovine oocytes using ethylene glycol (EG associated with trehalose and polivinylpyrrolidone on the percentage of recovered oocytes with normal morphology and nuclear maturation, fecundation and cleavage rates for in vitro cultivated embryos. Ovary oocytes of slaughtered cows were randomly allotted to three treatments (T: TI - oocytes neither undenuded nor vitrified, TII - vitrified oocytes with cumulus oophorus, TIII - undenuded vitrified oocytes. The percentage of recovered oocytes and oocytes with normal morphology after vitrification was different for TII and TIII (92.2 and 72.6%, 79.0 and 63.3% for TII and TIII, respectively. All normal oocytes were cultivated

Highly efficient vitrification method for cryopreservation of human oocytes.

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

2005-09-01

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.

Assessment of different methods of bovine oocytes collection, maturation and in vitro fertilization of abattoir specimens

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W.M. Saleh

2017-06-01

Full Text Available The aim of this study is designed to evaluate the best methods for cow oocytes collection from abattoir specimens which is the cheapest, easily obtained and bulky number. Forty five fresh cow genitalia specimens and testicle were collected directly after slaughter from Al-Shoáalla abattoir north-west of Baghdad the capital early morning, transported in cool box under (4-8 °C to the laboratory of theriogenology in the College of Veterinary Medicine/Baghdad University during the period from November 2016 to February 2017. Ovaries were separated from the surrounding tissues, washed thoroughly with dis. water repeatedly, then with normal saline and finally with MEM medium containing Antibiotics and Nystatin for contaminant elimination. Oocytes were collected with four methods aspiration, slashing, slicing after aspiration and slicing. The result showed that; the collected oocytes were 55, 68, 87 and 106 oocytes respectively; slicing methods yield more oocytes count. Period of time between slaughtering and samples processing significantly affect oocytes collected percentage and quality, periods as 2, 6, 12 and 24 hours yield 75%, 68%, 61% and 55% oocytes counts of good, fair, poor to aged and bad quality oocytes respectively. Two hours period yield an elevated oocytes count with good quality. Maturation index of oocytes according to the type of collected methods showed 44, 37, 39 and 42 with 12, 8, 6 and 6 good oocyte quality for the four methods respectively. In conclusion slicing methods yield more oocytes count with a moderate quality and embryos production while aspiration methods yield a moderate oocytes count with an elevated quality and good embryos production.

How do oocytes disappear?

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Bonilla-Musoles, F; Renau, J; Hernandez-Yago, J; Torres, J

1975-07-29

It has been study using transmission and scanner electron microscopy the mean procedures of dessaparence of the oocytes. On described three methods: 1. The necrosis of the oocytes. 2. The autolysis and fagocitosis by granulosa cells. 3. The migration of those to the superphicie and fall into the peritoneal cavity. Using the scanner electron microscopy in ovaries of fetus and newborn it seems the latest method to bee the most important during the intrauterine life. After the birth, this last phenomenon seems to disappear.

Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts from In Vitro Maturation of Bovine Oocytes

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Jolanta Opiela

2014-01-01

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.

Function of JARID2 in bovines during early embryonic development

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Yao Fu

2017-12-01

Full Text Available Histone lysine modifications are important epigenetic modifications in early embryonic development. JARID2, which is a member of the jumonji demethylase protein family, is a regulator of early embryonic development and can regulate mouse development and embryonic stem cell (ESC differentiation by modifying histone lysines. JARID2 can affect early embryonic development by regulating the methylation level of H3K27me3, which is closely related to normal early embryonic development. To investigate the expression pattern of JARID2 and the effect of JARID2-induced H3K27 methylation in bovine oocytes and early embryonic stages, JARID2 mRNA expression and localization were detected in bovine oocytes and early embryos via qRT-PCR and immunofluorescence in the present study. The results showed that JARID2 is highly expressed in the germinal vesicle (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell and blastocyst stages, but the relative expression level of JARID2 in bovine GV oocytes is significantly lower than that at other oocyte/embryonic stages (p < 0.05, and JARID2 is expressed primarily in the nucleus. We next detected the mRNA expression levels of embryonic development-related genes (OCT4, SOX2 and c-myc after JARID2 knockdown through JARID2-2830-siRNA microinjection to investigate the molecularpathwayunderlying the regulation of H3K27me3 by JARID2 during early embryonic development. The results showed that the relative expression levels of these genes in 2-cell embryos weresignificantly higher than those in the blastocyst stage, and expression levels were significantly increased after JARID2 knockdown. In summary, the present study identified the expression pattern of JARID2 in bovine oocytes and at each early embryonic stage, and the results suggest that JARID2 plays a key role in early embryonic development by regulating the expression of OCT4, SOX2 and c-myc via modification of H3K27me3 expression. This work provides new data for improvements in the

Cytoarchitecture of Caudiverbera caudiverbera stage VI oocytes: a light and electron microscope study.

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Dabiké, M; Preller, A

1999-06-01

The general characteristics and salient features of the full-grown stage VI Caudiverbera caudiverbera oocyte at the light and electron microscopy level are described. The oocyte is a huge cell with radial symmetry and distinct polarity. A black animal hemisphere, rich in pigment granules and containing the nucleus, is clearly distinguished from the unpigmented white-yellowish vegetal hemisphere. The cell is surrounded by a highly invaginated plasma membrane, with numerous microvilli. The cortex underlying the plasma membrane contains cortical and pigment granules, mitochondria, rough endoplasmic reticulum and coated vesicles. Cytoskeletal components, such as actin filaments and microtubules, are also found in this region. The predominant structures, distributed throughout the cell, are the yolk platelets, which show a gradient in size with small platelets in the animal half and very large ones in the vegetal zone. Mitochondria are also very abundant in both hemispheres and clouds of these organelles are found in the perinuclear region, frequently associated with microtubules. Developed Golgi complexes are present in the cytoplasm and occasionally, annulate lamellae appear towards the inner zones. The nucleus is a large structure containing numerous nucleoli. The nuclear envelope is highly invaginated, especially at the side facing the vegetal pole. It is regularly perforated by large nuclear pores. Our results show that the structural organization of Caudiverbera oocytes, although similar to that of other amphibian oocytes, differs from them especially concerning the spatial distribution of several structural components.

On-Chip Enucleation of Bovine Oocytes using Microrobot-Assisted Flow-Speed Control

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Akihiko Ichikawa

2013-06-01

Full Text Available In this study, we developed a microfluidic chip with a magnetically driven microrobot for oocyte enucleation. A microfluidic system was specially designed for enucleation, and the microrobot actively controls the local flow-speed distribution in the microfluidic chip. The microrobot can adjust fluid resistances in a channel and can open or close the channel to control the flow distribution. Analytical modeling was conducted to control the fluid speed distribution using the microrobot, and the model was experimentally validated. The novelties of the developed microfluidic system are as follows: (1 the cutting speed improved significantly owing to the local fluid flow control; (2 the cutting volume of the oocyte can be adjusted so that the oocyte undergoes less damage; and (3 the nucleus can be removed properly using the combination of a microrobot and hydrodynamic forces. Using this device, we achieved a minimally invasive enucleation process. The average enucleation time was 2.5 s and the average removal volume ratio was 20%. The proposed new system has the advantages of better operation speed, greater cutting precision, and potential for repeatable enucleation.

Viabilidade e fertilização in vitro de oócitos bovinos após vitrificação Viability and in vitro fertilization of bovine oocytes after vitrification

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Sérgio Galbinski

2003-09-01

ários para proteção da zona pelúcida e do oolema.PURPOSE: to verify vitrification techniques using 6 M DMSO to cryopreserve in vitro matured bovine oocytes, and to assess the effects of the time of exposure to vitrification solutions (VS. METHODS: dilutions of VS were prepared from the stock VS (VS 100% consisting of 6 M DMSO to give 25 and 65% DMSO solutions. Bovine oocytes were in vitro matured for 18-22 h. Matured oocytes were placed first into 25% VS, at room temperature for 5 min, then transferred to 65% VS, before being pipetted into the 100% VS in plastic straws. Three experimental groups were formed: in the first group, time of pipetting through 65% VS and loading the straw took up to 60 s, in the second group it did not exceed 30 s. For thawing, straws were held in air for 10 s and then in a water bath for 10 s. The contents of each straw were expelled in sucrose solution and held for 5 min. In the third experimental group, oocytes went through all VS, but were not vitrified. All retrieved oocytes were inseminated. For control, fresh, in vitro matured oocytes were inseminated. RESULTS: after vitrification, 69.1 and 59.8% of the oocytes were retrieved from the 30 s and 60 s groups, respectively, and 93 and 89% of these oocytes appeared morphologically normal 24 h after insemination, respectively. In the group of oocytes exposed without vitrification, 75.6% were retrieved and 84.7% were morphologically viable, 24 h after insemination. No fertilization was observed in the experimental groups. Among controls, 65.4% were fertilized. CONCLUSIONS: the vitrification technique using 6 M DMSO is not a feasible approach to cryopreserve in vitro matured bovine oocytes. Decreasing the time of exposure to VS did not overcome deleterious effects of the procedure on the fertilizability of oocytes. Improvements in the technique are needed to protect the zona pellucida and oolemma.

Addition of granulosa cell mass to the culture medium of oocytes derived from early antral follicles increases oocyte growth, ATP content, and acetylation of H4K12.

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Sugiyama, Miyako; Sumiya, Mei; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

2016-12-01

The main aim of the present study was to examine the hypothesis that an increase in the number of granulosa cells surrounding developing bovine oocytes results in both high ATP levels and an increase in the acetylation level of H4K12 in oocytes grown in vitro. Oocyte-granulosa cell complexes (OGCs) were collected from early antral follicles (EAFs, 0.4-0.7 mm in diameter), and individually cultured on 96-well plates with or without additional granulosa cell mass that had been prepared from other OGCs. After 16 days of culture, we examined: (i) the rate of antrum formation of the OGCs; (ii) the diameter, maturation, and fertilization rate of the oocytes; and (iii) the ATP content and acetylation level of H4K12 in the oocytes grown in vitro. Granulosa cell mass added to the culture medium contributed to the development of OGCs with a higher rate of antrum formation and oocyte growth. Furthermore, the addition of granulosa cells increased the ATP content and acetylation level of H4K12 in oocytes grown in vitro compared with those developed without addition of granulosa cells. In addition, there was a positive correlation between the ATP content in oocytes grown in vitro and the number of granulosa cells in the corresponding OGCs. The results suggest that granulosa cells play a role not only in the development of OGCs and the growth of oocytes, but also in the determination of ATP content and the acetylation of H4K12 in the oocytes developed in vitro.

Bovine somatic cell nuclear transfer.

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Ross, Pablo J; Cibelli, Jose B

2010-01-01

Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes.

Three-step in vitro maturation culture of bovine oocytes imitating temporal changes of estradiol-17β and progesterone concentrations in preovulatory follicular fluid

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M. Matsuo

2017-10-01

Full Text Available The objective of the article is to evaluate the effect of three-step in vitro maturation (IVM culture system imitating estradiol-17β (E2 and progesterone (P4 concentrations in preovulatory follicles on in vitro bovine embryo production. The cumulus–oocyte complexes (COCs were collected from follicles (2 to 8 mm in diameter of bovine ovaries obtained from a local slaughterhouse. For IVM, the COCs were cultured for 22 h in a three-step system: (1 culture in medium 199, containing 700 ng mL−1 E2 and 50 ng mL−1 P4, for 5 h, followed by the medium containing 150 ng mL−1 E2 and 150 ng mL−1 P4 for 11 h, and then the medium containing 20 ng mL−1 E2 and 300 ng mL−1 P4 for 6 h (EP group; (2 culture in the medium containing 700 ng mL−1 E2 for 5 h, followed by the medium containing 150 ng mL−1 E2 for 11 h, and then the medium containing 20 ng mL−1 E2 for 6 h (E group; or (3 culture in the medium containing 50 ng mL−1 P4 for 5 h, followed by the medium containing 150 ng mL−1 P4 for 11 h, and then the medium containing 300 ng mL−1 P4 for 6 h (P group. The COCs were cultured in the medium containing 1000 ng mL−1 E2 for 22 h (control group. After IVM, the COCs were co-incubated with sperm and further cultured. At 48 h after insemination, the cleavage rate of embryos was not different among the groups. At 192 h after insemination, the blastocyst formation rate of EP group was significantly higher than that of the other groups. The total cell number of blastocysts did not differ among the groups. In conclusion, these results demonstrate that the three-step IVM culture system of bovine oocytes imitating temporal changes of E2 and P4 concentrations in preovulatory follicular fluid improves the developmental potential of embryos in vitro.

Metabolic requirements associated with GSH synthesis during in vitro maturation of cattle oocytes.

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Furnus, C C; de Matos, D G; Picco, S; García, P Peral; Inda, A M; Mattioli, G; Errecalde, A L

2008-12-01

Glutathione (GSH) concentration increases in bovine oocytes during in vitro maturation (IVM). The constitutive amino acids involved in GSH synthesis are glycine (Gly), glutamate (Glu) and cysteine (Cys). The present study was conducted to investigate the effect of the availability of glucose, Cys, Gly and Glu on GSH synthesis during IVM. The effect of the amino acid serine (Ser) on intracellular reduced/oxidized glutathione (GSH/GSSG) content in both oocytes and cumulus cells was also studied. Cumulus-oocyte complexes (COC) of cattle obtained from ovaries collected from an abattoir were matured in synthetic oviduct fluid (SOF) medium containing 8 mg/ml bovine serum albumin-fatty acid-free (BSA-FAF), 10 microg/ml LH, 1 microg/ml porcine FSH (pFSH) and 1 microg/ml 17 beta-estradiol (17beta-E2). GSH/GSSG content was measured using a double-beam spectrophotometer. The COC were cultured in SOF supplemented with 1.5mM or 5.6mM glucose (Exp. 1); with or without Cys+Glu+Gly (Exp. 2); with the omission of one constitutive GSH amino acid (Exp. 3); with 0.6mM Cys or Cys+Ser (Exp. 4). The developmental capacity of oocytes matured in IVM medium supplemented with Cys and the cell number per blastocyst were determined (Exp. 5). The results reported here indicate (1) no differences in the intracellular GSH/GSSG content at any glucose concentrations. Also, cumulus cell number per COC did not differ either before or after IVM (Exp. 1). (2) Glutathione content in oocytes matured in SOF alone were significantly different from oocytes incubated with SOF supplemented with Cys+Glu+Gly (Exp. 2). (3) Addition of Cys to maturation medium, either with or without Gly and Glu supplementation resulted in an increase of GSH/GSSG content. However, when Cys was omitted from the IVM medium intracellular GSH in oocytes or cumulus cells was less but not significantly altered compared to SOF alone (Exp. 3). (4) Glutathione content in both oocytes and cumulus cells was significantly reduced by

In Vitro Testing of the Insecticide Reldan 22 on Swine Oocyte Maturation

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Ileana Miclea

2016-11-01

Full Text Available Chlorpyrifos (Reldan 22 is an widely used insecticide for the control of insect pests in agricultureand in residential areas. It is classified as moderately toxic by the United States Environmental Protection Agency and has been quantified in human biological fluids. Given that the use of porcine and bovine models for testing chemicals has increased recently we designed an experiment to test the toxicity of several Chlorpyrifos concentrations and investigate its effects on maturation of swine oocytes. Swine oocytes from ovaries harvested in a commercial slaughterhouse were cultured for 44-45h in M199 supplemented with the following Reldan 22 concentrations: 0.1, 0.5, 1 or 2 µg/ml. Cumulus oophorous expansion was assessed and oocytes were denuded and stained with 1 µg/ml fluorescein diacetate to estimate viability. Afterwards, oocytes were fixed in a 60% methanol/DPBS solution and stained with 50 µg/ml propidium iodide to observe the DNA stage. Differences were analysed by the analysis of variance and interpreted using the Tuckey test. Our research shows that the insecticide Reldan 22® stimulated cumulus expansion to an extent but reduced oocyte viability which was accompanied by an increase in the number of immature oocytes and a decrease in the percentages of gametes that resumed meiosis. This leads us conclude that its presence in the oocyte environment is toxic for development at concentrations 0.5, 1 and 2 µg/ml.

RESEARCHES REGARDING THE INFLUENCE OF THE NUMBER OF CUMULAR CELLS LAYER OVER THE OOCYTE MATURATION EFFICIENCY

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V. CARABĂ

2009-05-01

Full Text Available During the experiments we have carried out with imature oocyte collected from the ovarian follicles, wefound a variety of oocyte-cumulus complexes. We got the following experiment in order to understand therole of cumular cells on the achievement of the cytoplasma and oocyte nucleus maturation. We select theoocyte-cumulus complexes collected both from cows and sows according to the number of cumular celllayers and we watched their development to the blastocyst stade. Thus, we achieved three groups of COC(oocyte-cumulus complexes.One group was made of oocyte without cumular cells, the second group had a layer of cumular cells andthe third group had many layers of cumular cells. we performed an incubation of all these types of COCin TCM-199 enriched with 20% of bovine fetal serum. Because only 1,2 oocyte of the ones who lack thecumular cells layer had maturation signs during cultivation in the thermostat versus 55 and 115,respectively, of the ones that had many cellular layers, presents a solid evidence that cumular cells areindispensable for the maturation and even to the fecundation process. The cumular cells perform adecisive role on the cytoplasma and oocyte nucleus maturation process.

EFFECT OF COLLING IN DIPLOID OOCYTES AFTER IN VITRO MATURATION EFEITO DO RESFRIAMENTO NA PLOIDIA DE OVÓCITOS BOVINOS MATURADOS IN VITRO

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Iris Ferrari

2007-12-01

Full Text Available

The present study aimed to verify the incidence of bovine diploid oocytes when cooled at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a local slaughterhouse and divided into five groups: control group (uncooled oocytes, 0/4 h group (oocytes cooled at 4ºC before the onset of maturation, 0/29 (composed of oocytes cooled at 29ºC before the onset of maturation, 12/4 (cooled 12 h at 4ºC after the onset of maturation, and 12/29 h group (cooled 12 h at 29ºC after the onset of maturation. the oocytes remained cooled for 45 min. in all groups, the oocytes completed 24 h maturation. Subsequently, the cumulus cells were removed, and the denuded oocytes fixed on slides and stained with aceto-orcein. No differences (P > 0.05 in the incidence of diploid metaphase ii oocytes were observed between the control group (6.0% and oocytes cooled at 4ºC and 29ºC before (8.9% and 8.0% and after 12 h the onset of maturation (3.9% and 0.0%. These results suggest that the nuclear stage at which bovine oocytes are cooled does not affect the incidence of diploid oocytes after 24 h maturation.

Key-Words: Bovine, cooling, diploid, in vitro maturation, oocytes.

O presente estudo objetivou verificar o efeito do resfriamento de ovócitos bovinos em diferentes estágios de maturação na ploidia. Ovócitos bovinos foram obtidos de ovários de abatedouro e divididos em cinco grupos: grupo-controle (ovócitos não resfriados; grupo 0/4 (ovócitos resfriados a 4ºC antes do inicio da maturação; grupo 0/29 (ovócitos resfriados a 29ºC antes do início da maturação; grupo 12/4 (ovócitos resfriados a 4ºC após doze horas de maturação; e grupo 12/29 (ovócitos resfriados a 29ºC após doze horas de maturação. Os ovócitos permaneceram resfriados por 45 minutos. Em todos os grupos os ovócitos completaram 24 horas de maturação. Em seguida, as células da

Human oocyte cryopreservation and the fate of cortical granules.

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Ghetler, Yehudith; Skutelsky, Ehud; Ben Nun, Isaac; Ben Dor, Liah; Amihai, Dina; Shalgi, Ruth

2006-07-01

To examine the effect of the commonly used oocyte cryopreservation protocol on the cortical granules (CGs) of human immature germinal vesicle (GV) and mature metaphase II (MII) oocytes. Laboratory study. IVF unit. Unfertilized, intracytoplasmic sperm injected (ICSI) oocytes, and immature oocytes were cryopreserved using a slow freezing-rapid thawing program with 1,2-propanediol (PROH) as a cryoprotectant. Cortical granule exocytosis (CGE) was assessed by either confocal microscopy or transmission electron microscopy (TEM). The survival rates of frozen-thawed oocytes (mature and immature) were significantly lower compared with zygotes. Both mature and immature oocytes exhibited increased fluorescence after cryopreservation, indicating the occurrence of CGE. Mere exposure of oocytes to cryoprotectants induced CGE of 70% the value of control zygotes. The TEM revealed a drastic reduction in the amount of CGs at the cortex of frozen-thawed GV and MII oocytes, as well as appearance of vesicles in the ooplasm. The commonly used PROH freezing protocol for human oocytes resulted in extensive CGE. This finding explains why ICSI is needed to achieve fertilization of frozen-thawed human oocytes.

Quantitative analysis of bone morphogenetic protein 15 (BMP15 and growth differentiation factor 9 (GDF9 gene expression in calf and adult bovine ovaries

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Hayashi Ken-go

2011-03-01

Full Text Available Abstract Background It has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15 and growth and differentiation factor 9 (GDF9 play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of BMP15 and GDF9 in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes. Methods Bovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR and in situ hybridization. Immunohistochemical analysis was also performed. Results The expression of BMP15 and GDF9 in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. GDF9 expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, BMP15 expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry. Conclusions Our result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. BMP15 and GDF9 mRNA expression in oocytes and cumulus cells was different in calves and cows.

Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes.

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Morovic, Martin; Strejcek, Frantisek; Nakagawa, Shoma; Deshmukh, Rahul S; Murin, Matej; Benc, Michal; Fulka, Helena; Kyogoku, Hirohisa; Pendovski, Lazo; Fulka, Josef; Laurincik, Jozef

2017-12-01

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.

From fresh heterologous oocyte donation to autologous oocyte banking.

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Stoop, D

2012-01-01

Today, oocyte donation has become well established, giving rise to thousands of children born worldwide annually. The introduction of oocyte cryopreservation through vitrification allows the introduction of egg banking, improving the efficiency and comfort of oocyte donation. Moreover, the vitrification technique can now enable autologous donation of oocytes to prevent future infertility. We evaluated fresh heterologous oocyte donation in terms of obstetrical and perinatal outcome as well as of the reproductive outcome of past donors. We then evaluated the efficiency of a closed vitrification device and its clinical applications within ART. Thirdly, we evaluated the opinion of women with regard to preventive egg freezing and the efficiency of a human oocyte in relation to age. Oocyte donation is associated with an increased risk of first trimester bleeding and pregnancy induced hypertension. Donating oocytes does not seem to increase the likelihood for a later need of fertility treatment. The chance of an oocyte to result in live birth (utilization rate) in women women would consider safeguarding their reproductive potential through egg freezing or are at least open to the idea. The introduction of efficient oocyte cryopreservation has revolutionized oocyte donation through the establishment of eggbank donation. The technique also enables women to perform autologous donation after preventive oocyte storage in order to circumvent their biological clock.

Viable calves produced by somatic cell nuclear transfer using meiotic-blocked oocytes.

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De Bem, Tiago H C; Chiaratti, Marcos R; Rochetti, Raquel; Bressan, Fabiana F; Sangalli, Juliano R; Miranda, Moysés S; Pires, Pedro R L; Schwartz, Kátia R L; Sampaio, Rafael V; Fantinato-Neto, Paulo; Pimentel, José R V; Perecin, Felipe; Smith, Lawrence C; Meirelles, Flávio V; Adona, Paulo R; Leal, Cláudia L V

2011-10-01

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585±34,775 vs. 595,579±31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179±45,617 vs. 498,771±33,231) and blastocysts (816,627±40,235 vs. 765,332±51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.

Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes

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Carbone Maria

2006-09-01

Full Text Available Abstract Background Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC and the exocytosis of cortical granules in mouse oocytes. Methods An In-Vitro-Fertilization assay (IVF was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val, was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val, were evaluated. Results The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P Conclusion The presents results provide evidence that a cyclic RGD peptide highly effective in inhibiting sperm-oocyte interaction stimulates in mouse oocytes the activation of PKC and the exocytosis of cortical granules. These data support the view that RGD-binding receptors may function as signalling receptors giving rise integrated signalling not sufficient for

Meat and Livestock Association Plenary Lecture 2005. Oocyte signalling molecules and their effects on reproduction in ruminants.

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McNatty, Kenneth P; Lawrence, Stephen; Groome, Nigel P; Meerasahib, Mohammed F; Hudson, Norma L; Whiting, Lynda; Heath, Derek A; Juengel, Jennifer L

2006-01-01

Sheep (Ovis aries) are a highly diverse species, with more than 900 different breeds that vary significantly in their physiological characteristics, including ovulation rate and fecundity. From examination of inherited patterns of ovulation rate, several breeds have been identified with point mutations in two growth factor genes that are expressed in oocytes. Currently, five different point mutations have been identified in the BMP15 (GDF9b) gene and one in GDF9. Animals heterozygous for the GDF9 and/or the BMP15 mutations have higher ovulation rates than their wild-type counterparts. In contrast, those homozygous for any of the aforementioned BMP15 or GDF9 mutations are sterile owing to arrested follicular development. In bovine and ovine ovaries, GDF9 was expressed exclusively in oocytes throughout follicular growth from the primordial stage of development, whereas in sheep BMP15 was expressed exclusively in oocytes from the primary stage: no data for the ontogeny of BMP15 expression are currently available for cattle. In vitro, ovine growth differentiation factor 9 (oGDF9) has no effect on (3)H-thymidine incorporation by either bovine or ovine granulosa cells, whereas ovine bone morphogenetic protein 15 (oBMP15) has modest (1.2- to 1.6-fold; P reproduction in mammals, including rodents, humans and ruminants. Moreover, in vivo manipulation of these oocyte signalling molecules provides new opportunities for the management of the fertility of ruminants.

The effects of melatonin on bovine uniparental embryos development in vitro and the hormone secretion of COCs

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Shujuan Wang

2017-07-01

Full Text Available Melatonin is a unique multifunctional molecule that mediates reproductive functions in animals. In this study, we investigated the effects of melatonin on bovine parthenogenetic and androgenetic embryonic development, oocyte maturation, the reactive oxygen species (ROS levels in parthenogenetic and androgenetic embryos and cumulus—oocyte complexes (COCs hormone secretion with melatonin supplementation at four concentrations (0, 10, 20, and 30 pmol/mL, respectively. The results showed that melatonin significantly promoted the rates of bovine parthenogenetic and androgenetic embryonic cleavage and morula and blastocysts development (P < 0.05. The rate of cleavage was higher in the androgenetic embryo than that in the parthenogenetic embryo. Compared with the parthenogenetic embryos, the androgenetic embryos had a poor developmental competence from morula to blastocyst stage. Moreover, the levels of ROS were significantly lower in the parthenogenetic and androgenetic embryoes with melatonin-treated group than that of the control group (P < 0.05. Melatonin supplemented significantly increased the maturation rate of oocyte in vitro (P < 0.05. More importantly, melatonin significantly promoted the secretion of progesterone and estradiol by COCs (P < 0.05. To reveal the regulatory mechanism of melatonin on steroids synthesis, we found that steroidogenic genes (CYP11A1, CYP19A1 and StAR were upregulated, suggesting that melatonin regulated estradiol and progesterone secretion through mediating the expression of steroidogenic genes (CYP11A1, CYP19A1 and StAR. In addition, MT1 and MT2 were identified in bovine early parthenogenetic and androgenetic embryos using western blot. It could be concluded that melatonin had beneficial effects on bovine oocyte in vitro maturation, COC hormone secretion, early development of subsequent parthenogenetic and androgenetic embryos. It is inferred that melatonin could be used to enhance the efficiency of in

The effects of platelet lysate on maturation, fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage.

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Pazoki, Hassan; Eimani, Hussein; Farokhi, Farah; Shahverdi, Abdol-Hossein; Tahaei, Leila Sadat

2016-04-01

Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored. Oocytes at germinal vesicle stage with cumulus cells (cumulus-oocyte complex) and without cumulus cells (denuded oocytes) were obtained from mature female mice. The maturation medium was enriched by 5 and 10 % PL and 5 % PL + 5 % fetal bovine serum (FBS) as experimental groups; the control groups' media consisted of 5 and 10 % FBS. After 18 h, the matured oocytes were collected and, after fertilization, subsequent development was monitored. The rates of maturation, fertilization and 2-cell embryo development for the denuded oocyte groups in experimental media 5 % PL and 5 % PL + 5 % FBS were significantly higher than those of the control groups ( P platelet lysate could improve the maturation rate in the absence of granulosa cells compared to media with FBS. This extract also had positive effects on fertilization and embryo development.

Good Egg or Bad Egg: Developing markers of oocyte competence for Assisted Reproductive Interventions.

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Trudee Fair

2016-06-01

Full Text Available The oocyte is the foundation of life. It develops from a single fertilized cell to a multicellular organism capable of an independent existence. Competence to achieve this maximum potential is acquired following the protracted, but, highly co-ordinated process of growth and subsequently, maturation. The environment in which all or part of these processes occur, can ultimately have long-term consequences for female fertility and the health of resulting offspring. The pressure to identify and select oocytes or embryos with the highest developmental potential has intensified as the number of patients and the range of options available to them have increased. In particular, as in vitro maturation and single embryo transfer become more routine in assisted reproductive technology, selection is critical to a successful outcome. Moreover, the identification of markers of oocyte health and quality is essential to monitor the impact of these technologies on gametes and embryos. Technologies, such as transcriptomics, proteomics and metabolomics, offer more sophisticated methods for oocyte and embryo selection, with the emphasis on the predictive value of non-invasive protocols which profile follicular fluid, follicle/ granulosa cells and cumulus cells, for assessment of oocyte quality. Using the bovine as a model, we have employed a range of approaches and identified many potential markers, such as oocyte and cumulus candidate proteins and transcripts and follicular fluid fatty acids and amino acids. The models, technologies, and future strategies will be discussed in detail.

In vitro oocyte culture and somatic cell nuclear transfer used to produce a live-born cloned goat.

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Ohkoshi, Katsuhiro; Takahashi, Seiya; Koyama, Shin-Ichiro; Akagi, Satoshi; Adachi, Noritaka; Furusawa, Tadashi; Fujimoto, Jun-Ichiro; Takeda, Kumiko; Kubo, Masanori; Izaike, Yoshiaki; Tokunaga, Tomoyuki

2003-01-01

The use of an in vitro culture system was examined for production of somatic cells suitable for nuclear transfer in the goat. Goat cumulus-oocyte complexes were incubated in tissue culture medium TCM-199 supplemented with 10% fetal bovine serum (FBS) for 20 h. In vitro matured (IVM) oocytes were enucleated and used as karyoplast recipients. Donor cells obtained from the anterior pituitary of an adult male were introduced into the perivitelline space of enucleated IVM oocytes and fused by an electrical pulse. Reconstituted oocytes were cultured in chemically defined medium for 9 days. Two hundred and twenty-eight oocytes (70%) were fused with donor cells. After in vitro culture, seven somatic cell nuclear transfer (SCNT) oocytes (3%) developed to the blastocyst stage. SCNT embryos were transferred to the oviducts of recipient females (four 8-cell embryos per female) or uterine horn (two blastocysts per female). One male clone (NT1) was produced at day 153 from an SCNT blastocyst and died 16 days after birth. This study demonstrates that nuclear transferred goat oocytes produced using an in vitro culture system could develop to term and that donor anterior pituitary cells have the developmental potential to produce term offspring. In this study, it suggested that the artificial control of endocrine system in domestic animal might become possible by the genetic modification to anterior pituitary cells.

Cryopreservation of oocytes

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Jadoon, S.

2015-01-01

Various approaches have been utilized in attempting to cryopreserve oocytes, beginning with slow cooling and more recently the advent of technique of vitrification. Now it seems that oocyte cryopreservation is no longer an experimental technique and it is being increasingly utilized in clinics around the world. As successful outcome in oocyte cryopreservation can be assessed by survival through the freeze-thaw process, potential for fertilization, embryo development and dynamics of meiotic spindles. This study aimed to analyse these features in context of vitrification and slow freezing. Methods: In this laboratory based study, mature MII mouse oocytes from F1(C57BL6/J X CBA) mice (n=43) were divided randomly into two groups of equal numbers and were cryopreserved by slow freezing and by vitrification. Upon re-warming these oocytes were assessed for survival and for fertilization potential. Oocytes were fixed and stained to compare the effect of both protocols on spindle reassembly and chromosome configuration 10min, 1h and 3h after warming. Unfrozen oocytes were used as controls. Results: A greater number of vitrified oocytes survived cryopreservation than slow frozen oocytes (70.3% vs. 12.5%, p=0.024). After insemination, fertilization rates were higher for vitrified oocytes as compared to slow frozen oocytes (15.86% vs. 4.6%, p=0.046). Morphology of the meiotic spindle was found to be in a disorganized configuration in slow frozen oocytes at all-time points 10 mins, 1 h and 3h), whereas in vitrified oocytes the spindles were found to be aligned at all-time points. Chromosomes were seen to be displaced from equatorial region in both groups. Conclusion: Cryopreservation of mouse oocytes was conducted with greater success using vitrification, compared to slow freezing, with survival, fertilization, and spindle assembly more favourable to a successful outcome in this model. (author)

Ovarian Grafts 10 Days after Xenotransplantation: Folliculogenesis and Recovery of Viable Oocytes.

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Paulo Henrique Almeida Campos-Junior

Full Text Available Ovarian xenotransplantation is a promising alternative to preserve fertility of oncologic patients. However, several functional aspects of this procedure remained to be addressed. The aim of this study was evaluate the feasibility of xenotransplantation as a strategy to maintain bovine ovarian grafts and produce oocytes. Adult ovarian cortical pieces were xenotransplanted to the dorsal subcutaneous of female NOD-SCID mice (n = 62. Grafts were recovered ten days after xenotransplantation. Host and graft weights; folliculogenesis progression; blood perfusion, relative gene expression and number of macrophage and neutrophil of xenografts; in vitro developmental competence of graft-derived oocytes were evaluated. Folliculogenesis was supported in the grafts, as indicated by the presence of primordial, primary, secondary, antral, and atretic follicles. The xenografts showed a greater volumetric density of atretic follicles and higher hyperemia and number of host-derived macrophage and neutrophil (P<0.05, when compared to non-grafted fragments. There was a higher blood perfusion under the back skin in the transplantation sites of host animals than in control and non-grafted (P<0.01. BAX and PRDX1 genes were up-regulated, while BCL2, FSHR, IGF1R and IGF2R were down-regulated, when compared to the control (P<0.01. Twenty seven oocytes were successfully harvested from grafts, and some of these oocytes were able to give rise to blastocysts after in vitro fertilization. However, cleavage and blastocyst rates of xenograft derived oocytes were lower than in control (P<0.01. Despite showing some functional modifications, the ovarian xenografts were able to support folliculogenesis and produce functional oocytes.

Milrinone treatment of bovine oocytes during in vitro maturation benefits production of nuclear transfer embryos by improving enucleation rate and developmental competence.

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Naruse, Kenji; Iga, Kosuke; Shimizu, Manabu; Takenouchi, Naoki; Akagi, Satoshi; Somfai, Tamas; Hirao, Yuji

2012-01-01

In the production of cattle nuclear transfer embryos, the production efficiency is affected by the oocyte developmental competence and successful enucleation rate. This study investigated the effect of treating oocytes with milrinone, a phosphodiesterase inhibitor, on these two characteristics. When cumulus-oocyte complexes (COCs) were cultured for 19 h with 0, 50 or 100 μM of milrinone, the enucleation rate was significantly improved by 100 μM milrinone. However, milrinone treatment during in vitro maturation (IVM) also delayed meiotic progression by at least 2 h, which would affect the examination of enucleation rate and developmental competence of oocytes. Thus, in the second experiment, meiotic resumption was temporarily inhibited with butyrolactone I (BL-I; 100 μM, 18 h) to decrease the delayed maturation caused by milrinone; this enabled a more accurate comparison of the effects of milrinone after oocyte maturation. In nuclear transfer embryo production, oocytes treated with milrinone (100 μM, 20 h) showed a significantly higher rate of enucleation compared with that of control oocytes. This improved enucleation rate was associated with a closer location of the metaphase plate to the first polar body in the treated oocytes compared with that in control oocytes. Furthermore, milrinone improved the frequency of development to the blastocyst stage in the resulting embryos. In conclusion, milrinone supplementation during IVM improved enucleation rates by rendering the metaphase plate in close proximity to the first polar body, and this treatment also improved oocyte developmental competence. These benefits additively improved the yield of cloned embryos that developed to the blastocyst stage.

Oil-free culture system for in vitro bovine embryo production

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Paulo B.D. Gonçalves

2010-04-01

Full Text Available The use of oil to avoid water evaporation from cell culture has several disadvantages, amongst which there is the migration of compounds from media to oil and from oil to media. The aim of this study was to evaluate the osmolality of a culture system using four-well plates with water in the central hole as an alternative to in vitro bovine embryo production (IVP. In addition, the osmolality changes of the oocyte washing medium were assessed in 35mm dishes with or without 2 mL of silicon oil overlay. Osmolality of oocyte washing medium changed a great deal over time after 60 minutes on a 39°C heated plate (291 mOsm kg-1, which was not detected when the medium was overlaid with silicon oil (280 mOsm kg-1; P0.05. Blastocyst rates were higher when embryos were cultured in presence of water or oil (29.7 and 29.9% for water and 33% in oil conventional microdrop system, except in the group that oocytes were washed in hyperosmotic washing medium (15.1%; P<0.05. Groups cultured in absence of water in the central hole had lower blastocyst rates (P<0.05 independently of exposure (15.5% or not (16.2 and 16.8% to hyperosmotic washing medium. In conclusion, four-well plates with water in the central hole can be an alternative to replace oil overlay for bovine IVP, maintaining stable osmolality and embryo development rates.

Toxic effects of ethylene oxide residues on bovine embryos in vitro.

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Holyoak, G R; Wang, S; Liu, Y; Bunch, T D

1996-04-15

The potential of ethylene oxide (EtO) residues in exposed plastic tissue culture dishes to adversely affect bovine oocyte maturation, fertilization and subsequent embryonic development was monitored. In experiment 1, the effects of aeration time and aeration combined with washing of EtO-gassed culture dishes on the extent of residual toxicity were investigated. There was no cleavage in any treatment in which oocytes were matured and fertilized in dishes exposed to EtO. EtO residues caused functional degeneration of oocytes even when culture dishes were aerated for more than 12 days post EtO-exposure and repeatedly washed. In experiment 2, the residual toxicity of EtO gas on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) were evaluated. Cleavage rate significantly decreased and post-cleavage development was retarded in ova maintained in dishes treated with EtO either during IVM or IVF. EtO residues may be more detrimental to spermatozoa than to oocytes which may have been the primary cause of fertilization failure during IVF.

Establishment of pregnancy after the transfer of nuclear transfer embryos produced from the fusion of argali (Ovis ammon) nuclei into domestic sheep (Ovis aries) enucleated oocytes.

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White, K L; Bunch, T D; Mitalipov, S; Reed, W A

1999-01-01

Cloning mammalian species from cell lines of adult animals has been demonstrated. Aside from its importance for cloning multiple copies of genetically valuable livestock, cloning now has the potential to salvage endangered or even extinct species. The aim of this study was to investigate the effect of the bovine and domestic (Ovis aries) ovine oocyte cytoplasm on the nucleus of an established cell line from an endangered argali wild sheep (Ovis ammon) after nuclear transplantation. A fibroblast cell line was established from skin biopsies from an adult argali ram from the People's Republic of China. Early karyotype analysis of cells between 3-6 passages revealed a normal diploid chromosome number of 56. The argali karyotype consisted of 2 pairs of biarmed and 25 pairs of acrocentric autosomes, a large acrocentric and minute biarmed Y. Bovine ovaries were collected from a local abattoir, oocytes aspirated, and immediately placed in maturation medium consisting of M-199 containing 10% fetal bovine serum, 100 IU/mL penicillin, 100 microg/mL streptomycin, 0.5 microg/mL follicle-stimulating hormone (FSH), 5.0 microg/mL luetinizing hormone (LH) and 1.0 microg/mL estradiol. Ovine (O. aries) oocytes were collected at surgery 25 hours postonset of estrus from the oviducts of superovulated donor animals. All cultures were carried out at 39 degrees C in a humidified atmosphere of 5% CO2 and air. In vitro matured MII bovine oocytes were enucleated 16-20 hours after onset of maturation and ovine oocytes within 2-3 hours after collection. Enucleation was confirmed using Hoechst 33342 and UV light. The donor argali cells were synchronized in G0-G1 phase by culturing in Dulbecco's modified Eagle's medium (DMEM) plus 0.5% fetal bovine serum for 5-10 days. Fusion of nuclear donor cell to an enucleated oocyte (cytoplast) to produce nuclear transfer (NT) embryos was induced by 2 electric pulses of 1.4 kV/cm for 30 microsc. Fused NT embryos were activated after 24 hours of maturation

Nucleoli from growing oocytes inhibit the maturation of enucleolated, full-grown oocytes in the pig.

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Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi; Fulka, Josef

2011-06-01

In mammals, the nucleolus of full-grown oocyte is essential for embryonic development but not for oocyte maturation. In our study, the role of the growing oocyte nucleolus in oocyte maturation was examined by nucleolus removal and/or transfer into previously enucleolated, growing (around 100 µm in diameter) or full-grown (120 µm) pig oocytes. In the first experiment, the nucleoli were aspirated from growing oocytes whose nucleoli had been compacted by actinomycin D treatment, and the enucleolated oocytes were matured in vitro. Most of non-treated or actinomycin D-treated oocytes did not undergo germinal vesicle breakdown (GVBD; 13% and 12%, respectively). However, the GVBD rate of enucleolated, growing oocytes significantly increased to 46%. The low GVBD rate of enucleolated, growing oocytes was restored again by the re-injection of nucleoli from growing oocytes (23%), but not when nucleoli from full-grown oocytes were re-injected into enucleolated, growing oocytes (49%). When enucleolated, full-grown oocytes were injected with nucleoli from growing or full-grown oocytes, the nucleolus in the germinal vesicle was reassembled (73% and 60%, respectively). After maturation, the enucleolated, full-grown oocytes injected with nucleoli from full-grown oocytes matured to metaphase II (56%), whereas injection with growing-oocyte nucleoli reduced this maturation to 21%. These results suggest that the growing-oocyte nucleolus is involved in the oocyte's meiotic arrest, and that the full-grown oocyte nucleolus has lost the ability. Copyright © 2011 Wiley-Liss, Inc.

