An, Ran; Turek, John; Machaty, Zoltan; Nolte, David
Freshly-harvested porcine oocytes are invested with cumulus granulosa cells in cumulus-oocyte complexes (COCs). The cumulus cell layer is usually too thick to image the living oocyte under a conventional microscope. Therefore, it is difficult to assess the oocyte viability. The low success rate of implantation is the main problem for in vitro fertilization. In this paper, we demonstrate our dynamic imaging technique called motility contrast imaging (MCI) that provides a non-invasive way to monitor the COCs before and after maturation. MCI shows a change of intracellular activity during oocyte maturation, and a measures dynamic contrast between the cumulus granulosa shell and the oocytes. MCI also shows difference in the spectral response between oocytes that were graded into quality classes. MCI is based on shortcoherence digital holography. It uses intracellular motility as the endogenous imaging contrast of living tissue. MCI presents a new approach for cumulus-oocyte complex assessment.
Interactions between oocytes and cumulus cells during in vitro maturation of porcine cumulus-oocyte complexes in a chemically defined medium: effect of denuded oocytes on cumulus expansion and oocyte maturation.
Appeltant, R; Somfai, T; Nakai, M; Bodó, S; Maes, D; Kikuchi, K; Van Soom, A
The aim of the present study was to clarify interactions between oocytes and cumulus cells (CCs) on the level of cumulus expansion and oocyte maturation during IVM of cumulus-oocyte complexes (COCs) in a chemically defined medium using a system that allows individual tracking of oocytes. Especially, the influence of oocyte-secreted factors was investigated by the aid of addition of denuded oocytes (DOs) as a possible approach to improve the IVM system. The basic maturation medium was porcine oocyte medium with addition of gonadotropins only during the first 20 hours of IVM. During IVM, COCs were kept fixed to the bottom of culture dish by adhesive Cell-Tak coating, which enabled individual tracking of COCs during IVM. Size changes in COCs during IVM were measured by digital image analysis. Cumulus expansion in a porcine oocyte medium of intact COCs increased in a typical manner until 20 hours and decreased in size subsequently until 48 hours of IVM (P 0.05) but did enhance cumulus expansion of oocytectomized complexes (P medium: carbenoxolone repressed the expansion of COCs at 20 hours of IVM. In conclusion, the porcine oocyte enhances cumulus expansion both by gap junctional communications and presumably by oocyte-secreted factor production. Nevertheless, the presence of oocytes is not a prerequisite for this process. In return, CCs maintain meiotic arrest in cumulus-enclosed oocytes during the initial culture through gap junctions. On the basis of these findings, future research could investigate if coculture with DOs during IVM is beneficial for fertilization and embryo development. Copyright © 2015 Elsevier Inc. All rights reserved.
Su, You-Qiang; Sugiura, Koji; Li, Qinglei; Wigglesworth, Karen; Matzuk, Martin M.; Eppig, John J.
LH triggers the maturation of the cumulus-oocyte complex (COC), which is followed by ovulation. These ovarian follicular responses to LH are mediated by epidermal growth factor (EGF)-like growth factors produced by granulosa cells and require the participation of oocyte-derived paracrine factors. However, it is not clear how oocytes coordinate with the EGF receptor (EGFR) signaling to achieve COC maturation. The aim of the present study was to test the hypothesis that oocytes promote the expression of EGFR by cumulus cells, thus enabling them to respond to the LH-induced EGF-like peptides. Egfr mRNA and protein expression were dramatically reduced in cumulus cells of mutant mice deficient in the production of the oocyte-derived paracrine factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). Moreover, microsurgical removal of oocytes from wild-type COCs dramatically reduced expression of Egfr mRNA and protein, and these levels were restored by either coculture with oocytes or treatment with recombinant GDF9 or GDF9 plus recombinant BMP15. Blocking Sma- and Mad-related protein (SMAD)2/3 phosphorylation in vitro inhibited Egfr expression in wild-type COCs and in GDF9-treated wild-type cumulus cells, and conditional deletion of Smad2 and Smad3 genes in granulosa cells in vivo resulted in the reduction of Egfr mRNA in cumulus cells. These results indicate that oocytes promote expression of Egfr in cumulus cells, and a SMAD2/3-dependent pathway is involved in this process. At least two oocyte-derived growth factors, GDF9 and BMP15, are required for EGFR expression by cumulus cells. PMID:20382892
Sudarshan, Viswanath P; Weiser, Tobias; Chintala, Phalgun; Mandal, Subhamoy; Dutta, Rahul
Visual observation of Cumulus Oocyte Complexes provides only limited information about its functional competence, whereas the molecular evaluations methods are cumbersome or costly. Image analysis of mammalian oocytes can provide attractive alternative to address this challenge. However, it is complex, given the huge number of oocytes under inspection and the subjective nature of the features inspected for identification. Supervised machine learning methods like random forest with annotations...