Transporte de oócitos bovinos em meio de maturação sem controle de atmosfera gasosa Transport of bovine oocytes in maturation medium without a controlled gaseous atmosphere

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Fábio Gallas Leivas

2004-02-01

aspirated from 2 to 8mm follicles obtained from bovine slaughterhouse ovaries (11 replications were randomly distributed in four treatments. Oocytes were matured for 24h with modified TCM-199 Earle salts, plus 25mM bicarbonate, 25 mM HEPES, rFSH-h, Estrus Cow Serum (ECS, and piruvate at 39ºC, in incubator with 5% CO2 and saturated humidity (Control Group, n=296 or exposed to a simulated transport for 6 (T6, n=286, 12 (T12, n=294 or 18h (T18, n=301 in maturation medium containing TCM + HEPES, in a 39ºC water bath, with the same components used in the Control Group, but with 1mM bicarbonate. At the conclusion of each transport period, oocytes were transferred to dishes with maturation medium to reach 24h in incubator, under the same conditions described for the Control group. Fertilization was accomplished during 18h, with the same temperature and gaseous atmosphere, in FERT-TALP plus heparin. The insemination dose was 1x106 spermatozoa/mL, sorted by swim-up. Presumptive zygotes were cultured in SOF medium + 5% ECS for 8 days, in incubator at 39ºC using gasified bags with 5% CO2, 5% O2 and 90% N2. Cleavage rates did not differ between treatments. Embryonic development rates at D7 were similar for Control (20.9%, T6 (19.2% and T12 (21.4% groups, with a reduction (P0.05 in hatched blastocyst rate. The average number of cells of hatched blastocysts was similar (P>0.05 in Control (136, T6 (125.5 and T12 (126.8 groups. These results indicate the possibility of transporting bovine oocytes in maturation medium containing TCM + HEPES, without controlled gaseous atmosphere environment, at 39ºC, for up to 12 hours. This technique offers a practical and efficient alternative for the transport of bovine oocytes for in vitro production of bovine embryos (IVP.

Increase of mitochondrial DNA content and transcripts in early bovine embryogenesis associated with upregulation of mtTFA and NRF1 transcription factors

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Heyman Yvan

2005-11-01

Full Text Available Abstract Background Recent work has shown that mitochondrial biogenesis and mitochondrial functions are critical determinants of embryonic development. However, the expression of the factors controlling mitochondrial biogenesis in early embryogenesis has received little attention so far. Methods We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA in bovine oocytes and in various stages of in vitro produced embryos. To investigate the molecular mechanisms responsible for the replication and the transcriptional activation of mtDNA, we quantified the mRNA corresponding to the mtDNA-encoded cytochrome oxidase 1 (COX1, and two nuclear-encoded factors, i.e. the Nuclear Respiratory Factor 1 (NRF1, and the nuclear-encoded Mitochondrial Transcription Factor A (mtTFA. Results Unlike findings reported in mouse embryos, the mtDNA content was not constant during early bovine embryogenesis. We found a sharp, 60% decrease in mtDNA content between the 2-cell and the 4/8-cell stages. COX1 mRNA was constant until the morula stage after which it increased dramatically. mtTFA mRNA was undetectable in oocytes and remained so until the 8/16-cell stage; it began to appear only at the morula stage, suggesting de novo synthesis. In contrast, NRF1 mRNA was detectable in oocytes and the quantity remained constant until the morula stage. Conclusion Our results revealed a reduction of mtDNA content in early bovine embryos suggesting an active process of mitochondrial DNA degradation. In addition, de novo mtTFA expression associated with mitochondrial biogenesis activation and high levels of NRF1 mRNA from the oocyte stage onwards argue for the essential function of these factors during the first steps of bovine embryogenesis.

Morphological markers to select populations of oocytes with different cultural needs for dedicated pre-maturation protocols

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Cecilia Dieci

2017-05-01

Full Text Available Oocyte’s chromatin gradually becomes more compacted during the final stage of oocyte development and the level of chromatin compaction is considered a marker of oocyte differentiation [Luciano et al, 2014]. Moreover, several studies demonstrate that in vitro pre-maturation treatments (Pre-IVM, aimed to improve the developmental capability of immature oocytes, might behave differently depending on the oocyte metabolic status, when it is isolated from follicle [Luciano et al., 2011]. This study aims at identifying correlations between cumulus-oocyte complex (COC morphology and oocyte chromatin configuration and secondly at testing the hypothesis that only fully grown oocytes at earlier stages of differentiation with loosely compacted chromatin  (GV1 can benefit from Pre-IVM treatment.   COCs were collected from bovine 2-6mm ovarian follicles, and further divided in three groups according to their morphology (Class-1, 2 and 3 as previously described [Blondin & Sirard, 1995]. Analysis of chromatin configuration revealed that only Class-1 COC was enriched in GV1 oocyte, while Class-2 and 3 presented a similar distribution of GV1, GV2 and GV3 oocytes, where GV2 and 3 oocytes are characterized by increased chromatin compaction [Lodde et al., 2007]. Then COCs were divided into two groups, one containing Class-1 COCs and the other containing Class-2 and 3 COCs and subjected to pre-IVM for 6 hours in presence of cilostamide and 10-4 UI/ml rhFSH. Finally, COCs underwent standard in vitro maturation (IVM for 22 hours, in vitro fertilization and embryo culture. Blastocyst rate and embryos cell number were assessed at day 7. Pre-IVM positively affected developmental competences of Class-1, while in Classes 2 and 3 Pre-IVM had detrimental effects.In conclusion COCs morphology could be used as a non-invasive approach to select population of oocyte with different cultural needs. These data could be useful in setting-up dedicated IVM protocols considering

Nucleoli from growing oocytes support the development of enucleolated full-grown oocytes in the pig.

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Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

2010-02-01

Recent research has shown that the maternal nucleolus is essential for embryonic development. The morphology of the nucleolus in growing oocytes differs from that in full-grown oocytes. We determined the ability of nucleoli from growing oocytes to substitute for nucleoli of full-grown oocytes in terms of supporting embryonic development in this study. Growing (around 100 microm in diameter) and full-grown porcine oocytes (120 microm) were collected from small (0.6-1.0 mm) and large antral follicles (4-5 mm), respectively. The nucleolus was aspirated from full-grown oocytes by micromanipulation, and the resulting enucleolated oocytes were matured to metaphase II; the nucleoli originating from full-grown and growing oocytes were then injected into the oocytes. The Chromatin of growing oocytes was aspirated with the nucleolus during the enucleolation process. Growing oocytes were thus treated with actinomycin D to release the chromatin from their nucleoli, and the nucleoli were collected and transferred to the enucleolated and matured full-grown oocytes. After activation by electro-stimulation, nucleoli were formed in pronuclei of sham-operated oocytes. Enucleolated oocytes that had been injected with nucleoli from either full-grown or growing, however, did not form any nucleoli in the pronuclei. No enucleolated oocytes developed to blastocysts, whereas enucleolated oocytes injected with nucleoli from full-grown oocytes (15%) or growing oocytes (18%) developed to blastocysts. These results indicate that the nucleoli from growing oocytes can substitute for nucleoli from full-grown oocytes during early embryonic development. (c) 2009 Wiley-Liss, Inc.

Effect of Leptin on In Vitro Nuclear Maturation and Apoptosis of Buffalo (Bubalus bubalis Oocyte

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Amir Khaki

2014-03-01

Full Text Available Background: Leptin, as a 16 kDa adipokine, is a pleiotropic cytokine-like hormone that primarily secreted from adipose tissue. It also involves in the regulation of energy homeostasis, neuroendocrine function, immunity, lipid and glucose homeostasis, fatty acid oxidation, angiogenesis, puberty and reproduction. The aim of this study was to investigate the effects of in vitro addition of leptin to in vitro maturation (IVM medium on buffalo oocyte maturation and apoptosis. Materials and Methods: In this experimental study, Ovaries from apparently normal reproductive organs of slaughtered adult buffaloes (Bubalus bubalis with unknown breeding history were collected from Urmia Abattoir, Urmia, Iran, and were transported immediately to the laboratory in a thermos flask containing sterile normal saline with added antibiotics. Oocytes were aspirated from 2-8 mm visible follicles of the ovaries using an 18-G needle attached to a 10 ml syringe. IVM medium included tissue culture medium-199 (TCM-199, 10% fetal bovine serum (FBS, 22 μg/ml sodium pyruvate, 0.5 IU/ml ovine follicle-stimulating hormone (oFSH, 0.5 IU/ml ovine luteinizing hormone (oLH, 1 μg/ml oestradiol, 50 μg/ml gentamycin, and leptin [0 (control, 10, 50, and 100 ng/ml]. The good quality buffalo oocytes (batches of 10 oocytes were placed in a culture plate containing six 50 μl droplets of maturation medium, covered with sterilized mineral oil, and then incubated at 38.5˚C with 5% CO2 in air for 24 hours. The maturation of oocytes was evaluated under a stereomicroscope by detecting the first polar body extrusion of oocytes. FITC-Annexin V - propidium iodide (PI staining method was used to detect oocyte apoptosis. Results: From a total of 115 collected ovaries, 1100 oocytes were recovered among which 283 oocyte were suitable for IVM. In the groups of leptin treated with 0 (control, 10, 50 and 100 ng/ml, the percentage of oocytes maturation was 74.65, 83.81, 77.85, and 75.40%, while the

The effect of IVM and IVC media on in vitro development of bovine embryos

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E.T Mergawati

2000-12-01

Full Text Available The purpose of this study was to examine the effect of medium combination of IVM and IVC on the in vitro development of bovine embryos. The study involved 4 groups in a 2 (IVM medium x 2 (IVC medium factorial in a randomized block design. Each group was replicated for 5 times. The treatments were as follows: TCM-199/CR1aa (T1; TCM-199/SOF (T2; B- 199/CR1aa (T3 and B-199/SOF (T4. Oocytes were aspirated from ovaries collected at local abattoirs using aspiration medium of PBS supplemented with 3% FCS and 0.1% Penicillin and Streptomycin. The oocytes were matured in medium of TCM-199 or B-199 supplemented with 10% FCS, hormones: 10μg/ml FSH+ 10μg/ml hCG+ 1μg/ml Estradiol. Maturation was maintained at 37oC for 22 hours in 5% CO2 incubator with high humidity. A method of BRACKETT & Oliphant (BO was used to fertilize the matured oocytes. The fertilization was incubated for 7 hours in the 5% CO2 incubator. Two culture media of CR1aa or SOF/AA/BSA were used to develop the fertilized oocytes undergo to morula and blastocyst embryos. The findings showed that the proportion of oocytes cleaved and formation of blastocysts were affected significantly by a combination of IVM and IVC media (P<0.05. A combination of B-199/SOF (T4 resulted in a higher blastocyst rate (32% than others (T3= 29%; T2=T1= 23%. This study suggests that either SOF/AA/BSA or CR1aa has similar competence in development of bovine embryos in vitro.

Association of the transcription profile of bovine oocytes and embryos with developmental potential

Czech Academy of Sciences Publication Activity Database

Kaňka, Jiří; Němcová, Lucie; Toralová, Tereza; Vodičková Kepková, Kateřina; Vodička, Petr; Jeseta, M.; Machatková, M.

2012-01-01

Roč. 134, 1-2 (2012), 29-35 ISSN 0378-4320. [Embryo Genomics Meeting /3./. Bonn, 20.08.2012-22.08.2012] R&D Projects: GA ČR GA523/09/1035; GA MZe QI91A018 Institutional support: RVO:67985904 Keywords : oocyte * in vitro maturation * pre-implantation development Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.897, year: 2012

Relocalization of nuclear DNA helicase II during the growth period of bovine oocytes

Czech Academy of Sciences Publication Activity Database

Baran, V.; Kovářová, Hana; Klíma, Jiří; Hozák, Pavel; Motlík, Jan

2006-01-01

Roč. 125, 1-2 (2006), s. 155-164 ISSN 0948-6143 R&D Projects: GA ČR GA523/03/0857 Grant - others:Slovenská Akademie věd(SK) VEGA 2/3065/23 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50390512 Keywords : DNA helicase II * fibroblasts * oocytes Subject RIV: EB - Genetics ; Molecular Biology Impact factor : 3.220, year: 2006

Molecular Characterization of the First Bovine Herpesvirus 4 (BoHV-4 Strains Isolated from In Vitro Bovine Embryos production in Argentina.

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Erika González Altamiranda

Full Text Available Bovine herpesvirus 4 (BoHV-4 is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as "starting material" for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these

Carbonate radical anion-induced electron transfer in bovine serum albumin

Energy Technology Data Exchange (ETDEWEB)

Joshi, Ravi [Chemistry Group, Bhabha Atomic Research Centre, Mumbai 400 085 (India)]. E-mail: rjudrin@yahoo.com; Mukherjee, T. [Chemistry Group, Bhabha Atomic Research Centre, Mumbai 400 085 (India)

2006-07-15

Reaction of native and thermally denatured bovine serum albumin (BSA) with carbonate radical anion (CO{sub 3}{sup -} radical) has been studied using pulse radiolysis technique. Scavenging of CO{sub 3}{sup -} radical by native BSA and consequent electron transfer from tyrosine to tryptophan radical has been observed to occur with almost same rate constant (k{approx}1.7x10{sup 8} dm{sup 3} mol{sup -1} s{sup -1}) at pH 8.8. Effect of structural changes, due to thermal denaturation, on scavenging of CO{sub 3}{sup -} radical and the electron transfer process have been studied and discussed in this paper.

Genetic parameters for oocyte number and embryo production within a bovine ovum pick-up-in vitro production embryo-production program.

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Merton, J S; Ask, B; Onkundi, D C; Mullaart, E; Colenbrander, B; Nielen, M

2009-10-15

Genetic factors influencing the outcome of bovine ovum pick-up-in vitro production (OPU-IVP) and its relation to female fertility were investigated. For the first time, genetic parameters were estimated for the number of cumulus-oocyte complexes (Ncoc), quality of cumulus-oocyte complexes (Qcoc), number and proportion of cleaved embryos at Day 4 (Ncleav(D4), Pcleav(D4)), and number and proportion of total and transferable embryos at Day 7 of culture (Nemb(D7), Pemb(D7) and NTemb(D7), PTemb(D7), respectively). Data were recorded by CRV (formally Holland Genetics) from the OPU-IVP program from January 1995 to March 2006. Data were collected from 1508 Holstein female donors, both cows and pregnant virgin heifers, with a total of 18,702 OPU sessions. Data were analyzed with repeated-measure sire models with permanent environment effect using ASREML (Holstein Friesian). Estimates of heritability were 0.25 for Ncoc, 0.09 for Qcoc, 0.19 for Ncleav(D4), 0.21 for Nemb(D7), 0.16 for NTemb(D7), 0.07 for Pcleav(D4), 0.12 for Pemb(D7), and 0.10 for PTemb(D7). Genetic correlation between Ncoc and Qcoc was close to zero, whereas genetic correlations between Ncoc and the number of embryos were positive and moderate to high for Nemb(D7) (0.47), NTemb(D7) (0.52), and Ncleav(D4) (0.85). Genetic correlations between Ncoc and percentages of embryos (Pcleav(D4), Pemb(D7), and PTemb(D7)) were all close to zero. Phenotypic correlations were in line with genetic correlations. Genetic and phenotypic correlations between Qcoc and all other traits were not significant except for the phenotypic correlations between Qcoc and number of embryos, which were negative and low to moderate for Nemb(D7) (-0.20), NTemb(D7) (-0.24), and Ncleav(D4) (-0.43). Results suggest that cumulus-oocyte complex (COC) quality, based on cumulus investment, is independent from the total number of COCs collected via OPU and that in general, a higher number of COCs will lead to a higher number of embryos produced. The

Bovine ovarian cells have (pro)renin receptors and prorenin induces resumption of meiosis in vitro.

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Dau, Andressa Minussi Pereira; da Silva, Eduardo Pradebon; da Rosa, Paulo Roberto Antunes; Bastiani, Felipe Tusi; Gutierrez, Karina; Ilha, Gustavo Freitas; Comim, Fabio Vasconcellos; Gonçalves, Paulo Bayard Dias

2016-07-01

The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10(-10), 10(-9), and 10(-8)M incubated with oocytes co-cultured with follicular hemisections for 15h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10(-7), 10(-5), and 10(-3)M blocked this effect (Pmeiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200μM) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (Pmeiosis in cattle. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts.

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Lu, F; Jiang, J; Li, N; Zhang, S; Sun, H; Luo, C; Wei, Y; Shi, D

2011-09-15

The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P fusion (P fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation. Copyright © 2011 Elsevier Inc. All rights reserved.

Gene expression and apoptosis in bovine embryos during in vitro culture and in vivo development

NARCIS (Netherlands)

Knijn, H.W.

2004-01-01

The first attempts to fertilise in vitro bovine oocytes were done in the late sixties but only in 1982 the first calf was born after transplantation of a complete in vitro produced embryo. Since then the in vitro production system improved a lot but it is still impossible to mimic the in vivo

Localization of DNA methyltransferase-1 during oocyte differentiation, in vitro maturation and early embryonic development in cow

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A. M. Luciano

2009-12-01

Full Text Available DNA methyltransferase-1 (Dnmt1 is involved in the maintenance of DNA methylation patterns and is crucial for normal mammalian development. The aim of the present study was to assess the localization of Dnmt1 in cow, during the latest phases of oocyte differentiation and during the early stages of segmentation. Dnmt1 expression and localization were assessed in oocytes according to the chromatin configuration, which in turn provides an important epigenetic mechanism for the control of global gene expression and represents a morphological marker of oocyte differentiation.We found that the initial chromatin condensation was accompanied by a slight increase in the level of global DNA methylation, as assessed by 5-methyl-cytosine immunostaining followed by laser scanning confocal microscopy analysis (LSCM. RT-PCR confirmed the presence of Dnmt1 transcripts throughout this phase of oocyte differentiation. Analogously, Dnmt1 immunodetection and LSCM indicated that the protein was always present and localized in the cytoplasm, regardless the chromatin configuration and the level of global DNA methylation. Moreover, our data indicate that while Dnmt1 is retained in the cytoplasm in metaphase II stage oocytes and zygotes, it enters the nuclei of 8-16 cell stage embryos. As suggested in mouse, the functional meaning of the presence of Dnmt1 in the bovine embryo nuclei could be the maintainement of the methylation pattern of imprinted genes. In conclusion, the present work provides useful elements for the study of Dnmt1 function during the late stage of oocyte differentiation, maturation and early embryonic development in mammals.

Ovarian morphometric characterization and in vitro maturation of oocytes obtained from buffalo (Bubalus bubalis ovaries – partial results

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F.C. Landim-Alvarenga

2010-02-01

Full Text Available Buffalo ovaries were collected from a slaughterhouse (Frigol, Brazil and transported to the laboratory in saline solution at 36º C. The ovaries were dissected to realize the evaluations (weight, length, width and height of the ovary; corpus luteum and dominant follicle diameters. The Cumulus-oocyte complexes (COCs were recovered by aspiration of 2-8 mm follicles. Selected COCs were matured in TCM 199 supplemented with 10% fetal bovine serum, sodium pyruvate, LH, FSH, estradiol and gentamicin. In vitro maturation was carried out at 38.5° C for 22-24 h and 34-36 h. For the evaluation of the nuclear maturation the oocytes were placed in TCM 199 medium added with type v hialuronidase where the granulosa cells were extracted. The denuded oocytes were transferred to 10 μl of Hoescht 33342 and the chromosomic configuration was evaluated. The oocytes were classified according to meiosis stage in: Germinal Vesicle, Germinal Vesicle Breakdown, Metaphase I, Metaphase II and Degenerated. The means of weight, length, width and height of the ovary were 3.83 g, 2.27 cm, 1.08 cm and 1.56 cm, respectively. The means of corpus luteum and dominant follicle diameters were 1.40 cm and 7.77 mm. The proportion of oocytes that reached metaphase II stage was: 36.68%.

MITOCHONDRIAL DYNAMICS IN PRE- AND POSTPUBERTAL PIG OOCYTES BEFORE AND AFTER IN VITRO MATURATION

DEFF Research Database (Denmark)

Pedersen, H. S.; Løvendahl, P.; Nikolaisen, N. K.

2013-01-01

Oocytes from prepubertal (PRE) or postpubertal (POST) pigs are used in, for example, somatic cell nuclear transfer and in vitro fertilization. Here we describe mitochondrial dynamics in pig oocytes of different sizes before and after in vitro maturation (IVM), isolated from PRE or POST animals....... In PRE oocytes, inside-zona pellucida diameter was measured before and after IVM (μm; small: ≤110, medium: >110, large: ≥120) and used for evaluation of (1) mitochondrial numbers before maturation and (2) mitochondrial morphology and location before and after maturation in comparison with POST oocytes....... Oocytes were processed for transmission electron microscopy (Acta Anat. 129:12). For assessment of mitochondrial numbers, paired dissector sections were collected at uniform intervals throughout the oocyte, and in each set of dissector sections a known area fraction was sampled for mitochondrial counting...

Criopreservação de ovócitos de bovinos imaturos desnudados ou não, utilizando o etilenoglicol pelo método da vitrificação Cryopreservation of bovines immature oocytes desnudes or not, by the ethylene glycol vitrification method

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Eduardo Paulino da Costa

2002-06-01

Full Text Available Objetivou-se avaliar os efeitos da vitrificação em ovócitos de bovinos após o cultivo in vitro, utilizando o etilenoglicol como crioprotetor. Ovócitos obtidos de ovários de vacas abatidas em matadouro foram distribuídos aleatoriamente em três tratamentos. Tratamento 0 (testemunha: ovócitos não-desnudados e não-congelados. Tratamento 1: vitrificação de ovócitos imaturos não desnudados, desidratados previamente por cinco minutos em três soluções contendo 20, 20 e 40% de etilenoglicol, acrescidas de 0,3 mol L-1 de trehalose e 20% de PVP, em meio de Talp Hepes. Tratamento 2: vitrificação de ovócitos imaturos desnudados, conforme o Tratamento 1. Após o descongelamento (imersão em banho-maria a 30ºC por 20 segundos, os ovócitos foram reidratados gradativamente, mantendo-os por 6 minutos em cada uma das soluções a seguir, sucessivamente: meio Talp Hepes com 20% de etilenoglicol + 0,3 mol L-1 de trehalose + 10% de PVP e meio Talp Hepes sem etilenoglicol, trehalose e PVP, onde foram lavados três vezes. Posteriormente, os ovócitos foram cultivados a 38,5ºC, com 95% de umidade e atmosfera de 5% de CO2 por 24 horas. Após o cultivo, os ovócitos foram fecundados e os embriões cultivados in vitro por sete dias. Foi encontrada uma taxa de maturação nuclear de 81 (68/84, 19 (7/36 e 0% (0/31, nos Tratamentos 0, 1 e 2, respectivamente. As taxas de clivagem e de desenvolvimento embrionário foram de 56,4 (102/181 e 54,9% (56/102, 1,7 (1/60 e 0,0% (1/60, 0,0 (0/71 e 0,0% (0/71, nos Tratamentos 0, 1 e 2, respectivamente. Esses resultados indicam que o procedimento de vitrificação, segundo os protocolos utilizados, não é indicado para a criopreservação de ovócitos de bovinos.The objective was to evaluate the effects of vitrification of immature bovine oocytes after in vitro culture, by the use of cryoprotectors ethylene glycol. Oocytes from cows ovaries from slaughters houses were randomly alocated into three treatments

Diving into the oocyte pool

DEFF Research Database (Denmark)

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

2017-01-01

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...... for revitalizing deficient oocytes may transform ART, and potentially enhance both quantity and quality of fertilizable oocytes; hereby augmenting the pregnancy potential of women with poor reproductive performance....

Potential role for MATER in cytoplasmic lattice formation in murine oocytes.

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Boram Kim

2010-09-01

Full Text Available Mater and Padi6 are maternal effect genes that are first expressed during oocyte growth and are required for embryonic development beyond the two-cell stage in the mouse. We have recently found that PADI6 localizes to, and is required for the formation of, abundant fibrillar Triton X-100 (Triton insoluble structures termed the oocyte cytoplasmic lattices (CPLs. Given their similar expression profiles and mutant mouse phenotypes, we have been testing the hypothesis that MATER also plays a role in CPL formation and/or function.Herein, we show that PADI6 and MATER co-localize throughout the oocyte cytoplasm following Triton extraction, suggesting that MATER co-localizes with PADI6 at the CPLs. Additionally, the solubility of PADI6 was dramatically increased in Mater(tm/tm oocytes following Triton extraction, suggesting that MATER is involved in CPL nucleation. This prediction is supported by transmission electron microscopic analysis of Mater(+/+ and Mater(tm/tm germinal vesicle stage oocytes which illustrated that volume fraction of CPLs was reduced by 90% in Mater(tm/tm oocytes compared to Mater(+/+ oocytes.Taken together, these results suggest that, similar to PADI6, MATER is also required for CPL formation. Given that PADI6 and MATER are essential for female fertility, these results not only strengthen the hypothesis that the lattices play a critical role in mediating events during the oocyte-to-embryo transition but also increase our understanding of the molecular nature of the CPLs.

Oocyte activation and preimplantation development of bovine embryos obtained by specific inhibition of cyclin-dependent kinases Ativação oocitária e desenvolvimento pré-implantação de embriões bovinos obtidos com o uso de inibidores específicos das quinases dependentes de ciclina

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F. Perecin

2007-04-01

Full Text Available The efficiency of bohemine and roscovitine in combination with ionomycin on parthenogenetic activation and initial embryonic development of bovine oocytes was studied. Two experiments were performed: in the first, different concentrations (0, 50, 75 or 100µM and different exposure periods (2, 4 or 6 hours to bohemine or roscovitine were tested for activation rates of in vitro matured (IVM bovine oocytes, which were pre-exposed to ionomycin. The best treatments, 75µM bohemine and 50µM roscovitine, both for 6h, were used in the second experiment, in which IVM bovine oocytes were exposed to ionomycin, followed or not by bohemine or roscovitine treatment, and evaluated for nuclear status, activation rate and blastocyst development were assessed. The combined treatments (ionomycin + cyclin-dependent kinases inhibitors - CDKIs showed better results for activation rates (77.3% and initial embryonic development (35.2% than the single ionomycin treatment (69.4% for activation and 21.9% for development; and also lead to a more uniform activation (nearly 90% single pronucleus development. The results showed that CDKIs improve the effects of ionomycin on parthenogenetic activation and blastocyst development in bovine oocytes and could help to achieve more efficient activation protocols, increasing the developmental competence of embryos obtained by reproductive biotechniques.Realizaram-se dois experimentos para avaliar a eficiência da bohemina e roscovitina associadas à ionomicina para ativação partenogenética e desenvolvimento embrionário inicial de bovinos. No primeiro, foram testadas diferentes concentrações (0, 50, 75 ou 100µM e diferentes tempos de exposição (2, 4 ou 6 horas à bohemina ou à roscovitina na ativação de oócitos bovinos maturados in vitro (MIV pré-expostos à ionomicina. Os melhores tratamentos, bohemina 75µM e roscovitina 50µM, ambos por seis horas, foram utilizados no segundo experimento, no qual oócitos bovinos

Influence of maternal nutrition and heat stress on bovine oocyte and embryo development

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Alzahraa M. Abdelatty

Full Text Available The global population is expected to increase from 7.6 to 9.6 billion people from 2017 to 2050. Increased demand for livestock production and rising global temperatures have made heat stress (HS a major challenge for the dairy industry. HS been shown to have negative effects on production parameters such as dry matter intake, milk yield, and feed efficiency. In addition to affecting production parameters, HS has also been shown to have negative effects on the reproductive functions of dairy cows. Mitigation of HS effects on dairy cow productivity and fertility necessitate the strategic planning of nutrition, and environmental conditions. The current review will discuss the potential nutriepigenomic strategies to mitigate the effect of HS on bovine embryo. Keywords: Bovine embryo, Dairy cow, Fertility, Heat stress, Maternal nutrition, Oocyst

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

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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.

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

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

2017-01-01

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...... 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....

Electron microscopic observation of radiation effects on the oocytes of killfish

Energy Technology Data Exchange (ETDEWEB)

Yamamoto, M; Egami, N [Tokyo Univ. (Japan). Faculty of Science

1974-06-01

The morphological changes of the ovaries, pituitary body and liver after the 2KR of X-ray irradiation were observed in female killifish. In the ovary, some the follicular cells which enclose the oocytes were destroyed in 10 hours after the irradiation. The oocytes during the period of yolk formation began to show a remarkable increase of mitochondria in the cytoplasm. On the 2nd day, myelin began to be observed. On the 3rd day, each of the organelles became completely degenerated and the cells were destroyed later. In the pituary body, an increased number of secretary granules was observed to accumulate in the cytoplasm during the 3rd day and the 10th day. In the liver cells, most of the glycogen granules disappeared during the 1st day and the 5th day. The volume of cells decreased and irregular intracellular spaces appeared.

A feasibility study of prepubertal and over mature aged local goat in relation to results of In Vitro growth culture to obtain additional M-II oocyte resources

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Ciptadi, Gatot; Ihsan, M. Nur; Rahayu, Sri; Widjaja, D. H. K.; Mudawamah, Mudawamah

2017-11-01

The aims of this research are to study the potential source of mature (M-II) oocytes of domestic animals using follicles isolated from prepubertal and over mature aged Indonesian local goats, resulting from an in vitro growth (IVG) method. This method of IVG could provide a new source of M-II oocytes for embryo production. In Indonesia, a very limited number of a good quality oocytes are available for research purposes, as there is a limited number of reproductive females slaughtered, which is dominated by prepubertal and old mature aged animals. IVG culture systems could be improved as an alternative method to provide a new source of a good quality oocytes for in vitro maturation of M-II oocytes. From a number of prepubertal and mature aged goats slaughtered in a local abattoir, the small oocytes in the preantral follicles were cultured in vitro to normal oocyte growth. The methods used in this research are experimental. Follicles were isolated, cultured in vitro for 14 days individually using a sticky medium containing 4% (w/v) polyvinylpyrrolidone in TCM 199 10% Fetal Bovine Serum supplemented with Follicle Stimulating Hormone, which was then evaluated for their follicle development and oocyte quality. The research results showed that a minimum follicle size and oocyte diameter is needed (>100 um) for early evaluation of maturation to be achieved, meanwhile oocytes recovered from IVG after being cultured in vitro for maturation resulted in a very low rate of maturation. However, in the future, IVG of the preantral follicles of Indonesian local goat could be considered as an alternative source of oocytes for both research purposes and embryo production in vitro.

Characterization of x-ray diffraction and electron spin resonance: Effects of sintering time and temperature on bovine hydroxyapatite

International Nuclear Information System (INIS)

Kusrini, Eny; Sontang, Muhammad

2012-01-01

The physical and chemical properties of a hydroxyapatite produced by the sintering of bovine bone were investigated by powder x-ray diffraction (PXRD), electron spin resonance (ESR), energy dispersive x-ray spectroscopy (EDX), scanning electron microscopy (SEM), Fourier transform infrared (FTIR), and differential thermal analysis (DTA). A bovine bone powder was sintered at different temperatures ranging from 500 to 1400 °C. The influences of post-irradiation storage on the radiation ESR response of the bovine bone powder before and after sintering were also studied. The results indicate that the sintered bovine bone powder contained hydroxyapatite. Diffraction patterns were sharp and clear based on the (211), (300), and (202) reflections corresponding to bovine hydroxyapatite (BHA), which confirmed the phase purity and high crystalline grade of the BHA produced. The PXRD profile of BHA was dependent on sintering temperatures and times. The molecular formula of BHA was determined by Rietveld analysis showed a similar structure and composition to calcium hydroxyapatite in hexagonal P6 3 /m space group a=b=9.435 Å and c=6.895 Å. ESR data showed that the sintering process can decrease the number of free radicals in BHA; it also revealed that the number of free radicals is constant during long storage periods (75 days). The sintering technique described in this study may be used to extract hydroxyapatite from biowaste bovine bone, leading to its application as a bone filler. - Highlights: ► Natural hydroxyapatite was produced from the bio-wasting bovine bones by sintering method. ► PXRD profile of BHA is dependent on the different temperatures and times in sintering process. ► ESR data is useful to study the typical of free radicals formed in the samples after irradiation. ► Stability and physicochemical properties of BHA is dependent on the different storage times. ► Technique is able to be used to find the natural hydroxyapatite applicable for bone filler.

Stearoyl-CoA desaturase activity in bovine cumulus cells protects the oocyte against saturated fatty acid stress

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Aardema, Hilde; van Tol, Helena T A; Wubbolts, Richard W; Brouwers, Jos F H M; Gadella, Bart M; Roelen, Bernard A J

2017-01-01

Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a dose-dependent negative impact on oocyte developmental competence, while mono-unsaturated free fatty

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.

Mammalian oocyte growth and development in vitro.

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Eppig, J J; O'Brien, M; Wigglesworth, K

1996-06-01

This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes form preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk.

Enhancement of Bovine oocyte maturation by leptin is accompanied by an upregulation in mRNA expression of leptin receptor isoforms in cumulus cells

NARCIS (Netherlands)

van Tol, Helena T A; van Eerdenburg, Frank J C M; Colenbrander, Ben; Roelen, Bernard A J

In this study, the mechanisms of supposed leptin action on oocyte maturation were examined. Expression of leptin mRNA, as determined with RT-PCR, was present in oocytes but not in cumulus cells. The long isoform of the leptin receptor (ObR-L) was expressed exclusively in cumulus cells after 7 and 23

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

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Rinaudo Paolo F

2009-04-01

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.

Can you ever collect too many oocytes?

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Briggs, Rosalind; Kovacs, Gabor; MacLachlan, Vivien; Motteram, Caroline; Baker, H W Gordon

2015-01-01

Does the chance of pregnancy keep improving with increasing number of oocytes, or can you collect too many? Clinical pregnancy (CP) and live birth (LB) rates per embryo transfer varied from 10.2 and 9.2% following one oocyte collected to 37.7 and 31.3% when >16 oocytes were collected. Regression modelling indicated success rates increased or at least stayed the same with number of oocytes collected. It has been suggested that if >15 oocytes are collected, the success rate for fresh embryo transfers decreases. As this is counterintuitive, as more oocytes should result in more embryos, with a better choice of quality embryos, we decided to analyse the recent experience in a busy IVF unit. A retrospective analysis of clinical pregnancy and live birth outcome, with respect to number of oocytes collected at Monash IVF for the 2-year period between August 2010 and July 2012, where patients under the age of 45 years underwent a fresh embryo transfer. This included 7697 stimulated cycles for IVF and ICSI. Statistical analysis involved data tables and graphs comparing oocyte number with outcome. Results of women who had their first oocyte collection with an embryo transfer within the reference period were analysed by logistic regression analysis including other covariates that might influence pregnancy outcome. Analysis was also carried out of all the 7679 oocyte collections undertaken, resulting in fresh embryo transfers by generalized estimating equations to allow for the within subject correlation in outcomes for repeated treatments. The number of oocytes collected varied from 1 to 48. Clinical pregnancy and live birth rates per embryo transfer varied from 10.2 and 9.2% when only one oocyte was collected to 37.7 and 31.3% when >16 oocytes were collected. Regression modelling indicated success rates increased or at least stayed the same or with the number of oocytes collected. The percentage of women with embryos cryopreserved increased from under 20% with 16 oocytes

Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

DEFF Research Database (Denmark)

Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

2009-01-01

Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

The extracellular matrix of porcine mature oocytes: Origin, composition and presumptive roles

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Pivko Juraj

2003-12-01

Full Text Available Abstract The extracellular matrix (ECM of porcine mature oocytes was revealed by transmission electron microscopy (TEM after treatment with tannic acid and ruthenium red. Present in the perivitelline space (PVS and on the surface of the zona pellucida (ZP, it appeared to be composed of thin filaments and granules at the interconnections of the filaments, which were interpreted respectively as hyaluronic acid chains and bound proteoglycans. In order to determine whether this material is produced by the corona cells (the same ECM was found also on the surface of the zona pellucida and between cumulus cells or by the oocyte itself, the synthesis of glycoproteins and glycosaminoglycans was checked by autoradiography on semi-thin and thin sections observed by light and electron microscopy. Immature oocytes within or without cumulus cells, were incubated with L [3H-] fucose or L [3H-] glucosamine – precursors respectively of glycoproteins and hyaluronic acid or hyaluronan (HA bound to proteoglycans – for various times (with or without chase and at different stages during in vitro maturation. In the first case, incorporation was found in both cumulus cells and ooplasm (notably in the Golgi area for 3H-fucose and labeled material accumulated in the ECM of the PVS and of the ZP surface. Labeling in the PVS with both precursors was maximum between metaphase I (MI and metaphase II (MII and was partially extracted by hyaluronidase but not by neuraminidase. Tunicamycin, an inhibitor of glycoprotein synthesis, significantly decreased the amount of 3H-fucose labeled molecules in the PVS and increased the incidence of polyspermic penetration during subsequent in vivo fertilization. Since cumulus-free oocytes also secreted 3H-glucosamine containing compounds, both oocyte and cumulus cells probably contribute to the production of the ECM found in the PVS of mature oocytes. ECM and particularly its HA moiety present on both sides of the ZP may constitute a

The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice

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Cheng-Jie Zhou

2016-03-01

Full Text Available Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes, in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes, and in vitro-matured, denuded oocytes without cumulus cells (DOs. Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

Follicular flushing during oocyte retrieval: a systematic review and meta-analysis.

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Roque, Matheus; Sampaio, Marcos; Geber, Selmo

2012-11-01

The purpose of this systematic review and meta-analysis was to examine the literature and identify randomized controlled trials (RCTs), in order to answer if performing follicular flushing during the oocyte retrieval may improve the assisted reproductive technologies (ART) outcomes. An exhaustive electronic search was performed using MEDLINE and EMBASE databases. Only RCTs comparing follicular flushing to aspiration only during ART, were included. We included 5 trials, with a total of 482 patients randomized, with median ages ranging from 30.5 to 37.1. The data analyses did not show significant differences regarding live birth rate, clinical pregnancies rates, and the number of oocytes retrieved. The duration of oocyte retrieval was significantly increased in the follicular flushing group. The results from this systematic review and meta-analysis suggest that there is no advantage to use of routine follicular flushing during OR in an unselected group of patients.

Apoptosis in mammalian oocytes: a review.

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

2015-08-01

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.

Ribosomal RNA and nucleolar proteins from the oocyte are to some degree used for embryonic nucleolar formation in cattle and pig

DEFF Research Database (Denmark)

Maddox-Hyttel, Poul; Svarcova, Olga; Laurincik, Josef

2007-01-01

The nucleolus is the site of ribosomal RNA (rRNA) and ribosome production. In the bovine primordial follicle oocyte, this organelle is inactive, but in the secondary follicle an active fibrillo-granular nucleolus develops and proteins involved in rDNA transcription (topoisomerase I, RNA polymerase...... I and upstream binding factor) and early (fibrillarin) or late rRNA processing (nucleolin and nucleophosmin) localize to it. At the end of the oocyte growth phase, the nucleolus is inactivated again and transforms into a solid remnant. The nucleolar remnant is dissolved when meiosis is resumed. Upon...... fertilization, structures resembling the nucleolar remnant, now referred to as nucleolus precursor bodies (NPBs), are established in the pronuclei. These entities are engaged in the re-establishment of fibrilo-granular nucleoli at the major activation of the embryonic genome. This nucleolar formation can...

The dormant and the fully competent oocyte: comparing the transcriptome of human oocytes from primordial follicles and in metaphase II

DEFF Research Database (Denmark)

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

2013-01-01

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...... oocyte in the quiescent state and at the pinnacle of maturity at ovulation. In silico bioinformatic comparisons were made between the transcriptome of human oocytes from dormant primordial follicles and that of human metaphase II (MII) oocytes and granulosa cells and unique gene expression profiles were...... identified as well as functional and pathway enrichments associated with the oocytes from the two developmental hallmarks. A total of 729 genes were highly enriched in oocytes from primodial follicles and 1456 genes were highly enriched in MII oocytes (>10-fold, P...

Successful Oocyte Cryopreservation in Reproductive-Aged Cancer Survivors.

Science.gov (United States)

Druckenmiller, Sarah; Goldman, Kara N; Labella, Patty A; Fino, M Elizabeth; Bazzocchi, Antonia; Noyes, Nicole

2016-03-01

To demonstrate that oocyte cryopreservation is a feasible reproductive option for patients with cancer of childbearing age who require gonadotoxic therapies. This study is a university-based retrospective review of reproductive-aged cancer patient treatment cycles that included ovarian stimulation, transvaginal oocyte retrieval, oocyte cryopreservation, and, in some cases, subsequent oocyte thaw, in vitro fertilization, and embryo transfer. Outcome measures included ovarian stimulation response, number of oocytes retrieved, cryopreserved, and thawed, and pregnancy data. From 2005 to 2014, 176 reproductive-aged patients with cancer (median age 31 years, interquartile range 24-36) completed 182 oocyte cryopreservation cycles. Median time between consult request and oocyte retrieval was 12 days (interquartile range 10-14). Median peak stimulation estradiol was 1,446 pg/mL (interquartile range 730-2,687); 15 (interquartile range 9-23) oocytes were retrieved and 10 (interquartile range 5-18) metaphase II oocytes were cryopreserved per cycle. Ten patients (11 cycles) have returned to attempt pregnancy with their cryopreserved oocytes. Among thawed oocytes, the cryopreservation survival rate was 86% (confidence interval [CI] 78-94%). Nine of 11 thaw cycles resulted in embryos suitable for transfer. The embryo implantation rate was 27% (CI 8-46%) and the live birth rate was 44% (CI 12-77%) per embryo transfer. Chance for live birth with embryos created from cryopreserved oocytes was similar between the patients with cancer in this study and noncancer patients who underwent the same treatment at our center (44% [CI 12-77%] compared with 33% [CI 22-44%] per embryo transfer). Oocyte cryopreservation is now a feasible fertility preservation option for reproductive-aged patients with cancer who require gonadotoxic therapies.

Ethical issues in transnational "mail order" oocyte donation.

Science.gov (United States)

Heng, B C

2006-12-01

The rising demand for donor oocytes in developed countries has led to what is referred to as transnational or international oocyte donation, or the outsourcing of oocyte donation to poorer countries. In a further twist, frozen sperm from a recipient's partner can also be mailed to a foreign clinic to fertilize donor oocytes, and the resulting embryos are mailed back, cryopreserved, for transfer to the recipient. Among the numerous ethical concerns raised by this practice of mail order oocyte donation, the most obvious are that underprivileged women from poorer countries are often exploited; fertility physicians from richer counties abdicate responsibility for the welfare of donors; and responsibility could become an issue of contention if transmission of disease to the oocyte recipient or congenital defects in offspring born from such oocyte donation were to occur. Moreover, savings from utilizing donors from poorer countries ought to be shared with oocyte recipients.

Successful ongoing pregnancies after vitrification of oocytes.

Science.gov (United States)

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

2006-01-01

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.

Maturation arrest of human oocytes at germinal vesicle stage

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Zhi Qin Chen

2010-01-01

Full Text Available Maturation arrest of human oocytes may occur at various stages of the cell cycle. A total failure of human oocytes to complete meiosis is rarely observed during assisted conception cycles. We describe here a case of infertile couples for whom all oocytes repeatedly failed to mature at germinal vesicle (GV stage during in vitro fertilization/Intra cytoplasmic sperm injection (IVF/ICSI. The patient underwent controlled ovarian stimulation followed by oocyte retrieval and IVF/ICSI. The oocytes were stripped off cumulus cells prior to the ICSI procedure and their maturity status was defined. The oocyte maturation was repeatedly arrested at the GV. Oocyte maturation arrest may be the cause of infertility in this couple. The recognition of oocyte maturation arrest as a specific medical condition may contribute to the characterization of the currently known as "oocyte factor." The cellular and genetic mechanisms causing oocyte maturation arrest should be the subject for further investigation.

DNA Double-Strand Breaks Induce the Nuclear Actin Filaments Formation in Cumulus-Enclosed Oocytes but Not in Denuded Oocytes.

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Ming-Hong Sun

Full Text Available As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs and cumulus-enclosed oocytes (CEOs. We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells.

The human cumulus--oocyte complex gene-expression profile

Science.gov (United States)

Assou, Said; Anahory, Tal; Pantesco, Véronique; Le Carrour, Tanguy; Pellestor, Franck; Klein, Bernard; Reyftmann, Lionel; Dechaud, Hervé; De Vos, John; Hamamah, Samir

2006-01-01

BACKGROUND The understanding of the mechanisms regulating human oocyte maturation is still rudimentary. We have identified transcripts differentially expressed between immature and mature oocytes, and cumulus cells. METHODS Using oligonucleotides microarrays, genome wide gene expression was studied in pooled immature and mature oocytes or cumulus cells from patients who underwent IVF. RESULTS In addition to known genes such as DAZL, BMP15 or GDF9, oocytes upregulated 1514 genes. We show that PTTG3 and AURKC are respectively the securin and the Aurora kinase preferentially expressed during oocyte meiosis. Strikingly, oocytes overexpressed previously unreported growth factors such as TNFSF13/APRIL, FGF9, FGF14, and IL4, and transcription factors including OTX2, SOX15 and SOX30. Conversely, cumulus cells, in addition to known genes such as LHCGR or BMPR2, overexpressed cell-tocell signaling genes including TNFSF11/RANKL, numerous complement components, semaphorins (SEMA3A, SEMA6A, SEMA6D) and CD genes such as CD200. We also identified 52 genes progressively increasing during oocyte maturation, comprising CDC25A and SOCS7. CONCLUSION The identification of genes up and down regulated during oocyte maturation greatly improves our understanding of oocyte biology and will provide new markers that signal viable and competent oocytes. Furthermore, genes found expressed in cumulus cells are potential markers of granulosa cell tumors. PMID:16571642

The DNA damage response in mammalian oocytes

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John eCarroll

2013-06-01

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.

Apoptosis maintains oocyte quality in aging Caenorhabditis elegans females.

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Sara Andux

2008-12-01

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.

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.

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

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George W. Smith

2010-02-01

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.

77 FR 29914 - Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products

Science.gov (United States)

2012-05-21

... Spongiform Encephalopathy; Importation of Bovines and Bovine Products AGENCY: Animal and Plant Health... derived from bovines with regard to bovine spongiform encephalopathy. This action will allow interested... importation of live bovines and products derived from bovines with regard to bovine spongiform encephalopathy...

Cryopreservation of zebrafish (Danio rerio) oocytes by vitrification.

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

2010-01-01

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.

Functional and structural effects of amyloid-β aggregate on Xenopus laevis oocytes.

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Parodi, Jorge; Ochoa-de la Paz, Lenin; Miledi, Ricardo; Martínez-Torres, Ataúlfo

2012-10-01

Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of "spontaneous" blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca(2+) was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca(2+)-dependent Cl(-) currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (T(out)) and the serum-activated, oscillatory Cl(-) currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca(2+)-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication.

Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by Laser Capture Microdissection

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Bonnet Agnes

2011-08-01

Full Text Available Abstract Background Successful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult. The recently developed laser capture microdissection (LCM technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA. Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis. Results We developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15 and three granulosa cell-specific genes (KL, GATA4, AMH. A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte. Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA. Conclusions

Sucrose/bovine serum albumin mediated biomimetic crystallization

Indian Academy of Sciences (India)

To understand the role of the sucrose/bovine serum albumin system in the biomineralization process, we have tested the influence of different concentration of the sucrose/bovine serum albumin (BSA) on calcium carbonate (CaCO3) precipitation. The CaCO3 crystals were characterized by scanning electron microscope ...

309 proteomic analysis of the blastocoel fluid and remaining cells of bovine blastocysts

DEFF Research Database (Denmark)

Jensen, P L; Groendahl, M L; Beck, Helle

2012-01-01

Human embryonic stem cells (hESC) are derived from the human blastocyst and possess the potential to differentiate into any cell type present in the adult human body. Human ESC are considered to have great potential in regenerative medicine for the future treatment of severe diseases and conditions...... such as Parkinson's disease, diabetes, and spinal cord injury. One of today's challenges in regenerative medicine is to define proper culture conditions for hESC. The natural milieu in the blastocyst may provide clues on how to improve culture conditions, and the aim of the present study was to determine...... the proteome of the blastocoel fluid and the remaining cells of bovine blastocysts. Bovine blastocysts were produced by in vitro fertilization of oocytes retrieved from slaughterhouse ovaries. The blastocoel from 195 blastocysts (1-8nL per blastocyst) were isolated by micromanipulation and analysed by nano...

The total pregnancy potential per oocyte aspiration after assisted reproduction-in how many cycles are biologically competent oocytes available?

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

2016-07-01

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.

Effect of mating between the donor cow and bull (Holstein versus Gyr on the in vitro production of bovine embryos

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Ana Paula Toledo Barbosa da Silva

2015-03-01

Full Text Available The objective of this study was to evaluate the effect of the breed of the oocyte donor cow and bull (Holstein versus Gyr on in vitro production (IVP parameters of bovine embryos comparing the mean number of recovered oocytes and oocytes suitable for culture, the rate of suitable oocytes, and cleavage and blastocyst rates. Data from 1,000 follicular aspiration sessions (OPU, including 500 in donor cows of the Holstein breed and 500 of the Gyr breed, were collected. The results were analyzed by the unpaired Student t-test and chi-square test, adopting a level of significance of 5%. The mean number and standard deviation of recovered oocytes and oocytes suitable for culture were 15.1±13.0 and 8.7±7.6 for the Holstein breed and 15.5±11.9 and 9.1±7.9 for the Gyr breed. The rates of suitable oocytes were 57.7% and 58.5% for Holstein and Gyr breeds, respectively. A significant difference between breeds was observed for the number of oocytes suitable for culture (P<0.05, but not for the number of recovered oocytes or rates of suitable oocytes (P>0.05. Similarly, the breed of the oocyte donor cow and bull influenced cleavage and blastocyst rates (P<0.05. The cleavage rates were 65.7, 60.3, 59.6 and 56.5% for the combinations (donor breed x bull breed Holstein x Holstein (G1, Holstein x Gyr (G2, Gyr x Holstein (G3 and Gyr x Gyr (G4, respectively, with G1>G2, G1>G3, G1>G4, G2=G3, G2>G4, and G3>G4. The blastocyst rates were 28.1, 33.3, 26.8 and 31.0%, respectively, with G1>G2, G1=G3, G1oocytes suitable for culture in the Gyr breed.

Effect of concentration and exposure period to butyrolactone I on meiosis progression in bovine oocytes Efeito de concentração e tempo de exposição à butirolactona I na progressão da meiose de oócitos bovinos

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P.R. Adona

2006-06-01

Full Text Available The effect of concentration and exposure period of bovine oocytes to butyrolactone I (BLI on meiotic block and in vitro maturation (IVM kinetics was studied. In experiment 1, all oocytes were at germinal vesicle stage (GV, after 6h in culture with 0, 50 and 100µM BLI. After 12h, all oocytes cultured with 50 and 100µM BLI remained in GV. After 24h, less oocytes were in GV with 50µM (82% than with 100µM BLI (99%, P0.05. After 18h IVM, metaphase II (MII rates were similar for all groups (76-81%. In experiment 3, after 6h IVM, 74% of treated oocytes (50 or 100µM BLI for 12h were in GV. This rate was lower than for control oocytes (97.3%, P0.05 were in MII with BLI than for control (73%, PEstudou-se o efeito da concentração e do tempo de exposição à butirolactona I (BLI no bloqueio meiótico e na cinética da maturação in vitro (MIV de oócitos bovinos. No experimento 1, todos os oócitos encontravam-se em vesícula germinativa (VG após 6h de cultivo nas concentrações de 0,50 e 100µM BLI. Após 12h, somente oócitos cultivados com BLI (50 e 100µM estavam em VG. Após 24h, menos oócitos tratados com 50µM (82% estavam em VG em relação a 100µM (99%, P0,05. A taxa de metáfase II (MII, 76-81% foi similar para todos os tempos de exposição, após 18h de MIV. No experimento 3, após 6h de MIV, menos oócitos tratados (74% para 50 ou 100µM BLI por 12h estavam em VG comparados aos controles (97%, P0,05 do que os controles (73%, P<0.05. Conclui-se que para cultivos mais curtos, a concentração mais baixa de BLI bloqueia a meiose a cinética da maturação nuclear é acelerada em oócitos expostos à BLI e isso é afetado pelo tempo de cultivo, mas não pela concentração da droga.

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

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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

2016-09-01

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: journals.permissions@oup.com.

Neonatal oocyte development and selective oocyte-killing by X-rays in the Chinese hamster, Cricetulus griseus

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Tateno, H.; Mikamo, K. (Asahikawa Medical Coll. (Japan). Dept. of Biological Sciences)

1984-02-01

The process of ovarian development in neonatal Chinese hamsters aged between 0 and 16 days was studied histologically and quantitatively in both a non-irradiated group and an irradiated group. In the latter, ovaries were exposed to a single dose of 1 Gy X-rays on days 0, 2, 4, 6, 8, 10, 12 and 14 after birth. All oocytes on day 0 were at pachytene, and nearly all of them seemed to develop to dictyate by day 10. A quantitative analysis of age-dependent changes in the distribution of oocytes showed that a marked spontaneous degeneration of oocytes took place twice, i.e. during pachytene (day 0 to day 4) and dictyate (day 12 to day 14). Oocytes of this species were found to be very radioresistant at pachytene, but to become sharply sensitive during the phases between diplotene and early dictyate, suffering an almost complete oocyte-killing after 1 Gy. However, they recovered radioresistance after the onset of the resting stage. The changing aspects of radiosensitivity in the Chinese hamster were shown to be far more marked than in the mouse and the rat, which have been observed by previous investigators.

Oocyte-specific gene Oog1 suppresses the expression of spermatogenesis-specific genes in oocytes.

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Honda, Shinnosuke; Miki, Yuka; Miyamoto, Yuya; Kawahara, Yu; Tsukamoto, Satoshi; Imai, Hiroshi; Minami, Naojiro

2018-05-03

Oog1, an oocyte-specific gene that encodes a protein of 425 amino acids, is present in five copies on mouse chromosomes 4 and 12. In mouse oocytes, Oog1 mRNA expression begins at embryonic day 15.5 and almost disappears by the late two-cell stage. Meanwhile, OOG1 protein is detectable in oocytes in ovarian cysts and disappears by the four-cell stage; the protein is transported to the nucleus in late one-cell to early two-cell stage embryos. In this study, we examined the role of Oog1 during oogenesis in mice. Oog1 RNAi-transgenic mice were generated by expressing double-stranded hairpin Oog1 RNA, which is processed into siRNAs targeting Oog1 mRNA. Quantitative RT-PCR revealed that the amount of Oog1 mRNA was dramatically reduced in oocytes obtained from Oog1-knockdown mice, whereas the abundance of spermatogenesis-associated transcripts (Klhl10, Tekt2, Tdrd6, and Tnp2) was increased in Oog1 knockdown ovaries. Tdrd6 is involved in the formation of the chromatoid body, Tnp2 contributes to the formation of sperm heads, Tekt2 is required for the formation of ciliary and flagellar microtubules, and Klhl10 plays a key role in the elongated sperm differentiation. These results indicate that Oog1 down-regulates the expression of spermatogenesis-associated genes in female germ cells, allowing them to develop normally into oocytes.

Caffeine and oocyte vitrification: Sheep as an animal model

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Adel R. Moawad

Full Text Available Oocyte cryopreservation is valuable way of preserving the female germ line. Vitrification of immature ovine oocytes decreased the levels of both maturation promoting factor (MPF and mitogen-activated protein kinase (MAPK in metaphase II (MII oocytes after IVM. Our aims were 1 to evaluate the effects of vitrification of ovine GV-oocytes on spindle assembly, MPF/MAP kinases activities, and preimplantation development following IVM and IVF, 2 to elucidate the impact of caffeine supplementation during IVM on the quality and development of vitrified/warmed ovine GV-oocytes. Cumulus-oocyte complexes (COCs from mature ewes were divided into vitrified, toxicity and control groups. Oocytes from each group were matured in vitro for 18 h in caffeine free IVM medium and denuded oocytes were incubated in maturation medium supplemented with 10 mM (+ or without (− caffeine for another 6 h. At 24 h.p.m., oocytes were evaluated for spindle configuration, MPF/MAP kinases activities or fertilized and cultured in vitro for 7 days. Caffeine supplementation did not significantly affect the percentages of oocytes with normal spindle assembly in all the groups. Caffeine supplementation during IVM did not increase the activities of both kinases in vitrified groups. Cleavage and blastocyst development were significantly lower in vitrified groups than in control. Caffeine supplementation during the last 6 h of IVM did not significantly improve the cleavage and blastocyst rates in vitrified group. In conclusion, caffeine treatment during in vitro maturation has no positive impact on the quality and development of vitrified/warmed ovine GV-oocytes after IVM/IVF and embryo culture. Keywords: Caffeine, GV, MPF/MAPK, Oocytes, Ovine, Vitrification

Ultrastructure and mitochondrial numbers in pre- and postpubertal pig oocytes

DEFF Research Database (Denmark)

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

2016-01-01

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...... 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......, but no differences were observed in mitochondrial densities between groups. Mature postpubertal oocytes adhered to the following characteristics: presence of metaphase II, lack of contact between cumulus cells and oocyte, absence of rough endoplasmic reticulum and Golgi complexes, peripheral location of cortical...

[Wide support for oocyte donation and banking in the Netherlands].

Science.gov (United States)

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

2012-01-01

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.

IL-6 and mouse oocyte spindle.

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Jashoman Banerjee

Full Text Available Interleukin 6 (IL-6 is considered a major indicator of the acute-phase inflammatory response. Endometriosis and pelvic inflammation, diseases that manifest elevated levels of IL-6, are commonly associated with higher infertility. However, the mechanistic link between elevated levels of IL-6 and poor oocyte quality is still unclear. In this work, we explored the direct role of this cytokine as a possible mediator for impaired oocyte spindle and chromosomal structure, which is a critical hurdle in the management of infertility. Metaphase-II mouse oocytes were exposed to recombinant mouse IL-6 (50, 100 and 200 ng/mL for 30 minutes and subjected to indirect immunofluorescent staining to identify alterations in the microtubule and chromosomal alignment compared to untreated controls. The deterioration in microtubule and chromosomal alignment were evaluated utilizing both fluorescence and confocal microscopy, and were quantitated with a previously reported scoring system. Our results showed that IL-6 caused a dose-dependent deterioration in microtubule and chromosomal alignment in the treated oocytes as compared to the untreated group. Indeed, IL-6 at a concentration as low as 50 ng/mL caused deterioration in the spindle structure in 60% of the oocytes, which increased significantly (P<0.0001 as IL-6 concentration was increased. In conclusion, elevated levels of IL-6 associated with endometriosis and pelvic inflammation may reduce the fertilizing capacity of human oocyte through a mechanism that involves impairment of the microtubule and chromosomal structure.

OBSERVATIONS REGARDING OOCYTES STORAGE POST MENDING FROM SLAUGHTER FEMALES

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CARABA V.

2008-01-01

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

Effect of B-mercaptoethanol on the viability of IVM/IVF/IVC bovine embryos during long-distance transportation in plastic straws.

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Takahashi, H; Kuwayama, M; Hamano, S; Takahashi, M; Okano, A; Kadokawa, H; Kariya, T; Nagai, T

1996-10-15

Experiments were conducted to assess the effect of beta-mercaptoethanol (beta-ME) on the quality and viability of bovine blastocysts derived from in-vitro culture (IVC) of in-vitro matured and fertilized (TVM-IVF) oocytes during their transport between 2 distant places. Follicular oocytes were collected from ovaries obtained at a slaughterhouse and were cultured for 20 to 21 h in modified TCM-199. The IVM oocytes were fertilized in vitro with frozen-thawed spermatozoa. Fertilized oocytes were cultured for 7 d, and embryos that developed to the blastocyst stage were used for the experiments. The blastocysts, packed in straws with transportation medium that consisted of modified TCM-199 with HEPES equilibrated in air and supplemented with 20 % calf serum and 0, 10, 50, 100 or 150 microM beta-ME, were transported at 37 degrees C from Tokyo to Sapporo by air (18.3 h). The quality of blastocysts was assessed and ranked as excellent (A), good (B), fair (C) or poor (D) after transportation. The percentages of blastocysts ranked as A or B were significantly higher (P plastic straws for several hours without control of CO2 and that the concentration of beta-ME used in this experiment is not detrimental to the blastocysts.

Detection of Taste Change of Bovine and Goat Milk in Room Ambient Using Electronic Tongue

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Imam Tazi

2017-11-01

Full Text Available An electronic tongue (e-tongue based on an array of lipid/polymer membranes has been successfully developed for measuring the taste evolution of natural milk. The e-tongue consisted of 16 different lipid/polymer membranes combined with or without a pH sensor. The natural milk of bovine and goat were purchased from the local farming store in Malang-Indonesia. The taste measurement was carried out, from fresh (0 h to stale (12 h, every two hours under room ambient without any treatment. The responses of the e-tongue were evaluated using a Principal Component Analysis (PCA and a Linear Discriminant Analysis (LDA. From PCA results, the taste of both milk samples tends to change by time although some groups show a partial overlapping. LDA results show the high precision of the e-tongue in clustering taste evolution. The correctly classified groups after the cross-validation procedure were achieved 95.7 and 87.1% for bovine and goat milk, respectively. The improvement of the classification using LDA was obtained by adding data from a pH sensor of each measurement as 100 and 98.6% for bovine and goat milk, respectively. This work indicates that the lab-made e-tongue may be useful to predict the quality of natural milk for the food industry.

Human oocyte calcium analysis predicts the response to assisted oocyte activation in patients experiencing fertilization failure after ICSI.

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Ferrer-Buitrago, M; Dhaenens, L; Lu, Y; Bonte, D; Vanden Meerschaut, F; De Sutter, P; Leybaert, L; Heindryckx, B

2018-01-10

Can human oocyte calcium analysis predict fertilization success after assisted oocyte activation (AOA) in patients experiencing fertilization failure after ICSI? ICSI-AOA restores the fertilization rate only in patients displaying abnormal Ca2+ oscillations during human oocyte activation. Patients capable of activating mouse oocytes and who showed abnormal Ca2+ profiles after mouse oocyte Ca2+ analysis (M-OCA), have variable responses to ICSI-AOA. It remains unsettled whether human oocyte Ca2+ analysis (H-OCA) would yield an improved accuracy to predict fertilization success after ICSI-AOA. Sperm activation potential was first evaluated by MOAT. Subsequently, Ca2+ oscillatory patterns were determined with sperm from patients showing moderate to normal activation potential based on the capacity of human sperm to generate Ca2+ responses upon microinjection in mouse and human oocytes. Altogether, this study includes a total of 255 mouse and 122 human oocytes. M-OCA was performed with 16 different sperm samples before undergoing ICSI-AOA treatment. H-OCA was performed for 11 patients who finally underwent ICSI-AOA treatment. The diagnostic accuracy to predict fertilization success was calculated based on the response to ICSI-AOA. Patients experiencing low or total failed fertilization after conventional ICSI were included in the study. All participants showed moderate to high rates of activation after MOAT. Metaphase II (MII) oocytes from B6D2F1 mice were used for M-OCA. Control fertile sperm samples were used to obtain a reference Ca2+ oscillation profile elicited in human oocytes. Donated human oocytes, non-suitable for IVF treatments, were collected and vitrified at MII stage for further analysis by H-OCA. M-OCA and H-OCA predicted the response to ICSI-AOA in 8 out of 11 (73%) patients. Compared to M-OCA, H-OCA detected the presence of sperm activation deficiencies with greater sensitivity (75 vs 100%, respectively). ICSI-AOA never showed benefit to overcome

GGPP-Mediated Protein Geranylgeranylation in Oocyte Is Essential for the Establishment of Oocyte-Granulosa Cell Communication and Primary-Secondary Follicle Transition in Mouse Ovary.

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Chen Jiang

2017-01-01

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.

[Investigation of follicular development and oocyte maturation after cryopreservation and xenograft of newborn mouse ovaries].

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Qin, Bo-Lin; Chen, Xue-Jin; Shi, Zhen-Dan; Li, Wan-Li; Tian, Yun-Bo

2006-02-25

In order to explore the feasibility of cryopreserving primordial follicles in attaining their developmental competence following freezing and thawing, ovaries from newborn mice were cryopreserved and the thawed ovaries were xenografted into kidney capsules of adult female mice. Ovaries were isolated from newborn B6C2F(1) female mice, infiltrated by Leibovitz 15 (L-15) medium containing 10% (V/V) fetal bovine serum (FBS) and 1.5 mol/L dimethylsulfoxide (DMSO), and then packed into 0.25 ml plastic straws. The ovaries contained in straws were frozen under nitrogen vapour at -40 degrees C in Cryocell 1200 programmable freezer, and stored in liquid nitrogen for periods ranging from 1 week to 6 months. Upon thawing, the straws were dipped into room temperature water for 10~20 s, after which the ovaries were collected and washed in L-15 buffer containing 10% (V/V) FBS without DMSO to remove cryoprotectant. The thawed ovaries were transplanted into kidney capsules of 8~12-week old adult B6C2F(1) female recipient mice by two protocols, with either 1 or 2 ovaries in each capsule. Upon withdrawal after at least 14 d of transplantation, only 45.00% (72/160) of the ovaries were recovered from 40 recipients transplanted with 2 ovaries in each capsule, compared to 82.50% (33/40) in 20 recipients with only 1 ovary in each capsule. The grafted ovaries exhibited similar follicular developmental progression to that of natural ovaries. There were antral follicles present in the transplanted ovaries on day 14, whose number increased more substantially on day 19 after transplantation. Following stimulation of the recipient mice with 10 IU PMSG on day 19 after xenografting, follicles further developed to preovulatory stage with appearance of cumulus oocytes and enlarged antrum. Oocytes from these fully grown antral follicles were collected and matured in vitro in modified essential medium-alpha (MEMalpha). After 16~17 h of culture, 40.90% of the oocytes exhibited germinal vesicle

Fertilization and Embryo Development of Fresh and Cryopreserved Sibling Oocytes

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Robert F. Casper

2010-01-01

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.

Human oocyte chromosome analyses need a standardized ...

Indian Academy of Sciences (India)

Studies of DNA polymorphisms in human trisomic abor- tions and liveborn have ... Keywords. human oocyte chromosomes; cytogenetic analysis; aneuploidy; nondisjunction; predivision. Journal of .... oocytes and giant embryos. Hum. Reprod.

Age-dependent radiosensitivity of mouse oocytes

International Nuclear Information System (INIS)

Koehler, C.

1976-01-01

It has been shown that there are three distinct phases of radiosensitivity in oocytes of prepubertal mice: a period of rapidly increasing sensitivity between 0 and 4 days of age; a period of consistent, high sensitivity between 5 and 18 days of age; and a period of decreasing sensitivity from 19 to at least 21 days of age. Two distinct phases have been demonstrated for the rate of population decline of the oocytes of primary follicles: an initial period of rapid loss from 0 to 4 days of age; and a period of much slower loss from 5 through 23 days of age. Correlations have been drawn between the first two phases of radiosensitivity and morphological changes in the oocyte, and between the third phase of radiosensitivity and endocrinological changes in the maturing animal. The reaction of oocytes to radiation has been separated into two categories: immediate death (within 24 hours); and delayed death (over the entire lifespan of the animal)

Oocyte-like cells induced from mouse spermatogonial stem cells.

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Wang, Lu; Cao, Jinping; Ji, Ping; Zhang, Di; Ma, Lianghong; Dym, Martin; Yu, Zhuo; Feng, Lixin

2012-08-06

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. 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. 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.

Oocyte-like cells induced from mouse spermatogonial stem cells

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Wang Lu

2012-08-01

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.

[Satisfaction survey on the use of autologous oocyte vitrification in Assisted Reproductive Technologies].

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Boyer, P; Lefebvre, F; Siraudin, C; Montjean, D; Le Coz, P; Gervoise-Boyer, M

2018-04-02

We organized a survey for in-vitro fertilization couples who beneficiated on self-preservation of part of the oocyte cohort. The main objective was to measure couples' satisfaction. Secondary objectives were; to identify how patients had been informed; to verify that the use of the ART technique met their expectations, and how they felt about oocyte or embryo freezing. The data were collected by a questionnaire sent electronically to couples who had undergone partial vitrification of the oocyte cohort and at least one warming cycle. The questionnaire consisted of 2 components; one for the women and another one for their husband. Eighty-eight women and 62 men responded to the survey respectively, representing 50.86% and 35.84% of the targeted patients. They were satisfied with a 90% rate, men and women combined. The information we give in the center is heard by couples and is part of the trust in the medical staff. Men are more worried than women about the risks of stimulation or ovarian pick-up and are not displeased to be called upon for sperm recovery for every attempt at oocyte warming. The ambiguity of the answers on the representation of the embryo confirms what is already described, but is independent of the acceptance of freezing whether it is oocyte or embryo. In the same way as evaluating the results, the evaluation of patient satisfaction is useful for the implementation of therapeutic strategies and care pathways. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Ethical Dilemmas for Oocyte Donations: Slippery Slope for Conflicts of Interest.

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Tulay, Pinar

2016-01-01

Oocyte donations have increased with improvements in oocyte cryopreservation procedures in recent years. Women with medical conditions that require chemotherapy or radiotherapy have begun to opt for oocyte cryo¬preservation prior to their treatment or to enroll in an oocyte donation program. Alternatively, some women apply for "third-party" oocyte donation programs for nonmedical reasons such as delayed childbearing. Although society seems to accept oocyte donations for medical reasons, it appears that there are still some moral issues surrounding nonmedical oocyte donations. In this review, the ethical aspects of oocyte donations and donors' perspectives are discussed. With developing technologies, the genetic screening of donors has expanded to include diseases. This review explores the ethical issues involved in genetic screening of gamete donors.

Maturation of human oocytes in vitro

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Mojca Čižek-Sajko

2007-01-01

Full Text Available Background: Immature oocyte retrieval followed by in vitro maturation is a promising infertility treatment option. In patients with morphologically normal ovaries and regular menstrual cycles and in patients with polycystic ovary syndrome (PCOS we attempted to assess the success of oocyte in vitro maturation in in vitro fertilization (IVF procedures.Methods: Retrospectively we analyzed 87 IVF procedures with in vitro maturation of oocytes carried out in 73 infertile couples treated at the Maribor Teaching Hospital. We compared the success following three different hormone priming protocols: regular cycling patients with normal ovaries and without hormone priming (Group A, n = 27; patients with PCOS and hormone priming with follitropin (follicle stimulating hormone, FSH (Group B, n = 22; patients with PCOS and hormone priming with human chorionic gonadotrophin (hCG (Group C, n = 38. Success of the procedure was evaluated on the basis of the ability of oocytes to mature, fertilize and develop into embryos, and on the basis of the quality of embryos and their ability to implant in the uterus.Results: In regular cycling patients with normal ovaries (n = 27 we obtained a significantly lower number of immature oocytes (3.2 ± 2.5 compared with patients with PCOS and FSH priming (11.7 ± 7.2 or those with PCOS and hCG priming (10.4 ± 7.2. The oocyte maturation rate, the fertilization rate and the embryo cleavage rate were as follows: in Group A 57.7 %, 63.2 % and 91.7 %, in Group B 57.6 %, 66.2 % and 90.0 %, and in Group C 58.0 %, 66.2 % and 91.0 % (the differences between groups were not statistically significant. Six pregnancies were recorded only in patients with PCOS. The pregnancy rate per embryo transfer was 1/20 (5.0 % in patients with FSH priming, and 5/33 (15.2 % in patients with hCG priming.Conclusions: Oocyte in vitro maturation is successful in patients with normal ovaries and regular menstrual cycle as well as in those with polycystic

Pulse radiolysis investigation of the reaction of the electronic adduct of bovine serum albumin with oxygen. Polychromatic kinetics of the reaction with adsorbed oxygen

International Nuclear Information System (INIS)

Pribush, A.G.

1986-01-01

The method of pulse radiolysis was used to investigate the reaction of the electronic adduct of bovine serum albumin with oxygen. It was suggested that the disappearance of the electronic adduct of the protein occurs in the course of its interaction with oxygen adsorbed on the globular protein molecule

Biomolecule screening for efficient attachment of biofunctionalized microparticles to the zona pellucida of mammalian oocytes and embryos.

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Novo, Sergio; Ibáñez, Elena; Barrios, Leonardo; Castell, Onofre; Nogués, Carme

2013-10-01

Individual tagging of oocytes and embryos through the attachment of micrometer-sized polysilicon barcodes to their zona pellucida (ZP) is a promising approach to ensure their correct identification and traceability in human assisted reproduction and in animal production programs. To provide barcodes with the capacity of binding to the ZP, they must be first biofunctionalized with a biomolecule capable of binding to the ZP of both oocytes and embryos. The aim of this work was to select, among an anti-ZP2 antibody and the two lectins wheat germ agglutinin (WGA) and phytohemagglutinin-L, the most optimal biomolecule for the eventual biofunctionalization of barcodes, using mouse oocytes and embryos and commercially available microspheres as a model. Despite the anti-ZP2 antibody showed the highest number of binding sites onto the ZP surface, as determined by field emission scanning electron microscopy, the binding of anti-ZP2-biofunctionalized microspheres to the ZP of cultured oocytes and embryos was less robust and less stable than the binding of lectin-biofunctionalized ones. WGA proved to be, among the three candidates tested, the most appropriate biomolecule to biofunctionalize microparticles with the aim to attach them to the ZP of both oocytes and embryos and to maintain them attached through oocyte activation (zona reaction) and in vitro culture up to the blastocyst stage. As saccharides recognized by WGA are highly abundant in the ZP of most mammalian species, WGA-biofuncionalized microparticles would be able to attach to the ZP of oocytes/embryos of species other than the mouse, such as humans and farm animals.

Rapamycin Rescues the Poor Developmental Capacity of Aged Porcine Oocytes

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Seung Eun Lee

2014-05-01

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.

Attitude of Law and Medical Students to Oocyte Donation

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Samira Vesali

2018-05-01

Full Text Available Background Among the young generation, medical and law students’ attitude towards third party reproduction is very important because they will be directly involved in restricting or developing the programs that will support it in the future. The aim of this survey was to investigate attitude of law and medical students to oocyte donation and key aspects of this kind of third party. Materials and Methods In analytical cross-sectional study, 345 medical and law students were randomly selected using stratified sampling. Data was collected using attitude toward donation- oocyte (ATOD-O questionnaire. Re- sponses were on a 5-point Likert scale. Data were analyzed according to established statistical approach by Heeren and D'Agostino. Results The majority of the participants agreed with oocyte donation being the last choice for infertility treatment. There was a significant difference between medical students and law students regarding the acceptance of oocyte donation (3.23 vs. 3.53, P=0.025. In addition, female participants were more tolerant on receiving donated oocytes from their sisters than male participants (3.01 vs. 2.58, P=0.002 and finally, a higher number of the participants had a positive attitude towards anonymity of the donor and the recipient to one another (3.93 vs. 3.86, P=0.580. The vast majority of female students believed that the oocyte recipient naturally likes that child (P<0.0001. Conclusion In the current study, a great majority of law and medical students support oocyte donation as an alternative way of starting a family. There is an interest among female students in donating oocytes anonymously. The majority believed that the oocyte recipient family will like the donor oocyte child naturally.

Quality of common marmoset (Callithrix jacchus) oocytes collected after ovarian stimulation.

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Kanda, Akifumi; Nobukiyo, Asako; Yoshioka, Miyuki; Hatakeyama, Teruhiko; Sotomaru, Yusuke

2018-01-15

The common marmoset (Callithrix jacchus) is an experimental animal that is considered suitable for the creation of next-generation human disease models. It has recently been used in the reproductive technology field. Oocytes can be effectively collected from female marmosets via ovarian stimulation with injections of follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). The oocytes, collected about 28 h after the hCG injection, include both premature oocytes and postmature (in vivo matured; IVO) oocytes, and the premature oocytes can be matured by in vitro culture (in vitro matured; IVM). Although IVM and IVO oocytes are equivalent in appearance at the MII stage, it remains unclear whether there are differences in their properties. Therefore, we investigated their in vitro fertilization and developmental capacities and cytoskeletal statuses. Our findings revealed that the IVM and IVO oocytes had similar fertilization rates but that no IVO oocytes could develop to the blastocyst stage. Additionally, IVO oocytes showed abnormal cytoskeletal formation. It is concluded that IVM oocytes maintain normal function, whereas IVO oocytes would be affected by aging and other factors when they remain for a long time in the ovary. Copyright © 2017 Elsevier Inc. All rights reserved.

Data on endogenous bovine ovarian follicular cells peptides and small proteins obtained through Top-down High Resolution Mass Spectrometry

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Valérie Labas

2017-08-01

Full Text Available The endogenous peptides and small proteins extracted from bovine ovarian follicular cells (oocytes, cumulus and granulosa cells were identified by Top-down High Resolution Mass Spectrometry (TD-HR-MS/MS in order to annotate peptido- and proteoforms detected using qualitative and quantitative profiling method based on ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. The description and analysis of these Top-down MS data in the context of oocyte quality biomarkers research are available in the original research article of Labas et al. (2017 http://dx.doi.org/10.1016/j.jprot.2017.03.027 [1]. Raw data derived from this peptidomic/proteomic analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD004892. Here, we described the inventory of all identified peptido- and proteoforms including their biochemical and structural features, and functional annotation of correspondent proteins. This peptide/protein inventory revealed that TD-HR-MS/MS was appropriate method for both global and targeted proteomic analysis of ovarian tissues, and it can be further employed as a reference for other studies on follicular cells including single oocytes.

Oocyte cryopreservation for donor egg banking.

Science.gov (United States)

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

2011-09-01

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

Correlation between biological activity and electron transferring of bovine liver catalase: Osmolytes effects

International Nuclear Information System (INIS)

Tehrani, H. Sepasi; Moosavi-Movahedi, A.A.; Ghourchian, H.

2013-01-01

Highlights: • Proline increases ET in Bovine Liver Catalase (BLC) whereas histidine decreases it. • Proline also increased the biological activity, whereas histidine decreased it. • Electron transferring and biological activity for BLC are directly correlated. • Proline causes favorable ET for BLC shown by positive E 1/2 (E°′) and negative ΔG. • Histidine makes ET unfavorable for BLC, manifested by E 1/2 (E°′) 0. -- Abstract: Catalase is a crucial antioxidant enzyme that protects life against detrimental effects of H 2 O 2 by disproportionating it into water and molecular oxygen. Effect of proline as a compatible and histidine as a non compatible osmolyte on the electron transferring and midpoint potential of catalase has been investigated. Proline increases the midpoint potential (ΔE m > 0), therefore causing the ΔG ET to be less positive and making the electron transfer reaction more facile whereas histidine decreases the E m (ΔE m ET , thereby rendering the electron transfer reaction less efficient. These results indicate the inhibitory effect of histidine evident by a −37% decrease in the cathodic peak current compared to 16% increase in the case of proline indicative of activation. The insight paves the tedious way towards our ultimate goal of elucidating a correlation between biological activity and electron transferring

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

LENUS (Irish Health Repository)

2017-11-01

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.

Mitogenomes of polar bodies and corresponding oocytes.

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Luca Gianaroli

Full Text Available The objective of the present study was to develop an approach that could assess the chromosomal status and the mitochondrial DNA (mtDNA content of oocytes and their corresponding polar bodies (PBs with the goal of obtaining a comparative picture of the segregation process both for nuclear and mtDNA. After Whole Genome Amplification (WGA, sequencing of the whole mitochondrial genome was attempted to analyze the segregation of mutant and wild-type mtDNA during human meiosis. Three triads, composed of oocyte and corresponding PBs, were analyzed and their chromosome status was successfully assessed. The complete mitochondrial genome (mitogenome was almost entirely sequenced in the oocytes (95.99% compared to 98.43% in blood, while the percentage of sequences obtained in the corresponding PB1 and PB2 was lower (69.70% and 69.04% respectively. The comparison with the mtDNA sequence in blood revealed no changes in the D-loop region for any of the cells of each triad. In the coding region of blood mtDNA and oocyte mtDNA sequences showed full correspondence, whereas all PBs had at least one change with respect to the blood-oocyte pairs. In all, 9 changes were found, either in PB1 or PB2: 4 in MT-ND5, 2 in MT-RNR2, and 1 each in MT-ATP8, MT-ND4, MT-CYTB. The full concordance between oocyte and blood in the 3 triads, and the relegation of changes to PBs, revealed the unexpected coexistence of different variants, giving a refined estimation of mitochondrial heteroplasmy. Should these findings be confirmed by additional data, an active mechanism could be postulated in the oocyte to preserve a condition of 'normality'.

Social oocyte cryopreservation: a portrayal of Brazilian women.

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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

2017-06-01

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.

Dynamic distribution of spindlin in nucleoli, nucleoplasm and spindle from primary oocytes to mature eggs and its critical function for oocyte-to-embryo transition in gibel carp.

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Sun, Min; Li, Zhi; Gui, Jian-Fang

2010-10-01

Spindlin (Spin) was thought as a maternal-effect factor associated with meiotic spindle. Its role for the oocyte-to-embryo transition was suggested in mouse, but its direct evidence for the function had been not obtained in other vertebrates. In this study, we used the CagSpin-specific antibody to investigate CagSpin expression pattern and distribution during oogenesis of gibel carp (Carassius auratus gibelio). First, the oocyte-specific expression pattern and dynamic distribution was revealed in nucleoli, nucleoplasm, and spindle from primary oocytes to mature eggs by immunofluorescence localization. In primary oocytes and growth stage oocytes, CagSpin accumulates in nucleoli in increasing numbers along with the oocyte growth, and its disassembly occurs in vitellogenic oocytes, which implicates that CagSpin may be a major component of a large number of nucleoli in fish growth oocytes. Then, co-localization of CagSpin and β-tubulin was revealed in meiotic spindle of mature egg, indicating that CagSpin is one spindle-associated factor. Moreover, microinjection of CagSpin-specific antibody into the fertilized eggs blocked the first cleavage, and found that the CagSpin depletion resulted in spindle assembly disturbance. Thereby, our study provided the first direct evidence for the critical oocyte-to-embryo transition function of Spin in vertebrates, and confirmed that Spin is one important maternal-effect factor that participates in oocyte growth, oocyte maturation, and oocyte-to-embryo transition.

Ultrastructure of the human preovulatory oocyte.

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Szöllösi, D; Mandelbaum, J; Plachot, M; Salat-Baroux, J; Cohen, J

1986-08-01

The ultrastructure of preovulatory human oocyte-cumulus complexes was described after inducing maturation by clomiphene, human menopausal gonadotropin (hMG), human chorionic gonadotropin (hCG) treatment. The majority of the oocytes was at metaphase II of meiosis, with a radially orientated spindle. The oocyte surface was covered by a multitude of microvilli. Cortical granules were nonuniformly distributed along the cortex. A cytoplasmic polarization was observed. The cytoplasmic organelles were in general uniformly dispersed, with the exception of a narrow segment within which cytoplasmic membranes and mitochondria formed clusters. The spindle was usually found at the borderline between the two regions of the cytoplasm. The functional significance of this polarization is not yet known.

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

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S.M Banan Khojasteh

2009-02-01

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.

SIRT1, 2, 3 protect mouse oocytes from postovulatory aging.