SunQingyuan; FengHuailiang; 等
Bovine cumulus-oocyte complexes were cultured in the maturation medium containing 4 different concentrations of verapamil and trifluoperazine to testify the necessity of extracellular Ca2+ and Ca2+-calmodulin complex for the resumption and completion of meiosis as well as cumulus expansion.Ultrastructure of the treated oocytes was also observed to investigate the cytoplasm maturation.The results showed that verapamil didn't influence the cumulus expansion,meiosis resumption and completion and cytoplasm maturation significantly.TFP inhibited cumulus expansion in a dose-dependent manner.25um trifluoperazine significantly inhibited the GVBD and maturation (P<0.01),wherease 1um TFP had no effect,Both oocytes and cumulus cells treated with 25um TFP severely degenerated.Our observtions suggest that the resumption and completion of meiosis and cumulus expansion are Ca2+-CaM dependent and blocking membrane Ca2+ channel does not influence oocyte germinal vesicle breakdown,nuclear and cytoplasm maturation significantly in cattle.
Crocomo, L F; Marques Filho, W C; Ulian, C M V; Branchini, N S; Silva, D T; Ackermann, C L; Landim-Alvarenga, F C; Bicudo, S D
Inhibitors of cyclin-dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor-free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (-) at 38.5°C and 5% CO2 . At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/-) exhibited total expansion while in both Rosco groups (+/-) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression. © 2015 Blackwell Verlag GmbH.
Crocomo, Letícia Ferrari; Marques Filho, Wolff Camargo; Ackermann, Camila Louise; Paschoal, Daniela Martins; Guastali, Midyan Daroz; Dias Maziero, Rosiára Rosária; Sudano, Mateus José; Landim-Alvarenga, Fernanda da Cruz; Bicudo, Sony Dimas
Temporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus-oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.
Sudarshan, Viswanath P; Chintala, Phalgun; Mandal, Subhamoy; Dutta, Rahul
Visual observation of Cumulus Oocyte Complexes provides only limited information about its functional competence, whereas the molecular evaluations methods are cumbersome or costly. Image analysis of mammalian oocytes can provide attractive alternative to address this challenge. However, it is complex, given the huge number of oocytes under inspection and the subjective nature of the features inspected for identification. Supervised machine learning methods like random forest with annotations from expert biologists can make the analysis task standardized and reduces inter-subject variability. We present a semi-automatic framework for predicting the class an oocyte belongs to, based on multi-object parametric segmentation on the acquired microscopic image followed by a feature based classification using random forests.
Choi, Jung Kyu; He, Xiaoming
Background 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. Objective 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). Methods 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. Results 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. Significance 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. PMID:23457518
Jung Kyu Choi
Full Text Available BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. SIGNIFICANCE: 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.
Kylie R Dunning
Full Text Available Fatty acid oxidation is an important energy source for the oocyte; however, little is known about how this metabolic pathway is regulated in cumulus-oocyte complexes. Analysis of genes involved in fatty acid oxidation showed that many are regulated by the luteinizing hormone surge during in vivo maturation, including acyl-CoA synthetases, carnitine transporters, acyl-CoA dehydrogenases and acetyl-CoA transferase, but that many are dysregulated when cumulus-oocyte complexes are matured under in vitro maturation conditions using follicle stimulating hormone and epidermal growth factor. Fatty acid oxidation, measured as production of ³H₂O from [³H]palmitic acid, occurs in mouse cumulus-oocyte complexes in response to the luteinizing hormone surge but is significantly reduced in cumulus-oocyte complexes matured in vitro. Thus we sought to determine whether fatty acid oxidation in cumulus-oocyte complexes could be modulated during in vitro maturation by lipid metabolism regulators, namely peroxisome proliferator activated receptor (PPAR agonists bezafibrate and rosiglitazone. Bezafibrate showed no effect with increasing dose, while rosiglitazone dose dependently inhibited fatty acid oxidation in cumulus-oocyte complexes during in vitro maturation. To determine the impact of rosiglitazone on oocyte developmental competence, cumulus-oocyte complexes were treated with rosiglitazone during in vitro maturation and gene expression, oocyte mitochondrial activity and embryo development following in vitro fertilization were assessed. Rosiglitazone restored Acsl1, Cpt1b and Acaa2 levels in cumulus-oocyte complexes and increased oocyte mitochondrial membrane potential yet resulted in significantly fewer embryos reaching the morula and hatching blastocyst stages. Thus fatty acid oxidation is increased in cumulus-oocyte complexes matured in vivo and deficient during in vitro maturation, a known model of poor oocyte quality. That rosiglitazone further
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...
Cumulus-oocyte complexes were obtained from cows by aspiration of small (1-6 mm in diameter) antral follicles after slaughter. Complexes with a compact multilayered cumulus investment were cultured and processed for transmission electron microscopy after different periods of culture including a 0...... frequency of gap junctions was maintained until 12-18 h of culture where the junctional contact was completely disrupted. This decrease in intercellular communication was parallelled by resumption of oocyte meiosis....
Full Text Available Transforming growth factor-β (TGF-β is a two-chain polypeptide with molecular weight of 25 kDa which takes significant role in the steroidogenesis process. In the ovarian oocyte in particular, TGF-β has an important role in regulating reproductive function. TGF-β represents a key intrafollicular protein that regulates follicle development and aromatization process. The purpose of this research was to characterize and identify a protein fraction of TGF-β from the bovine isolated oocytes, which is synthesized during in vitro oocyte maturation process. Oocytes were collected from follicles with diameter of 3-8 mm. Oocytes were then matured in TCM 199 media supplemented with 5 μg/mg LH, 3% BSA, and 50 μg/ml gentamicin sulfate, and cultured in CO2 incubator (5%, 38.5 oC for 20 hours. TGF-β receptors were identified immunohistochemically. Characteristics of the TGF-β protein were determined using SDS PAGE and TGF-β specification was tested using Western Blotting. The results showed that TGF-β receptors were identified and found in cumulus oocyte complexes (COCs. TGF-β protein was isolated from bovine oocytes with molecular weight 25 kDa and it was identified by Western blotting methods in the same molecular weight.
Matsunaga, R; Funahashi, H
The present study was conducted to examine the supplemented effect of cumulus cell masses (CCMs) derived from middle follicle (MF; 3-6 mm diameter) on the morphology and the meiotic or developmental competence of oocytes from small follicles (SF; 1-2 mm diameter). The number of cumulus cells surrounding oocytes just after collection was also lower in cumulus-oocyte complexes (COCs) from SF than MF. The ooplasmic diameter of oocytes was significantly smaller in SF-derived oocytes than MF-derived ones before and after in vitro maturation (IVM), whereas the diameter significantly increased during the culture. Co-culture of SF-derived COCs with MF-derived CCMs during IVM significantly improved the meiotic competence of the oocytes to the metaphase-II stage. Furthermore, the ooplasmic diameter of SF-derived COCs during IVM was increased to the similar size of MF-derived those in the presence of MF-derived CCMs. The abilities of oocytes to be penetrated, to form male pronuclear formation and to cleave or develop to the blastocyst stage were not affected by the co-culture with CCMs. Electrophoretic analysis of CCM secretions clearly showed the presence of more protein(s) approximately 27.6 kDa in the conditioned medium when supplemented with MF-derived CCMs. In conclusion, we demonstrate that supplementation with MF-derived CCMs improves the ooplasmic diameter and meiotic competence of SF-derived oocytes.
Greco, F; Perrin, J; Auffan, M; Tassistro, V; Orsière, T; Courbiere, B
Conventional genotoxicity tests are technically difficult to apply to oocytes, and results obtained on somatic cells cannot be extrapolated to gametes. We have previously described a comet assay (original-CA) on denuded mouse oocytes, but, in vivo, oocytes are not isolated from their surrounding follicular cells. Our objective was to develop a comet assay on cumulus-oocyte complexes (COC-CA) for a more physiological approach to study the genotoxicity of environmental factors on oocytes. For COC-CA, whole COC were exposed directly to exogenous agents after ovulation and removal from oviducts. Three conditions were studied: a negative control group, and two positive control groups, one of which was exposed to hydrogen peroxide (H2O2) and the other group was incubated with cerium dioxide nanoparticles (CeO2 NPs). With both tests, DNA damage was significant in the presence of both H2O2 and CeO2 NPs compared with the negative control. COC-CA offers an interesting tool for assaying the genotoxicity of environmental agents towards germinal cells. Furthermore, COC-CA is less time-consuming and simplifies the protocol of the original-CA, because COC-CA is easier to perform without the washing-out procedure.
Paulo Roberto Antunes da Rosa
Full Text Available The objective of this study was to investigate the mRNA expression and protein localization of Grb10 gene in bovine cumulus-oocyte complexes (COCs from different follicle sizes. Firstly, it was investigated the mRNA expression to correlate with maturation rates. COCs from follicles at 1-3, 4-6, 6-8 and >8mm were used to evaluate Grb10 gene expression by qRT-PCR assay and nuclear maturation rates. It was observed that more competent oocytes (from follicles at 6-8 and >8mm; P>0.05, had lower Grb10 mRNA expression levels when compared to the oocytes from follicles at 1-3 and 4-6mm (P>0.05. After it was performed an immunofluorescence analysis in COCs from different follicle sizes (1-3, 4-6, 6-8 and >8mm to investigate Grb10 protein localization. Samples were incubated with primary antibody: Polyclonal rabbit anti-Grb10 (1:100. Primary antibody was detected using goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:500. Positive fluorescence signal was detected in all analyzed samples but less evident in COCs from largest follicles. These results characterized Grb10 gene in bovine COC and provide evidences for its involvement during oocyte molecular maturation.
Pomar, F.J.; Roelen, B.A.J.; Slot, K.A.; Tol, van H.T.A.; Colenbrander, B.; Teerds, K.J.
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
A pre-in vitro maturation medium containing cumulus oocyte complex ligand-receptor signaling molecules maintains meiotic arrest, supports the cumulus oocyte complex and improves oocyte developmental competence.