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Zhang, Teng; Zhou, Yang; Li, Li; Wang, Hong-Hui; Ma, Xue-Shan; Qian, Wei-Ping; Shen, Wei; Schatten, Heide; Sun, Qing-Yuan

2016-04-01

The quality of metaphase II oocytes will undergo a time-dependent deterioration following ovulation as the result of the oocyte aging process. In this study, we determined that the expression of sirtuin family members (SIRT1, 2, 3) was dramatically reduced in mouse oocytes aged in vivo or in vitro. Increased intracellular ROS was observed when SIRT1, 2, 3 activity was inhibited. Increased frequency of spindle defects and disturbed distribution of mitochondria were also observed in MII oocytes aged in vitro after treatment with Nicotinamide (NAM), indicating that inhibition of SIRT1, 2, 3 may accelerate postovulatory oocyte aging. Interestingly, when MII oocytes were exposed to caffeine, the decline of SIRT1, 2, 3 mRNA levels was delayed and the aging-associated defective phenotypes could be improved. The results suggest that the SIRT1, 2, 3 pathway may play a potential protective role against postovulatory oocyte aging by controlling ROS generation.

Vitrification affects nuclear maturation and gene expression of immature human oocytes

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Abbas Shahedi

2017-02-01

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. 

Molecular and structural aspects of oocyte maturation

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Hölzenspies, J.J.

2009-01-01

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

SPECIFICITY OF ANTIBODY BOVINE ZONNA PELLUCIDAE 3 (ANTI-bZP3 TO RABBIT ZP3 BASED ON bZP3 AS CONTRACEPTIVE ANTIGENS

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Edwin Widodo

2010-06-01

Full Text Available Zonna pellucidae can be develop as antigen potential candidates based on reversible immunocontraceptive vaccines. Immunogenic sites of bovine zonna pellucidae 3 (bZP3 could stimulated the presence of anti-bZP3 which be located on rabbit ZP and inhibit sperm-egg interaction on fertilization process. Purpose of this research is to detect spesific binding anti-bZP3 to rabbit oocytes using dot blotting and ELISA method. Sub cutan induction of bZP3 with Freund's adjuvant, CFA (Complete Freund's Adjuvant for initial immunization and following by IFA (Incomplete Freund's Adjuvant at the 14th day and 39th day. Control female rabbit injected by Tris-Cl buffer diluted in Freund's adjuvant without bZP3 antigen. Rabbit serum injected to rat for producing Rat Anti Rabbit Anti-bZP3. This research concludes spesific binding of anti-bZP3 with increasing purple colour on dot blotting methods. Anti-bZP3 increasing on 24th day and 31th day and still until 48th day. Measurement with ELISA methods showed increased titer on OD405. Highest titer showed on 31th day post immunization. Anti-bZP3 synthetized by bZP3 induced on rabbit detectable by immunohistochemistry methods on late primary oocytes, early secondary oocytes, growing secondary oocytes, and oocytes on de Graaf folicular phase. Keywords: Dot blotting, ELISA, bZP3, anti-bZP3

Interação entre células do cumulus e atividade da proteína quinase C em diferentes fases da maturação nuclear de oócitos bovinos Interaction between cumulus cells and the activity of protein kinase C at different stages of bovine oocyte nuclear maturation

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A.C. Bertagnolli

2004-08-01

Full Text Available Verificou-se a influência da proteína quinase C (PK-C no reinício e na progressão da meiose em oócitos bovinos, determinando se as células do cumulus são mediadoras da PK-C na regulação da maturação dos oócitos. Complexos cumulus-oócitos (CCO e oócitos desnudos (OD, distribuídos aleatoriamente em seis tratamentos (T com base na presença de um ativador da PK-C (PMA (T1 e T2, de um forbol éster incapaz de ativar a PK-C (4alfa-PDD-controle (T3 e T4 ou de apenas o meio básico (TCM-199-controle (T5 e T6, foram cultivados por 7, 9, 12, 18 e 22 horas. A percentagem de rompimento da vesícula germinativa no grupo cultivado com PMA foi maior do que nos dois grupos controle, com e sem células do cumulus. O cultivo de CCO e OD por 12 e 18 horas demonstrou que a PK-C influencia a progressão para os estádios de metáfase I (MI e metáfase II (MII de maneira dependente das células do cumulus. Nos períodos de 9 e 22 horas, não foi possível observar diferença entre os grupos quanto aos diferentes estádios de maturação. A ativação da PK-C acelera o reinício da meiose independentemente das células somáticas e acelera a progressão até os estádios de MI e MII na dependência das células do cumulus.The aim of this study was to evaluate the effect of protein kinase C (PK-C on the meiotic resumption and progression in bovine oocyte, and to determine if the cumulus cells mediate the PK-C action in the regulation of bovine oocyte nuclear maturation. Cumulus-oocyte complexes (COC and denuded oocytes (DO, randomly allotted to 6 treatments (T based on the presence of an activator of PK-C (PMA (T1 and T2, or a phorbol ester unable to activate PK-C (4alphaPDD-control (T3 and T4 or a basic culture medium (T5 and T6, were cultivated for 7, 9, 12, 18 and 22 hours. The percentage of germinal vesicle breakdown (GVBD was higher when the oocytes were cultured with PMA than in the control groups with and without cumulus cells. However, PK-C was

Fate and role of macromolecules synthesized during mammalian oocyte meiotic maturation. II. - Autoradiographic topography of (/sup 3/H)-fucose incorporation in pig oocytes cultured in vitro

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Pivko, J. (Animal Production Research Institute, Nitra (Czechoslovakia)); Motlik, J. (Institute of Animal Physiology and Genetics, Libechov (Czechoslovakia)); Kopecny, V. (University J.E. Purkyne, Brno (Czechoslovakia)); Flechon, J.E. (I.N.R.A., Station Centrale de Physiologie Animale, Jouy-en-Josas (France))

1982-01-01

Pig oocytes in different maturational stages--germinal vesicle (GV), metaphase I (MI) and metaphase II (MII)- were cultured in vitro with (/sup 3/H)-fucose. The incorporation of the precursor was followed by LM or EM autoradiography on air-dried preparations and on semithin or thin sections. The cumulus cells connected with oocytes at the GV stage were intensely labelled, while the labelling of the cumulus of MI and MII oocytes was lower. The cytoplasm of oocytes in the GV stage was characterized by nests of silver grains located mainly in a juxtanuclear position. The accumulation of label in the cortical region, observed in oocytes cultured with an intact cumulus, was less evident in cumulus-deprived oocytes. Lower labelling of the ooplasm, together with uniform distribution of the grains, was observed in later stages of meiosis. EM autoradiographs demonstrated the main localization, at the GV stage, of label in the Golgi apparatus and near the cell surface of oocytes and cumulus cells, as well as in the cytoplasmic processes of corona radiata cells. It is concluded that a relatively intense glycoprotein synthesis takes place in pig oocytes and cumulus cells during resumption of meiosis, at least before GV breakdown. Metabolic cooperation may occur as long as oocytes and cumulus cells keep membrane junctions.

[Recovery and light microscopic evaluation of follicular oocytes of swine and relationship between the degeneration rate of oocytes and the estrus phase].

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Schnurrbusch, U; Schmette, C; Elze, K

1990-10-01

Cumulus-oocyte complexes were recovered from 25 gilts by aspiration of follicular fluid or cutting of follicles from all Graafian follicles of greater than or equal to 3 mm in diameter during diestrus, proestrus or estrus. In 5 gilts the oocytes were collected post ovulation by flushing of oviducts. The recovery rate of follicular oocytes differed between 75.5% during the late diestrus (days 13-17) and 43.5% during the proestrus (days 18-21). During the proestrus and on day 1 of the estrus the recovery of oocytes was more difficult as a result of the higher viscosity of follicular fluid and the mucification of cumulus-oocyte complexes. The degeneration rate of oocytes was high during the diestrus with a peak at the time of regression of corpora lutea. From diestrus to the estrus the degeneration rate decreased. Following degeneration rates were found in the oocytes during the cycle: days 7-12: 38.8%, days 13-17: 50.0%, days 18-21: 29.6%, day 1 of the estrus: 10.8%, day 2 of the estrus ante ovulation: 11.8%, day 2 of the estrus post ovulation: 6.2%. Signs of degeneration were: Loss of cumulus cells (during diestrus and proestrus), damaged zona pellucida, enlargement of perivitelline space, deformation of oocyte, alteration of structure of the ooplasm, diameter of vitellus less than 100 microns. It was concluded that the selection of dominant follicles takes place in pigs during a long time of the cycle, especially during the diestrus. There were not any indications of a 2-wave hypothesis of follicular growth during the cycle in pig.

PTK2b function during fertilization of the mouse oocyte

International Nuclear Information System (INIS)

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

2014-01-01

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

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: wkinsey@kumc.edu [Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

2014-08-01

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.

Establishment of an oocyte donor program. Donor screening and selection.

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Quigley, M M; Collins, R L; Schover, L R

1991-01-01

IVF with donated oocytes, followed by embryo placement in the uterus of a recipient who has been primed with exogenous steroids, is a successful treatment for special cases of infertility. Preliminary results indicate that the success rate in this situation is even greater than that usually seen with normal IVF (with placement of the embryos back into the uteri of the women from whom the oocytes were recovered). Although different sources for donated oocytes have been identified, the use of "excess" oocytes from IVF cycles and the attempted collection of oocytes at the time of otherwise indicated pelvic surgery have ethical and practical problems associated with their use. We have herein described the establishment of a successful program relying on anonymous volunteers who go through ovarian stimulation, monitoring, and oocyte recovery procedures solely to donate oocytes. The potential donors go through an exhaustive screening and education process before they are accepted in the program. Psychological evaluation of our potential donors indicated a great degree of turmoil in their backgrounds and a wide variety of motivations for actually participating. Despite the extensive educational and screening process, a substantial percentage of the donors did not complete a donation cycle, having either voluntarily withdrawn or been dropped because of lack of compliance. Further investigation of the psychological aspects of participating in such a program is certainly warranted. The use of donated oocytes to alleviate specific types of infertility is quite successful, but the application of this treatment is likely to be limited by the relative unavailability of suitable oocyte donors.

Melatonin prevents postovulatory oocyte aging and promotes subsequent embryonic development in the pig.

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Wang, Tao; Gao, Ying-Ying; Chen, Li; Nie, Zheng-Wen; Cheng, Wei; Liu, Xiaoyan; Schatten, Heide; Zhang, Xia; Miao, Yi-Liang

2017-06-26

Oxidative stress is known as a major contributing factor involved in oocyte aging, which negatively affects oocyte quality and development after fertilization. Melatonin is an effective free radical scavenger and its metabolites AFMK and AMK are powerful detoxifiers that eliminate free radicals. In this study, we used porcine oocytes to test the hypothesis that melatonin could scavenge free radicals produced during oocyte aging, thereby maintaining oocyte quality. We compared reactive oxygen species levels, apoptosis levels, mitochondrial membrane potential ratios, total glutathione contents and expression levels in fresh, aged and melatonin-treated aged porcine oocytes and observed the percentage of blastocyst formation following parthenogenetic activation. We found that melatonin could effectively maintain the morphology of oocytes observed in control oocytes, alleviate oxidative stress, markedly decrease early apoptosis levels, retard the decline of mitochondrial membrane potential and significantly promote subsequent embryonic development in oocytes aged for 24 hr in vitro . These results strongly suggest that melatonin can prevent postovulatory oocyte aging and promote subsequent embryonic development in the pig, which might find practical applications to control oocyte aging in other mammalian species including humans to maintain the quality of human oocytes when performing clinical assisted reproductive technology.

Induction and inhibition of oocyte maturation by EDCs in zebrafish

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Tokumoto Mika

2005-12-01

Full Text Available Abstract Background Oocyte maturation in lower vertebrates is triggered by maturation-inducing hormone (MIH, which acts on unidentified receptors on the oocyte surface and induces the activation of maturation-promoting factor (MPF in the oocyte cytoplasm. We previously described the induction of oocyte maturation in fish by an endocrine-disrupting chemical (EDC, diethylstilbestrol (DES, a nonsteroidal estrogen. Methods In this study, stimulatory and inhibitory effects of EDCs and natural steroids on oocyte maturation were examined in zebrafish. For effective agents, some details about the mechanism in induction or inhibition of maturation were examined. Possible groups of DES interacting with the MIH receptor are discussed based on relative potency of steroids to induce maturation. Results Among agents tested, tamoxifen (TAM and its metabolite 4-hydroxytamoxifen (4-OHT showed stimulatory activity similar to DES. The time courses of the change in germinal vesicle breakdown and an intracellular molecular event (the synthesis of cyclin B induced by TAM were indistinguishable from those induced by MIH. In contrast, pentachlorophenol (PCP had a potent inhibitory effect on MIH-induced oocyte maturation. PCP inhibited not only MIH-induced maturation but also DES- and TAM-induced maturation. Methoxychlor also inhibited maturation when oocytes were pre-treated with this agent. Conclusion These results suggest that EDCs act as agonists or antagonists in the induction of oocyte maturation in fish.

Short-term preservation of porcine oocytes in ambient temperature: novel approaches.

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Cai-Rong Yang

Full Text Available The objective of this study was to evaluate the feasibility of preserving porcine oocytes without freezing. To optimize preservation conditions, porcine cumulus-oocyte complexes (COCs were preserved in TCM-199, porcine follicular fluid (pFF and FCS at different temperatures (4°C, 20°C, 25°C, 27.5°C, 30°C and 38.5°C for 1 day, 2 days or 3 days. After preservation, oocyte morphology, germinal vesicle (GV rate, actin cytoskeleton organization, cortical granule distribution, mitochondrial translocation and intracellular glutathione level were evaluated. Oocyte maturation was indicated by first polar body emission and spindle morphology after in vitro culture. Strikingly, when COCs were stored at 27.5°C for 3 days in pFF or FCS, more than 60% oocytes were still arrested at the GV stage and more than 50% oocytes matured into MII stages after culture. Almost 80% oocytes showed normal actin organization and cortical granule relocation to the cortex, and approximately 50% oocytes showed diffused mitochondria distribution patterns and normal spindle configurations. While stored in TCM-199, all these criteria decreased significantly. Glutathione (GSH level in the pFF or FCS group was higher than in the TCM-199 group, but lower than in the non-preserved control group. The preserved oocytes could be fertilized and developed to blastocysts (about 10% with normal cell number, which is clear evidence for their retaining the developmental potentiality after 3d preservation. Thus, we have developed a simple method for preserving immature pig oocytes at an ambient temperature for several days without evident damage of cytoplasm and keeping oocyte developmental competence.

Short-term preservation of porcine oocytes in ambient temperature: novel approaches.

Science.gov (United States)

Yang, Cai-Rong; Miao, De-Qiang; Zhang, Qing-Hua; Guo, Lei; Tong, Jing-Shan; Wei, Yanchang; Huang, Xin; Hou, Yi; Schatten, Heide; Liu, ZhongHua; Sun, Qing-Yuan

2010-12-07

The objective of this study was to evaluate the feasibility of preserving porcine oocytes without freezing. To optimize preservation conditions, porcine cumulus-oocyte complexes (COCs) were preserved in TCM-199, porcine follicular fluid (pFF) and FCS at different temperatures (4°C, 20°C, 25°C, 27.5°C, 30°C and 38.5°C) for 1 day, 2 days or 3 days. After preservation, oocyte morphology, germinal vesicle (GV) rate, actin cytoskeleton organization, cortical granule distribution, mitochondrial translocation and intracellular glutathione level were evaluated. Oocyte maturation was indicated by first polar body emission and spindle morphology after in vitro culture. Strikingly, when COCs were stored at 27.5°C for 3 days in pFF or FCS, more than 60% oocytes were still arrested at the GV stage and more than 50% oocytes matured into MII stages after culture. Almost 80% oocytes showed normal actin organization and cortical granule relocation to the cortex, and approximately 50% oocytes showed diffused mitochondria distribution patterns and normal spindle configurations. While stored in TCM-199, all these criteria decreased significantly. Glutathione (GSH) level in the pFF or FCS group was higher than in the TCM-199 group, but lower than in the non-preserved control group. The preserved oocytes could be fertilized and developed to blastocysts (about 10%) with normal cell number, which is clear evidence for their retaining the developmental potentiality after 3d preservation. Thus, we have developed a simple method for preserving immature pig oocytes at an ambient temperature for several days without evident damage of cytoplasm and keeping oocyte developmental competence.

Apoptosis in mouse fetal and neonatal oocytes during meiotic prophase one

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Hartshorne Geraldine M

2007-07-01

Full Text Available Abstract Background The vast majority of oocytes formed in the fetal ovary do not survive beyond birth. Possible reasons for their loss include the elimination of non-viable genetic constitutions arising through meiosis, however, the precise relationship between meiotic stages and prenatal apoptosis of oocytes remains elusive. We studied oocytes in mouse fetal and neonatal ovaries, 14.5–21 days post coitum, to examine the relationship between oocyte development and programmed cell death during meiotic prophase I. Results Microspreads of fetal and neonatal ovarian cells underwent immunocytochemistry for meiosis- and apoptosis-related markers. COR-1 (meiosis-specific highlighted axial elements of the synaptonemal complex and allowed definitive identification of the stages of meiotic prophase I. Labelling for cleaved poly-(ADP-ribose polymerase (PARP-1, an inactivated DNA repair protein, indicated apoptosis. The same oocytes were then labelled for DNA double strand breaks (DSBs using TUNEL. 1960 oocytes produced analysable results. Oocytes at all stages of meiotic prophase I stained for cleaved PARP-1 and/or TUNEL, or neither. Oocytes with fragmented (19.8% or compressed (21.2% axial elements showed slight but significant differences in staining for cleaved PARP-1 and TUNEL to those with intact elements. However, fragmentation of axial elements alone was not a good indicator of cell demise. Cleaved PARP-1 and TUNEL staining were not necessarily coincident, showing that TUNEL is not a reliable marker of apoptosis in oocytes. Conclusion Our data indicate that apoptosis can occur throughout meiotic prophase I in mouse fetal and early postnatal oocytes, with greatest incidence at the diplotene stage. Careful selection of appropriate markers for oocyte apoptosis is essential.

Frequency of aneuploidy related to age in porcine oocytes.

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Miroslav Hornak

Full Text Available 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.

First attempts to cryopreserve red abalone (Haliotis rufescens oocytes

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Ramírez Torrez, A.

2015-01-01

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.

Spectroscopic study of gamma irradiated bovine hemoglobin

International Nuclear Information System (INIS)

Maghraby, Ahmed Mohamed; Ali, Maha Anwar

2007-01-01

In the present study, the effects of ionizing radiation of Cs-137 and Co-60 from 4.95 to 743.14 Gy and from 40 Gy to 300 kGy, respectively, on some bovine hemoglobin characteristics were studied. Such an effect was evaluated using electron paramagnetic resonance (EPR) spectroscopy, and infra-red (IR) spectroscopy. Bovine hemoglobin EPR spectra were recorded and analyzed before and after irradiation and changes were explained in detail. IR spectra of unirradiated and irradiated Bovine hemoglobin were recorded and analyzed also. It was found that ionizing radiation may lead to the increase of free radicals production, the decrease in α-helices contents, which reflects the degradation of hemoglobin molecular structure, or at least its incomplete performance. Results also show that the combined application of EPR and FTIR spectroscopy is a powerful tool for determining structural modification of bovine hemoglobin samples exposed to gamma irradiation

Intraovarian markers of follicular and oocyte maturation.

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Pellicer, A; Diamond, M P; DeCherney, A H; Naftolin, F

1987-08-01

The use of ovulation induction for multiple follicular growth in in vitro fertilization (IVF) has introduced the problem of follicular asynchrony. As a consequence of the asynchrony, the parameters most commonly used by IVF groups to assess follicular and oocyte quality within those follicles are not sufficiently sensitive or specific. Thus, each follicle must be considered separately, and specific markers of follicular and/or oocyte maturation must be sought from within the follicle. In this review we analyze previous reports of potential markers of follicular and oocyte maturation. In regards to the follicular fluid constituents, the level of estradiol in follicular fluid correlates with fertilization and pregnancy in stimulated cycles. Other steroids are only helpful when specific stimulation protocols are used. The level of some follicular proteins such as alpha-1-antitrypsin and fibrinogen also correlates with fertilization and pregnancy outcome. Cyclic AMP levels in follicular fluid are significantly reduced in follicles leading to conception. Regulators of oocyte maturation, such as the Oocyte Maturation Inhibitor (OMI) or the Meiosis Inducing Substance (MIS) have also been correlated with IVF outcome, but their exact structure remains still unknown. In addition, other sophisticated parameters, such as chemotactic activity of human leukocytes, or simple methods, such as the presence of intrafollicular echoes, have also been used as successful markers in predicting IVF outcome.

First delivery of healthy offspring after freezing and thawing of oocytes in Switzerland.

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De Geyter, Maria; Steimann, Sabine; Holzgreve, Wolfgang; De Geyter, Christian

2007-08-11

The interest in long-term storage of uninseminated oocytes through cryopreservation has seen a recent upsurge, because it provides the potential to assist young women to postpone childbirth after having overcome a malignant disease or delaying childbirth until after management of a professional career. The low fertilisation rate of frozen/thawed oocytes in earlier feasibility trials can now be improved by using intracytoplasmic sperm injection (ICSI) for assisting the penetration of the spermatozoon through the oocyte's hardened zona pellucida. Another reason for the reported low success rates of oocyte cryopreservation in earlier studies may have been the low developmental potential of spare oocytes, which were available for experimental cryopreservation. Oocytes retrieved from supernumerary follicles in women treated with gonadotropins for ovulation induction and intrauterine insemination can be used for the optimisation of cryostorage of uninseminated oocytes. We intended to investigate to what extent the well-established and successful cryopreservation protocols for pronucleate oocytes are also applicable for the cryopreservation of uninseminated oocytes. We herewith report the first successful pregnancy and delivery of frozen/thawed oocytes in Switzerland, which were inseminated with ICSI. In unbiased treatment groups the freezing and thawing of uninseminated oocytes and pronucleate oocytes give comparable results, if the additional manipulation during ICSI was taken into account.

Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics

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Massobrio Marco

2009-05-01

Full Text Available Abstract The assessment of oocyte quality in human in vitro fertilization (IVF is getting increasing attention from embryologists. Oocyte selection and the identification of the best oocytes, in fact, would help to limit embryo overproduction and to improve the results of oocyte cryostorage programs. Follicular fluid (FF is easily available during oocyte pick-up and theorically represents an optimal source on non-invasive biochemical predictors of oocyte quality. Unfortunately, however, the studies aiming to find a good molecular predictor of oocyte quality in FF were not able to identify substances that could be used as reliable markers of oocyte competence to fertilization, embryo development and pregnancy. In the last years, a well definite trend toward passing from the research of single molecular markers to more complex techniques that study all metabolites of FF has been observed. The metabolomic approach is a powerful tool to study biochemical predictors of oocyte quality in FF, but its application in this area is still at the beginning. This review provides an overview of the current knowledge about the biochemical predictors of oocyte quality in FF, describing both the results coming from studies on single biochemical markers and those deriving from the most recent studies of metabolomics

Microstructure and hardness of bovine enamel in roselle extract solution

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Dame, M. T.; Noerdin, A.; Indrani, D. J.

2017-08-01

The aim of this study was to analyze the effect of roselle extract solution on the microstructure and hardness of bovine enamel. Ten bovine teeth and a 5% concentration of roselle extract solution were prepared. Immersions of each bovine tooth in roselle extract solution were conducted up to 60 minutes. The bovine enamel surface was characterized in hardness and microscopy. It was apparent that the initial hardness was 328 KHN, and after immersion in 15 and 60 min, the values decrease to 57.4 KHN and 11 KHN, respectively. Scanning electron microscopy (SEM) revealed changes in enamel rods after immersion in the roselle extract solution.

Kit ligand promotes first polar body extrusion of mouse preovulatory oocytes

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Ye Yinghui

2009-04-01

Full Text Available Abstract Background Shortly after stimulation by the preovulatory surge of luteinizing hormone (LH, oocytes arrested at the late prophase I resume meiosis characterized by germinal vesicle breakdown (GVBD, chromosome condensation, and extrusion of the first polar body in preparation for fertilization and early embryonic development. However, oocytes express few or no LH receptors and are insensitive to direct LH stimulation. Thus, factors released by granulosa or theca cells expect to convey the LH stimuli to oocytes. To identify candidate ligand-receptor pairs potentially involved in the process of oocyte maturation, we performed DNA microarray analyses of ovarian transcripts in mice and identified Kit ligand (Kitl as an ovarian factor stimulated by the LH/hCG surge. The purpose of this study is to investigate the roles of KITL in the nuclear and cytoplasmic maturation of preovulatory mouse oocytes. Methods The levels of Kitl and c-kit transcripts in mouse ovaries and isolated ovarian cells were determined by real-time RT-PCR, while expression of KITL protein was examined by immunohistochemistry. Follicle culture, cumulus-oocyte complexes (COC and denuded oocytes culture were used to evaluate the effect of KITL on mouse oocyte nuclear maturation. To assess the effect of KITL treatment on the cytoplasmic maturation of preovulatory oocytes, we performed in vitro maturation of oocytes followed by in vitro fertilization. Results Major increase of Kitl transcripts in granulosa cells and mouse ovaries, and predominant expression of c-kit in preovulatory oocytes were identified by real-time RT-PCR. Predominant expression of KITL protein was found in granulosa cells of preovulatory and small antral follicles at 4 h after hCG treatment. In vitro cultures demonstrated that treatment with KITL enhanced first polar body extrusion in a dose-dependent manner. Moreover, treatment of COC with KITL enhanced first polar body extrusion with increase in cyclin B1

In vitro maturation of human oocytes for assisted reproduction.

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Jurema, Marcus W; Nogueira, Daniela

2006-11-01

To describe and evaluate the current practice of in vitro maturation of oocytes for assisted reproduction. Review of the available and relevant literature regarding in vitro maturation of oocytes. In vitro maturation of human oocytes retrieved from antral ovarian follicles is an emerging procedure quickly being incorporated into the realm of assisted reproductive technologies. This new technology has several potential advantages over traditional controlled ovarian hyperstimulation for IVF, such as reduction of costs by minimizing gonadotropin and GnRH analogue use, elimination of ovarian hyperstimulation syndrome, and simplicity of protocol. In vitro maturation of oocytes for assisted reproduction in human beings still is undergoing refinement but currently is providing efficacy and safety outcome comparable to that of traditional IVF in recent selected studies. Implementing in vitro maturation into an established IVF practice is feasible and requires only a few simple adjustments. Crucial to the advancement and optimization of the technology is a better understanding of how to maximize immature oocyte developmental competence and endometrial receptivity.

PREVALENCE OF BOVINE HERPESVIRUS-1,PARAINFLUENZA-3,BOVINE ROTAVIRUS, BOVINE VIRAL DIARRHEA, BOVINE ADENOVIRUS-7,BOVINE LEUKEMIA VIRUS AND BLUETONGUE VIRUS ANTIBODIES IN CATTLE IN MEXICO

OpenAIRE

SUZAN, Victor M.; ONUMA, Misao; AGUILAR, Romero E.; MURAKAMI, Yosuke

1983-01-01

Sera were collected from dairy and beef cattle in 19 different states of Mexico. These sera were tested for bovine herpesvirus-1 (BHV-1), parainfluenza-3 virus (PIV-3), bovine rotavirus (BRV), bovine leukemia virus (BLV), bovine adenovirus-7 (BAV-7), bluetongue virus (BTV) and bovine viral diarrhea virus (BVDV). Seropositive rates for each virus for dairy cattle tested were 158/277(57.0%) for BHV-1,217/286(75.0%) for PIV-3,541/1498(36.1%) for BLV, 134/144(93.1%) for BRV, 39/90(43.3%) for BTV,...

Gene expression and maturation evaluation of sheep oocytes ...

African Journals Online (AJOL)

associated X protein (Bax) of matured sheep oocytes. To carry out this study, cumulus-oocyte complexes (COCs) aspirated from sheep ovaries were cultured in TCM-199 medium supplemented with various concentrations of FSE (0, 1 and 10 μg/mL).

Effect of cumulus-oocyte complexes (COCs) culture duration on in ...

African Journals Online (AJOL)

We investigated and optimized the cumulus-oocyte complexes (COCs) culture duration for pig oocyte in vitro maturation and produced a number of high-quality metaphase-II (M-II) oocytes for generation of parthenotes. The present study graded the COCs into levels A, B and C according to layers of cumulus cells, which ...

Characterization of oocyte retrieval cycles with empty zona pellucida.

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Oride, Aki; Kanasaki, Haruhiko; Hara, Tomomi; Ohta, Hiroko; Kyo, Satoru

2018-01-01

To identify the factors that characterize cycles with empty zona pellucida (EZP). Thirty-six oocyte retrieval cycles from which EZP were collected and another 36 cycles from which no EZP was collected were compared. The patients were divided into three groups: those with no EZP collected during any cycle, those with EZP collected during all cycles, and those experiencing cycles both with and without EZP. The mean number of oocytes collected per cycle was higher in the cycles with EZP than without EZP. The fertilization rate of the collected oocytes and the rate of good embryo formation were significantly lower in the cycles with EZP. No significant difference was observed between the three groups in terms of age, number of oocytes collected, or hormone levels before and after the oocyte retrieval. The fertilization and pregnancy rates were highest in the patients with no EZP being collected during any cycle, followed by those experiencing cycles both with and without EZP, and then by those with EZP collected during all cycles. The observation of lower fertilization, poor embryo formation, and a low pregnancy rate in the patients with EZP suggests the poor quality of oocytes that were collected with EZP in the same cycle.

The signaling pathways by which the Fas/FasL system accelerates oocyte aging.

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Zhu, Jiang; Lin, Fei-Hu; Zhang, Jie; Lin, Juan; Li, Hong; Li, You-Wei; Tan, Xiu-Wen; Tan, Jing-He

2016-02-01

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.

Effect of gonadotropins on oocyte maturation in vitro: an animal model.

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Sha, Wei; Xu, Bao-Zeng; Li, Mo; Liu, Di; Feng, Huai L; Sun, Qing-Yuan

2010-03-15

Analysis of the effects of human-derived gonadotropin drugs, FSH and LH (Repronex) and hCG (Novarel), on oocyte maturation, using a porcine oocyte in vitro maturation system as a culture model. Randomized research experimental study. Academic basic research laboratory. Prepubertal gilts that were slaughtered in the local slaughter house. Oocytes will be exposed to immunofluorescent staining and confocal laser scanning microscopy: Western blot analysis on cumulus-oocyte-complexes following treatment with different concentrations of the gonadotropin drugs Repronex, Novarel, and a Repronex and Novarel combination. Analysis of porcine oocyte spindle and chromosomal configuration with alpha-tubulin-fluorescein isothiocyanate antibody and propidium iodide staining. Porcine oocyte mitochondrial distribution and aggregation pattern staining was assessed with Mito Tracker Red CMXRox probe. Porcine oocyte cortical granule distribution was observed via peanut agglutinin-fluorescein isothiocyannate staining; Western blot analysis detected extra-cellular signal-regulated kinase 1/2 activation in cumulus cells. An increase of gonadotropin concentration in the culture medium resulted in an increase in the following: the percentage of oocytes reaching metaphase II, normal configuration of the spindle, normal chromosomal alignment, cortical granule migration, and mitochondrial aggregation. Levels of nuclear and cytoplasmic maturation peaked as the concentration of gonadotropins approached its threshold level. Addition of a threshold concentration of the gonadotropin drugs Repronex, Novarel, and a combination of the two can significantly improve porcine oocyte maturation in vitro. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Cytoskeleton and Cytoskeleton-Bound RNA Visualization in Frog and Insect Oocytes.

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Kloc, Malgorzata; Bilinski, Szczepan; Kubiak, Jacek Z

2016-01-01

The majority of oocyte functions involves and depends on the cytoskeletal elements, which include microtubules and actin and cytokeratin filaments. Various structures and molecules are temporarily or permanently bound to the cytoskeletal elements and their functions rely on cytoskeleton integrity and its timely assembly. Thus the accurate visualization of cytoskeleton is often crucial for studies and analyses of oocyte structure and functions. Here we describe several reliable methods for microtubule and/or microfilaments preservation and visualization in Xenopus oocyte extracts, and in situ in live and fixed insect and frog (Xenopus) oocytes. In addition, we describe visualization of cytoskeleton-bound RNAs using molecular beacons in live Xenopus oocytes.

Consequences of metaphase II oocyte cryopreservation on mRNA content.

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Chamayou, S; Bonaventura, G; Alecci, C; Tibullo, D; Di Raimondo, F; Guglielmino, A; Barcellona, M L

2011-04-01

We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte. Copyright © 2011 Elsevier Inc. All rights reserved.

Nicotine supplementation blocks oocyte maturation in Rattus norvegicus

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Meitria Syahadatina Noor

2013-08-01

Full Text Available Background Indonesia has the third largest tobacco consumption in the world after China and India. Nicotine as the main component of cigarette smoke has negative effects on the reproductive system, such as oocyte maturation, ovulation, and fertilization, and increasing the diploidy of oocytes. The goal of this research was to evaluate the effect of nicotine on oocyte maturation in Rattus norvegicus. Methods This was an experimental study with post test only control group design. The subjects were 40 rats selected homogenously and randomly. They were divided into a control group (receiving carboxy-methyl-cellulose sodium and 3 treatment groups (I-III receiving nicotine subcutaneously for 7 days at dosages of 21 mg/kgBW, 41 kg/kgBW and 84/kgBW, respectively. The observations comprised oocyte maturation stage, viz. germinal vesicle (GV, germinal vesicle breakdown (GVBD, metaphase I and metaphase II. Data were analyzed by one-way Anova with á=0.05, followed by Tukey’s HSD test. Results One-way Anova showed significant differences in oocyte maturation in all groups. Tukey’s HSD test showed that for GV, the differing groups were control and I, control and II, I and III. For GVBD, the differing groups were control and I, I and II, I and III. For metaphase I, the differing groups were control with I, II, and III, I and II, I and III. For metaphase II, the differing groups were control versus I, II, and III, I and II, I and III. Conclusion Low dose of nicotine is capable of affecting oocyte maturation in Rattus norvegicus.

Mature Oocyte Cryopreservation for Fertility Preservation.

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Liang, Tina; Motan, Tarek

2016-01-01

In recent decades, advances in cancer treatment have led to a dramatic improvement in long term survival. This has led to an increasing focus on quality of life after surviving cancer treatment, with fertility being an important aspect. Given the known reproductive risks of cancer therapies, there has been a growing interest in the field of fertility preservation (also referred to as oncofertility). Mature oocyte cryopreservation is no longer considered experimental and has become a realistic option for reproductive aged women prior to undergoing cancer treatment. Additionally, as cryopreservation techniques improve, mature oocyte cryopreservation is increasing being marketed to healthy women without cancer wishing to delay child bearing, also termed "social egg freezing". This chapter provides a review of the current technology, use, and outcomes of mature oocyte cryopreservation. It also outlines the ethical debate surrounding social egg freezing and directions for future research in female fertility preservation.

Intact fetal ovarian cord formation promotes mouse oocyte survival and development

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Pera Renee

2010-01-01

Full Text Available Abstract Background Female reproductive potential, or the ability to propagate life, is limited in mammals with the majority of oocytes lost before birth. In mice, surviving perinatal oocytes are enclosed in ovarian follicles for subsequent oocyte development and function in the adult. Before birth, fetal germ cells of both sexes develop in clusters, or germline cysts, in the undifferentiated gonad. Upon sex determination of the fetal gonad, germ cell cysts become organized into testicular or ovarian cord-like structures and begin to interact with gonadal somatic cells. Although germline cysts and testicular cords are required for spermatogenesis, the role of cyst and ovarian cord formation in mammalian oocyte development and female fertility has not been determined. Results Here, we examine whether intact fetal ovarian germ and somatic cell cord structures are required for oocyte development using mouse gonad re-aggregation and transplantation to disrupt gonadal organization. We observed that germ cells from disrupted female gonad prior to embryonic day e13.5 completed prophase I of meiosis but did not survive following transplantation. Furthermore, re-aggregated ovaries from e13.5 to e15.5 developed with a reduced number of oocytes. Oocyte loss occurred before follicle formation and was associated with an absence of ovarian cord structure and ovary disorganization. However, disrupted ovaries from e16.5 or later were resistant to the re-aggregation impairment and supported robust oocyte survival and development in follicles. Conclusions Thus, we demonstrate a critical window of oocyte development from e13.5 to e16.5 in the intact fetal mouse ovary, corresponding to the establishment of ovarian cord structure, which promotes oocyte interaction with neighboring ovarian somatic granulosa cells before birth and imparts oocytes with competence to survive and develop in follicles. Because germline cyst and ovarian cord structures are conserved in the

RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study

Energy Technology Data Exchange (ETDEWEB)

Motlik, J. (Institute of Animal Physiology and Genetics, Czechoslovak Academy of Sciences, CS, Libechov); Kopecny, V.; Travnik, P. (Faculty of Medecine, J.E. Purkyne University, Brno (Czechoslovakia)); Pivko, J. (Animal Production Research Institute, Nitra (Czechoslovakia))

1984-01-01

RNA synthesis in pig oocytes was studied using autoradiography and silver staining of the nucleolus organizing region. Both methods confirmed that oocytes from the smallest follicles (0.5-0.7 mm in diam.) very intensely synthesize nuclear and nucleolar RNA. The nucleolar area of oocytes originating from follicles of 1.6-2.2 mm in diam. was labelled mainly on its periphery. After short pulse labelling (15 min) of oocytes from follicles of 5-6 mm in diam. only the nucleoplasm was labelled. The nucleolus had no significant labelling. The possibility that labelling of the compact nucleolus after a longer pulse represents migration of the newly synthesized nuclear RNA into the compact nucleolus, is discussed. The quantity of silver-positive material in dictyate oocytes significantly decreased as pig follicles enlarged in diam. from 2 mm to 5-6 mm.

RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study

International Nuclear Information System (INIS)

Motlik, J.; Pivko, J.

1984-01-01

RNA synthesis in pig oocytes was studied using autoradiography and silver staining of the nucleolus organizing region. Both methods confirmed that oocytes from the smallest follicles (0.5-0.7 mm in diam.) very intensely synthesize nuclear and nucleolar RNA. The nucleolar area of oocytes originating from follicles of 1.6-2.2 mm in diam. was labelled mainly on its periphery. After short pulse labelling (15 min) of oocytes from follicles of 5-6 mm in diam. only the nucleoplasm was labelled. The nucleolus had no significant labelling. The possibility that labelling of the compact nucleolus after a longer pulse represents migration of the newly synthesized nuclear RNA into the compact nucleolus, is discussed. The quantity of silver-positive material in dictyate oocytes significantly decreased as pig follicles enlarged in diam. from 2 mm to 5-6 mm

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

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

2014-11-01

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.

Maturation of pig oocytes in vitro in a medium with pyruvate

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H. Gonzales-Figueroa

2005-06-01

Full Text Available The aim of in vitro maturation oocyte systems is to produce oocytes of comparable quality to those derived in vivo. The present study was designed to examine the surface morphological changes of the cumulus-oocyte complex (COC and nuclear maturation in a culture system containing pyruvate. Ovaries were obtained from a slaughterhouseand transported to the laboratory within 2 h at 35-39ºC,and rinsed three times in 0.9% NaCl. The COCs were harvested from the ovaries and in vitro maturation was evaluated in San Marcos (SM medium, a chemically defined culture system containing 22.3 mM sodium pyruvate. Oocytes were cultured in SM, SM + porcine follicular fluid (pFF and in SM + pFF + gonadotropins (eCG and hCG for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Oocytes matured in SM (40.9% and SM + pFF (42.9% showed moderate cumulus expansion, whereas oocytes matured in SM + pFF + gonadotropins (54.6% showed high cumulus expansion. The maturation rate of cultured oocytes, measured in function of the presence of the polar corpuscle, did not differ significantly between SM (40.9 ± 3.6% and SM + pFF (42.9 ± 3.7%. These results indicate that pig oocytes can be successfully matured in a chemically definedmedium and suggest a possible bifunctional role of pyruvate as an energy substrate and as an antioxidant protecting oocytes against the stress of the in vitro environment.

Factors affecting the outcome of in vitro bovine embryo production using ovum pick-up-derived cumulus oocyte complexes

NARCIS (Netherlands)

Merton, J.S.

2013-01-01

Optimization of bovine ovum pick up (OPU) followed by in vitro embryo production (IVP) has been driven by the desire of both beef and dairy cattle breeders to enhance genetic improvement. The work presented in this thesis focuses on optimizing the efficiency and efficacy of the OPU-IVP program.

Embryo apoptosis identification: Oocyte grade or cleavage stage?

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Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

2015-01-01

Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

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

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Valeriu Carabă

2011-05-01

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.

Influence of Meiotic Stages on Developmental Competence of Goat’ Oocyte After Vitrification

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Wahyuningsih, S.; Ihsan, M. N.

2018-02-01

This objective of this research was to investigate effect of goat oocyte meiotic stages on developmental competence after cryopreservation. Ovaries were collected from slaugterhouse and oocytes was aspirated from2-6 mm of follicles. Oocyte with compacted cumulus cells and evenly granulated ooplasm were selected for this experiment. The lenght of in vitro maturation before vitrification was 8 or 22 h in IVM media TCM 199 + FCS 10 % + PMSG 10 IU + hCG 10 IU at 38.5 °C in a humidified atmosphere of 5 % CO2 in air and were vitrified. After vitrification process, GVBD and MII oocyte were matured for 18 or 4 h to fullfill 26 h maturation requirement and then oocytes were subjected to IVF and culture. Cleavage and blastocyst formation rate were to asses their developmental competence. Cleavage rates were obtained for both GVBD ( 56.78 %) and MII (69.64 % ) oocytes (PGoat oocytes in different maturation stages response to vitrification differently and MII stages have better developmental competence than GVBD.

In vitro production of buffalo embryos from stepwise vitrified immature oocytes.

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Abd-Allah, Saber Mohammed

2009-01-01

This study was conducted to produce buffalo embryos in vitro from stepwise vitrified immature oocytes. Cumulus oocyte complexes (COCs) were obtained from the ovaries of slaughtered buffalo and were collected from the local abattoir. Selected COCs were exposed to a vitrification solution consisting of 40% ethylene glycol (EG) plus 0.3 M trehalose and 20% polyvinyl pyrrolidone (PVP) for 1 min and loaded in 0.25 ml plastic mini-straws containing 100 microl of 10% sucrose. The loaded cryostraws were cryopreserved by stepwise vitrification and were stored in liquid nitrogen for 4 to 6 months. Data analysis revealed a high percentage of post-thawing morphologically normal immature oocytes (80.7%) with a low percentage of damaged oocytes. There were no significant differences in the maturation (82.1%), cleavage (47.6%) and buffalo embryo development (15.4%) produced by the stepwise vitrified immature oocytes in comparison to the three observations in fresh oocytes (88.3%, 50.4% and 19.4%, respectively, p<0.05).

In vitro production of buffalo embryos from stepwise vitrified immature oocytes

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Saber Mohammed Abd-Allah

2009-09-01

Full Text Available This study was conducted to produce buffalo embryos in vitro from stepwise vitrified immature oocytes. Cumulus oocyte complexes (COCs were obtained from the ovaries of slaughtered buffalo and were collected from the local abattoir. Selected COCs were exposed to a vitrification solution consisting of 40% ethylene glycol (EG plus 0.3 M trehalose and 20% polyvinyl pyrrolidone (PVP for 1 min and loaded in 0.25 ml plastic mini-straws containing 100 µl of 10% sucrose. The loaded cryostraws were cryopreserved by stepwise vitrification and were stored in liquid nitrogen for 4 to 6 months. Data analysis revealed a high percentage of post-thawing morphologically normal immature oocytes (80.7% with a low percentage of damaged oocytes. There were no significant differences in the maturation (82.1%, cleavage (47.6% and buffalo embryo development (15.4% produced by the stepwise vitrified immature oocytes in comparison to the three observations in fresh oocytes (88.3%, 50.4% and 19.4%, respectively, p<0.05.

Meiotic recombination in human oocytes.

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Edith Y Cheng

2009-09-01

Full Text Available Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1 in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of "vulnerable" crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.

RNA-Seq analysis uncovers transcriptomic variations between morphologically similar in vivo- and in vitro-derived bovine blastocysts

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Driver Ashley M

2012-03-01

Full Text Available Abstract Background A valuable tool for both research and industry, in vitro fertilization (IVF has applications range from gamete selection and preservation of traits to cloning. Although IVF has achieved worldwide use, with approximately 339,685 bovine embryos transferred in 2010 alone, there are still continuing difficulties with efficiency. It is rare to have more than 40% of fertilized in vitro cattle oocytes reach blastocyst stage by day 8 of culture, and pregnancy rates are reported as less than 45% for in vitro produced embryos. To investigate potential influences in-vitro fertilization (IVF has on embryonic development, this study compares in vivo- and in vitro-derived bovine blastocysts at a similar stage and quality grade (expanded, excellent quality to determine the degree of transcriptomic variation beyond morphology using RNA-Seq. Results A total of 26,906,451 and 38,184,547 fragments were sequenced for in vitro and in vivo embryo pools, respectively. We detected expression for a total of 17,634 genes, with 793 genes showing differential expression between the two embryo populations with false discovery rate (FDR Conclusions Thus, our results support that IVF may influence at the transcriptomic level and that morphology is limited in full characterization of bovine preimplantation embryos.

Melatonin accelerates maturation inducing hormone (MIH): induced oocyte maturation in carps.

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Chattoraj, Asamanja; Bhattacharyya, Sharmistha; Basu, Dipanjan; Bhattacharya, Shelley; Bhattacharya, Samir; Maitra, Saumen Kumar

2005-02-01

The present communication is an attempt to demonstrate the influence of melatonin on the action of maturation inducing hormone (MIH) on the maturation of oocytes in carps. The oocytes from gravid female major carp Labeo rohita were isolated and incubated separately in Medium 199 containing (a) only MIH (1 microg/ml), (b) only melatonin (at concentrations of 50, 100 or 500 pg/ml), and (c) both melatonin and MIH, but at different time intervals. In the latter group, melatonin was added to the incubating medium either (i) 4 h before addition of MIH, (ii) 2 h before addition of MIH, (iii) co-administered with MIH (0 h interval) or (iv) 2 h after addition of MIH. In each case, oocytes were further incubated for 4, 8, 12 or 16 h post- administration of MIH, and the effects of treatment on oocyte maturation were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). Incubation of oocytes in a medium containing only melatonin did not result in GVBD of any oocyte. Nearly all the oocytes underwent GVBD when incubated with MIH for 16 h. Administration of melatonin along with MIH (at 0 h interval) or 2 h after addition of MIH did not result in any significant change in the rate of GVBD compared to that in a medium containing only MIH. However, it was quite interesting to observe that incubation of oocytes with melatonin especially 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD in the oocytes. Experiments with the oocytes of another major carp Cyprinus carpio following an identical schedule depicted similar results except a difference in the optimum melatonin dose. In L. rohita, 50 pg/ml melatonin had maximum acceleratory effect on MIH-induced GVBD of oocytes, while it was 100 pg/ml in C. carpio. Further study revealed that pre-incubation with melatonin accelerates the action of MIH on the formation of a complex of two proteins (MPF), a regulatory component called cyclin B and the catalytic component protein kinase known as

Oocyte vitrification as an efficient option for elective fertility preservation.

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Cobo, Ana; García-Velasco, Juan A; Coello, Aila; Domingo, Javier; Pellicer, Antonio; Remohí, José

2016-03-01

To provide a detailed description of the current oocyte vitrification status as a means of elective fertility preservation (EFP). Retrospective observational multicenter study. Private university-affiliated center. A total of 1,468 women who underwent EFP because of age or having associated a medical condition other than cancer (January 2007 to April 2015). None. Survival and cumulative live birth rate (CLBR) per consumed oocyte. Mean age was higher with EFP due to age versus having an associated medical reason (37.7 y [95% confidence interval (CI) 36.5-37.9] vs. 35.7 y [95% CI 34.9-36.3]). In total, 137 patients (9.3%) returned to use their oocytes. Overall survival rate was 85.2% (95% CI 83.2-87.2). Live birth rate per patient was higher in women ≤35 years old than ≥36 years old (50% [95% CI 32.7-67.3] vs. 22.9% [95% CI 14.9-30.9]). CLBR was higher and increased faster in younger women. The gain in CLBR was sharp from 5 (15.4%, 95% CI -4.2 to 35.0) to 8 oocytes (40.8%, 95% CI 13.2-68.4), with an 8.4% gain per additional oocyte, in the ≤35-year-old group. The increase was slower with 10-15 oocytes, reaching a plateau CLBR of 85.2%. A milder increase (4.9% gain) was observed in the ≥36-year-old group (from 5.1% [95% CI -0.6 to 10.7] to 19.9% [95% CI 8.7-31.1] when 5-8 oocytes were consumed), reaching the plateau with 11 oocytes (CLBR 35.6%). Forty babies were born. At least 8-10 metaphase II oocytes are necessary to achieve reasonable success. Numbers should be individualized in women >36 years old. We suggest encouraging women who are motivated exclusively by a desire to postpone childbearing because of age, to come at younger ages to increase success possibilities. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Artificial intelligence techniques for embryo and oocyte classification.

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Manna, Claudio; Nanni, Loris; Lumini, Alessandra; Pappalardo, Sebastiana

2013-01-01

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

Vitrification of human germinal vesicle oocytes; before or after in vitro maturation?

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Evangelia Kasapi

2017-03-01

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

RESULTS OF IN VITRO MATURATION OF MEIOTICALLY IMMATURE HUMAN OOCYTES IN A SIMPLE MEDIUM

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Borut Kovačič

2002-12-01

Full Text Available Background. Among oocytes obtained during aspiration of preovulatory ovarian follicles in hormonally stimulated cycles, we ascertained the percentage of immature oocytes with the nucleus in the metaphase (M I oocytes or even in the prophase (GV oocytes of the first meiotic division and their capacity to mature in vitro in a simple medium without hormonal supplements.Methods. In 818 women, stimulated by gonadotropin releasing hormone agonist (GnRHa and gonadotropins, aspiration of preovulatory size follicles yielded 4972 oocytes. From these we denuded cells of cumulus oophorus and corona, meiotic maturity was evaluated under a microscope. Cells in the metaphase of the second meiotic division (M II oocytes and those maturing after 5 hours were used clinically in the intracytoplasmic sperm injection (ICSI procedure. Immature cells were left in the simple medium. The degree of their nuclear maturity was evaluated after one and after two days of culture. In vitro maturation was clinically used also in 14 cycles with no mature oocytes.Results. Among 4731 oocytes with denuded corona and cumulus, 4199 (88.8% were mature M II oocytes, 295 (6.2% immature M I oocytes and 237 (5% immature GV oocytes. Under in vitro conditions, 68.7% (90/131 GV oocytes attained maturity. Among M I oocytes, 63.6% (136/214 cells matured already after 5 hours and 26.6% (57/214 until the next day. In all 14 women with only immature oocytes, the embryos for embryotransfer were obtained after in vitro maturation and ICSI procedure. The result was four pregnancies and two deliveries.Conclusions. Immature oocytes, obtained in hormonally stimulated cycles, may become clinically applicable if left to mature in vitro in a simple medium without supplementation of growth factors and hormones.

Oocyte cryopreservation beyond cancer: tools for ethical reflection.

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Linkeviciute, Alma; Peccatori, Fedro A; Sanchini, Virginia; Boniolo, Giovanni

2015-08-01

This article offers physicians a tool for structured ethical reflection on challenging situations surrounding oocyte cryopreservation in young healthy women. A systematic literature review offers a comprehensive overview of the ethical debate surrounding the practice. Ethical Counseling Methodology (ECM) offers a practical approach for addressing ethical uncertainties. ECM consists of seven steps: (i) case presentation; (ii) analysis of possible implications; (iii) presentation of ethical question(s); (iv) explanation of ethical terms; (v) presentation of the ethical arguments in favor of and against the procedure; (vi) examination of the individual patient's beliefs and wishes; and (vii) conclusive summary. The most problematic aspects in the ethical debate include the distinction between medical and non-medical use of oocyte cryopreservation, safety and efficiency of the procedure, and marketing practices aimed at healthy women. Female empowerment and enhanced reproductive choices (granted oocyte cryopreservation is a safe and efficient technique) are presented as ethical arguments supporting the practice, while ethical reservations towards oocyte cryopreservation are based on concerns about maternal and fetal safety and wider societal implications. Oocyte cryopreservation is gaining popularity among healthy reproductive age women. However, despite promised benefits it also involves risks that are not always properly communicated in commercialized settings. ECM offers clinicians a tool for structured ethical analysis taking into consideration a wide range of implications, various ethical standpoints, and patients' perceptions and beliefs.

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.

Vitrification versus slow freezing for women undergoing oocyte cryopreservation

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Glujovsky, Demián; Riestra, Barbara; Sueldo, Carlos; Fiszbajn, Gabriel; Repping, Sjoerd; Nodar, Florencia; Papier, Sergio; Ciapponi, Agustín

2014-01-01

Oocyte cryopreservation is a technique with considerable potential in reproductive medicine, including fertility preservation, as a way of delaying childbearing and as part of oocyte donation programs. Although the technique was relatively ineffective at first more recently numerous modifications

The Relationship Between Transcript Expression Levels of Nuclear Encoded (TFAM, NRF1 and Mitochondrial Encoded (MT-CO1 Genes in Single Human Oocytes During Oocyte Maturation

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Ghaffari Novin M.

2015-06-01

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.

Reproductive experiences of women who cryopreserved oocytes for non-medical reasons.

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Hammarberg, Karin; Kirkman, Maggie; Pritchard, Natasha; Hickey, Martha; Peate, Michelle; McBain, John; Agresta, Franca; Bayly, Chris; Fisher, Jane

2017-03-01

What are the reproductive experiences of women who cryopreserve oocytes for non-medical reasons? One in three women had been pregnant at some stage in their lives and while most still wanted to have a child or another child, very few had used their stored oocytes, predominantly because they did not want to be single parents. The number of healthy women who freeze oocytes to avoid age-related infertility is increasing. Evidence about reproductive outcomes after oocyte cryopreservation for non-medical reasons is needed to help women make informed decisions. A cross-sectional survey was carried out. Study packs which included a self-administered questionnaire were mailed by clinic staff to 193 eligible women. Women who had stored oocytes for non-medical reasons at Melbourne IVF, a private ART clinic, between 1999 and 2014 were identified from medical records and invited to complete an anonymous questionnaire about their reproductive histories and experience of oocyte cryopreservation. A total of 10 survey packs were returned to the clinic marked 'address unknown'. Of the 183 potential respondents, 96 (53%) returned the questionnaire. One respondent provided only free-text comments, thus data from 95 respondents were compiled. The mean age at the time of freezing oocytes was 37.1 years (SD ± 2.6, range: 27-42) and the average number of oocytes stored was 14.2 (SD ± 7.9, range: 0-42); 2% had attempted to store oocytes but had none suitable for freezing, 24% had stored 23 oocytes. About one-third of respondents (34%) had been pregnant at some point in their lives. Six women (6%) had used their stored oocytes and three of them had given birth as a result. The main reason for not using stored oocytes was not wanting to be a single parent. Of the 87 (91%) women who still had oocytes stored, 21% intended to use them while 69% indicated that their circumstances would determine usage. The mean number of children respondents would ideally have liked to have was significantly

Truths and myths of oocyte sensitivity to controlled rate freezing.

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Coticchio, G; Bonu, M A; Sciajno, R; Sereni, E; Bianchi, V; Borini, A

2007-07-01

The mammalian oocyte is especially sensitive to cryopreservation. Because of its size and physiology, it can easily undergo cell death or sub-lethal damage as a consequence of intracellular ice formation, increase in the concentration of solutes and other undesired effects during the conversion of extracellular water into ice. This has generated the belief that oocyte storage cannot be achieved with the necessary efficiency and safety. However, many concerns raised by oocyte freezing are the result of unproven hypotheses or observations conducted under sometimes inappropriate conditions. For instance, spindle organization can undergo damage under certain freezing conditions but not with other protocols. The controversial suggestion that cryopreservation induces cortical granule discharge and zona pellucida hardening somehow questions the routine use of sperm microinjection. Damage to mouse oocytes caused by solute concentration is well documented but, in the human, there is no solid evidence that modifications of freezing mixtures, to prevent this problem, provide an actual advantage. The hope of developing oocyte cryopreservation as a major IVF option is becoming increasingly realistic, but major efforts are still required to clarify the authentic implications of oocyte cryopreservation at the cellular level and identify freezing conditions compatible with the preservation of viability and developmental ability.

Regulation of ALF promoter activity in Xenopus oocytes.

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Dan Li

Full Text Available BACKGROUND: In this report we evaluate the use of Xenopus laevis oocytes as a matched germ cell system for characterizing the organization and transcriptional activity of a germ cell-specific X. laevis promoter. PRINCIPAL FINDINGS: The promoter from the ALF transcription factor gene was cloned from X. laevis genomic DNA using a PCR-based genomic walking approach. The endogenous ALF gene was characterized by RACE and RT-PCR for transcription start site usage, and by sodium bisulfite sequencing to determine its methylation status in somatic and oocyte tissues. Homology between the X. laevis ALF promoter sequence and those from human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis was relatively low, making it difficult to use such comparisons to identify putative regulatory elements. However, microinjected promoter constructs were very active in oocytes and the minimal promoter could be narrowed by PCR-mediated deletion to a region as short as 63 base pairs. Additional experiments using a series of site-specific promoter mutants identified two cis-elements within the 63 base pair minimal promoter that were critical for activity. Both elements (A and B were specifically recognized by proteins present in crude oocyte extracts based on oligonucleotide competition assays. The activity of promoter constructs in oocytes and in transfected somatic Xenopus XLK-WG kidney epithelial cells was quite different, indicating that the two cell types are not functionally equivalent and are not interchangeable as assay systems. CONCLUSIONS: Overall the results provide the first detailed characterization of the organization of a germ cell-specific Xenopus promoter and demonstrate the feasibility of using immature frog oocytes as an assay system for dissecting the biochemistry of germ cell gene regulation.

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

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Joanna Budna

2017-12-01

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.

Ultrastructural analysis of bovine oocytes from ovarian follicles with different diametersAnálise ultra-estrutural de oócitos bovinos provenientes de folículos ovarianos com diferentes diâmetros

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Cesar Roberto Esper

2011-10-01

Full Text Available In vitro embryo production is an important technique for facilitating the reproduction of animals with high genetic merit. The greatest challenge for the reproducibility of this technique is the quality of the oocyte that is submitted for in vitro maturation. The aim of this work was to evaluate the ultrastructural characteristics of oocytes from follicles of different diameters using transmission electron microscopy. The animals were divided into 2 groups and were given a single i.m. injection of 250 IU FSH (Pluset, Serono, Italy. To synchronize the follicular growth, all follicles > 2 mm were aspirated during the estrous cycle, which was considered day zero (D0. Group 1 (G1; n = 4 received FSH on day 1 (D1, and the 2- to 5-mm follicles were aspirated on day 2 (D2. The animals in group 2 (G2; n = 5 received FSH on day 2 (D2, and their 10- to 15-mm follicles were aspirated on day 5 (D5. After aspiration, the oocytes from both groups were fixed and prepared for ultrastructural analysis. The oocytes analyzed from both groups had similar ultrastructural characteristics. The presence and distribution of organelles in the cytoplasm of the oocytes did not differ between groups, suggesting that, in relation to the ultrastructural characteristics, oocytes from 2 to 5 mm and 10 to 15 mm follicles are similar.A produção in vitro de embriões é uma técnica importante para facilitar a reprodução de animais com elevado mérito genético. O maior desafio para a reprodutibilidade desta técnica é a qualidade do oócito destinado à maturação in vitro. O objetivo deste trabalho foi avaliar as características ultra-estruturais de oócitos provenientes de folículos com diferentes diâmetros por microscopia eletrônica de transmissão. Os animais foram divididos em dois grupos e receberam uma única injeção im de 250 UI de FSH (Pluset, Serono, Itália. Para sincronizar o crescimento folicular, todos os folículos > 2 mm foram aspirados durante o ciclo

Somatic cell cloning in Buffalo (Bubalus bubalis): effects of interspecies cytoplasmic recipients and activation procedures.

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Kitiyanant, Y; Saikhun, J; Chaisalee, B; White, K L; Pavasuthipaisit, K

2001-01-01

Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p > 0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.

Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

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Bo Fu

2015-12-01

Full Text Available The in vitro maturation (IVM efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+ oocytes with low glucose-6-phosphate dehydrogenase (G6PDH activity have shown superior quality than BCB negative (− oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9 and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

Oocyte batch development and enumeration in the European anchovy (Engraulis encrasicolus

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R. FERRERI

2016-09-01

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.

[Meiotic abnormalities of oocytes from patients with endometriosis submitted to ovarian stimulation].

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Barcelos, Ionara Diniz Evangelista Santos; Vieira, Rodolpho Cruz; Ferreira, Elisa Melo; Araújo, Maria Cristina Picinato Medeiros de; Martins, Wellington de Paula; Ferriani, Rui Alberto; Navarro, Paula Andrea de Albuquerque Salles

2008-08-01

to evaluate the meiotic spindle and the chromosome distribution of in vitro mature oocytes from stimulated cycles of infertile women with endometriosis, and with male and/or tubal infertility factors (Control Group), comparing the rates of in vitro maturation (IVM) between the two groups evaluated. fourteen patients with endometriosis and eight with male and/or tubal infertility factors, submitted to ovarian stimulation for intracytoplasmatic sperm injection have been prospectively and consecutively selected, and formed a Study and Control Group, respectively. Immature oocytes (46 and 22, respectively, from the Endometriosis and Control Groups) were submitted to IVM. Oocytes presenting extrusion of the first polar corpuscle were fixed and stained for microtubules and chromatin evaluation through immunofluorescence technique. Statistical analysis has been done by the Fisher's exact test, with statistical significance at pControl Groups, respectively). The chromosome and meiotic spindle organization was observed in 18 and 11 oocytes from the Endometriosis and Control Groups, respectively. In the Endometriosis Group, eight oocytes (44.4%) presented themselves as normal metaphase II (MII), three (16.7%) as abnormal MII, five (27.8%) were in telophase stage I and two (11.1%) underwent parthenogenetic activation. In the Control Group, five oocytes (45.4%) presented themselves as normal MII, three (27.3%) as abnormal MII, one (9.1%) was in telophase stage I and two (18.2%) underwent parthenogenetic activation. There was no significant difference in meiotic anomaly rate between the oocytes in MII from both groups. the present study data did not show significant differences in the IVM or in the meiotic anomalies rate between the IVM oocytes from stimulated cycles of patients with endometriosis, as compared with controls. Nevertheless, they have suggested a delay in the outcome of oocyte meiosis I from patients with endometriosis, shown by the higher proportion of oocytes in

Kif4 Is Essential for Mouse Oocyte Meiosis.

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

2017-01-01

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.

Mural granulosa cell gene expression associated with oocyte developmental competence

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Jiang Jin-Yi

2010-03-01

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

Oocyte quality in mice is affected by a mycotoxin-contaminated diet.

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Hou, Yan-Jun; Xiong, Bo; Zheng, Wei-Jiang; Duan, Xing; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Qiang; Xu, Yin-Xue; Sun, Shao-Chen

2014-05-01

Mycotoxins, such as deoxynivalenol (DON), zearalenone (ZEN), and aflatoxin (AF), are commonly found in many food commodities and may impair the growth and reproductive efficiency of animals and humans. We investigated the effects of a mycotoxin-contaminated diet on mouse oocyte quality. Maize contaminated with DON (3.875 mg/kg), ZEN (1,897 μg/kg), and AF (806 μg/kg) was incorporated into a mouse diet at three different levels (0, 15, and 30% w/w). After 4 weeks, ovarian and germinal vesicle oocyte indices decreased in mycotoxin-fed mice. Oocytes from these mice exhibited low developmental competence with reduced germinal vesicle breakdown and polar body extrusion rates. Embryo developmental competence also showed a similar pattern, and the majority of embryos could not develop to the morula stage. Actin expression was also reduced in both the oocyte cortex and cytoplasm, which was accompanied by decreased expression of the actin nucleation factors profilin-1 and mDia1. Moreover, a large percentage of oocytes derived from mice that were fed a mycotoxin-contaminated diet exhibited aberrant spindle morphology, a loss of the cortical granule-free domain, and abnormal mitochondrial distributions, which further supported the decreased oocyte quality. Thus, our results demonstrate that mycotoxins are toxic to the mouse reproductive system by affecting oocyte quality. Copyright © 2013 Wiley Periodicals, Inc.

Anandamide levels fluctuate in the bovine oviduct during the oestrous cycle.

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Maria Gracia Gervasi

Full Text Available Mammalian oviduct acts as a reservoir for spermatozoa and provides an environment in which they may compete for the opportunity to fertilize the oocyte. Whilst in the oviduct spermatozoa undergo capacitation essential for fertilization. Sperm-oviduct interaction is essential for sperm capacitation and is a tightly regulated process influenced by the local microenvironment. Previously we reported that the endocannabinoid anandamide (AEA regulates sperm release from epithelial oviductal cells by promoting sperm capacitation. The aims of this work were to measure the AEA content and to characterize the main AEA metabolic pathway in the bovine oviduct and determine how these change through the oestrous cycle. In this study, the levels of AEA and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in bovine oviduct collected during different stages of oestrous cycle by ultra high performance liquid chromatography tandem mass spectrometry. Results indicated that intracellular oviductal epithelial levels of all three N-acylethanolamines fluctuate during oestrous cycle. Anandamide from oviductal fluid also varied during oestrous cycle, with the highest values detected during the periovulatory period. Endocannabinoid levels from ipsilateral oviduct to ovulation were higher than those detected in the contralateral one, suggesting that levels of oviductal AEA may be regulated by ovarian hormones. The expression and localization of N-acylethanolamines metabolizing enzymes in bovine oviduct were also determined by RT-PCR, Western blot, and immunohistochemistry but no change was found during the oestrous cycle. Furthermore, nanomolar levels of AEA were detected in follicular fluids, suggesting that during ovulation the mature follicle may contribute to oviductal AEA levels to create an endocannabinoid gradient conducive to the regulation of sperm function for successful fertilization.

Investigations of oocyte in vitro maturation within a mouse model.

Science.gov (United States)

Chin, Alexis Heng Boon; Chye, Ng Soon

2004-02-01

This study attempted to develop a 'less meiotically competent' murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze-thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage.

PP2A regulates kinetochore-microtubule attachment during meiosis I in oocyte.

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Tang, An; Shi, Peiliang; Song, Anying; Zou, Dayuan; Zhou, Yue; Gu, Pengyu; Huang, Zan; Wang, Qinghua; Lin, Zhaoyu; Gao, Xiang

2016-06-02

Studies using in vitro cultured oocytes have indicated that the protein phosphatase 2A (PP2A), a major serine/threonine protein phosphatase, participates in multiple steps of meiosis. Details of oocyte maturation regulation by PP2A remain unclear and an in vivo model can provide more convincing information. Here, we inactivated PP2A by mutating genes encoding for its catalytic subunits (PP2Acs) in mouse oocytes. We found that eliminating both PP2Acs caused female infertility. Oocytes lacking PP2Acs failed to complete 1(st) meiotic division due to chromosome misalignment and abnormal spindle assembly. In mitosis, PP2A counteracts Aurora kinase B/C (AurkB/C) to facilitate correct kinetochore-microtubule (KT-MT) attachment. In meiosis I in oocyte, we found that PP2Ac deficiency destabilized KT-MT attachments. Chemical inhibition of AurkB/C in PP2Ac-null oocytes partly restored the formation of lateral/merotelic KT-MT attachments but not correct KT-MT attachments. Taken together, our findings demonstrate that PP2Acs are essential for chromosome alignments and regulate the formation of correct KT-MT attachments in meiosis I in oocytes.

A Flexure-Guided Piezo Drill for Penetrating the Zona Pellucida of Mammalian Oocytes.

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Johnson, Wesley; Dai, Changsheng; Liu, Jun; Wang, Xian; Luu, Devin K; Zhang, Zhuoran; Ru, Changhai; Zhou, Chao; Tan, Min; Pu, Huayan; Xie, Shaorong; Peng, Yan; Luo, Jun; Sun, Yu

2018-03-01

Mammalian oocytes such as mouse oocytes have a highly elastic outer membrane, zona pellucida (ZP) that cannot be penetrated without significantly deforming the oocyte, even with a sharp micropipette. Piezo drill devices leverage lateral and axial vibration of the micropipette to accomplish ZP penetration with greatly reduced oocyte deformation. However, existing piezo drills all rely on a large lateral micropipette vibration amplitude ( 20 ) and a small axial vibration amplitude (0.1 ). The very large lateral vibration amplitude has been deemed to be necessary for ZP penetration although it also induces larger oocyte deformation and more oocyte damage. This paper reports on a new piezo drill device that uses a flexure guidance mechanism and a systematically designed pulse train with an appropriate base frequency. Both simulation and experimental results demonstrate that a small lateral vibration amplitude (e.g., 2 ) and an axial vibration amplitude as large as 1.2 were achieved. Besides achieving 100% effectiveness in the penetration of mouse oocytes (n = 45), the new piezo device during ZP penetration induced a small oocyte deformation of 3.4 versus larger than 10 using existing piezo drill devices.

Increased incidence of preeclampsia in mothers of advanced age conceiving by oocyte donation.

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Dior, Uri P; Laufer, Neri; Chill, Henry H; Granovsky-Grisaru, Sorina; Yagel, Simcha; Yaffe, Haim; Gielchinsky, Yuval

2018-05-01

The aim of this study was to evaluate the risk of preeclampsia in women of advanced age who conceived through donated oocytes as compared with natural conceptions. A historical prospective study of singleton live births of parturients ≥ 45 years of age at four university hospitals was conducted. For the purpose of the study, the population was divided by the mode of conception into two groups: oocyte donation and natural conception. The main outcome variable in this study was preeclampsia. Secondary outcomes included pregnancy-induced hypertension and Small for Gestational Age. Two hundred and seventy pregnancies were achieved naturally and 135 women conceived by oocyte donation. Mean age at delivery for the natural conception and oocyte donation groups was 45.7 and 47.8, respectively. Preeclampsia complicated 3 out of 270 (1.1%) natural conception pregnancies and 17 out of 135 (12.6%) oocyte donation conceptions. After adjusting for confounders, oocyte donation pregnancies were found to be associated with a 12-fold increased risk for preeclampsia (P = 0.001). Among oocyte donation pregnancies, the risk of preeclampsia was not affected by parity or age. A substantially increased risk for preeclampsia was found in oocyte donation pregnancies, suggesting that the foreign oocyte may play a specific biologic role in the development of preeclampsia after the age of 45.

Biotin-deficient diet induces chromosome misalignment and spindle defects in mouse oocytes.

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Tsuji, Ai; Nakamura, Toshinobu; Shibata, Katsumi

2015-01-01

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.

Triplet pregnancy after intracytoplasmic sperm injection of cryopreserved oocytes: case report.

Science.gov (United States)

Young, E; Kenny, A; Puigdomenech, E; Van Thillo, G; Tiverón, M; Piazza, A

1998-08-01

To report a triplet pregnancy that occurred after intracytoplasmic injection of sperm into cryopreserved oocytes. Case report. Instituto de Ginecología y Fertilidad (IFER), Buenos Aires, Argentina. A 36-year-old infertile patient with premature ovarian failure and a previous term pregnancy with fresh donated oocytes. We administered leuprolide acetate for pituitary down-regulation followed by E2 valerianate in incremental doses until an endometrial lining of >8 mm was observed by ultrasound. Thawing of frozen donated oocytes, intracytoplasmic sperm injection (ICSI), and translaparoscopic fallopian tube ET also were performed. Natural micronized progesterone was administered intravaginally (600 mg/d) before ET. Ultrasound at the 8th week of gestation revealed a triplet pregnancy with active fetal heartbeats. A triple intrauterine gestation was achieved with the use of microinjection into cryopreserved oocytes. This case illustrates the feasibility of oocyte cryopreservation for clinical use in the era of ICSI.

Experience with Conscious sedation for Oocyte Retrieval in Nigeria

African Journals Online (AJOL)

elearning

The aim of this study was to assess clients' pain experience, acceptance of conscious sedation and correlates of pain during oocyte retrieval ... Conscious sedation and analgesia are one of several methods used to relieve pain during oocyte retrieval in. IVF procedures. .... relieves anxiety and reduces the patient's memory.

Effect of Antioxidant Flavonoids (Quercetin and Taxifolin on Maturation of Porcine Oocytes

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Jung-Taek Kang

2016-03-01

Full Text Available Quercetin (QT and taxifolin (TF are structurally similar plant-derived flavonoids that have antioxidant properties and act as free radical scavengers. The objective of this study was to investigate effects of QT and TF on nuclear maturation of porcine oocytes. Effects of TF at 0, 1, 10, and 50 μg/mL on oocyte nuclear maturation (polar body extrusion were investigated. After incubation for 44 h, there were no significant differences between the treatment and control groups except in the 50 μg/mL group which was significantly lower (59.2%, p80%. After parthenogenetic activation, further in vitro development of QT- or TF-treated vs control oocytes was investigated. A significantly higher proportion of QT-treated (1 μg/mL oocytes developed into blastocysts compared to controls (24.3% vs 16.8%, respectively; however, cleavage rate and blastocyst cell number were not affected. The TF-treated group was not significantly different from controls. Levels of reactive oxygen species (ROS and intracellular glutathione (GSH in oocytes and embryos in a culture medium supplemented with QT or TF were measured. Both treatment groups had significantly lower (p<0.05 levels of ROS than controls, however GSH levels were different only in QT-treated oocytes. We conclude that exogenous flavonoids such as QT and TF reduce ROS levels in oocytes. Although at high concentration (50 μg/mL both QT and TF appear to be toxic to oocytes.

In Vitro Maturation and Embryo Development to blastocyst Mouse Germinal Vesicle Oocytes after Vitrification

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M Nikseresht

2013-05-01

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

Selective Regulation of Oocyte Meiotic Events Enhances Progress in Fertility Preservation Methods

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Onder Celik

2015-01-01

Full Text Available Following early embryonic germ cell migration, oocytes are surrounded by somatic cells and remain arrested at diplotene stage until luteinizing hormone (LH surge. Strict regulation of both meiotic arrest and meiotic resumption during dormant stage are critical for future fertility. Intercellular signaling system between the somatic compartment and oocyte regulates these meiotic events and determines the follicle quality. As well as the collected number of eggs, their qualities are also important for in vitro fertilization (IVF outcome. In spontaneous and IVF cycles, germinal vesicle (GV–stage oocytes, premature GV breakdown, and persistence of first meiotic arrest limit the reproductive performance. Likewise, both women with premature ovarian aging and young cancer women are undergoing chemoradiotherapy under the risk of follicle loss because of unregulated meiotic events. Understanding of oocyte meiotic events is therefore critical for the prevention of functional ovarian reserve. High levels of cyclic guanosine monophophate (cGMP, cyclic adenosine monophophate (cAMP and low phosphodiesterase (PDE 3A enzyme activity inside the oocyte are responsible for maintaining of meiotic arrest before the LH surge. cGMP is produced in the somatic compartment, and natriuretic peptide precursor C (Nppc and natriuretic peptide receptor 2 (Npr2 regulate its production. cGMP diffuses into the oocyte and reduces the PDE3A activity, which inhibits the conversion of cAMP to the 5′AMP, and cAMP levels are enhanced. In addition, oocyte itself has the ability to produce cAMP. Taken together, accumulation of cAMP inside the oocyte induces protein kinase activity, which leads to the inhibition of maturation-promoting factor and meiotic arrest also continues. By stimulating the expression of epidermal growth factor, LH inhibits the Nppc/Npr2 system, blocks cGMP synthesis, and initiates meiotic resumption. Oocytes lacking the functional of this pathway may lead to

DNA comet assay as a rapid detection method of irradiated bovine meat by electron beam

International Nuclear Information System (INIS)

Marin-Huachaca, Nelida Simona; Villavicencio, Anna Lucia C.H.

2001-01-01

Full text: Introduction: The presence in food of pathogenic microorganisms, such as Salmonella species, Escherichia coli 0157:H7, Listeria Monocytogenes or Yersinia enterolitica, is a problem of growing concern to public health authorities all over the world. Thus, irradiation of certain prepackaged meat products such as ground beef, minced meat, and hamburgers may help in controlling meatborne pathogens and parasites. Pathogenic microorganisms and parasites in meat products, which are commonly consumed raw, are of particular importance, Up to now, only electron-beam accelerators and gamma-ray cells have been used for commercial applications. At the international conference on 'The Acceptance, Control of, and Trade in Irradiated Food', it was recommended that governments should encourage research into detection methods (Anon, 1989), Already five international standards are available to food control agencies. A number of physical, chemical, and biological techniques of detection of irradiated foods have been discussed in the literature. A rapid and inexpensive screening test employing DNA Comet Assay to identify radiation treatment of food has been described by Cerda et al. (1997). This method is restricted to foods that have not been subjected to heat or other treatments, which also induce DNA fragmentation. Advantages are its simplicity, low cost and speed of measurement. This method was proposed to the European Committee for Standardization (CEN) as a screening protocol (presumptive) and not as a proof (definitive). The DNA comet assay have been yielded good results with chicken, pork, fish meat, exotic meat, hamburgers, fruits and cereals. In this work we studied a DNA fragmentation of bovine meat irradiated by electron beam. Experimental: Bovine meat was purchased in local shops in Sao Paulo. Irradiation was performed with electron beam of accelerator facility of Radiation Dynamics Inc., USA (E=1,5 MeV, l=25 mA). The irradiation doses were 3,5; 4,5, 5,5, and 7

Effect of Nanoparticles on the Survival and Development of Vitrified Porcine GV Oocytes.

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Li, W J; Zhou, X L; Liu, B L; Dai, J J; Song, P; Teng, Y

BACKGROUND: Some mammalian oocytes have been successfully cryopreserved by vitrification. However, the survival and developmental rate of vitrified oocytes is still low. The incorporation of nanoparticles into cryoprotectant (CPA) may improve the efficiency of vitrification by changing the properties of solutions. The toxicity of different concentrations of hydroxy apatite (HA), silica dioxide (SO 2 ), aluminum oxide (Al 2 O 3 ) and titanium dioxide (TiO 2 ) nanoparticles (20 nm in diameter) to oocytes was tested and the toxicity threshold value of each nanoparticle was determined. Porcine GV oocytes were vitrified in optimized nano-CPA, and effects of diameter and concentration of nanoparticles on the survival rate and developmental rate of porcine GV oocytes were compared. HA nanoparticles have demonstrated the least toxicity among four nanoparticles and the developmental rate of GV-stage porcine oocytes was 100% when its concentration was lower than 0.5%. By adding 0.1% HA into VS, the developmental rate of GV-stage porcine oocytes (22%) was significantly higher than other groups. The effect of vitrification in nano-CPA on oocytes was related to the concentration of HA nanoparticles rather than their size. By adding 0.05% HA nanoparticles (60nm in diameter), the developmental rate increased dramatically from 14.7% to 30.4%. Nano-cryopreservation offers a new way to improve the effect of survival and development of oocytes, but the limitation of this technology shall not be ignored.

Ascorbic acid effects on in vitro maturation of mouse oocyte with or ...

African Journals Online (AJOL)

Ascorbic acid has long been associated with fertility. This study was designed to determine the effects of ascorbic acid on in vitro maturation of mouse oocyte with or without cumulus cells. In this study, 508 denuded oocytes (DOs) and 527 cumulus–oocyte complexes (COCs) from mice stimulated with pregnant mare's serum ...

Perinatal outcomes in 375 children born after oocyte donation

DEFF Research Database (Denmark)

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

2013-01-01

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)....

Oogenesis, follicular development and reproductive performance in the prenatally irradiated bovine

International Nuclear Information System (INIS)

Erickson, B.H.; Reynolds, R.A.

1978-01-01

To determine the radioresponse of the various developmental stages of the prenatal bovine germ cell, cows bearing foetuses varying in age from 40+-5 (point of gonadal sex differentiation) to 270+-10 (approximately 13 days before parturition) days of gestation were irradiated with 300 R of 60 Co gamma radiation at 25-50 R/min, a level just below that likely to cause maternal deaths and gross foetal abnormalities at some developmental stages. Dose to the foetal gonad was approximately 100 rad. When the prenatally irradiated heifers were approximately 10 months old their ovaries were recovered at slaughter, serially sectioned, and prepared for microscopic analysis. A complete quantitative analysis of oocytes in primordial, growing and vesicular follicles was effected. Follicular development (reflected by counts of growing and vesicular follicles) was apparently unaffected at all ages tested (P>0.25), whereas oogenesis (reflected by counts of oocytes in primordial follicles) was significantly impeded (64% of control) only between 70 and 90 days of gestation (P<0.05). The germ-cell population at this period differs from later developmental stages principally by the presence of a high proportion of mitotically active oogonia, hence it appears that the oogonium is the most vulnerable cell type. Neither the viability nor reproductive performance of 60 cows that were prenatally irradiated (100 rad) at either 80+-10 or 130+-20 days of gestation was affected through a minimum of 5 years of postnatal life and three pregnancies. (author)

Rac1 is dispensable for oocyte maturation and female fertility in vivo.

Science.gov (United States)

Hao, Jian-Xiu; Meng, Tie-Gang; Fan, Li-Hua; Yao, Yuan-Qing

2017-01-01

Oocyte maturation, the important process to produce female haploid gamete, accompanies with polarity establishment and highly asymmetric cell division to emit minor polar body within little cytoplasm. Microfilaments play central roles in polarity establishment and asymmetric cell division. Several actin regulators like WASP protein family as well as small GTPases function in microfilament dynamics, involving the process. Rac1, one member of RhoGTPases, has been reported to regulate the polarity and asymmetric cell division in mouse oocytes in vitro. The physiological role of Rac1 in mouse oocyte remains unknown. By conditional knockout technology, we specifically deleted Rac1 gene in mouse oocyte, and found that Rac1 deletion exerted little effect on mouse oocyte maturation including polarity establishment and asymmetric division, and the mutant mice showed normal fertility.

Genetic influences on ovulation of primary oocytes in LT/Sv strain mice.

Science.gov (United States)

Everett, Clare A; Auchincloss, Catherine A; Kaufman, Matthew H; Abbott, Catherine M; West, John D

2004-11-01

A high proportion of LT/Sv strain oocytes arrest in meiotic metaphase I (MI) and are ovulated as diploid primary oocytes rather than haploid secondary oocytes. (Mus musculus castaneus x LT/SvKau)F1 x LT/SvKau backcross females were analysed for the proportion of oocytes that arrested in MI and typed by PCR for a panel of microsatellite DNA sequences (simple sequence repeat polymorphisms) that differed between strain LT/SvKau and M. m. castaneus. This provided a whole genome scan of 86 genetic markers distributed over all 19 autosomes and the X chromosome, and revealed genetic linkage of the MI arrest phenotype to markers on chromosomes 1 and 9. Identification of these two chromosomal regions should facilitate the identification of genes involved in mammalian oocyte maturation and the control of meiosis.

Radiation- and drug-induced DNA repair in mammalian oocytes and embryos

International Nuclear Information System (INIS)

Pedersen, R.A.; Brandriff, B.

1979-01-01

A review of studies showing ultraviolet- or drug-induced unscheduled DNA synthesis in mammalian oocytes and embryos suggests that the female gamete has an excision repair capacity from the earliest stages of oocyte growth. The oocyte's demonstrable excision repair capacity decreases at the time of meiotic maturation for unknown reasons, but the fully mature oocyte maintans a repair capacity, in contrast to the mature sperm, and contributes this to the zygote. Early embryo cells maintain relatively constant levels of excision repair until late fetal stages, when they lose their capacity for excision repair. These apparent changes in excision repair capacity do not have a simple relationship to known differences in radiation sensitivity of germ cells and embryos

In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes

International Nuclear Information System (INIS)

Sakai, Tomomi; Tochio, Hidehito; Tenno, Takeshi; Ito, Yutaka; Kokubo, Tetsuro; Hiroaki, Hidekazu; Shirakawa, Masahiro

2006-01-01

In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. 15 N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional 1 H- 15 N correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein-protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions

Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence

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Yokoi Hayato

2011-04-01

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.