Santiquet, Nicolas W; Greene, Alison F; Becker, John; Barfield, Jennifer P; Schoolcraft, William B; Krisher, Rebecca L
Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following
Wani, N A; Skidmore, J A
In Experiment 1, studies were conducted to apply the transvaginal ultrasound guided ovum pick-up (OPU) technique in dromedary camels after their ovarian super-stimulation and in vivo oocyte maturation. In Experiment 2, the developmental potential of two commonly used oocyte types, i.e., in vivo matured oocytes collected by OPU and abattoir derived in vitro-matured oocytes was compared after their chemical activation. In Experiment 3, developmental competence of oocytes collected from super-stimulated camels by OPU, matured either in vivo or in vitro, was compared after their chemical activation. Mature female dromedary camels super-stimulated with a combination of eCG and pFSH were given an injection of 20 microg of the GnRH analogue, buserelin 24, 26, or 28 h before the scheduled OPU. For collection of cumulus oocyte complexes (COCs) the transducer was guided through the vulva into the cranial most portion of the vagina and 17-gauge, 55 cm single-lumen needle was placed in the needle guide of the ultrasound probe and advanced through the vaginal fornix and into the follicle. Follicular fluid was aspirated using a regulated vacuum pump into tubes containing embryo-flushing media. Aspirates were searched for COCs using a stereomicroscope, and they were then denuded of cumulus cells by hyaluronidase and repeated pipetting. The oocytes were classified as mature (with a visible polar body), immature (with no visible polar body), activated (with divided or fragmented ooplasm) and others (degenerated and abnormal). Overall an average of 12.12 +/- 7.9 COCs were aspirated per animal with an oocyte recovery rate from the aspirated follicles of about 77%. The majority (> 90%) of the collected COCs by OPU were with loose and expanded cumulus cells. The proportion of matured oocytes obtained at 28-29 h (91.2 +/- 4.1) and 26-27 h (82.1 +/- 3.4) were higher (P dromedary camels 26-28 h after GnRH administration. The developmental response, to chemical activation, of in vivo
Del Campo, M R; Del Campo, C H; Mapletoft, R J; Ginther, O J
Morphology and location of the attached cumulus-oocyte complex (COC) were studied in slaughter-house ovaries in horses (49 follicles, 9 to 44 mm), cattle (68 follicles, 6 to 18 mm), and llamas (38 follicles, 3 to 14 mm). The expected point of ovulation was marked, using the ovulation fossa in mares and the center of the projecting follicular surface in cattle and llamas. A follicle was dissected from an ovary, and tissue was removed from the follicle until the COC became visible by transillumination. However, most llama follicles protruded prominently from the ovarian surface so that dissection was not required to locate the COC. The COC was more readily recognized from the external follicular surface in mares and llamas than in cattle, primarily because of a dark oocyte. Compact COC's projected into the antrum with a smooth dome-shape in horses. The COC's in cattle were also dome-shaped but were more irregular and a few contained prominent processes. The mean diameter of the isolated follicle was calculated from 3 planes, except that in llamas the follicles were spherical so that the 3 dimensions were identical. The angle between a straight line connecting the expected ovulation site and the opposite pole and a straight line from the ovulation site to the COC was defined as the COC-location angle. This angle was chosen because it is unaltered by size of a sphere (45 degrees for a COC at the equator). The mean (+/-SEM) COC-location angle differed (P < 0.01) among horses (39.9 +/- 3.3), cattle (50.0 +/- 2.5), and llamas (64.8 +/- 2.1). In mares, the locations of the COC's did not differ from equality between follicular hemispheres, but in cattle and llamas the COC's were located with greater frequency (P < 0.05) in the hemisphere containing the expected ovulation site (cattle, 65%; llamas, 91%).
Okudaira, Yuichi; Wakai, Takuya; Funahashi, Hiroaki
The objective of this study was to compare the cAMP and cGMP levels in cumulus-oocyte complexes (COCs) derived from the middle follicles (MFs, 3-6 mm in diameter) and small follicles (SFs, 1-3 mm in diameter) of pre-pubertal gilts during the first 24-h period of maturation in vitro (IVM). Both cAMP and cGMP levels in MF- and SF-derived oocytes did not change during this period. Although the cAMP levels increased in the COCs at 10 and 20 h after the start of IVM, the levels of cAMP were significantly higher in MF-derived COCs than in SF-derived COCs at 20 h after the start of IVM. On the other hand, the cGMP levels in COCs decreased to basal levels between 10 and 20 h after the start of the IVM, whereas cGMP levels were lower in SF-derived COCs than in MF-derived COCs during the first 10 h. The number of cumulus cells was larger in the MF-derived COCs than in the SF-derived COCs during the first 20-h period of IVM. The estimated cAMP level per cumulus cell at 10 h after the start of the IVM was higher in SF-derived COCs than in MF-derived COCs, whereas the estimated cGMP level per cumulus cell was no different between MF- and SF-derived COCs. From these results, we conclude that cAMP and cGMP levels in COCs, but not in oocytes, drastically change during the first 20-h period of IVM, and that both cAMP and cGMP levels significantly differ between MF- and SF-derived COCs.
Færge, Inger; Strejcek, Frantisek; Laurincik, Jozef
Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturaion is improved....... The aim of the present study was to investigate the effects of FF-MAS on porcien oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/1 cysteine, 10 ng....../ml epidermal growth factor and 50µM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 µM, 3 µM, 10 µM, 30 µM or 100 µM FF-MAS dissolved in ethanol. After maturation the COCs were denuded...
Sutton-McDowall, Melanie L; Mottershead, David G; Gardner, David K; Gilchrist, Robert B; Thompson, Jeremy G
Bidirectional communication between cumulus cells and the oocyte is necessary to achieve oocyte developmental competence. The aim of the present study was to examine the effects of recombinant human bone morphogenetic protein 15 (rhBMP15) and follicle-stimulating hormone (FSH) supplementation on bovine cumulus-oocyte complex (COC) metabolism during maturation. Bovine COCs were matured in the presence of absence of FSH, rhBMP15, or both for 23 h. The addition of FSH and rhBMP15 increased blastocyst development (without rhBMP15 and FSH, 28.4% ± 7.4%; with FSH and rhBMP15, 51.5% ± 5.4%; P levels. rhBMP15 supplementation (regardless of FSH) significantly decreased ADP levels in COCs, leading to an increase in ATP:ADP ratios (P Indicators of mitochondrial activity and cellular REDOX, oxidized flavin adenine dinucleotide (FAD(++)) and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), levels within the oocyte of COCs were significantly higher with rhBMP15 alone, whereas the presence of FSH diminished the rhBMP15 effect. Regardless of treatment, no changes in REDOX state (FAD(++):NAD(P)H). The significant increase in FAD(++) and NAD(P)H in COCs with rhBMP15 was mediated via cumulus cells, because no differences were found in denuded oocytes cultured in the presence or absence of FSH, rhBMP15, or both. The present study demonstrates that a principal metabolic consequence of FSH supplementation of COCs is to alter the glycolytic rate of cumulus cells, whereas that of rhBMP15 is to regulate oxidative phosphorylation in the oocyte, even though it acts via cumulus cells. These effects are tempered when FSH and rhBMP15 are present together but, nonetheless, yield the best oocyte developmental competence.
Schoenfelder, M; Schams, D; Einspanier, R
Steroids are known as important factors on the route of oocytes development and cumulus oocyte complexes (COC) as well as follicular granulosa cells (GC) are suggested to be themselves involved in steroidogenesis. The aim of this study was to characterize such a local sex steroidogenic system during in vitro maturation (IVM) of bovine COCs according to the production of estradiol (E), testosterone (T) and progesterone (P). The expression of two steroid-converting key-enzymes was measured in parallel by quantitative RT-PCR. Furthermore, possible effects of the environmental pollutant tri-butyltin (TBT) were elucidated for the first time on bovine COC and GC in vitro concerning that steroidogenic system. During IVM of bovine COCs concentrations of P increased continuously, corresponding with steady-state levels of 3-beta-hydroxy-steroid-dehydrogenase (HSD) transcripts. In contrast, E together with P450 aromatase mRNA (ARO) increased in the first hours of IVM but declining thereafter, whereas T reached almost balanced levels. However, TBT showed only slight effects during IVM of COC. In cultured GC, LH caused highest P- and E-production within 24h and treatment with 50pM TBT induced a significant decrease of E in contrast to 100pM TBT and the control. These results indicate, that (1) COCs were able to modulate their steroidogenic environment in vitro and that (2) TBT may possibly influence or disturb steroidogenesis in the cows reproductive tract shown here for GC.
Kitahara, Go; Kamimura, Shunichi; Hamana, Katsumi
The relationship between the peripheral concentrations of estradiol-17β (E(2)) and the preovulatory characteristics of cumulus oocyte complexes (COCs) during superovulation treatment was investigated in Japanese Black cows. A superovulation regimen with FSH treatment in a descending manner was commenced on day 7 (n=3) or day 10 (n=2) of the estrous cycle (day 0=estrus). Peripheral blood was collected to measure E(2) concentrations twice a day throughout the treatment. Ovariectomies were performed at 100 h after the initial FSH treatment in five cows. Every follicle more than 8 mm in diameter was isolated from the ovaries, and cumulus-oocyte complexes (COCs) were gently aspirated. The COCs were then separated into three groups based on the characteristics of the cumulus (compact, expanded and denuded) and subgrouped based on the stage of the nucleus in the oocytes (GV, GVBD). Plasma E(2) concentrations tended to increase gradually and reached the peak level at around 84 h (E(2)-84: n=3) or 96 h (E(2)-96: n=2) after the initial FSH treatment. The ratio of COCs with expanded cumulus was significantly higher in E(2)-84 than in E(2)-96 (Psuperovulation treatment in Japanese Black cows.
Faerge, Inger; Strejcek, Frantisek; Laurincik, Jozef; Rath, Detlef; Niemann, Heiner; Schellander, Karl; Rosenkranz, Christine; Hyttel, Poul Maddox; Grøndahl, Christian
Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.