In-vitro maturation and cryopreservation of oocytes at the time of oophorectomy

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Melanie L. Walls

2015-08-01

Full Text Available A 27 year old female presented for fertility preservation prior to undergoing pelvic radiotherapy. She had previously undergone a radical laparoscopic hysterectomy for cervical carcinoma seven months earlier. A trans-vaginal oocyte aspiration was not advisable due to a vaginal recurrence of the disease. Due to a polycystic ovarian morphology (PCO, follicle stimulating hormone (FSH priming with no human chorionic gonadotrophin (hCG trigger was performed prior to oophorectomy followed by ex-vivo oocyte aspiration and in vitro maturation (IVM. All visualized follicles were punctured and follicular fluid aspirated. There were 22 immature oocytes identified and placed into maturation culture for 24 h. After this time, 15 oocytes were deemed to be mature and suitable for vitrification. Following an additional 24 h in maturation culture of the remaining 7 oocytes, three more were suitable for cryopreservation. The patient recovered well and progressed to radiotherapy three days later. This report demonstrates the use of IVM treatment to store oocytes for oncology patients in time-limited circumstances.

Small GTPases and formins in mammalian oocyte maturation: cytoskeletal organizers.

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Kwon, Sojung; Lim, Hyunjung J

2011-03-01

The maturation process of mammalian oocytes accompanies an extensive rearrangement of the cytoskeleton and associated proteins. As this process requires a delicate interplay between the cytoskeleton and its regulators, it is often targeted by various external and internal adversaries that affect the congression and/or segregation of chromosomes. Asymmetric cell division in oocytes also requires specific regulators of the cytoskeleton, including formin-2 and small GTPases. Recent literature providing clues regarding how actin filaments and microtubules interact during spindle migration in mouse oocytes are highlighted in this review.

Inherited effects from mouse immature oocytes following low-dose irradiation

International Nuclear Information System (INIS)

Straume, T.; Khan, R.; Raabe, O.G.; Walsh, K.J.; Wiley, L.M.

1992-07-01

Immature oocytes represent the genetic pool in female mice as well as in women and therefore are principal cells of concern for genetic studies. Previous studies have demonstrated that genetic effects in female mice can be masked by the hypersensitive plasma membrane lethality target of immature oocytes. Studies have also shown that genetic effects can be detected when the plasma mambrane is sufficiently spared. Here, new data obtained using the mouse preimplantation embryo chimera assay are presented and discussed in light of previous findings for irradiated mouse oocytes

Sheep oocyte expresses leptin and functional leptin receptor mRNA

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Seyyed Jalil Taheri

2016-09-01

Conclusions: The result of present study reveals that leptin and its functional receptor (Ob-Rb mRNA are expressed in sheep oocyte and further studies should investigate the role(s of leptin on sheep oocyte physiology and embryo development.

Utilization of endogenous fatty acid stores for energy production in bovine preimplantation embryos.

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Sutton-McDowall, Melanie L; Feil, Deanne; Robker, Rebecca L; Thompson, Jeremy G; Dunning, Kylie R

2012-05-01

Although current embryo culture media are based on carbohydrate metabolism of embryos, little is known about metabolism of endogenous lipids. L-carnitine is a β-oxidation cofactor absent in most culture media. The objective was to investigate the influence of L-carnitine supplementation on bovine embryo development. Abattoir-derived bovine cumulus oocyte complexes were cultured and fertilized. Post-fertilization, presumptive zygotes were transferred into a basic cleavage medium ± carbohydrates (glucose, lactate and pyruvate) ± 5 mm L-carnitine and cultured for 4 days in vitro. In the absence of carbohydrates during culture, embryos arrested at the 2- and 4-cell stages. Remarkably, +L-carnitine increased development to the morula stage compared to +carbohydrates alone (P levels were higher and ATP: ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared to embryos cultured in -L-carnitine. Therefore, we inferred that +L-carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. In conclusion, L-carnitine supplementation supported precompaction embryo development and there was an additive effect of +L-carnitine +carbohydrates on early embryo development, most likely through increased β-oxidation within embryos. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Evidence for an absence of deleterious effects of ultrasound on human oocytes.

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Mahadevan, M; Chalder, K; Wiseman, D; Leader, A; Taylor, P J

1987-10-01

Animal and human data would suggest that ultrasound causes deleterious effects to oocytes during meiosis. We directly compared the fertilization rate and embryonic development following in vitro fertilization and embryo transfer of those oocytes exposed to ultrasound and those not exposed in the same patient. In 39 unscreened patients a combination of laparoscopy and ultrasound was used for oocyte recovery. Laparoscopy was performed first on the most accessible ovary (usually the right) and at least one oocyte was obtained. Ultrasound-guided oocyte recovery was successful in the other inaccessible ovary. To assess how oocytes obtained by ultrasound or laparoscopy related to the pregnancy rate, two groups of patients were evaluated in whom the embryos transferred either had been exposed to ultrasound or had not been. The fertilization and the embryo cleavage rates were not significantly different between the ultrasound-exposed and the unexposed groups. The pregnancy rate was also not significantly different [9 of 49 (18.4%) for ultrasound exposed versus 14 of 74 (18.9%) for unexposed]. There was one early spontaneous abortion in each group. Further analysis of a group of 40 patients, in whom the oocytes were exposed to ultrasound in situ, after the endogenous luteinizing hormone (LH) surge had begun 1-27 hr earlier, revealed that 6 became pregnant (15%). This preliminary study suggests that exposure of human oocytes to ultrasonic waves, either during the different phases of meiosis or after the completion of meiosis, did not significantly influence the developmental potential of the in vitro fertilized embryos.

Role of animal pole protuberance and microtubules during meiosis in sea cucumber Apostichopus japonicus oocytes

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

2010-05-01

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.

Optimization of cryoprotectant loading into murine and human oocytes.

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Karlsson, Jens O M; Szurek, Edyta A; Higgins, Adam Z; Lee, Sang R; Eroglu, Ali

2014-02-01

Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethyl sulfoxide (Me(2)SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me(2)SO exposure time, revealing that neither shrinkage nor Me(2)SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me(2)SO addition appears to result from interactions between the effects of Me(2)SO toxicity and osmotic stress. We also investigated Me(2)SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me(2)SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me(2)SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach. Copyright © 2013 Elsevier Inc. All rights reserved.

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

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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.

Maternal aging affects oocyte resilience to carbonyl cyanide-m-chlorophenylhydrazone -induced mitochondrial dysfunction in cows.

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Kazuki Kansaku

Full Text Available Mitochondrial quality control is important for maintaining cellular and oocyte viability. In addition, aging affects mitochondrial quality in many cell types. In the present study, we examined how aging affects oocyte mitochondrial biogenesis and degeneration in response to induced mitochondrial dysfunction. Cumulus oocyte complexes were harvested from the ovaries of young (21‒45 months and aged (≥120 months cows and treated for 2 hours with 10 μM carbonyl cyanide-m- chlorophenylhydrazone (CCCP, or a vehicle control, after which cumulus oocyte complexes were subjected to in vitro fertilization and culture. CCCP treatment reduced ATP content and increased reactive oxygen species (ROS levels in the oocytes of both young and aged cows. When CCCP-treated cumulus oocyte complexes were subsequently cultured for 19 hours and/or subjected to fertilization, high ROS levels in oocytes and a low rate of blastocyst development was observed in oocytes derived from aged cows. In addition, we observed differential responses in mitochondrial biogenesis to CCCP treatment between young and aged cows. CCCP treatment enhanced mitochondrial biogenesis concomitant with upregulation of SIRT1 expression in oocytes of young, but not aged, cows. In conclusion, aging affects mitochondrial quality control and recuperation of oocytes following CCCP-induced mitochondrial dysfunction.

Effect of milrinone on the developmental competence of growing lamb oocytes identified with brilliant cresyl blue.

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Wang, Liqin; Jiang, Xiangjiu; Wu, Yangsheng; Lin, Jiapeng; Zhang, Li; Yang, Nan; Huang, Juncheng

2016-11-01

Juvenile in vitro embryo transfer is a novel technique that can be used to increase the rate of genetic gain in a population and presents an alternative to embryo technologies on the basis of adult animals. However, oocytes from prepubertal animals have a lower viability than those obtained from adult ewe oocyte donors. In this research, we aimed to determine the optimum concentration and time of treatment of oocytes from prepubertal lambs with brilliant cresyl blue (BCB) stain and milrinone during IVM. This would improve the developmental rate of lamb oocytes and embryos after IVF. First, lamb cumulus-oocyte complexes were cultured under different concentrations (13 or 26 μM) of BCB staining. Treated lamb oocytes were then divided into BCB- (colorless cytoplasm) and BCB+ (colored cytoplasm) groups on the basis of their glucose-6-phosphate dehydrogenase activity. The blastocyst efficiency rate of BCB+ oocytes treated with 13 μM BCB (37.03%) was significantly higher than that of BCB+ oocytes treated with 26 μM BCB (23.25%) and that of nontreated BCB control oocytes (15.37%), as well as that of BCB- oocytes (6.28%). Both control oocytes and BCB+ oocytes exhibited significantly higher cleavage rates (60.15% and 73.44%, respectively) than that of BCB- oocytes (36.19%). Moreover, the diameter and glutathione content of BCB+ oocytes were found to be significantly greater than those of BCB- oocytes (163.37 vs. 159.25 μm and 6.39 vs. 0.26 pM, respectively). After culturing BCB- oocytes in different concentrations of milrinone (0, 50, 75, and 100 μM) for 3, 6, or 9 hours, results reported that supplementation of IVM medium with 75 μM milrinone for 6 hours yielded a significantly higher proportion of blastocysts than the other treatments. These results show that the staining of lamb cumulus-oocyte complexes with 13 μM BCB before IVM may be used to select developmentally competent lamb oocytes. Furthermore, they suggest that milrinone can be used to promote

Influence of follicular fluid and cumulus cells on oocyte quality: clinical implications.

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Da Broi, M G; Giorgi, V S I; Wang, F; Keefe, D L; Albertini, D; Navarro, P A

2018-03-02

An equilibrium needs to be established by the cellular and acellular components of the ovarian follicle if developmental competence is to be acquired by the oocyte. Both cumulus cells (CCs) and follicular fluid (FF) are critical determinants for oocyte quality. Understanding how CCs and FF influence oocyte quality in the presence of deleterious systemic or pelvic conditions may impact clinical decisions in the course of managing infertility. Given that the functional integrities of FF and CCs are susceptible to concurrent pathological conditions, it is important to understand how pathophysiological factors influence natural fertility and the outcomes of pregnancy arising from the use of assisted reproduction technologies (ARTs). Accordingly, this review discusses the roles of CCs and FF in ensuring oocyte competence and present new insights on pathological conditions that may interfere with oocyte quality by altering the intrafollicular environment.

Isolation and characterization of mesenchymal stem cells derived from bovine Wharton's jelly and their potential for use in cloning by nuclear transfer

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Carolina Gonzales da Silva

Full Text Available ABSTRACT: Wharton's jelly is a source of mesenchymal stem cells (MSCs that had not yet been tested for bovine embryo production by nuclear transfer (NT. Thus, the objective of this study was to isolate, characterize and test MSCs derived from Wharton's jelly for embryo and pregnancy production by NT in cattle. The umbilical cord was collected during calving and cells derived from Wharton's jelly (WJCs were isolated by explant and cultured in Dulbecco's Modified Eagle Medium. Skin Fibroblasts (FB were isolated after 6 months of life. Morphological analysis was performed by bright field and scanning electron microscopy (SEM during cell culture. Phenotypic and genotypic characterization by flow cytometry, immunocytochemistry, RT-PCR and differentiation induction in cell lineages were performed for WJC. In the NT procedure, oocytes at the arrested metaphase II stage were enucleated using micromanipulators, fused with WJCs or FB and later activated artificially. SEM micrographs revealed that WJCs have variable shape under culture. Mesenchymal markers of MSCs (CD29+, CD73+, CD90+ and CD105+ were expressed in bovine-derived WJC cultures, as evidenced by flow cytometry, immunocytochemistry and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes and adipocytes. After classification, the WJCs were used in NT. Blastocyst formation rate by NT with WJCs at day 7 was 25.80±0.03%, similar to blatocyst rate with NT using skin fibroblasts (19.00±0.07%. Pregnancies were obtained and showed that WJCs constitute a new cell type for use in animal cloning.

Conforming with current regulation in Turkey regarding the freezing of oocytes: A case report of the first pregnancy in Turkey achieved through oocyte vitrification

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Semra Kahraman MD

2017-01-01

Full Text Available Objectives: To present the first pregnancy achieved in Turkey with frozen–warmed oocytes in a case with previous nine unsuccessful assisted reproductive technology (ART attempts. Methods: The clinical follow-up of a 33-year-old female applying to our ART centre after a long and complicated history of infertility is described. Results: In April 2013, the woman attempted our centre for her 10th ART trial. She informed us on oocyte pick-up (OPU day that her husband had been hospitalized following a car crush in Albania and was unable to travel to our clinic to give a sperm sample. We were therefore placed in the position of having to make an emergency decision. OPU was done and seven oocytes were retrieved. Six metaphase II (MII oocytes out of seven Cumulus Oocyte Complexes (COCs were vitrified using the Kitazato Vitrification Cryotop Kit. Six months later, in November 2013, the patient applied for transfer. Two blastocysts were transferred and the ART trial resulted with a singleton pregnancy and the birth of a healthy new-born at term via cesarean section. Conclusion: Regulation Codes on Assisted Reproductive Procedures and Assisted Reproductive Technology Centres, published in the Official Gazette of the Republic of Turkey, on 6 March 2010 forbade the freezing of gonad cells and tissues except when essential for medical reasons and stated that this would be specified later. However, the Regulation Codes published in the Official Gazette of the Republic of Turkey, on 30 September 2014 provided no further clarification. Unfortunately, the wording of the regulations did not specifically address this unexpected emergency situation. However, we saw our decision to cryopreserve the oocytes as a valid interpretation of the regulations, bearing in mind also the requirement that sperm and oocyte in the IVF process must be those of a married couple. Turkish medicolegal regulations should be revised to increase the chances of more women taking advantage

Cumulus cells steroidogenesis is influenced by the degree of oocyte maturation

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Barboni Barbara

2003-05-01

Full Text Available Abstract Background The possibility to predict the ability of a germ cell to properly sustain embryo development in vitro or in vivo as early as possible is undoubtedly the main problem of reproductive technologies. To date, only the achievement of nuclear maturation and cumulus expansion is feasible, as all the studies on cytoplasmic maturation are too invasive and have been complicated by the death of the cells analyzed. The authors studied the possibility to test the cytoplasmic quality of pig oocytes by evaluating their ability to produce steroidogenesis enabling factor(s. To this aim, oocytes matured under different culture conditions that allowed to obtain gradable level of cytoplasmic maturation, were used to produce conditioned media (OCM. The secretion of the factor(s in conditioned media was then recorded by evaluating the ability of the spent media to direct granulosa cells (GC steroidogenesis. Methods In order to obtain germ cells characterized by a different degree of developmental competence, selected pig oocytes from prepubertal gilts ovaries were cultured under different IVM protocols; part of the matured oocytes were used to produce OCM, while those remaining were submitted to in vitro fertilization trials to confirm their ability to sustain male pronuclear decondensation. The OCM collected were finally used on cumulus cells grown as monolayers for 5 days. The demonstration that oocytes secreted factor(s can influence GC steroidogenesis in the pig was confirmed in our lab by studying E2 and P4 production by cumulus cells monolayers using a radioimmunoassay technique. Results Monolayers obtained by growing GC surrounding the oocytes for five days represent a tool, which is practical, stable and available in most laboratories; by using this bioassay, we detected the antiluteal effect of immature oocytes, and for the first time, demonstrated that properly matured germ cells are able to direct cumulus cells steroidogenesis by

Ethical issues in paying for long-distance travel and accommodation expenses of oocyte donors.

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Heng, Boon Chin

2005-11-01

In many countries where the sale and purchase of donor oocytes is banned, a legal loophole often exploited is the use of free air tickets and hotel stay to entice prospective oocyte donors, in lieu of monetary payment. Such a means of procuring much-needed donor oocytes is ethically unsound. There is a lack of transparency and the personal motivation of the oocyte donor may be clouded by the desire for a 'free' holiday. Moreover, such a system is open to abuse by medical professionals. Private fertility clinics may source for oocyte donors to attract patients. The oocyte donor is paid nothing (except free travel and hotel stay), while the medical professional makes a handsome profit from treating infertile patients, which is not equitable. Medical professionals can also easily make a profit by marking up the price of air tickets and hotel stay to the patient (oocyte recipient). This would be thoroughly unprofessional, since the money earned is not directly related to the medical skills and expertise of the fertility specialist. Hence, it is imperative that various regulatory authorities should critically re-examine the giving of free travel and accommodation to oocyte donors, instead of monetary compensation.

Melatonin improves the quality of in vitro produced (IVP bovine embryos: implications for blastocyst development, cryotolerance, and modifications of relevant gene expression.

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Feng Wang

Full Text Available To evaluate the potential effects of melatonin on the kinetics of embryo development and quality of blastocyst during the process of in vitro bovine embryo culture. Bovine cumulus-oocyte complexes (COCs were fertilized after in vitro maturation. The presumed zygotes were cultured in in vitro culture medium supplemented with or without 10(-7 M melatonin. The cleavage rate, 8-cell rate and blastocyst rate were examined to identify the kinetics of embryo development. The hatched blastocyst rate, mortality rate after thawing and the relevant transcript abundance were measured to evaluate the quality of blastocyst. The results showed that melatonin significantly promoted the cleavage rate and 8-cell embryo yield of in vitro produced bovine embryo. In addition, significantly more blastocysts were observed by Day 7 of embryo culture at the presence of melatonin. These results indicated that melatonin accelerated the development of in vitro produced bovine embryos. Following vitrification at Day 7 of embryo culture, melatonin (10(-7 M significantly increased the hatched blastocyst rate from 24 h to 72 h and decreased the mortality rate from 48 h to 72 h after thawing. The presence of melatonin during the embryo culture resulted in a significant increase in the gene expressions of DNMT3A, OCC, CDH1 and decrease in that of AQP3 after thawing. In conclusion, melatonin not only promoted blastocyst yield and accelerated in vitro bovine embryo development, but also improved the quality of blastocysts which was indexed by an elevated cryotolerance and the up-regulated expressions of developmentally important genes.

Meiotic maturation and developmental capability of ovine oocytes at germinal vesicle stage following vitrification using different cryodevices.

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

2016-02-01

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.

Efficacy of Tuohy Needle in Oocytes Collection from Excised Mare Ovaries

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F. Cremonesi

2010-01-01

Full Text Available Two methods have been described to recover oocytes from equine follicles in excised ovaries: aspiration and scraping. Aim of this work was to develop an effective method for collecting equine oocytes using Tuohy needle and comparing this technique to aspiration and scraping, with or without tunica albuginea removal. This hollow hypodermic needle, usually employed for inserting epidural catheters, is designed with a slightly curved tip, shaped similar to a small curette. In unpeeled ovaries, the recovery rates of Tuohy needle group was higher (<.05 than in the 16 g needle aspiration and in the scraped ovaries (57% versus 36% and 47% while the rate of cumulus-intact oocytes was higher than aspiration (46.9% versus 39.36% but lower than scraping (46.97% (<.001. In unpeeled ovaries there was significant difference in maturation rate of oocytes recovered by Tuohy needle in respect to peeled ovaries (58.54% versus 50.17%, resp.. Combination of aspiration and scraping by Tuohy needle allows a faster and reliable collection of oocytes suitable for horse IVM.

Merotelic kinetochore attachment in oocyte meiosis II causes sister chromatids segregation errors in aged mice.

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Cheng, Jin-Mei; Li, Jian; Tang, Ji-Xin; Hao, Xiao-Xia; Wang, Zhi-Peng; Sun, Tie-Cheng; Wang, Xiu-Xia; Zhang, Yan; Chen, Su-Ren; Liu, Yi-Xun

2017-08-03

Mammalian oocyte chromosomes undergo 2 meiotic divisions to generate haploid gametes. The frequency of chromosome segregation errors during meiosis I increase with age. However, little attention has been paid to the question of how aging affects sister chromatid segregation during oocyte meiosis II. More importantly, how aneuploid metaphase II (MII) oocytes from aged mice evade the spindle assembly checkpoint (SAC) mechanism to complete later meiosis II to form aneuploid embryos remains unknown. Here, we report that MII oocytes from naturally aged mice exhibited substantial errors in chromosome arrangement and configuration compared with young MII oocytes. Interestingly, these errors in aged oocytes had no impact on anaphase II onset and completion as well as 2-cell formation after parthenogenetic activation. Further study found that merotelic kinetochore attachment occurred more frequently and could stabilize the kinetochore-microtubule interaction to ensure SAC inactivation and anaphase II onset in aged MII oocytes. This orientation could persist largely during anaphase II in aged oocytes, leading to severe chromosome lagging and trailing as well as delay of anaphase II completion. Therefore, merotelic kinetochore attachment in oocyte meiosis II exacerbates age-related genetic instability and is a key source of age-dependent embryo aneuploidy and dysplasia.

Patterns of oocyte development in natural habitat and captive Salminus hilarii Valenciennes, 1850 (Teleostei: Characidae).

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Honji, R M; Narcizo, A M; Borella, M I; Romagosa, E; Moreira, R G

2009-03-01

Fecundity and oocyte development in Salminus hilarii female brood stock were analyzed with the aim of investigating the impact of migration impediment on oogenesis. Histological analyses of the ovaries were performed in adult females caught in two different environments--the Tietê River (natural) and captivity--and the gonadossomatic index, oocyte diameter and fecundity determined. Five germ cell development stages (oogonium, perinucleolar, cortical alveoli, vitellogenic, ripe) and two other structures (postovulatory follicles and atretic oocytes) were observed in females caught in the river. Captive animals lacked the ripe oocytes and postovulatory follicles and had a relatively higher number of atretic oocytes. Females in captivity are known to produce larger oocytes, and they release fewer eggs in each spawn (absolute fecundity) when compared with animals that are able to migrate. Our results suggest that the Tietê River is undergoing alterations which are being reflected in the reproductive performance of S. hilarii, mainly due to the presence of atretic oocytes in females caught in the river. The lack of postovulatory follicles and ripe oocytes in captive animals reveals that migratory impediment negatively impacts final oocyte maturation. However, the stage of maturation reached is adequate for ovulation induction with hormone manipulation.

Expanding reproductive lifespan: a cost-effectiveness study on oocyte freezing

NARCIS (Netherlands)

van Loendersloot, L. L.; Moolenaar, L. M.; Mol, B. W. J.; Repping, S.; van der Veen, F.; Goddijn, M.

2011-01-01

The average age of women bearing their first child has increased strongly. This is an important reproductive health problem as fertility declines with increasing female age. Unfortunately, IVF using fresh oocytes cannot compensate for this age-related fertility decline. Oocyte freezing could be a

Two Dimensional Finite Element Model to Study Calcium Distribution in Oocytes

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Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2015-06-01

Cytosolic free calcium concentration is a key regulatory factor and perhaps the most widely used means of controlling cellular function. Calcium can enter cells through different pathways which are activated by specific stimuli including membrane depolarization, chemical signals and calcium depletion of intracellular stores. One of the important components of oocyte maturation is differentiation of the Ca2+ signaling machinery which is essential for egg activation after fertilization. Eggs acquire the ability to produce the fertilization-specific calcium signal during oocyte maturation. The calcium concentration patterns required during different stages of oocyte maturation are still not completely known. Also the mechanisms involved in calcium dynamics in oocyte cell are still not well understood. In view of above a two dimensional FEM model has been proposed to study calcium distribution in an oocyte cell. The parameters such as buffers, ryanodine receptor, SERCA pump and voltage gated calcium channel are incorporated in the model. Based on the biophysical conditions the initial and boundary conditions have been framed. The model is transformed into variational form and Ritz finite element method has been employed to obtain the solution. 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, SERCA pump and VGCC on calcium distribution in an oocyte cell.

Integration of immunodeficiency virus in oocytes via intracytoplasmic injection: possible but extremely unlikely

NARCIS (Netherlands)

Steenvoorden, Marjan M. C.; Cornelissen, Marion; van Leeuwen, Elisabeth; Schuurman, Nancy M.; Egberink, Herman F.; Berkhout, Ben; van der Veen, Fulco; Repping, Sjoerd

2012-01-01

Objective: To determine if human oocytes can be infected with HIV-1 via intracytoplasmic injection and to determine the infection threshold. Design: Twenty-eight donated immature and unfertilized human oocytes from HIV-negative women were injected with 4 x 10(4) HIV-1 virions and 13 oocytes were

Ovarian ageing: the role of mitochondria in oocytes and follicles.

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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

2016-11-01

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

Production of giant mouse oocyte nucleoli and assessment of their protein content.

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Fulka, Helena; Martinkova, Stanislava; Kyogoku, Hirohisa; Langerova, Alena; Fulka, Josef

2012-01-01

Compared with advanced developmental stage embryos and somatic cells, fully grown mammalian oocytes contain specific nucleolus-like structures (NPB - nucleolus precursor bodies). It is commonly accepted that they serve as a store of material(s) from which typical nucleoli are gradually formed. Whilst nucleoli from somatic cells can be collected relatively easily for further biochemical analyses, a sufficient number of oocyte nucleoli is very difficult to obtain. We have found that isolated oocytes nucleoli fuse very efficiently when contact is established between them. Thus, well visible giant nucleoli can be obtained, relatively easily handled and then used for further biochemical analyses. With the use of colloidal gold staining, we estimated that a single fully grown mouse oocyte nucleolus contains approximately 1.6 ng of protein. We do believe that this approach will accelerate further research aiming at analyzing the composition of oocyte nucleoli in more detail.

Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation

International Nuclear Information System (INIS)

Han, Jun; Wang, Qiao-Chu; Zhu, Cheng-Cheng; Liu, Jun; Zhang, Yu; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

2016-01-01

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications. - Highlights: • DON exposure disrupted meiotic spindle by reducing p-MAPK expression. • DON exposure caused retardation of cell cycle progression in porcine oocytes. • DON triggered autophagy and early-apoptosis in porcine oocytes. • DON exposure led to aberrant epigenetic modifications in porcine oocytes.

Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation

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Han, Jun; Wang, Qiao-Chu; Zhu, Cheng-Cheng; Liu, Jun; Zhang, Yu [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Cui, Xiang-Shun; Kim, Nam-Hyung [Department of Animal Science, Chungbuk National University, Cheongju 361-763 (Korea, Republic of); Sun, Shao-Chen, E-mail: sunsc@njau.edu.cn [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China)

2016-06-01

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications. - Highlights: • DON exposure disrupted meiotic spindle by reducing p-MAPK expression. • DON exposure caused retardation of cell cycle progression in porcine oocytes. • DON triggered autophagy and early-apoptosis in porcine oocytes. • DON exposure led to aberrant epigenetic modifications in porcine oocytes.

Stimulation of mitochondria during maturation influences development and gene expression in bovine embryos derived from oocytes with differenet meiotic competence.

Czech Academy of Sciences Publication Activity Database

Hulínská, P.; Hanzalová, K.; Knitlová, D.; Němcová, Lucie; Kaňka, Jiří; Jeseta, M.; Machatková, M.

2016-01-01

Roč. 49, č. 4 (2016), s. 174-174 ISSN 1335-3683. [International Scientific Conference "Animal Biotechnology " /4./. 08.12.2016-08.12.2016, Nitra] R&D Projects: GA MZe(CZ) QJ1510138 Institutional support: RVO:67985904 Keywords : bovine embryos Subject RIV: EB - Genetics ; Molecular Biology

The influence of ovarian hyperstimulation drugs on morphometry and morphology of human oocytes in ICSI program.

Science.gov (United States)

Taheri, Fatemeh; Alemzadeh Mehrizi, Arezoo; Khalili, Mohammad Ali; Halvaei, Iman

2018-04-01

To compare the influences of controlled ovarian hyperstimulation (COH) drugs using recombinant follicular stimulating hormone (rFSH) versus human menopausal gonadotropins (hMG) on morphometry and morphology of MII oocytes in ICSI cycles. In this prospective study, 363 MII oocytes from 50 ICSI cycles with male factor infertility were evaluated. The patients were divided into two groups according to the protocols of COH: I- rFSH and II- hMG. The immature oocytes were excluded from the study. All oocytes were categorized into four morphological groups of normal, and those with single, double, or multiple defects. The inclusive morphometrical criteria were: areas and diameters of oocyte, ooplasm, and zona pellucida (ZP). Also, circumferences of oocyte and ooplasm were assessed. The ZP area and ooplasm diameter for both normal and abnormal oocytes were significantly higher in group I (P: .05; P: .028, respectively) compared to group II (P: .023; P: .003, respectively). In abnormal oocytes, ooplasm diameter was higher in group I compared to group II. Furthermore, ooplasm area for abnormal oocytes was significantly higher in group I compared to group II. There was an increasing trend for number of mature oocytes, in abnormal oocytes, for group I (5.53 ± 3.1) in comparison with group II (4.4 ± 2.97; P = .25). The rate of oocytes with normal morphology was significantly higher in hMG, when compared to rFSH groups. Morphometrical parameters were increased in rFSH group, but the normal morphology of oocytes were significantly enhanced in hMG group. Treatment with proper dosage of ovulation induction drugs may enhance the number of normal sized oocytes. Copyright © 2018. Published by Elsevier B.V.

Effects of Aroclor 1254 on in vivo oocyte maturation in the mouse.

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ShuZhen Liu

Full Text Available Polychlorinated biphenyls (PCBs are stable, lipophilic compounds that accumulate in the environment and in the food chain. Though some studies provided evidence that PCBs had adverse effects on reproductive function, most of these results were from in vitro models. Therefore we investigated the effect of Aroclor 1254 (a commercial PCBs mixture treatments on in vivo maturation and developmental potential of mouse oocytes. In the present study, female ICR mice were treated with different doses (12.5, 25 and 50 mg/kg of Aroclor 1254 (a commercial PCB mixture once every 72 hours by intraperitoneal injection for 9 days. After three treatments of Aroclor 1254, the mice were superovulated to collect oocytes one day after the last exposure. The effects of Aroclor 1254 on oocyte maturation, fertilization, and preimplantation embryonic development were investigated. Immunofluorescence-stained oocytes were observed under a confocal microscope to assess the effects of Aroclor 1254 on spindle morphology. Parthenogenic activation and the incidence of cumulus apoptosis in cumulus-oocyte complexes were observed as well. Oocytes exposed to different doses of Aroclor 1254 in vivo were associated with a significant decrease in outgrowth potential, abnormal spindle configurations, and the inhibition of parthenogenetic activation of ovulated oocytes. Furthermore, the incidence of apoptosis in cumulus cells was increased after exposed to Aroclor 1254. These results may provide reference for the treatment of reproductive diseases such as infertility or miscarriage caused by environmental contaminants.

Fasciola hepatica: a light and electron microscope study of the ovary and of the development of oocytes within eggs in the uterus provides an insight into reproductive strategy.

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Hanna, R E B; Moffett, D; Forster, F I; Trudgett, A G; Brennan, G P; Fairweather, I

2016-05-15

The ultrastructure of the ovary of Fasciola hepatica collected from field-infected sheep, was compared with that of flukes from laboratory-infected rats harbouring the Oberon or the Cullompton fluke isolate. At the periphery of the ovarian tubules, in all flukes, interstitial tissue was identified that appears to provide physical support and facilitate the metabolism of the germinal-line cells. Oogonia undergo mitotic division to maintain the cell population and to produce oocytes. Early oocytes feature conspicuous synaptonemal complexes in the nucleoplasm, and these become less evident as the oocytes grow in size, move towards the core of the ovarian tubule, and synthesise osmiophilic bodies. The latter may represent cortical granules, and serve to block polyspermy. The identity of the synaptonemal complexes was confirmed by immunocytochemical labelling of synaptonemal proteins. The occurrence of synaptonemal complexes in the oocytes of all fluke types examined indicates that pairing of bivalent chromosomes, with the potential for genetic recombination and chiasmata formation, is a feature of the triploid aspermic parthenogenetic Cullompton flukes, as well as of the wild-type out-breeding field-derived and Oberon isolate flukes. In oocytes within shelled eggs in the proximal uterus of all flukes, condensed chromosomes align at meiotic metaphase plates. Following the reduction division, two equal pronuclei appear in each oocyte in the distal uterus. On the basis of these observations, a mechanism of facultative parthenogenesis for F. hepatica is proposed that accommodates the survival and clonal expansion of triploid aspermic isolates. Copyright © 2016 Elsevier B.V. All rights reserved.

Follicle Size on Day of Trigger Most Likely to Yield a Mature Oocyte

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Ali Abbara

2018-04-01

Full Text Available ObjectiveTo identify follicle sizes on the day of trigger most likely to yield a mature oocyte following hCG, GnRH agonist (GnRHa, or kisspeptin during IVF treatment.DesignRetrospective analysis to determine the size of follicles on day of trigger contributing most to the number of mature oocytes retrieved using generalized linear regression and random forest models applied to data from IVF cycles (2014–2017 in which either hCG, GnRHa, or kisspeptin trigger was used.SettingHCG and GnRHa data were collected at My Duc Hospital, Ho Chi Minh City, Vietnam, and kisspeptin data were collected at Hammersmith Hospital, London, UK.PatientsFour hundred and forty nine women aged 18–38 years with antral follicle counts 4–87 were triggered with hCG (n = 161, GnRHa (n = 165, or kisspeptin (n = 173.Main outcome measureFollicle sizes on the day of trigger most likely to yield a mature oocyte.ResultsFollicles 12–19 mm on the day of trigger contributed the most to the number of oocytes and mature oocytes retrieved. Comparing the tertile of patients with the highest proportion of follicles on the day of trigger 12–19 mm, with the tertile of patients with the lowest proportion within this size range, revealed increases of 4.7 mature oocytes for hCG (P < 0.0001 and 4.9 mature oocytes for GnRHa triggering (P < 0.01. Using simulated follicle size profiles of patients with 20 follicles on the day of trigger, our model predicts that the number of oocytes retrieved would increase from a mean 9.8 (95% prediction limit 9.3–10.3 to 14.8 (95% prediction limit 13.3–16.3 oocytes due to the difference in follicle size profile alone.ConclusionFollicles 12–19 mm on the morning of trigger administration were most likely to yield a mature oocyte following hCG, GnRHa, or kisspeptin.

Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro.

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Shuzhen Liu

Full Text Available 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.

Prooxidant Effects of Verbascoside, a Bioactive Compound from Olive Oil Mill Wastewater, on In Vitro Developmental Potential of Ovine Prepubertal Oocytes and Bioenergetic/Oxidative Stress Parameters of Fresh and Vitrified Oocytes

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Dell'Aquila, M. E.; Bogliolo, L.; Russo, R.; Martino, N. A.; Filioli Uranio, M.; Ariu, F.; Amati, F.; Sardanelli, A. M.; Linsalata, V.; Ferruzzi, M. G.; Cardinali, A.; Minervini, F.

2014-01-01

Verbascoside (VB) is a bioactive polyphenol from olive oil mill wastewater with known antioxidant activity. Oxidative stress is an emerging problem in assisted reproductive technology (ART). Juvenile ART is a promising topic because, in farm animals, it reduces the generation gap and, in human reproductive medicine, it helps to overcome premature ovarian failure. The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. In fresh oocytes, VB exerted prooxidant short-term effects, that is, catalase activity increase and uncoupled increases of mitochondria and reactive oxygen species (ROS) fluorescence signals, and long-term effects, that is, reduced blastocyst formation rate. In vitrified oocytes, VB increased ROS levels. Prooxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of in vitro maturation culture, may have contributed to significant increase of oocyte oxidation. Further studies are needed to identify lower concentrations and/or shorter exposure times to figure out VB antioxidant effects in juvenile ARTs. PMID:24719893

Prooxidant Effects of Verbascoside, a Bioactive Compound from Olive Oil Mill Wastewater, on In Vitro Developmental Potential of Ovine Prepubertal Oocytes and Bioenergetic/Oxidative Stress Parameters of Fresh and Vitrified Oocytes

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M. E. Dell'Aquila

2014-01-01

Full Text Available Verbascoside (VB is a bioactive polyphenol from olive oil mill wastewater with known antioxidant activity. Oxidative stress is an emerging problem in assisted reproductive technology (ART. Juvenile ART is a promising topic because, in farm animals, it reduces the generation gap and, in human reproductive medicine, it helps to overcome premature ovarian failure. The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. In fresh oocytes, VB exerted prooxidant short-term effects, that is, catalase activity increase and uncoupled increases of mitochondria and reactive oxygen species (ROS fluorescence signals, and long-term effects, that is, reduced blastocyst formation rate. In vitrified oocytes, VB increased ROS levels. Prooxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of in vitro maturation culture, may have contributed to significant increase of oocyte oxidation. Further studies are needed to identify lower concentrations and/or shorter exposure times to figure out VB antioxidant effects in juvenile ARTs.

Cytoplasmic movement profiles of mouse surrounding nucleolus and not-surrounding nucleolus antral oocytes during meiotic resumption.

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Bui, Thi Thu Hien; Belli, Martina; Fassina, Lorenzo; Vigone, Giulia; Merico, Valeria; Garagna, Silvia; Zuccotti, Maurizio

2017-05-01

Full-grown mouse antral oocytes are classified as surrounding nucleolus (SN) or not-surrounding nucleolus (NSN), depending on the respective presence or absence of a ring of Hoechst-positive chromatin surrounding the nucleolus. In culture, both types of oocytes resume meiosis and reach the metaphase II (MII) stage, but following insemination, NSN oocytes arrest at the two-cell stage whereas SN oocytes may develop to term. By coupling time-lapse bright-field microscopy with image analysis based on particle image velocimetry, we provide the first systematic measure of the changes to the cytoplasmic movement velocity (CMV) occurring during the germinal vesicle-to-MII (GV-to-MII) transition of these two types of oocytes. Compared to SN oocytes, NSN oocytes display a delayed GV-to-MII transition, which can be mostly explained by retarded germinal vesicle break down and first polar body extrusion. SN and NSN oocytes also exhibit significantly different CMV profiles at four main time-lapse intervals, although this difference was not predictive of SN or NSN oocyte origin because of the high variability in CMV. When CMV profile was analyzed through a trained artificial neural network, however, each single SN or NSN oocyte was blindly identified with a probability of 92.2% and 88.7%, respectively. Thus, the CMV profile recorded during meiotic resumption may be exploited as a cytological signature for the non-invasive assessment of the oocyte developmental potential, and could be informative for the analysis of the GV-to-MII transition of oocytes of other species. © 2017 Wiley Periodicals, Inc.

Relationship between time post-ovulation and progesterone on oocyte maturation and pregnancy in canine cloning.

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Kim, Joung Joo; Park, Kang Bae; Choi, Eun Ji; Hyun, Sang Hwan; Kim, Nam-Hyung; Jeong, Yeon Woo; Hwang, Woo Suk

2017-10-01

Canine oocytes ovulated at prophase complete meiosis and continue to develop in presence of a high progesterone concentration in the oviduct. Considering that meiotic competence of canine oocyte is accomplished in the oviductal environment, we postulate that hormonal milieu resulting from the circulating progesterone concentration may affect oocyte maturation and early development of embryos. From 237 oocyte donors, 2620 oocytes were collected and their meiotic status and morphology were determined. To determine optimal characteristics of the mature oocytes subjected to nuclear transfer, a proportion of the meiotic status of the oocytes were classified in reference to time post-ovulation as well as progesterone (P4) level. A high proportion of matured oocytes were collected from >126h (55.5%) post-ovulation or 40-50ngmL -1 (46.4%) group compared to the other groups. Of the oocyte donors that provided mature oocytes in vivo, there was no correlation between serum progesterone of donors and time post ovulation, however, time post-ovulation were significantly shorter for cloned embryos were reconstructed and transferred into 77 surrogates. In order to determine the relationship between pregnancy performance and serum progesterone level, embryos were transferred into surrogates showing various P4 serum levels. The highest pregnancy (31.8%) and live birth cloning efficacy (2.2%) rates were observed when the embryos were transferred into surrogates with circulating P4 levels were from 40 to 50ngmL -1 . In conclusion, measurement of circulating progesterone of female dog could be a suitable an indicator of the optimal time to collect quality oocyte and to select surrogates for cloning. Copyright © 2017 Elsevier B.V. All rights reserved.

Obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy.

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

2017-06-01

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.

Daam1 regulates fascin for actin assembly in mouse oocyte meiosis.

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Lu, Yujie; Zhang, Yu; Pan, Meng-Hao; Kim, Nam-Hyung; Sun, Shao-Chen; Cui, Xiang-Shun

2017-07-18

As a formin protein, Daam1 (Dishevelled-associated activator of morphogenesis 1) is reported to regulate series of cell processes like endocytosis, cell morphology and migration via its effects on actin assembly in mitosis. However, whether Daam1 plays roles in female meiosis remains uncertain. In this study, we investigated the expression and functions of Daam1 during mouse oocyte meiosis. Our results indicated that Daam1 localized at the cortex of oocytes, which was similar with actin filaments. After Daam1 morpholino (MO) microinjection, the expression of Daam1 significantly decreased, which resulted in the failure of oocyte polar body extrusion. These results might be due to the defects of actin assembly, since the decreased fluorescence intensity of actin filaments in oocyte cortex and cytoplasm were observed. However, Daam1 knockdown seemed not to affect the meiotic spindle movement. In addition, we found that fascin might be the down effector of Daam1, since the protein expression of fascin decreased after Daam1 knockdown. Thus, our data suggested that Daam1 affected actin assembly during oocyte meiotic division via the regulation of fascin expression.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs.

Science.gov (United States)

Peter, A B; Schittny, J C; Niggli, V; Reuter, H; Sigel, E

1991-08-01

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Wolbachia utilizes host microtubules and Dynein for anterior localization in the Drosophila oocyte.