Corbett, Hannah E; Dubé, Chantal D; Slow, Sandy; Lever, Michael; Trasler, Jacquetta M; Baltz, Jay M
Betaine (N,N,N-trimethylglycine) has previously been shown to function in cell volume homeostasis in early mouse embryos and also to be a key donor to the methyl pool in the blastocyst. A betaine transporter (SLC6A20A or SIT1) has been shown to be activated after fertilization, but there is no saturable betaine uptake in mouse oocytes or eggs. Unexpectedly, the same high level of betaine is present in mature metaphase II (MII) eggs as is found in one-cell embryos despite the lack of transport in oocytes or eggs. Significant saturable betaine transport is, however, present in intact cumulus-oocyte complexes (COCs). This transport system has an affinity for betaine of ∼227 μM. The inhibition profile indicates that betaine transport by COCs could be completely blocked by methionine, proline, leucine, lysine, and arginine, and transport is dependent on Na(+) but not Cl(-). This is consistent with transport by a y+L-type amino acid transport system. Both transcripts and protein of one y+L isoform, SLC7A6 (y+LAT2), are present in COCs, with little or no expression in isolated germinal vesicle (GV)-stage oocytes, MII eggs, or one-cell embryos. Betaine accumulated by COCs is transferred into the enclosed GV oocyte, which requires functional gap junctions. Thus, at least a portion of the endogenous betaine in MII eggs could be derived from transport into cumulus cells and subsequent transfer into the enclosed oocyte before gap junction closure during meiotic maturation. The oocyte-derived betaine then could be regulated and supplemented by the SIT1 transporter that arises in the embryo after fertilization.
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.
Morselli, M G; Luvoni, G C; Comizzoli, P
The objective of the study was to assess the efficacy of coculture with conspecific cumulus-denuded oocytes (CDOs) during in vitro maturation in a three-dimensional system of barium alginate microcapsules on the in vitro embryo development of domestic cat cumulus-oocyte complexes (COCs). In Experiment I, COCs were cocultured with conspecific CDOs or cultured separately in a 3D system for 24 hr of in vitro maturation, before assessing the meiotic progression. In Experiment II, the in vitro fertilization of COCs and CDOs was carried out with chilled epididymal spermatozoa and the presumptive zygotes were cultured in vitro separately for 7 days in 3D microcapsules before assesment of embryonic development. The results showed that the viability was maintained and that meiosis was resumed in the 3D culture system. The presence of CDOs during in vitro maturation improved the meiotic competence of the COCs, since the proportions of telophase I/metaphase II were higher than that in the groups cultured separately. The enrichment of the maturation system by companion oocytes also enhanced the ability of COCs to develop into embryos, and increased the percentages of morula and blastoycst stages. The COCs cocultured with CDOs developed at higher rates than the COCs cultured separately and the CDOs themselves. The beneficial effects of coculture with conspecific CDOs were presumably due to the paracrine action of some secreted factors that enhanced many molecular patterns related to the complex of cumulus oophorous cells. Further investigations to understand how the 3D microenvironment can influence the features of oocytes and embryos are required. © 2016 Blackwell Verlag GmbH.
Full Text Available Cumulus expansion of the cumulus-oocyte complex is necessary for meiotic maturation and acquiring developmental competence. Cumulus expansion is based on extracellular matrix synthesis by cumulus cells. Hyaluronic acid is the most abundant component of this extracellular matrix. Cumulus expansion takes place during meiotic oocyte maturation under in vivo and in vitro conditions. Quantification and measurement of cumulus expansion intensity is one possible method of determining oocyte quality and optimizing conditions for in vitro cultivation. Currently, subjective methods of expanded area and more exact cumulus expansion measurement by hyaluronic acid assessment are available. Among the methods of hyaluronic acid measurement is the use of radioactively labelled synthesis precursors. Alternatively, immunological and analytical methods, including enzyme-linked immunosorbent assay (ELISA, spectrophotometry, and high-performance liquid chromatography (HPLC in UV light, could be utilized. The high sensitivity of these methods could provide a precise analysis of cumulus expansion without the use of radioisotopes. Therefore, the aim of this review is to summarize and compare available approaches of cumulus expansion measurement, respecting special biological features of expanded cumuli, and to suggest possible solutions for exact cumulus expansion analysis.
Liu, Shuzhen; Jiang, Ligang; Zhong, Tao; Kong, Shuhui; Zheng, Rongbin; Kong, Fengyun; Zhang, Cong; Zhang, Lei; An, Liguo
Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus–oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus–oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro. PMID:26275143
Full Text Available Objective: The purpose of this study is to investigate the effect of cumulus cells on maturation,fertilization and subsequent development of mouse germinal vesicle oocytes.Materials and Methods: A total of 470 germinal vesicle (GV oocytes were obtained from26 ovaries of 3- 4 week old ICR female mice 48 hours after injection of 5 IU pregnant mareserum gonadotropin (PMSG. Collected oocytes were divided into two groups; group I: GVoocytes without cumulus cells (denuded oocyte, group II: GV oocytes with cumulus cells(cumulus-oocyte complex. The oocytes in both groups were cultured in TCM-199 mediumsupplemented with 10% fetal bovine serum (FBS for 22- 24 hours in a humidified atmosphereof 5% CO2 in air at 37°C. Oocyte maturation was scored under inverted microscope.To do in vitro fertilization, matured oocytes from each group were placed in T6 mediumand capacitated spermatozoa were added. Then the fertilized oocytes were cultured andassessed for cleavage to the 2-cell stage 24 hours and production of blastocyst 120 hoursafter fertilization. Data was analyzed by chi-square test and differences in the values wereconsiderable significant when p<0.05.Results: Maturation, fertilization, cleavage and blastocyst rates in denuded oocytes were:76.32%, 57.49%, 51.15% and 19.14% respectively. In the cumulus-oocyte complex rateswere: 89.41%, 80.76%, 75.58% and 45.62% respectively; all in the cumulus-oocyte complexwere significantly higher than those of denuded oocytes (p<0.05.Conclusion: The present study indicates that cumulus cells have important role duringmaturation, fertilization and subsequent embryo development to the blastocyst stage.
Adam F Summers
Full Text Available Aspiration of bovine follicles 12-36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High androstenedione (A4; >40 ng/ml; mean = 102 and another with Low A4 (<20 ng/ml; mean = 9 in follicular fluid. We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance. To test this hypothesis, estrous cycles of cows were synchronized and ovariectomy was performed 36 hours later. HPLC MS/MS analysis of follicular fluid showed increased dehydroepiandrosterone (6-fold, A4 (158-fold and testosterone (31-fold in the dominant follicle of High A4 cows. However, estrone (3-fold and estradiol (2-fold concentrations were only slightly elevated, suggesting a possible inefficiency in androgen to estrogen conversion in High A4 cows. Theca cell mRNA expression of LHCGR, GATA6, CYP11A1, and CYP17A1 was greater in High A4 cows. Furthermore, abundance of ZAR1 was decreased 10-fold in cumulus oocyte complexes from High A4 cows, whereas NLRP5 abundance tended to be 19.8-fold greater (P = 0.07. There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired. High A4 cows tended (P<0.07 to have a 17% reduction in calving rate compared with Low A4 cows suggesting reduced fertility in the High A4 population. These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development.
Sugiura, Koji; Su, You-Qiang; Li, Qinglei; Wigglesworth, Karen; Matzuk, Martin M.; Eppig, John J.
The differentiation and function of cumulus cells depend upon oocyte-derived paracrine factors, but studies on the estrogen receptor knockout mice suggested that estrogen also participates in these processes. This study investigates the possible coordination of estrogen and oocytes in the development and function of cumulus cells using cumulus expansion and the expression of transcripts required for expansion as functional endpoints. Preantral granulosa cell-oocyte complexes developed in vitro with 17β-estradiol (E2) exhibited increased levels of cumulus expansion and Has2 transcripts, encoding hyaluronan synthase 2, compared with those developed without E2. Moreover, cumulus cell-oocyte complexes (COCs) isolated from antral follicles and maintained in culture without E2 exhibited reduced cumulus expansion and Has2 mRNA levels compared with freshly isolated COCs. Exogenous E2, provided during the maintenance culture, alleviated these deficiencies. However, when oocytes were removed from COCs, E2 supplementation did not maintain competence to undergo expansion; the presence in culture of either fully grown oocytes or recombinant growth differentiation factor 9 (GDF9) was required. Recombinant bone morphogenetic protein 15, but not fibroblast growth factor 8, augmented the GDF9 effect. Oocytes or GDF9 suppressed cumulus cell levels of Nrip1 transcripts encoding nuclear receptor-interacting protein 1, a potential inhibitor of estrogen receptor signals. Therefore, E2 and oocyte-derived paracrine factors GDF9 and bone morphogenetic protein 15 coordinate to promote the development of cumulus cells and maintain their competence to undergo expansion. Furthermore, suppression of Nrip1 expression in cumulus cells by oocyte may be one mechanism mediating cross talk between oocyte and E2 signals that promotes follicular development. PMID:21047911
Cervantes, Miriam P; Palomino, J Manuel; Anzar, Muhammad; Mapletoft, Reuben J; Mastromonaco, Gabriela F; Adams, Gregg P
Experiments were conducted in wood bison to determine the effect of additional maturation time on embryo development of in vivo matured oocytes. In experiment 1, cumulus-oocyte complexes (COC) were collected 30 hours after hCG treatment in superstimulated wood bison, and expanded COC were fertilized immediately or after 4 hours of additional in vitro maturation. Embryo development was assessed on Days 3, 7, and 8 (Day 0 = day of fertilization). No difference in cleavage rate was detected (55.3% vs. 60.5%, P = 0.82), but the Day 8 blastocyst rate was higher after an additional 4 hours of in vitro maturation time (44.7 vs. 18.4%, P = 0.03). In experiment 2, COC were collected at either 30 hours or 34 hours after hCG treatment. Expanded COC from the 30 hours group were fertilized after 4 hours of in vitro maturation, whereas those from the 34 hours group were fertilized immediately. A higher cleavage rate (74.3 vs. 57.0%) and blastocyst rate (54.1 vs. 37.2%) were found in the 34 hours group versus the 30 hours group (P vitro maturation, or an extended period of in vivo maturation are beneficial for in vitro embryo production in wood bison. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.