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Patrick M Ferree

2005-10-01

Full Text Available To investigate the role of the host cytoskeleton in the maternal transmission of the endoparasitic bacteria Wolbachia, we have characterized their distribution in the female germ line of Drosophila melanogaster. In the germarium, Wolbachia are distributed to all germ cells of the cyst, establishing an early infection in the cell destined to become the oocyte. During mid-oogenesis, Wolbachia exhibit a distinct concentration between the anterior cortex and the nucleus in the oocyte, where many bacteria appear to contact the nuclear envelope. Following programmed rearrangement of the microtubule network, Wolbachia dissociate from this anterior position and become dispersed throughout the oocyte. This localization pattern is distinct from mitochondria and all known axis determinants. Manipulation of microtubules and cytoplasmic Dynein and Dynactin, but not Kinesin-1, disrupts anterior bacterial localization in the oocyte. In live egg chambers, Wolbachia exhibit movement in nurse cells but not in the oocyte, suggesting that the bacteria are anchored by host factors. In addition, we identify mid-oogenesis as a period in the life cycle of Wolbachia in which bacterial replication occurs. Total bacterial counts show that Wolbachia increase at a significantly higher rate in the oocyte than in the average nurse cell, and that normal Wolbachia levels in the oocyte depend on microtubules. These findings demonstrate that Wolbachia utilize the host microtubule network and associated proteins for their subcellular localization in the Drosophila oocyte. These interactions may also play a role in bacterial motility and replication, ultimately leading to the bacteria's efficient maternal transmission.

The Effects of Progesterone on Oocyte Maturation and Embryo Development

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Saeed Zavareh

2013-01-01

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.

Number and Quality of Oocytes Collected from Heterotopic Autografted Mice Ovary after PMSG Induction

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NURBARIAH

2011-12-01

Full Text Available Heterotopic grafting sites can be useful in producing oocytes for in vitro Fertilization, therefore, maximising the oocyte yield from the graft by gonadotrophin stimulation would be advantageous. The aim of this study was to investigate the number and quality of oocytes collected from heterotopic autografted ovary after Pregnant Mare Serum Gonadothropin (PMSG induction. Graft recipients were treated either with or without PMSG stimulation 48 hours prior to graft collection. Ovarian tissue from four weeks old mice (DDY strain were autotransplanted under the kidney capsule of the same ovariectomized mice and the oocytes were collected 21 days after autotransplantation. The results showed that the average number of oocytes collected from autografted ovaries without PMSG induction were 9.0. ± 2.8 not significantly different with those received PMSG induction, 10.9 ± 5.1. The percentage of matured and fertilized oocytes and the developed embryos from the autografted ovaries without PMSG induction were 52.4, 33.4, and 26.0%, respectively not significantly different with those received PMSG induction, 53.2, 35.1, and 29.9%, respectively. The number of oocytes and the capacity to matured, fertilized and developed were significantly lower (P < 0.05 compared to the superovulated nongrafted (control ovaries. In conclusion, PMSG induction on the graft recipients did not significantly increase oocytes yield from grafted heterotopic ovaries. The number and quality of oocytes produced from the autografted ovaries were lower than the superovulated nongrafted ovaries, but still can be used for in vitro embryo production after sequential in vitro maturation and fertilization.

Generation of meiomaps of genome-wide recombination and chromosome segregation in human oocytes

DEFF Research Database (Denmark)

Ottolini, Christian S; Capalbo, Antonio; Newnham, Louise

2016-01-01

We have developed a protocol for the generation of genome-wide maps (meiomaps) of recombination and chromosome segregation for the three products of human female meiosis: the first and second polar bodies (PB1 and PB2) and the corresponding oocyte. PB1 is biopsied and the oocyte is artificially......-nucleotide polymorphisms (SNPs) genome-wide by microarray. Informative maternal heterozygous SNPs are phased using a haploid PB2 or oocyte as a reference. A simple algorithm is then used to identify the maternal haplotypes for each chromosome, in all of the products of meiosis for each oocyte. This allows mapping...

Protein incorporation by isolated amphibian oocytes. VI. Comparison of autologous and xenogeneic vitellogenins

Energy Technology Data Exchange (ETDEWEB)

Wallace, R A; Deufel, R A; Misulovin, Z

1980-01-01

1. Labeled vitellogenins were isolated from the sera of several amphibians, a turtle, and a pigeon, and were incubated in vitro with oocytes from Xenopus laevis and Rana pipiens. 2. Oocytes from X. laevis sequestered vitellogenin from salamanders, turtle, and pigeon at rates comparable to that for autologous vitellogenin, while anuran vitellogenins were sequestered at somewhat lower rates. 3. Oocytes from R. pipiens sequestered X. laevis vitellogenin at a rate comparable to autologous vitellogenin, while salamander, turtle, and pigeon vitellogenins were sequestered at faster rates. 4. All vitellogenins examined appear to be recognized and incorporated specifically by X. laevis and R. pipiens oocytes.

Cryopreservation of Mammalian Oocyte for Conservation of Animal Genetics

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Jennifer R. Prentice

2011-01-01

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.

Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm

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J. E. Hernández-Pichardo

2016-11-01

Full Text Available Abstract Background In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up (SU or swim up + zona pellucida (SU + ZP binding. Results Experiment 1, 4–20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation (precense of one PN. Treatments showed similar results (54, 47, 42 %, respectively but statistically differents (P  0.05. Conclusions Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU + ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU + ZP were significantly different than frozen-thawed sperm, but between sperm treatments no significant differences were obtained.

Molecular Mechanisms Responsible for Increased Vulnerability of the Ageing Oocyte to Oxidative Damage

Science.gov (United States)

Redgrove, Kate A.; McLaughlin, Eileen A.

2017-01-01

In their midthirties, women experience a decline in fertility, coupled to a pronounced increase in the risk of aneuploidy, miscarriage, and birth defects. Although the aetiology of such pathologies are complex, a causative relationship between the age-related decline in oocyte quality and oxidative stress (OS) is now well established. What remains less certain are the molecular mechanisms governing the increased vulnerability of the aged oocyte to oxidative damage. In this review, we explore the reduced capacity of the ageing oocyte to mitigate macromolecular damage arising from oxidative insults and highlight the dramatic consequences for oocyte quality and female fertility. Indeed, while oocytes are typically endowed with a comprehensive suite of molecular mechanisms to moderate oxidative damage and thus ensure the fidelity of the germline, there is increasing recognition that the efficacy of such protective mechanisms undergoes an age-related decline. For instance, impaired reactive oxygen species metabolism, decreased DNA repair, reduced sensitivity of the spindle assembly checkpoint, and decreased capacity for protein repair and degradation collectively render the aged oocyte acutely vulnerable to OS and limits their capacity to recover from exposure to such insults. We also highlight the inadequacies of our current armoury of assisted reproductive technologies to combat age-related female infertility, emphasising the need for further research into mechanisms underpinning the functional deterioration of the ageing oocyte. PMID:29312475

Possible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography.

Science.gov (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

2012-09-01

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.

Effect of Cumulus cell co-culture and Protein Supplement on Success of in vitro Fertilization and Development of Pre-implanted Embryos in mice

Directory of Open Access Journals (Sweden)

Muhammad-Baqir M-R. Fakhrildin

2005-06-01

Full Text Available Successful oocyte fertilization and normal embryonic development of mice were considered the most important diagnostic criteria for the safety of materials and tools used for human in vitro fertilization and embryo transfer (IVF-ET. Therefore, we studied the influence of cumulus cells co-culture and protein supplement within culture medium on percentages of in vitro fertilization (IVF and normal development of early stages of mouse embryo later. Oocytes were collected and treated with hyaluronidase to remove cumulus cells. Oocytes were divided into four groups namely: Group-1: Oocytes incubated within modified Earl’s medium (MEM supplied with 10% inactivated bovine amniotic fluid as a protein source and cumulus cells; Group-2: Oocytes incubated with MEM supplied with cumulus cells only; Group-3: Oocytes incubated with MEM supplied with 10% inactivated bovine amniotic fluid only; and Group-4: Oocytes  incubated with MEM free of both protein source and cumulus cells. For IVF, 5-6 oocytes were incubated with active spermatozoa under paraffin oil for 18-20 hours at 37° oC in 5% CO2. Percentages of IVF and embryonic development were then recorded. Best results for IVF and normal embryonic development were achieved from oocytes of Group-1 when compared to the other groups. As compared to Group-1, the percentage of IVF for Group-2 and Group-3 were decreased insignificantly and significantly (P<0.002, respectively. Significant (P<0.01 reduction in the percentages of IVF and normal embryonic development were reported in Group-4 as compared to Group-1. Therefore, it was concluded that the presence of cumulus cells co-culture and bovine amniotic fluid as a protein source within culture medium may have an important role on the fertilizing capacity of spermatozoa and oocytes and normal development of pre-implanted mouse embryo later.

Evaluation of zona pellucida birefringence intensity during in vitro maturation of oocytes from stimulated cycles.

Science.gov (United States)

Petersen, Claudia G; Vagnini, Laura D; Mauri, Ana L; Massaro, Fabiana C; Silva, Liliane F I; Cavagna, Mario; Baruffi, Ricardo L R; Oliveira, Joao B A; Franco, José G

2011-04-23

This study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF) intensity and the nuclear (NM) and cytoplasmic (CM) in vitro maturation of human oocytes from stimulated cycles. The ZP-BF was evaluated under an inverted microscope with a polarizing optical system and was scored as high/positive (when the ZP image presented a uniform and intense birefringence) or low/negative (when the image presented moderate and heterogeneous birefringence). CM was analyzed by evaluating the distribution of cortical granules (CGs) throughout the ooplasm by immunofluorescence staining. CM was classified as: complete, when CG was localized in the periphery; incomplete, when oocytes presented a cluster of CGs in the center; or in transition, when oocytes had both in clusters throughout cytoplasm and distributed in a layer in the cytoplasm periphery Nuclear maturation: From a total of 83 germinal vesicle (GV) stage oocytes, 58 of oocytes (69.9%) reached NM at the metaphase II stage. From these 58 oocytes matured in vitro, the high/positively scoring ZP-BF was presented in 82.7% of oocytes at the GV stage, in 75.8% of oocytes when at the metaphase I, and in 82.7% when oocytes reached MII. No relationship was observed between NM and ZP-BF positive/negative scores (P = 0.55). These variables had a low Pearson's correlation coefficient (r = 0.081). Cytoplasmic maturation: A total of 85 in vitro-matured MII oocytes were fixed for CM evaluation. Forty-nine oocytes of them (57.6%) showed the complete CM, 30 (61.2%) presented a high/positively scoring ZP-BF and 19 (38.8%) had a low/negatively scoring ZP-BF. From 36 oocytes (42.3%) with incomplete CM, 18 (50%) presented a high/positively scoring ZPBF and 18 (50%) had a low/negatively scoring ZP-BF. No relationship was observed between CM and ZP-BF positive/negative scores (P = 0.42). These variables had a low Pearson's correlation coefficient (r = 0.11). The current study demonstrated an absence of

Evaluation of zona pellucida birefringence intensity during in vitro maturation of oocytes from stimulated cycles

Directory of Open Access Journals (Sweden)

Silva Liliane FI

2011-04-01

Full Text Available Abstract Background This study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF intensity and the nuclear (NM and cytoplasmic (CM in vitro maturation of human oocytes from stimulated cycles. Results The ZP-BF was evaluated under an inverted microscope with a polarizing optical system and was scored as high/positive (when the ZP image presented a uniform and intense birefringence or low/negative (when the image presented moderate and heterogeneous birefringence. CM was analyzed by evaluating the distribution of cortical granules (CGs throughout the ooplasm by immunofluorescence staining. CM was classified as: complete, when CG was localized in the periphery; incomplete, when oocytes presented a cluster of CGs in the center; or in transition, when oocytes had both in clusters throughout cytoplasm and distributed in a layer in the cytoplasm periphery Nuclear maturation: From a total of 83 germinal vesicle (GV stage oocytes, 58 of oocytes (69.9% reached NM at the metaphase II stage. From these 58 oocytes matured in vitro, the high/positively scoring ZP-BF was presented in 82.7% of oocytes at the GV stage, in 75.8% of oocytes when at the metaphase I, and in 82.7% when oocytes reached MII. No relationship was observed between NM and ZP-BF positive/negative scores (P = 0.55. These variables had a low Pearson's correlation coefficient (r = 0.081. Cytoplasmic maturation: A total of 85 in vitro-matured MII oocytes were fixed for CM evaluation. Forty-nine oocytes of them (57.6% showed the complete CM, 30 (61.2% presented a high/positively scoring ZP-BF and 19 (38.8% had a low/negatively scoring ZP-BF. From 36 oocytes (42.3% with incomplete CM, 18 (50% presented a high/positively scoring ZPBF and 18 (50% had a low/negatively scoring ZP-BF. No relationship was observed between CM and ZP-BF positive/negative scores (P = 0.42. These variables had a low Pearson's correlation coefficient (r = 0.11. Conclusions The current

In vitro fertilization of in vitro matured canine oocytes using frozen-thawed dog semen.

Science.gov (United States)

De los Reyes, M; Carrion, R; Barros, C

2006-10-01

Experiments were conducted to evaluate in vitro fertilization (IVF) of in vitro matured (IVM) bitch oocytes using dog spermatozoa frozen in three different extenders. Sperm-rich fraction from eight ejaculates of five dogs was frozen in each one of three egg yolk Tris extenders with additional: (A) 1.4 g citric acid and 0.8 g glucose; (B) 0.7 g citric acid and 3.5 g glucose; or (C) 1.4 g citric acid and 0.8 g fructose (all with 5% glycerol in 100 mL milliQ water). Thawed sperm were co-incubated with IVM bitch oocytes for 6 h. Oocytes were fixed and evaluated under an epifluorescence microscope; penetrated oocytes were defined as those having sperm heads in the perivitelline space or in the oocyte cytoplasm. Higher penetration rates (P < 0.05) were obtained in oocytes cultured with spermatozoa frozen in extenders B and C than those frozen in extender A (33.1, 34.2 and 26.4%, respectively).

Public support for intergenerational oocyte donation in the United States.

Science.gov (United States)

Bortoletto, Pietro; Farland, Leslie V; Ginsburg, Elizabeth S; Goldman, Randi H

2018-02-01

To determine whether the general public supports intergenerational oocyte donation. Cross-sectional study. Not applicable. A nationally representative sample based on age distribution of United States residents. Not applicable. Characteristics of respondents who supported (strongly agree and agree) various oocyte donation practices were compared with participants who did not support them (disagree and strongly disagree) using log binomial regression to calculate risk ratios (RRs) and 95% confidence intervals of support (95% CIs). Models were adjusted for age, gender, and religion to yield adjusted risk ratios (aRR). A total of 1,915 people responded to the Web-based survey; 53% were female, and 24% were racial/ethnic minorities. Eighty-five percent had prior knowledge of oocyte donation, and 74% felt that a woman should be able to donate oocytes to a family member. The desire to help a family member was the most commonly perceived motivation for donors (79%). Christian-Catholics compared with Christian-non-Catholics (aRR 0.91, 95% CI 0.86-0.98), African Americans compared with non-Hispanic Caucasians (aRR 0.86, 95% CI 0.76-0.97), and Republicans compared with Democrats (RR 0.93, 95% CI 0.88-0.98) were less likely to support intergenerational oocyte donation. Respondents with three or more biological children (RR 1.06, 95% CI 1.00-1.11) compared with those with no children were less likely to support this practice. Eight percent of participants disapproved of donation to any family member. The most common reason for disapproval was the potential negative impact on the child (53%). A majority of Americans support the practice of intergenerational oocyte donation; however, support varies according to demographic characteristics. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Increased stress tolerance of matured pig oocytes after high hydrostatic presure

DEFF Research Database (Denmark)

Pribenszky, Cs; Du, Y; Molnár, M

2008-01-01

The present paper describes a method which uses high hydrostatic pressure as a pre-treatment to in vitro matured porcine oocytes to improve their survival rates in the subsequent processes including cryopreservation, parthenogenetic activation and embryo culture. In Experiment I oocytes were...... treated with different pressure impulses in the range of 20-80 MPa (200-800 times greater than atmospheric pressure) for 30-120 min at 24 °C. For parthenogenetic activation a single dc of 12.5 kV/cm was used, to test shock tolerance of the treated vs. control oocytes and also compare their developmental...... competence evaluated with continued in vitro development. The upper limit of pressure tolerance was found in the 40 MPa range. In Experiment II oocytes pre-treated with pressures in the 20-40 MPa range were vitrified with the Cryotop method, and parthenogenetically activated subsequently with combined...

In vitro production of bovine embryos: cumulus/granulosa cell gene expression patterns point to early atresia as beneficial for oocyte competence

DEFF Research Database (Denmark)

Mazzoni, Gianluca; Razza, Eduardo; Pedersen, Hanne S.

2017-01-01

In vitro production (IW) of bovine embryos has become widespread technology implemented in cattle breeding and production. Here, we review novel data on cumulus/granulosa cell gene expression, as determined by RNAseq on cellular material from pooled follicular fluids at the single animal level...

Epigenetic reprogramming of breast cancer cells with oocyte extracts

Directory of Open Access Journals (Sweden)

Kumari Rajendra

2011-01-01

Full Text Available Abstract Background Breast cancer is a disease characterised by both genetic and epigenetic alterations. Epigenetic silencing of tumour suppressor genes is an early event in breast carcinogenesis and reversion of gene silencing by epigenetic reprogramming can provide clues to the mechanisms responsible for tumour initiation and progression. In this study we apply the reprogramming capacity of oocytes to cancer cells in order to study breast oncogenesis. Results We show that breast cancer cells can be directly reprogrammed by amphibian oocyte extracts. The reprogramming effect, after six hours of treatment, in the absence of DNA replication, includes DNA demethylation and removal of repressive histone marks at the promoters of tumour suppressor genes; also, expression of the silenced genes is re-activated in response to treatment. This activity is specific to oocytes as it is not elicited by extracts from ovulated eggs, and is present at very limited levels in extracts from mouse embryonic stem cells. Epigenetic reprogramming in oocyte extracts results in reduction of cancer cell growth under anchorage independent conditions and a reduction in tumour growth in mouse xenografts. Conclusions This study presents a new method to investigate tumour reversion by epigenetic reprogramming. After testing extracts from different sources, we found that axolotl oocyte extracts possess superior reprogramming ability, which reverses epigenetic silencing of tumour suppressor genes and tumorigenicity of breast cancer cells in a mouse xenograft model. Therefore this system can be extremely valuable for dissecting the mechanisms involved in tumour suppressor gene silencing and identifying molecular activities capable of arresting tumour growth. These applications can ultimately shed light on the contribution of epigenetic alterations in breast cancer and advance the development of epigenetic therapies.

SIRT1 signalling protects mouse oocytes against oxidative stress and is deregulated during aging.

Science.gov (United States)

Di Emidio, Giovanna; Falone, Stefano; Vitti, Maurizio; D'Alessandro, Anna Maria; Vento, Marilena; Di Pietro, Cinzia; Amicarelli, Fernanda; Tatone, Carla

2014-09-01

Is SIRT1 involved in the oxidative stress (OS) response in mouse oocytes? SIRT1 plays a pivotal role in the adaptive response of mouse germinal vesicle (GV) oocytes to OS and promotes a signalling cascade leading to up-regulation of the MnSod gene. OS is known to continuously threaten acquisition and maintenance of oocyte developmental potential during in vivo processes and in vitro manipulations. Previous studies in somatic cells have provided strong evidence for the role of SIRT1 as a sensor of the cell redox state and a protector against OS and aging. GV oocytes obtained from young (4-8 weeks) and reproductively old (48-52 weeks) CD1 mice were blocked in the prophase stage by 0.5 µM cilostamide. Groups of 30 oocytes were exposed to 25 µM H2O2 and processed following different times for the analysis of intracellular localization of SIRT1 and FOXO3A, and evaluation of Sirt1, miRNA-132, FoxO3a and MnSod gene expression. Another set of oocytes was cultured in the presence or absence of the SIRT1-specific inhibitor Ex527, and exposed to H2O2 in order to assess the involvement of SIRT1 in the activation of a FoxO3a-MnSod axis and ROS detoxification. In the last part of this study, GV oocytes were maturated in vitro in the presence of different Ex527 concentrations (0, 2.5, 5, 10, 20 µM) and assessed for maturation rates following 16 h. Effects of Ex527 on spindle morphology and ROS levels were also evaluated. SIRT1 and FOXO3A intracellular distribution in response to OS was investigated by immunocytochemistry. Real-time RT-PCR was employed to analyse Sirt1, miR-132, FoxO3a and MnSod gene expression. Reactive oxygen species (ROS) production was evaluated by in vivo measurement of carboxy-H2DCF diacetate labelling. Spindle and chromosomal distribution in in vitro matured oocytes were analysed by immunocytochemistry and DNA fluorescent labelling, respectively. Specific changes in the intracellular localization of SIRT1 and up-regulation of Sirt1 gene were detected in

Role of ataxia-telangiectasia mutated (ATM) in porcine oocyte in vitro maturation.

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Lin, Zi-Li; Kim, Nam-Hyung

2015-06-01

Ataxia-telangiectasia mutated (ATM) is critical for the DNA damage response, cell cycle checkpoints, and apoptosis. Significant effort has focused on elucidating the relationship between ATM and other nuclear signal transducers; however, little is known about the connection between ATM and oocyte meiotic maturation. We investigated the function of ATM in porcine oocytes. ATM was expressed at all stages of oocyte maturation and localized predominantly in the nucleus. Furthermore, the ATM-specific inhibitor KU-55933 blocked porcine oocyte maturation, reducing the percentages of oocytes that underwent germinal vesicle breakdown (GVBD) and first polar body extrusion. KU-55933 also decreased the expression of DNA damage-related genes (breast cancer 1, budding uninhibited by benzimidazoles 1, and P53) and reduced the mRNA and protein levels of AKT and other cell cycle-regulated genes that are predominantly expressed during G2/M phase, including bone morphogenetic protein 15, growth differentiation factor 9, cell division cycle protein 2, cyclinB1, and AKT. KU-55933 treatment decreased the developmental potential of blastocysts following parthenogenetic activation and increased the level of apoptosis. Together, these data suggested that ATM influenced the meiotic and cytoplasmic maturation of porcine oocytes, potentially by decreasing their sensitivity to DNA strand breaks, stimulating the AKT pathway, and/or altering the expression of other maternal genes. © 2015 International Federation for Cell Biology.

Enucleolation of porcine oocytes

Czech Academy of Sciences Publication Activity Database

Fulka Jr., J.; Moor, R. M.; Loi, P.; Fulka, Josef

2003-01-01

Roč. 59, - (2003), s. 1879-1885 ISSN 0093-691X R&D Projects: GA ČR GA524/02/0032; GA MŠk LN00A065 Grant - others:Evropsá unie(XE) QKLCT-1999-00104 Institutional research plan: CEZ:AV0Z5045916 Keywords : oocyte * nucleous * maturation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.839, year: 2003

Rapid Prototyping of a Cyclic Olefin Copolymer Microfluidic Device for Automated Oocyte Culturing.

Science.gov (United States)

Berenguel-Alonso, Miguel; Sabés-Alsina, Maria; Morató, Roser; Ymbern, Oriol; Rodríguez-Vázquez, Laura; Talló-Parra, Oriol; Alonso-Chamarro, Julián; Puyol, Mar; López-Béjar, Manel

2017-10-01

Assisted reproductive technology (ART) can benefit from the features of microfluidic technologies, such as the automation of time-consuming labor-intensive procedures, the possibility to mimic in vivo environments, and the miniaturization of the required equipment. To date, most of the proposed approaches are based on polydimethylsiloxane (PDMS) as platform substrate material due to its widespread use in academia, despite certain disadvantages, such as the elevated cost of mass production. Herein, we present a rapid fabrication process for a cyclic olefin copolymer (COC) monolithic microfluidic device combining hot embossing-using a low-temperature cofired ceramic (LTCC) master-and micromilling. The microfluidic device was suitable for trapping and maturation of bovine oocytes, which were further studied to determine their ability to be fertilized. Furthermore, another COC microfluidic device was fabricated to store sperm and assess its quality parameters over time. The study herein presented demonstrates a good biocompatibility of the COC when working with gametes, and it exhibits certain advantages, such as the nonabsorption of small molecules, gas impermeability, and low fabrication costs, all at the prototyping and mass production scale, thus taking a step further toward fully automated microfluidic devices in ART.

Toxicity of beauvericin on porcine oocyte maturation and preimplantation embryo development

NARCIS (Netherlands)

Schoevers, Eric J; Santos, Regiane R; Fink-Gremmels, Johanna; Roelen, Bernard A J

2016-01-01

Beauvericin (BEA) is one of many toxins produced by Fusarium species that contaminate feed materials. The aim of this study was to assess its effects on porcine oocyte maturation and preimplantation embryo development. Cumulus-oocyte-complexes and developing embryos were exposed to BEA and cultured

IVF for premature ovarian failure: first reported births using oocytes donated from a twin sister.

LENUS (Irish Health Repository)

Sills, Eric Scott

2010-01-01

BACKGROUND: Premature ovarian failure (POF) remains a clinically challenging entity because in vitro fertilisation (IVF) with donor oocytes is currently the only treatment known to be effective. METHODS: A 33 year-old nulligravid patient with a normal karyotype was diagnosed with POF; she had a history of failed fertility treatments and had an elevated serum FSH (42 mIU\\/ml). Oocytes donated by her dizygotic twin sister were used for IVF. The donor had already completed a successful pregnancy herself and subsequently produced a total of 10 oocytes after a combined FSH\\/LH superovulation regime. These eggs were fertilised with sperm from the recipient\\'s husband via intracytoplasmic injection and two fresh embryos were transferred to the recipient on day three. RESULTS: A healthy twin pregnancy resulted from IVF; two boys were delivered by caesarean section at 39 weeks\\' gestation. Additionally, four embryos were cryopreserved for the recipient\\'s future use. The sister-donor achieved another natural pregnancy six months after oocyte retrieval, resulting in a healthy singleton delivery. CONCLUSION: POF is believed to affect approximately 1% of reproductive age females, and POF patients with a sister who can be an oocyte donor for IVF are rare. Most such IVF patients will conceive from treatment using oocytes from an anonymous oocyte donor. This is the first report of births following sister-donor oocyte IVF in Ireland. Indeed, while sister-donor IVF has been successfully undertaken by IVF units elsewhere, this is the only known case where oocyte donation involved twin sisters. As with all types of donor gamete therapy, pre-treatment counselling is important in the circumstance of sister oocyte donation.

Hanging drop monoculture for selection of optimal antioxidants during in vitro maturation of porcine oocytes.

Science.gov (United States)

Ishikawa, S; Machida, R; Hiraga, K; Hiradate, Y; Suda, Y; Tanemura, K

2014-04-01

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre-culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 μg/ml of L-carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non-presence of oxidant stress induced by H2O2 supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 μg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H2O2. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture. © 2014 Blackwell Verlag GmbH.

Effects of Mild and Severe Vitamin B Deficiencies on the Meiotic Maturation of Mice Oocytes

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Ai Tsuji

2017-03-01

Full Text Available We investigated the effects of vitamin B 1 deficiency on the meiosis maturation of oocytes. Female Crl:CD1 (ICR mice were fed a 20% casein diet (control group or a vitamin B 1 –free diet (test group. The vitamin B 1 concentration in ovary was approximately 30% lower in the test group than in the control group. Oocyte meiosis was not affected by vitamin B 1 deficiency when the deficiency was not accompanied by body weight loss. On the contrary, frequency of abnormal oocyte was increased by vitamin B 1 deficiency when deficiency was accompanied by body weight loss (referred to as severe vitamin B 1 deficiency; frequency of abnormal oocyte, 13.8% vs 43.7%, P  = .0071. The frequency of abnormal oocytes was decreased by refeeding of a vitamin B 1 –containing diet (13.9% vs 22.9%, P  = .503. These results suggest that severe vitamin B 1 deficiency inhibited meiotic maturation of oocytes but did not damage immature oocytes.

How oocytes try to get it right: spindle checkpoint control in meiosis.

Science.gov (United States)

Touati, Sandra A; Wassmann, Katja

2016-06-01

The generation of a viable, diploid organism depends on the formation of haploid gametes, oocytes, and spermatocytes, with the correct number of chromosomes. Halving the genome requires the execution of two consecutive specialized cell divisions named meiosis I and II. Unfortunately, and in contrast to male meiosis, chromosome segregation in oocytes is error prone, with human oocytes being extraordinarily "meiotically challenged". Aneuploid oocytes, that are with the wrong number of chromosomes, give rise to aneuploid embryos when fertilized. In humans, most aneuploidies are lethal and result in spontaneous abortions. However, some trisomies survive to birth or even adulthood, such as the well-known trisomy 21, which gives rise to Down syndrome (Nagaoka et al. in Nat Rev Genet 13:493-504, 2012). A staggering 20-25 % of oocytes ready to be fertilized are aneuploid in humans. If this were not bad enough, there is an additional increase in meiotic missegregations as women get closer to menopause. A woman above 40 has a risk of more than 30 % of getting pregnant with a trisomic child. Worse still, in industrialized western societies, child birth is delayed, with women getting their first child later in life than ever. This trend has led to an increase of trisomic pregnancies by 70 % in the last 30 years (Nagaoka et al. in Nat Rev Genet 13:493-504, 2012; Schmidt et al. in Hum Reprod Update 18:29-43, 2012). To understand why errors occur so frequently during the meiotic divisions in oocytes, we review here the molecular mechanisms at works to control chromosome segregation during meiosis. An important mitotic control mechanism, namely the spindle assembly checkpoint or SAC, has been adapted to the special requirements of the meiotic divisions, and this review will focus on our current knowledge of SAC control in mammalian oocytes. Knowledge on how chromosome segregation is controlled in mammalian oocytes may help to identify risk factors important for questions

Study on the immunogenic ability of Eimeria tenella oocytes following treatment with gamma rays

International Nuclear Information System (INIS)

Penev, P.; Stafanova, M.

1975-01-01

Studied was the immunizing capacity of Eimeria tenella oocytes, treated with gamma rays at the rate of 6000 R, in 10- and 20-day-old chickens. The oocytes sporulated after treatment. Applied at the rate of 50,000 R they showed lower virulence and were capable of inducing resistance to reinfection with non-irradiated oocytes at rates that were three times as much. Following reinfection some birds manifested subclinical coccidiosis but survived. This showed that the immunization with oocytes that had been irradiated with 6,000 R had its peculiar aspects. (author)

Ultra-structural study of Egyptian Buffalo oocytes before and after in ...

African Journals Online (AJOL)

The oocytes examined in this study showed normal ultra-structure of mitochondria, smooth endoplasmic reticulum (SER), zona pellucida (ZP), lipid droplets, vesicles and Golgi in the good type meanwhile, some differences and abnormalities in denuded oocytes were recorded. The most remarkable changes observed in the ...

DNA damage in the oocytes SACs

Czech Academy of Sciences Publication Activity Database

Macůrek, Libor

2016-01-01

Roč. 15, č. 4 (2016), s. 491-492 ISSN 1538-4101 Institutional support: RVO:68378050 Keywords : DNA damage response * oocyte * meiosis * checkpoint Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.530, year: 2016

RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production

Science.gov (United States)

Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

2015-01-01

We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

Effect of induced peritoneal endometriosis on oocyte and embryo quality in a mouse model.

Science.gov (United States)

Cohen, J; Ziyyat, A; Naoura, I; Chabbert-Buffet, N; Aractingi, S; Darai, E; Lefevre, B

2015-02-01

To assess the impact of peritoneal endometriosis on oocyte and embryo quality in a mouse model. Peritoneal endometriosis was surgically induced in 33 B6CBA/F1 female mice (endometriosis group, N = 17) and sham-operated were used as control (sham group, N = 16). Mice were superovulated 4 weeks after surgery and mated or not, to collect E0.5-embryos or MII-oocytes. Evaluation of oocyte and zygote quality was done by immunofluorescence under spinning disk confocal microscopy. Endometriosis-like lesions were observed in all mice of endometriosis group. In both groups, a similar mean number of MII oocytes per mouse was observed in non-mated mice (30.2 vs 32.6), with a lower proportion of normal oocytes in the endometriosis group (61 vs 83 %, p endometriosis group (21 vs 35.5, p = 0.02) without difference in embryo quality. Our results support that induced peritoneal endometriosis in a mouse model is associated with a decrease in oocyte quality and embryo number. This experimental model allows further studies to understand mechanisms of endometriosis-associated infertility.

SP-A binding sites on bovine alveolar macrophages.

Science.gov (United States)

Plaga, S; Plattner, H; Schlepper-Schaefer, J

1998-11-25

Surfactant protein A (SP-A) binding to bovine alveolar macrophages was examined in order to characterize SP-A binding proteins on the cell surface and to isolate putative receptors from these cells that could be obtained in large amounts. Human SP-A, unlabeled or labeled with gold particles, was bound to freshly isolated macrophages and analyzed with ELISA or the transmission electron microscope. Binding of SP-A was inhibited by Ca2+ chelation, by an excess of unlabeled SP-A, or by the presence of 20 mg/ml mannan. We conclude that bovine alveolar macrophages expose binding sites for SP-A that are specific and that depend on Ca2+ and on mannose residues. For isolation of SP-A receptors with homologous SP-A as ligand we isolated SP-A from bovine lung lavage. SDS-PAGE analysis of the purified SP-A showed a protein of 32-36 kDa. Functional integrity of the protein was demonstrated. Bovine SP-A bound to Dynabeads was used to isolate SP-A binding proteins. From the fractionated and blotted proteins of the receptor preparation two proteins bound SP-A in a Ca2+-dependent manner, a 40-kDa protein showing mannose dependency and a 210-kDa protein, showing no mannose sensitivity. Copyright 1998 Academic Press.

Effects of N, N-dimethylglycine on the development of in vitro produced bovine embryos.

Science.gov (United States)

Takahashi, Toshikiyo; Itoh, Ryu; Nagai, Takashi

2009-06-01

This study investigated the effects of N, N-Dimethylglycine (DMG) on the development of in vitro produced (IVP) bovine embryos. IVP embryos were obtained by in vitro fertilization of in vitro matured oocytes for 6 h. In Experiment 1, IVP embryos were cultured in mSOFaa supplemented with bovine serum albumin but without glucose (SOF1) for 4 days, transferred to mSOFaa (with 5% fetal bovine serum and 1.5 mM glucose; SOF2) supplemented with 0 (control), 0.1,1 or 10 microM DMG and cultured for an additional 7 days (11 days in total) to assess their development in vitro. When cultured in the medium with 0.1 microM DMG, a significantly higher number of IVP embryos developed to the blastocyst and hatched blastocyst stages (40.3 and 40.8%, respectively) compared with the other groups (18.7-31.0% and 15.0-28.7%, respectively; PDMG for 4 days, transferred to SOF2 with or without 0.1 microM DMG and further cultured as in Experiment 1; DMG was added to either SOF1 or SOF2 and to both of them to assess its exposure effects on embryo development. When cultured continuously with DMG for 11 days, significantly higher rates of IVP embryos developed into blastocyst and hatched blastocyst stages (39.0 and 47.7%, respectively) compared with the other groups (31.0-32.2% and 29.5-31.0%, respectively; PDMG to IVC medium after 7 days of IVC. When DMG was added to IVC medium, the ratio of embryos developed to advanced developmental stages (No. of embryos developed to the blastocyst and expanded blastocyst stages/No. of embryos developed to the morula stage) was 28.7% (86/3) and 7 times higher than that of those cultured without DMG, 4.0% (52/13). These results suggest that addition of 0.1 microM DMG to mSOFaa during IVC of IVP bovine embryos has a promoting effect on their development.

Localization of RNA transcription sites in insect oocytes using microinjections of 5-bromouridine 5'-triphosphate.

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Dmitry Bogolyubov

2007-06-01

Full Text Available In the present study we used 5-bromouridine 5'-triphosphate (BrUTP microinjections to localize the transcription sites in oocytes of insects with different types of the ovarium structure: panoistic, meroistic polytrophic, and meroistic telotrophic. We found that in an insect with panoistic ovaries (Acheta domesticus, oocyte nuclei maintain their transcription activity during the long period of oocyte growth. In insects with meroistic ovaries (Tenebrio molitor and Panorpa communis, early oocyte chromosomes were found to be transcriptionally active, and some transcription activity still persist while the karyosphere, a compact structure formed by all condensed oocyte chromosomes, begins to develop. At the latest stages of karyosphere development, no anti-Br-RNA signal was registered in the karyosphere.

Reduced developmental competence of immature, in-vitro matured and postovulatory aged mouse oocytes following IVF and ICSI

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Trounson Alan

2008-12-01

Full Text Available Abstract Background The present study highlights basic physiological differences associated with oocyte maturation and ageing. The study explores the fertilizing capacity and resistance to injury of mouse oocytes at different stages of maturation and ageing following IVF and ICSI. Also, the study examines the developmental competence of embryos obtained from these oocytes. The outcome of the study supports views that the mouse can be a model for human IVF suggesting that utilizing in-vitro matured and failed fertilized oocytes to produce embryos mainly when limited number of oocytes is retrieved in a specific cycle, should be carefully considered. Methods Hybrid strain mouse oocytes were inseminated by in-vitro fertilization (IVF or intracytoplasmic sperm injection (ICSI. Oocytes groups that were used were germinal vesicle (GV in-vitro matured metaphase II (IVM-MII, freshly ovulated MII (OV-MII, 13 hrs in-vitro aged MII (13 hrs-MII and 24 hrs in-vitro aged MII (24 hrs-MII. Fertilization and embryo development to the blastocyst stage were monitored up to 5 days in culture for IVF and ICSI zygotes. Sperm head decondensation and pronuclear formation were examined up to 9 hrs in oocytes following ICSI. Apoptotic events in blocked embryos were examined using the TUNNEL assay. Differences between females for the number and quality of GV and OV-MII oocytes were examined by ANOVA analyses. Differences in survival after ICSI, fertilization by IVF and ICSI and embryo development were analysed by Chi-square test with Yates correction. Results No differences in number and quality of oocytes were identified between females. The findings suggest that inability of GV oocytes to participate in fertilization and embryo development initiates primarily from their inability to support initial post fertilization events such as sperm decondensation and pronuclei formation. These events occur in all MII oocytes in similar rates (87–98% for IVF and ICSI. Following

The influence of N-acetyl-L-cysteine on damage of porcine oocyte exposed to zearalenone in vitro

Energy Technology Data Exchange (ETDEWEB)

Lai, Fang-Nong [College of Life Sciences, Qingdao Agricultural University, Qingdao 266109 (China); Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109 (China); Ma, Jun-Yu [Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109 (China); Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109 (China); Liu, Jing-Cai [College of Life Sciences, Qingdao Agricultural University, Qingdao 266109 (China); Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109 (China); Wang, Jun-Jie; Cheng, Shun-Feng [Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109 (China); Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109 (China); Sun, Xiao-Feng [College of Life Sciences, Qingdao Agricultural University, Qingdao 266109 (China); Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109 (China); Li, Lan [Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109 (China); Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109 (China); Li, Bo [Chengguo Station of Animal Husbandry and Veterinary, Laizhou 261437 (China); Nyachoti, Charles Martin [Department of Animal Science, University of Manitoba, Winnipeg, MB R3T 2N2 (Canada); Shen, Wei, E-mail: wshen@qau.edu.cn [Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109 (China); Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109 (China)

2015-12-01

Zearalenone (ZEA), one of the mycotoxins produced by Fusarium fungi, impacts porcine reproduction by interfering with the estrogen signaling pathway. Previous studies have shown that ZEA inhibits porcine oocyte maturation through the formation of aberrant spindle. To explore the effect of ZEA on porcine oocyte meiotic maturation, the extent of both nuclear and cytoplasmic maturation was examined in this study. Compared with control group, presence of ZEA (3 μM) during oocyte maturation, significantly inhibited the polar body extrusions from 71% to 51%, and significantly increased intracellular reactive oxygen species (ROS) level (12.01 vs. 5.89). Intracellular glutathione (GSH) content in ZEA treatment group was lower than in the control group (1.08 pmol/oocyte vs. 0.18 pmol/oocyte), and cortical granules of cortical area distributed oocytes were reduced (88% vs. 62%). ZEA decreases cumulus expansion in both morphology and mRNA level (HAS2, PTX3, TNFAIP6 and CX43). Addition of N-acetyl-L-cysteine (NAC) to the oocyte maturation media reversed the ZEA-induced inhibition of polar body extrusion (from 69% to 81%), up-regulated ROS (from 7.9 to 6.5), down-regulated GSH content (from 0.16 to 0.82 pmol/oocyte) and recovered cumulus cells expansion in morphology and mRNA level. It is concluded that ZEA affects both oocyte nucleus and cytoplasmic maturation during in vitro maturation, and NAC can reverse these damages to some extent. - Highlights: • ZEA significantly inhibited the polar body extrusions during oocyte maturation. • ZEA significantly increased intracellular ROS level and reduced GSH content. • ZEA disturbed cortical granules of cortical area distributed oocytes. • NAC reversed the ZEA-induced inhibition of oocyte maturation.

Cryopreservation of Embryos and Oocytes in Human Assisted Reproduction

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János Konc

2014-01-01

Full Text Available 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.

Cryopreservation of embryos and oocytes in human assisted reproduction.

Science.gov (United States)

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

2014-01-01

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.

Expression and putative function of fibronectin and its receptor (integrin alpha(5)beta(1)) in male and female gametes during bovine fertilization in vitro.

Science.gov (United States)

Thys, Mirjan; Nauwynck, Hans; Maes, Dominiek; Hoogewijs, Maarten; Vercauteren, Dries; Rijsselaere, Tom; Favoreel, Herman; Van Soom, Ann

2009-09-01

Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm-egg interaction in human. Recently, it has been demonstrated that Fn--when present during bovine IVF--strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (alpha(5)beta(1)) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin alpha(5) on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin alpha(5) at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm-oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin alpha(5)beta(1) receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin alpha(5) expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a 'velcro' interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm-egg binding.