Sananmuang, T; Techakumphu, M; Tharasanit, T
The developmental competence of cat oocytes matured in vitro is relatively poor when compared with that of in vivo oocytes. The study aimed to investigate the effect of roscovitine on the developmental competence of cat Felis catus oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were classified as Grade I and II to III. Groups of COCs were cultured in 0, 12.5, 25, 50, 100, and 200 microM roscovitine for 24h and were either fixed to assess the stages of nuclear maturation (Experiment 1) or additionally matured in vitro for 24h before fixation (Experiment 2). In Experiment 3, cumulus cells from the COCs treated with roscovitine were examined for apoptosis. Experiment 4 examined the developmental competence of cat oocytes after roscovitine treatment and in vitro fertilization in terms of cleavage and morula and blastocyst formation rates. Roscovitine reversibly arrested cat oocytes at an immature stage in a dose-dependent manner. Roscovitine at 12.5 and 25 microM demonstrated less efficiency compared with that of other doses. However, higher doses of roscovitine induced cumulus cell apoptosis and resulted in a high number of degenerated oocytes after in vitro maturation. Roscovitine at 12.5 and 25 microM were therefore used to evaluate their effect on embryo development. Pretreatment with 12.5 and 25 microM roscovitine prior to in vitro maturation decreased the developmental competence of cat oocytes compared with that of non-roscovitine-treated controls. In conclusion, roscovitine reversibly maintained cat oocytes at the germinal vesicle stage without detrimental effect on nuclear maturation. However, it negatively affected cumulus cell viability and developmental competence.
Appeltant, Ruth; Somfai, Tamás; Kikuchi, Kazuhiro; Maes, Dominiek; Van Soom, Ann
Co-culture of cumulus-oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte-secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β-mercaptoethanol. Cumulus-oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co-culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus-enclosed porcine oocytes in a defined system.
Shimada, M; Maeda, T; Terada, T
Mammalian oocytes are surrounded by numerous layers of cumulus cells, and the loss of gap junctional communication in the outer layers of cumulus cells induces meiotic resumption in oocytes. In this study, we investigated the dynamic changes in the gap junctional protein connexin-43 in cumulus cells during the meiotic resumption of porcine oocytes. The amount of connexin-43 in all layers of cumulus cells recovered from cumulus-oocyte complexes was increased after 4-h cultivation. However, at 12-h cultivation, the positive signal for connexin-43 immunoreactivity was markedly reduced in the outer layers of cumulus cells. When these reductions of connexin-43 were blocked by protein kinase C (PKC) or phosphatidylinositol (PI) 3-kinase inhibitor, networks of filamentous bivalents (i.e., advanced chromosomal status) were undetectable in the germinal vesicle of the oocyte. After 28-h cultivation, when the majority of oocytes were reaching the metaphase I (MI) stage, the connexin-43 in the inner layers of cumulus cells was phosphorylated, regardless of mitogen-activated protein (MAP) kinase activation. These results suggest that the initiation of meiotic resumption, namely, the formation of networks of filamentous bivalents in germinal vesicle, is associated with the reduction of gap junctional protein connexin-43 in the outer layers of cumulus cells via the PKC and/or PI 3-kinase pathway. Moreover, the connexin-43 in the inner layers of cumulus cells is phosphorylated during meiotic progression beyond the MI stage, regardless of MAP kinase activation in cumulus cells surrounding the oocyte.
Full Text Available The aim of this study was to establish and characterize the porcine luteal cells (PLC culture for the subsequent coculture with porcine COC. The final purpose is to promote the oocyte maturation. The PLC was established using corpora lutea obtained from slaughterhouse ovaries. Corpora lutea were dissected and luteal tissue submitted to a mechanical and enzymatic digestion with collagenase IV. The cell suspension was filtered and centrifuged and the cells obtained were diluted in 15 mL of DMEM-F12 supplemented media. Diluted cells were seeded in 3 culture flasks T25, staying in a controlled environment and changing the medium every 2 days. For the analysis and characterization, the cells were assessed by the Nile red staining to detect intracellular lipids, immunocytochemistry (ICC for 3β-hydroxy steroid dehidrogenase (3β-HSD and ELISA for P4 determination. We observed the presence of lipid intracellular droplets. Also, we observed an increase of P4 concentration at 48, 96 y 144 h of primary culture and almost all the cells were positive to the ICC evaluation for 3β-HSD, showing the steroidogenic capacity of the culture cells.
Hwang, Heekyung; Cheon, Yong-Pil
Intact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT-8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest.
Prates, E G; Alves, S P; Marques, C C; Baptista, M C; Horta, A E M; Bessa, R J B; Pereira, R M
The effect of maturation and of two lipid modulators supplementation along in vitro maturation (IVM) on fatty acid (FA) and dimethylacetal (DMA) composition of porcine cumulus oocyte complexes (COC) were studied. Abattoir-derived immature COC were analyzed for FA and DMA or submitted to IVM as follows: control group; t10,c12 CLA group, t10,c12 CLA supplementation for 44 h; Forskolin group, forskolin supplementation during the initial 2 h; t10,c12 CLA + forskolin group, t10,c12 CLA for 44 h and forskolin for just 2h. Each experimental group had five replicates. FA analysis of oocytes, cumulus cells (CC), follicular fluid, and culture media were performed by gas-liquid chromatography. Oocytes and their CC had different FA composition. Oocytes were richer in saturated FA (SFA) preferentially maintaining their FA profile during maturation. Mature CC had the highest polyunsaturated FA (PUFA) content. Five individual and total SFA, and monounsaturated FA (MUFA), notably oleic acid (c9-18:1), percentages were lower (P ≤ 0.023) in mature than in immature CC. t10,c12 CLA was accumulated by COC from t10,