Interaction of Water-Soluble CdTe Quantum Dots with Bovine Serum Albumin

Science.gov (United States)

2011-01-01

Semiconductor nanoparticles (quantum dots) are promising fluorescent markers, but it is very little known about interaction of quantum dots with biological molecules. In this study, interaction of CdTe quantum dots coated with thioglycolic acid (TGA) with bovine serum albumin was investigated. Steady state spectroscopy, atomic force microscopy, electron microscopy and dynamic light scattering methods were used. It was explored how bovine serum albumin affects stability and spectral properties of quantum dots in aqueous media. CdTe–TGA quantum dots in aqueous solution appeared to be not stable and precipitated. Interaction with bovine serum albumin significantly enhanced stability and photoluminescence quantum yield of quantum dots and prevented quantum dots from aggregating. PMID:27502633

Spectral studies of intermediate species formed in one-electron reactions of bovine liver catalase at room and low temperatures. A comparison with peroxidase reactions

International Nuclear Information System (INIS)

Metodiewa, D.; Dunford, H.B.

1992-01-01

The reactions of native bovine catalase with superoxide and solvated electrons have been investigated using three different methods for generating these reducing substrates: γ-radiolysis of oxygenated or deaerated buffer solutions in the presence of an OH radical scavenger; either xanthine or acetaldehyde with xanthine oxidase; and low-temperature (77 K) γ-radiolysis of buffered ethylene glycol/water solutions with subsequent annealing of samples at 183 K. (Author)

Perceptions of oocyte banking from women intending to circumvent age-related fertility decline.

Science.gov (United States)

de Groot, Marije; Dancet, Eline; Repping, Sjoerd; Goddijn, Mariette; Stoop, Dominic; van der Veen, Fulco; Gerrits, Trudie

2016-12-01

Women can now opt to bank their oocytes with the intention of increasing their chances of achieving a pregnancy after their fertility has declined. This exploratory study aimed to gain insight into how women, considering oocyte banking to circumvent age-related fertility decline, perceive this intervention. We conducted a qualitative study in a Dutch university medical center and held in-depth interviews with women on the waiting list for oocyte banking. We recorded the interviews, transcribed them verbatim and used thematic analysis. All women were financially independent and lived in single-person urban households. They opted for oocyte banking because they wished to share parenthood with a future partner rather than becoming a single parent. This strong desire was key in their interpretation of all aspects of the intervention. Women set aside information about the limited success rates and potential risks, as they were optimistic about their own prognosis, thought that the chances for success were equally likely as the chances it would fail, and because of "anticipatory regret". They perceived oocyte banking as a "helping hand" to achieve shared parenthood. Although women found the costs of the intervention high, they were willing to invest their money to increase their chances for shared parenthood. Oocyte banking allows women to circumvent age-related fertility decline. The prospect of potential shared parenthood overrules the perceived health risks and burden. Health professionals should take this into account when informing potential users of oocyte banking. © 2016 Nordic Federation of Societies of Obstetrics and Gynecology.

Evolution of sex-biased maternal effects in birds: I. Sex-specific resource allocation among simultaneously growing oocytes.

Science.gov (United States)

Young, R L; Badyaev, A V

2004-11-01

Females in species that produce broods of multiple offspring need to partition resources among simultaneously growing ova, embryos or neonates. In birds, the duration of growth of a single egg exceeds the ovulation interval, and when maternal resources are limited, a temporal overlap among several developing follicles in the ovary might result in a trade-off of resources among them. We studied growth of oocytes in relation to their future ovulation order, sex, and overlap with other oocytes in a population of house finches (Carpodacus mexicanus) where strongly sex-biased maternal effects are favoured by natural selection. We found pronounced differences in growth patterns between oocytes that produced males and females. Male oocytes grew up to five times faster and reached their ovulation size earlier than female oocytes. Early onset and early termination of male oocytes' growth in relation to their ovulation resulted in their lesser temporal overlap with other growing ova compared with female oocytes. Consequently, ovulation mass of female but not male oocytes was strongly negatively affected by temporal overlap with other oocytes. In turn, mass of male oocytes was mostly affected by the order of ovulation and by maternal incubation strategy. These results provide a mechanism for sex-biased allocation of maternal resources during egg formation and provide insights into the timing of the sex-determining meiotic division in relation to ovulation in this species.

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

Science.gov (United States)

Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

2016-01-01

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte

Science.gov (United States)

Baccetti, B; Benedetto, A; Burrini, AG; Collodel, G; Ceccarini, EC; Crisa, N; Di Caro, A; Estenoz, M; Garbuglia, AR; Massacesi, A; Piomboni, P; Renieri, T; Solazzo, D

1994-01-01

By immunocytochemistry and in situ hybridization at the electron microscopy level, and by the PCR technique, we have shown that HIV-1 binds and enters normal sperm; that viral particles, their antigens, and nucleic acid are present in sperm from HIV-1 infected men; and that such sperm can transfer HIV-1 like particles to normal human oocytes. We also present evidence that a galactosylceramide-like compound is present on the sperm membrane and could function as an alternative receptor for HIV. PMID:7962075

Oocyte toxicity: female germ-cell loss from radiation and chemical exposures

International Nuclear Information System (INIS)

Dobson, R.L.

1984-01-01

In some mammals, female germ cells are extraordinarily sensitive to killing by exposure to ionizing radiation, especially during development. Immature oocytes, which constitute the lifetime germ-cell pool of the female, have an LD 50 in juvenile mice of only 6 rad (compared with typical LD 50 s of 100-300 rad for most other cell types studied). Essentially, the entire germ-cell supply in female squirrel monkeys is destroyed prenatally by exposure of only 0.7 rad/day. Severe but lesser destruction has been found in other species. However, evidence suggests (though not ruled out for all developmental stages) that unusually high sensitivity probably does not occur in the human female. Germ cells can also be killed by certain chemicals, and similarities exist between chemical and radiation effects. More than 75 compounds have been quantitatively studied in mice, with determination of OTI values (OTI = oocyte toxicity index = mouse LD 50 /oocyte LD 50 ) to measure the degree of preferential oocyte killing. High sensitivity in mice does not mean necessarily high sensitivity in women. Of special interest is the recent discovery that the lethal target in the extremely sensitive mouse immature oocyte is probably the plasma membrane, not DNA. Since mouse data form the main basis from which human genetic hazard (for both radiation and chemicals) is estimated, this has important implications for the determination of genetic risk in women

[Comparison of the frequency of chromosomal disorders in populations of in vitro-matured and ovulating rat oocytes].

Science.gov (United States)

Kitaev, E M; Pimenova, M N

1980-12-01

The rat oocytes extracted from the rat ovaries and cultivated for 42-46 hours were compared with ovulated oocytes by the chromosomal aberration rate. The chromosomal aberration rate in the population of "follicular" oocytes was 8.2% on the average whereas in ovulated oocytes, it did not exceed 1.8%. Analysis of the chromosomal aberrations depending on the phase of the estral cycle suggests that the main portion of chromosomal aberrations in cultivated oocytes occurs during the physiological process of follicular atresia.

Cytoplasmic Streaming in the Drosophila Oocyte.

Science.gov (United States)

Quinlan, Margot E

2016-10-06

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.

Artificial oocyte activation with calcium ionophore for frozen sperm cycles.

Science.gov (United States)

Karabulut, Seda; Aksünger, Özlem; Ata, Can; Sağıroglu, Yusuf; Keskin, İlknur

2018-04-05

Fertilization problems are the major problems that may be faced in 30-55% of the patients during an intracytoplasmic sperm injection (ICSI) cycle. A successful oocyte activation depends on factors related to both sperm and oocyte, and one of the important factors that mediates the process is Ca 2+ concentration within the oocyte. Artificial oocyte activation (AOA) is a method used for fertilization problems that commonly involve the usage of Ca 2+ ionophores and is usually used in problems such as total fertilization failure (TFF) and globozoospermia. The aim of the present study was to investigate possible effects of AOA for different groups of patients with fertilization failure. Four groups of patients (previous TFF, low oocyte number, severe sperm quality, and frozen sperm (FS) group) that underwent ICSI with AOA were included in the study. All groups had similar control groups with same indications except TFF, where AOA was not performed. Fertilization rates were significantly higher in the TFF group than those observed in other AOA groups. Fertilization rates and quality of embryos observed in the remaining AOA groups were higher than those of the controls, which were statistically insignificant. Prgenancy rates were higher in all AOA groups compared to the controls, although the differences were significant in FS group only. Quality of embryos and pregnancy rates were lower in the TFF group compared to the remaining AOA groups indicating possible concomitant problems. Fertilization rates, quality of embryos and pregnancy rates seemed to be increased in all indication groups suggesting that not only TFF patients but also a wide variety of patients with different indications may benefit from AOA. ICSI: Intracytoplasmic sperm injection; ARTs: Assisted reproductive techniques; Ca: Calcium; AOA: Artificial oocyte activation; TFF: Total fertilization failures; OAT: Oligoasthenoteratozoospemia; IVF: In vitro fertilization; SOAT: Severe OAT; LON: Low ooctye number

Effect of glucose, lactate and pyruvate concentrations on in vitro ...

African Journals Online (AJOL)

Jane

2011-08-01

Aug 1, 2011 ... growth of oocytes and other follicular cells in vitro. The aim of this study ... major metabolite used in bovine cumulus oocyte complex in maturation .... implantation embryos, because it eliminates undefined factors present in ...

The RNA-binding protein, ZFP36L2, influences ovulation and oocyte maturation.

Directory of Open Access Journals (Sweden)

Christopher B Ball

Full Text Available ZFP36L2 protein destabilizes AU-rich element-containing transcripts and has been implicated in female fertility. In the C57BL/6NTac mouse, a mutation in Zfp36l2 that results in the decreased expression of a form of ZFP36L2 in which the 29 N-terminal amino acid residues have been deleted, ΔN-ZFP36L2, leads to fertilized eggs that arrest at the two-cell stage. Interestingly, homozygous ΔN-Zfp36l2 females in the C57BL/6NTac strain release 40% fewer eggs than the WT littermates (Ramos et al., 2004, suggesting an additional defect in ovulation and/or oocyte maturation. Curiously, the same ΔN-Zfp36l2 mutation into the SV129 strain resulted in anovulation, prompting us to investigate a potential problem in ovulation and oocyte maturation. Remarkably, only 20% of ΔN-Zfp36l2 oocytes in the 129S6/SvEvTac strain matured ex vivo, suggesting a defect on the oocyte meiotic maturation process. Treatment of ΔN-Zfp36l2 oocytes with a PKA inhibitor partially rescued the meiotic arrested oocytes. Furthermore, cAMP levels were increased in ΔN-Zfp36l2 oocytes, linking the cAMP/PKA pathway and ΔN-Zfp36l2 with meiotic arrest. Since ovulation and oocyte maturation are both triggered by LHR signaling, the downstream pathway was investigated. Adenylyl cyclase activity was increased in ΔN-Zfp36l2 ovaries only upon LH stimulation. Moreover, we discovered that ZFP36L2 interacts with the 3'UTR of LHR mRNA and that decreased expression levels of Zfp36l2 correlates with higher levels of LHR mRNA in synchronized ovaries. Furthermore, overexpression of ZFP36L2 decreases the endogenous expression of LHR mRNA in a cell line. Therefore, we propose that lack of the physiological down regulation of LHR mRNA levels by ZFP36L2 in the ovaries is associated with anovulation and oocyte meiotic arrest.

Dietary saccharides and sweet tastants have differential effects on colonization of Drosophila oocytes by Wolbachia endosymbionts

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Moises Camacho

2017-07-01

Full Text Available Wolbachia bacteria are widespread, maternally transmitted endosymbionts of insects. Maintenance of sufficient Wolbachia titer in maternal germline cells is required for transmission efficacy. The mechanisms that regulate Wolbachia titer are not well understood; however, dietary sucrose was reported to elevate oocyte Wolbachia titer in Drosophila melanogaster whereas dietary yeast decreased oocyte titer. To further investigate how oocyte Wolbachia titer is controlled, this study analyzed the response of wMel Wolbachia to diets enriched in an array of natural sugars and other sweet tastants. Confocal imaging of D. melanogaster oocytes showed that food enriched in dietary galactose, lactose, maltose and trehalose elevated Wolbachia titer. However, oocyte Wolbachia titers were unaffected by exposure to the sweet tastants lactulose, erythritol, xylitol, aspartame and saccharin as compared to the control. Oocyte size was generally non-responsive to the nutrient-altered diets. Ovary size, however, was consistently smaller in response to all sugar- and sweetener-enriched diets. Furthermore, most dietary sugars administered in tandem with dietary yeast conferred complete rescue of oocyte titer suppression by yeast. All diets dually enriched in yeast and sugar also rescued yeast-associated ovary volume changes. This indicates oocyte colonization by Wolbachia to be a nutritionally sensitive process regulated by multiple mechanistic inputs.

The Phosphatase Dusp7 Drives Meiotic Resumption and Chromosome Alignment in Mouse Oocytes

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Thomas Tischer

2016-10-01

Full Text Available Mammalian oocytes are stored in the ovary, where they are arrested in prophase for prolonged periods. The mechanisms that abrogate the prophase arrest in mammalian oocytes and reinitiate meiosis are not well understood. Here, we identify and characterize an essential pathway for the resumption of meiosis that relies on the protein phosphatase DUSP7. DUSP7-depleted oocytes either fail to resume meiosis or resume meiosis with a significant delay. In the absence of DUSP7, Cdk1/CycB activity drops below the critical level required to reinitiate meiosis, precluding or delaying nuclear envelope breakdown. Our data suggest that DUSP7 drives meiotic resumption by dephosphorylating and thereby inactivating cPKC isoforms. In addition to controlling meiotic resumption, DUSP7 has a second function in chromosome segregation: DUSP7-depleted oocytes that enter meiosis show severe chromosome alignment defects and progress into anaphase prematurely. Altogether, these findings establish the phosphatase DUSP7 as an essential regulator of multiple steps in oocyte meiosis.

Genetic hazard of 3H: estimation by oocyte uptake of 2H

International Nuclear Information System (INIS)

Slatkin, D.N.; Carsten, A.L.; Commerford, S.L.; Jones, K.W.; Kraner, H.W.

1979-01-01

Tritium can be incorporated biosynthetically from tritiated water into non-exchangeable C- 3 H bonds of deoxyribonucleotides during DNA synthesis. Thus, accidental ingestion of tritiated water during pregnance may result in some degree of tritiation of DNA in primary oocytes of female progeny. The genetic hazard posed by such incorporation would depend mainly upon the amount of tritium incorporated into long-lived primary oocytes and upon the persistence of tritium in those cells. To study this problem in mammals while avoiding tritium-induced inhibition of oogenesis, it is proposed to measure deuterium that persists in the primary oocytes of mice after exposure to deuterated water in utero. Data from such measurements could be extrapolated to estimate the fraction of tritium that persists in human primary oocytes after incorporation from tritiated water during prenatal oogenesis. Preliminary studies are described which demonstrate the feasibility of these stable isotope tracer measurements

mtDNA copy number in oocytes of different sizes from individual pre- and post-pubertal pigs

DEFF Research Database (Denmark)

Pedersen, Hanne Skovsgaard; Løvendahl, Peter; Larsen, Knud Erik

2014-01-01

from ovaries of 10 pre- and 10 post-pubertal pigs. Cumulus cells were removed and the oocytes were measured (inside-ZP-diameter). Oocytes were transferred to DNAase-free tubes, snap-frozen, and stored at –80°C. The genes ND1 and COX1 were used to determine the mtDNA copy number. Plasmid preparations...... Reproduction 131, 233–245). However, the correlation between size and mtDNA copy number in single oocytes has not been determined. This study describes the relation between oocytes of defined diameters from individual pre- and postpubertal pigs and mtDNA copy number. Cumulus-oocyte complexes were aspirated...

NH3 and NH4+ permeability in aquaporin-expressing Xenopus oocytes

DEFF Research Database (Denmark)

Holm, Lars M.; Jahn, Thomas Paul; Møller, Anders Laurell Blom

2005-01-01

We have shown recently, in a yeast expression system, that some aquaporins are permeable to ammonia. In the present study, we expressed the mammalian aquaporins AQP8, AQQP9, AQP3, AQP1 and a plant aquaporin TIP2;1 in Xenopus oocytes to study the transport of ammonia (NH3) and ammonium (NH4+) under...... inwards currents carried by NH4+. This conductivity increased as a sigmoid function of external [NH3]: for AQP8 at a bath pH (pH(e)) of 6.5, the conductance was abolished, at pH(e) 7.4 it was half maximal and at pH(e) 7.8 it saturated. NY4+ influx was associated with oocyte swelling. In comparison, native...... oocytes as well as AQP1 and tip2;1-expressing oocytes showed small currents that were associated with small and even negative volume changes. We conclude that AQP8, AQP9, AQP3, and TIP2;1, apart from being water channels, also support significant fluxes of NH3. These aquaporins could support NH4...

Cloned foal derived from in vivo matured horse oocytes aspirated by the short disposable needle system

OpenAIRE

Lee, Wonyou; Song, Kilyoung; Lee, Inhyung; Shin, Hyungdo; Lee, Byeong Chun; Yeon, Seongchan; Jang, Goo

2015-01-01

Transvaginal ultrasound-guided follicle aspiration is one method of obtaining recipient oocytes for equine somatic cell nuclear transfer (SCNT). This study was conducted: (1) to evaluate the possibility of oocyte aspiration from pre-ovulatory follicles using a short disposable needle system (14-G) by comparing the oocyte recovery rate with that of a long double lumen needle (12-G); (2) to investigate the developmental competence of recovered oocytes after SCNT and embryo transfer. The recover...

Effect of a supplementation with myo-inositol plus melatonin on oocyte quality in women who failed to conceive in previous in vitro fertilization cycles for poor oocyte quality: a prospective, longitudinal, cohort study.

Science.gov (United States)

Unfer, Vittorio; Raffone, Emanuela; Rizzo, Piero; Buffo, Silvia

2011-11-01

Several factors can affect oocyte quality and therefore pregnancy outcome in assisted reproductive technology (ART) cycles. Recently, a number of studies have shown that the presence of several compounds in the follicular fluid positively correlates with oocyte quality and maturation (i.e., myo-inositol and melatonin). In the present study, we aim to evaluate the pregnancy outcomes after the administration of myo-inositol combined with melatonin in women who failed to conceive in previous in vitro fertilization (IVF) cycles due to poor oocyte quality. Forty-six women were treated with 4 g/day myo-inositol and 3 mg/day melatonin (inofolic® and inofolic® Plus, Lo.Lipharma, Rome) for 3 months and then underwent a new IVF cycle. After treatment, the number of mature oocytes, the fertilization rate, the number of both, total and top-quality embryos transferred were statistically higher compared to the previous IVF cycle, while there was no difference in the number of retrieved oocyte. After treatment, a total of 13 pregnancies occurred, 9 of them were confirmed echographically; four evolved in spontaneous abortion. The treatment with myo-inositol and melatonin improves ovarian stimulation protocols and pregnancy outcomes in infertile women with poor oocyte quality.

Comparison of sucrose and trehalose media modification as an update of oocyte vitrification: A study of apoptotic level

Science.gov (United States)

Lestari, Silvia W.; Fitriyah, Nurin N.; Pangestu, Mulyoto; Pratama, Gita; Margiana, Ria

2018-02-01

Number of women who are not being able to have offspring in their reproductive life is increasing which might be influenced by several factors. As a consequence, oocyte cryopreservation could be an ensuring solution for women fertility preservation. A good vitrification could be conducted by combining an appropriate of type and concentration of cryoprotectants. One of the marks of successful vitrification is the vitrified oocytes could avoid apoptosis. This study aimed to evaluate the modification of cryoprotectant media as un update of oocyte vitrification as follow: the combination and the concentration of cryoprotectant media of oocytes vitrification, based on their effects on the apoptosis or DNA damage of oocytes. A total of 84 MII stage oocytes from adult female Deutschland, Denken and Yoken (DDY) mice (7-8 weeks old) were used in this study. Vitrification procedure was performed by using VS1 contained 15% EG, 15% DMSO, 0.5 mol/l sucrose (Merck, Darmstadt, Germany) and VS2 contained 15% EG, 15% DMSO, 0.5 mol/l trehalose (Merck, Darmstadt, Germany) in HM. Furthermore, warming solution (WS) was divided into four groups. There were: WS1a contained 0.3 mol/l sucrose, WS1b contained 0.15 mol/l sucrose, WS2a contained 0.3 mol/l trehalose, and WS2b contained 0.15 mol/l trehalose. Apoptotic level was performed by staining the oocytes with TUNEL and propidium iodide (PI) based on Brison and Schultz method then examined under confocal microscope. The rate of apoptosis in oocytes after vitrification and warming was higher compared to the fresh control oocytes. Furthermore, the rate of apoptosis in the vitrified oocytes by sucrose media (28%) was higher compared to the vitrified oocytes by trehalose media (16%). The results of this study indicated that vitrification increased apoptosis in the vitrified oocytes related to the oocyte injury after vitrification. Moreover, the vitrification increased apoptosis more in the vitrified oocytes by sucrose media than the vitrified

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

2013-01-01

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...

Influence of Insulin-like Growth Factor 1 on Nuclear Maturation of Germinal Vesicle Mouse Oocytes

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R mahmoudi

2014-09-01

Full Text Available Background & aim: In vitro maturation and fertilization of oocytes play an important role in reproductive biotechnology. The aim of this study is to define the IGF-1 effect on in vitro maturation, fertilization and development of mice immature oocytes to 2-cells in TCM199 medium cultures. Methods: In this study 4 week old NMRI mice were used. Ovaries stimulation carried out using PMSG. GV oocytes with or without cumulus cells were isolated from ovaries and cultured in TCM199 in presence of 100 ng IGF-1 for 24hr.The oocytes (MII were inseminated with sperm in T6 medium for fertilization and development of 2-cells stage and they were investigated under inverted microscope. Data analysis was performed by using Chi- 2 test. Results: In cumulus cell group and in the presence of insulin-like growth factor fertilization of oocytes, forming embryos and the formation of 2-cells compared to the group without cumulus cells significantly increased (p < 0.05. Conclusion: As the results showed oocytes with cumulus cells in the presence of insulin-like growth factor enhances maturation, fertilization and embryonic development in 2-cells oocytes compared to group without cumulus cells TCM199.

Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

International Nuclear Information System (INIS)

Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer; Campbell, Keith H.S.

2005-01-01

The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requires permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells

Light-induced, GTP-binding protein mediated membrane currents of Xenopus oocytes injected with rhodopsin of cephalopods.

Science.gov (United States)

Ando, H; Seidou, M; Kito, Y

1991-01-01

Xenopus oocytes that were injected with rhabdomeric membranes of squid and octopus photoreceptors acquired light sensitivity. The injected oocytes showed a light-induced current having characteristics similar to other G-protein-mediated Cl- currents induced by the activation of other membrane receptors. Pretreatment of the oocytes with pertussis toxin before the injection suppressed the generation of the light-induced current, indicating an ability of cephalopod rhodopsin to cross-react with an endogenous G-protein of Xenopus oocytes.

Phospholipid transfer activities in toad oocytes and developing embryos

International Nuclear Information System (INIS)

Rusinol, A.; Salomon, R.A.; Bloj, B.

1987-01-01

The role of lipid transfer proteins during plasma membrane biogenesis was explored. Developing amphibia embryos were used because during their growth an active plasma membrane biosynthesis occurs together with negligible mitochondrial and endoplasmic reticulum proliferation. Sonicated vesicles, containing 14 C-labeled phospholipids and 3 H-labeled triolein, as donor particles and cross-linked erythrocyte ghosts as acceptor particles were used to measure phospholipid transfer activities in unfertilized oocytes and in developing embryos of the toad Bufo arenarum. Phosphatidylcholine transfer activity in pH 5.1 supernatant of unfertilized oocytes was 8-fold higher than the activity found in female toad liver supernatant, but dropped steadily after fertilization. After 20 hr of development, at the stage of late blastula, the phosphatidylcholine transfer activity had dropped 4-fold. Unfertilized oocyte supernatant exhibited phosphatidylinositol and phosphatidylethanolamine transfer activity also, but at the late blastula stage the former had dropped 18-fold and the latter was no longer detectable under our assay conditions. Our results show that fertilization does not trigger a phospholipid transport process catalyzed by lipid transfer proteins. Moreover, they imply that 75% of the phosphatidylcholine transfer activity and more than 95% of the phosphatidylinositol and phosphatidylethanolamine transfer activities present in pH 5.1 supernatants of unfertilized oocytes may not be essential for toad embryo development. Our findings do not rule out, however, that a phosphatidylcholine-specific lipid transfer protein could be required for embryo early growth

Inherited effects from irradiated mouse immature oocytes detected in aggregation embryo chimeras

International Nuclear Information System (INIS)

Straume, T.; Raabe, O.G.; Walsh, K.J.; Wiley, L.M.

1993-01-01

Data obtained using the mouse-preimplantation-embryo-chimera assay are presented that show a transmitted effect following low-dose irradiation of immature oocytes in vivo. Six-week-old female mice were irradiated using 137 Cs-γ-rays (0.05 Gy, 0.15 Gy, and unexposed controls). At 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 weeks post exposure, the mice were mated and aggregation chimeras made from the 4-cell embryos. Three independent experiments have now been carried out, all showing a significant embryonic cell-proliferation disadvantage of the embryos obtained from the females treated 7 weeks previously, i.e., embryos from oocytes that were immature at the time of radiation exposure. No effect was detected at 1-6 weeks when embryos were obtained from maturing oocytes. Also, the effect was not seen at 8, 9, 10, 11, and 12 weeks post exposure. The implications of these results are discussed in the light of previous studies on mouse oocytes

Nucleoli from two-cell embryos support the development of enucleolated germinal vesicle oocytes in the pig.

Science.gov (United States)

Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

2012-11-01

Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.

Outcomes of pregnancies achieved by double gamete donation: A comparison with pregnancies obtained by oocyte donation alone.

Science.gov (United States)

Preaubert, Lise; Vincent-Rohfritsch, Aurélie; Santulli, Pietro; Gayet, Vanessa; Goffinet, François; Le Ray, Camille

2018-03-01

Women increasingly resort to oocyte donation to become pregnant. The high risk of preeclampsia found in oocyte donation pregnancies and the separate risk of preeclampsia associated with sperm donation may be cumulative in double donation pregnancies. We aimed to study the obstetrical and perinatal outcomes of pregnancies obtained by double donation (both oocyte and sperm) in comparison with those obtained by oocyte donation alone (oocyte donation and partner's sperm). This cohort study included all women aged 43 and older who became pregnant after oocyte donation and gave birth between 2010 and 2016 in a tertiary maternity center. Primary outcomes were preeclampsia and hypertensive gestational disorders. Secondary outcomes were gestational diabetes, placental abnormalities, postpartum hemorrhage, perinatal death, and preterm delivery. We used univariate and multivariate analysis to compare IVF with double donation and IVF with oocyte donation alone for obstetric and perinatal outcomes. 247 women, 53 with double donations and 194 with oocyte donations alone, gave birth to 339 children. We observed no significant differences between groups for any obstetric or perinatal complications, except for the risk of gestational diabetes, which was more frequent in women with double donations compared with oocyte donation alone (26.4% vs. 12.9%, P = 0.02) and remained significant after adjustment (aOR = 2.80 95%CI[1.26-6.17]). Rates of gestational hypertension and preeclampsia were high, but similar between groups (20.7% vs. 26.3%, P = 0.41, and 18.9% vs. 17.5%, P = 0.82). Women undergoing oocyte donation should be fully informed of its high rates of obstetric and perinatal risks. However, except for a higher observed risk of gestational diabetes, double donation does not appear to be associated with a higher risk of complications than oocyte donation alone. Copyright © 2017 Elsevier B.V. All rights reserved.

Error-prone meiotic division and subfertility in mice with oocyte-conditional knockdown of pericentrin.

Science.gov (United States)

Baumann, Claudia; Wang, Xiaotian; Yang, Luhan; Viveiros, Maria M

2017-04-01

Mouse oocytes lack canonical centrosomes and instead contain unique acentriolar microtubule-organizing centers (aMTOCs). To test the function of these distinct aMTOCs in meiotic spindle formation, pericentrin (Pcnt), an essential centrosome/MTOC protein, was knocked down exclusively in oocytes by using a transgenic RNAi approach. Here, we provide evidence that disruption of aMTOC function in oocytes promotes spindle instability and severe meiotic errors that lead to pronounced female subfertility. Pcnt-depleted oocytes from transgenic (Tg) mice were ovulated at the metaphase-II stage, but show significant chromosome misalignment, aneuploidy and premature sister chromatid separation. These defects were associated with loss of key Pcnt-interacting proteins (γ-tubulin, Nedd1 and Cep215) from meiotic spindle poles, altered spindle structure and chromosome-microtubule attachment errors. Live-cell imaging revealed disruptions in the dynamics of spindle assembly and organization, together with chromosome attachment and congression defects. Notably, spindle formation was dependent on Ran GTPase activity in Pcnt-deficient oocytes. Our findings establish that meiotic division is highly error-prone in the absence of Pcnt and disrupted aMTOCs, similar to what reportedly occurs in human oocytes. Moreover, these data underscore crucial differences between MTOC-dependent and -independent meiotic spindle assembly. © 2017. Published by The Company of Biologists Ltd.

Oocytes with a dark zona pellucida demonstrate lower fertilization, implantation and clinical pregnancy rates in IVF/ICSI cycles.

Directory of Open Access Journals (Sweden)

Wei Shi

Full Text Available The morphological assessment of oocytes is important for embryologists to identify and select MII oocytes in IVF/ICSI cycles. Dysmorphism of oocytes decreases viability and the developmental potential of oocytes as well as the clinical pregnancy rate. Several reports have suggested that oocytes with a dark zona pellucida (DZP correlate with the outcome of IVF treatment. However, the effect of DZP on oocyte quality, fertilization, implantation, and pregnancy outcome were not investigated in detail. In this study, a retrospective analysis was performed in 268 infertile patients with fallopian tube obstruction and/or male factor infertility. In 204 of these patients, all oocytes were surrounded by a normal zona pellucida (NZP, control group, whereas 46 patients were found to have part of their retrieved oocytes enclosed by NZP and the other by DZP (Group A. In addition, all oocytes enclosed by DZP were retrieved from 18 patients (Group B. No differences were detected between the control and group A. Compared to the control group, the rates of fertilization, good quality embryos, implantation and clinical pregnancy were significantly decreased in group B. Furthermore, mitochondria in oocytes with a DZP in both of the two study groups (A and B were severely damaged with several ultrastructural alterations, which were associated with an increased density of the zona pellucida and vacuolization. Briefly, oocytes with a DZP affected the clinical outcome in IVF/ICSI cycles and appeared to contain more ultrastructural alterations. Thus, DZP could be used as a potential selective marker for embryologists during daily laboratory work.

[CHALLENGING THE OPTIMAL NUMBER OF RETRIEVED OOCYTES AND ITS IMPACT ON PREGNANCY AND LIVE BIRTH RATES IN IVF/ICSI CYCLES].

Science.gov (United States)

Blais, Idit; Lahav-Baratz, Shirly; Koifman, Mara; Wiener-Megnazi, Zofnat; Auslender, Ron; Dirnfeld, Martha

2015-06-01

Large numbers of retrieved oocytes are associated with higher chances of having cryopreservation of embryos. However, the process entailed exposes women to increased risk for ovarian hyperstimulation syndrome. Furthermore, mild ovary stimulation protocols are more patient-friendly and with less adverse effects. Only limited reports exist on the significance of the number of retrieved oocytes achieved in a single stimulation cycle. To investigate the optimal number of retrieved oocytes to achieve pregnancy and live birth. This retrospective analysis included 1590 IVF cycles. Oocytes maturation, fertilization, cleavage, as well as pregnancy and live birth rates were analyzed according to the number of retrieved oocytes. Oocyte maturation, fertilization and cleavage rates were lower in cycles with more than 10 retrieved oocytes compared with other groups. Live birth rates were highest when the number of retrieved oocytes was 11-15. Retrieval of more than 15 oocytes was not associated with a significant increase in chances of conception and birth. The better oocyte quality with 10 or less oocytes retrieved could be the result of a possible interference with the natural selection, or the minimized exposure of growing follicles to the potentially negative effects of ovarian stimulation. Although the average number of available embryos was higher when more than 10 oocytes were retrieved, achievement of more than 15 oocytes did not improve IVF outcome in terms of pregnancy and delivery rates. Analysis of 1590 IVF cycles including the frozen-thawed transfers shows that the best outcomes were achieved with an optimal number of 11-15 oocytes.

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

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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.

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

DEFF Research Database (Denmark)

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

2013-01-01

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)?...

Fidelity in the translation of reovirus mRNA in oocytes of Xenopus laevis

International Nuclear Information System (INIS)

Opperman, D.P.J.; Van der Walt, M.P.K.; Reinecke, C.J.

1988-01-01

The translation products formed from reovirus mRNA micro-injected into oocytes of Xenopus laevis were compared with authentic reovirus proteins by polyacrylamide gel electrophoresis, immunoprecipition, isolation of immune complexes by affinity chromatography and peptide mapping using proteolytic digestion with Staphylococcus aureus V8 protease. Products from the s-, m- and l-class mRNAs were detectable in quantities comparable to those synthesized in vivo, confirming that the differences in the translational efficiencies in the oocyte system resemble those found in vivo. The experimental procedures during this study, include the labelling of these translation products with [ 35 S]methionine. Protein μ1C was formed in the oocytes by post-translational cleavage of its precursor, protein μ1. The V8 protease peptide profile of the translation product with the same electrophoretic mobility as protein, σ3, is identical to that of the authentic reovirus protein. All these observations indicate a high degree of fidelity in the translation of reovirus mRNA in the oocyte system. The fidelity in translation, ratios of the various translation products, as well as post-translational modification suggest that the oocyte system might provide a means for studying the mechanism of reovirus morphogenesis

Female Infertility Caused by Mutations in the Oocyte-Specific Translational Repressor PATL2

KAUST Repository

Maddirevula, Sateesh

2017-09-29

Infertility is a relatively common disorder of the reproductive system and remains unexplained in many cases. In vitro fertilization techniques have uncovered previously unrecognized infertility phenotypes, including oocyte maturation arrest, the molecular etiology of which remains largely unknown. We report two families affected by female-limited infertility caused by oocyte maturation failure. Positional mapping and whole-exome sequencing revealed two homozygous, likely deleterious variants in PATL2, each of which fully segregates with the phenotype within the respective family. PATL2 encodes a highly conserved oocyte-specific mRNP repressor of translation. Previous data have shown the strict requirement for PATL2 in oocyte-maturation in model organisms. Data gathered from the families in this study suggest that the role of PATL2 is conserved in humans and expand our knowledge of the factors that are necessary for female meiosis.

The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture

OpenAIRE

MORITA, Yasuhiro; TANIGUCHI, Masayasu; TANIHARA, Fuminori; ITO, Aya; NAMULA, Zhao; DO, Lanh Thi Kim; TAKAGI, Mitsuhiro; TAKEMOTO, Tatsuya; OTOI, Takeshige

2016-01-01

The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24...

Quercetin supplemented diet improves follicular development, oocyte quality, and reduces ovarian apoptosis in rabbits during summer heat stress.

Science.gov (United States)

Naseer, Zahid; Ahmad, Ejaz; Epikmen, Erkmen Tuğrul; Uçan, Uğur; Boyacioğlu, Murat; İpek, Emrah; Akosy, Melih

2017-07-01

The present study was designed to test the modulatory effect of dietary quercetin on follicle population, apoptosis, in vitro maturation rate and quality of oocytes in heat stressed female rabbits. A total of thirty-four New Zealand White heat stress (HS) exposed female rabbits were either fed with quercetin supplemented diet (QU-HS) or non-supplemented (HS) diet. Firstly, laparotomy was performed for oocyte retrieval and then, oocyte grading and COCs dimensional assessments were conducted. The A and B-grade oocytes were submitted for in vitro maturation. Thereafter, the ovaries were collected from rabbits and were processed for follicular population estimation and granulosa cells apoptosis. The results showed that follicle number, retrieved oocytes and A-grade oocytes were higher in QU-HS, comparatively. A significant difference was observed in A-grade oocytes dimensions between QU-HS and HS treatment groups. The oocyte maturation rate was same across the groups. The quercetin supplementation significantly improved primordial and antral stage follicles. A greater number of apoptotic cells were observed in primary and antral follicles in the HS group. In conclusion, the quercetin provision improves the follicular development, minimize granulosa cells apoptosis, and maintain the oocyte competence in HS rabbits. Copyright © 2017. Published by Elsevier Inc.

Regulation of the formin Cappuccino is critical for polarity of Drosophila oocytes

Science.gov (United States)

Bor, Batbileg; Bois, Justin S.; Quinlan, Margot E.

2014-01-01

The Drosophila formin Cappuccino (Capu) creates an actin mesh-like structure that traverses the oocyte during mid-oogenesis. This mesh is thought to prevent premature onset of fast cytoplasmic streaming which normally happens during late-oogenesis. Proper cytoskeletal organization and cytoplasmic streaming are crucial for localization of polarity determinants such as osk, grk, bcd and nanos mRNAs. Capu mutants disrupt these events, leading to female sterility. Capu is regulated by another nucleator, Spire, as well as by autoinhibition in vitro. Studies in vivo confirm that Spire modulates Capu’s function in oocytes; however, how autoinhibition contributes is still unclear. To study the role of autoinhibition in flies, we expressed a Capu construct that is missing the Capu Inhibitory Domain, CapuΔN. Consistent with a gain of activity due to loss of autoinhibition, the actin mesh was denser in CapuΔN oocytes. Further, cytoplasmic streaming was delayed and fertility levels decreased. Localization of osk mRNA in early stages, and bcd and nanos in late stages, were disrupted in CapuΔN-expressing oocytes. Finally, evidence that these phenotypes were due to a loss of autoinhibition comes from co-expression of the N-terminal half of Capu with CapuΔN, which suppressed the defects in actin, cytoplasmic streaming and fertility. From these results, we conclude that Capu can be autoinhibited during Drosophila oocyte development. PMID:25557988

Oocyte activation and phospholipase C zeta (PLCζ: diagnostic and therapeutic implications for assisted reproductive technology

Directory of Open Access Journals (Sweden)

Ramadan Walaa M

2012-07-01

Full Text Available Abstract Infertility affects one in seven couples globally and has recently been classified as a disease by the World Health Organisation (WHO. While in-vitro fertilisation (IVF offers effective treatment for many infertile couples, cases exhibiting severe male infertility (19–57% often remain difficult, if not impossible to treat. In such cases, intracytoplasmic sperm injection (ICSI, a technique in which a single sperm is microinjected into the oocyte, is implemented. However, 1–5% of ICSI cycles still fail to fertilise, affecting over 1000 couples per year in the UK alone. Pregnancy and delivery rates for IVF and ICSI rarely exceed 30% and 23% respectively. It is therefore imperative that Assisted Reproductive Technology (ART protocols are constantly modified by associated research programmes, in order to provide patients with the best chances of conception. Prior to fertilisation, mature oocytes are arrested in the metaphase stage of the second meiotic division (MII, which must be alleviated to allow the cell cycle, and subsequent embryogenesis, to proceed. Alleviation occurs through a series of concurrent events, collectively termed ‘oocyte activation’. In mammals, oocytes are activated by a series of intracellular calcium (Ca2+ oscillations following gamete fusion. Recent evidence implicates a sperm-specific phospholipase C, PLCzeta (PLCζ, introduced into the oocyte following membrane fusion as the factor responsible. This review summarises our current understanding of oocyte activation failure in human males, and describes recent advances in our knowledge linking certain cases of male infertility with defects in PLCζ expression and activity. Systematic literature searches were performed using PubMed and the ISI-Web of Knowledge. Databases compiled by the United Nations and World Health Organisation databases (UNWHO, and the Human Fertilization and Embryology Authority (HFEA were also scrutinised. It is clear that PLCζ plays a

Oocyte activation and phospholipase C zeta (PLCζ): diagnostic and therapeutic implications for assisted reproductive technology.

Science.gov (United States)

Ramadan, Walaa M; Kashir, Junaid; Jones, Celine; Coward, Kevin

2012-07-09

Infertility affects one in seven couples globally and has recently been classified as a disease by the World Health Organisation (WHO). While in-vitro fertilisation (IVF) offers effective treatment for many infertile couples, cases exhibiting severe male infertility (19-57%) often remain difficult, if not impossible to treat. In such cases, intracytoplasmic sperm injection (ICSI), a technique in which a single sperm is microinjected into the oocyte, is implemented. However, 1-5% of ICSI cycles still fail to fertilise, affecting over 1000 couples per year in the UK alone. Pregnancy and delivery rates for IVF and ICSI rarely exceed 30% and 23% respectively. It is therefore imperative that Assisted Reproductive Technology (ART) protocols are constantly modified by associated research programmes, in order to provide patients with the best chances of conception. Prior to fertilisation, mature oocytes are arrested in the metaphase stage of the second meiotic division (MII), which must be alleviated to allow the cell cycle, and subsequent embryogenesis, to proceed. Alleviation occurs through a series of concurrent events, collectively termed 'oocyte activation'. In mammals, oocytes are activated by a series of intracellular calcium (Ca2+) oscillations following gamete fusion. Recent evidence implicates a sperm-specific phospholipase C, PLCzeta (PLCζ), introduced into the oocyte following membrane fusion as the factor responsible. This review summarises our current understanding of oocyte activation failure in human males, and describes recent advances in our knowledge linking certain cases of male infertility with defects in PLCζ expression and activity. Systematic literature searches were performed using PubMed and the ISI-Web of Knowledge. Databases compiled by the United Nations and World Health Organisation databases (UNWHO), and the Human Fertilization and Embryology Authority (HFEA) were also scrutinised. It is clear that PLCζ plays a fundamental role in the

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes.

Science.gov (United States)

Capmany, G; Mart, M; Santaló, J; Bolton, V N

1998-10-01

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.

Anti-Bovine Programmed Death-1 Rat–Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle

Directory of Open Access Journals (Sweden)

Tomohiro Okagawa

2017-06-01

Full Text Available Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1/PD-ligand 1 (PD-L1, is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat–bovine chimeric monoclonal antibody 5D2 (Boch5D2 was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV. Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4+ T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals.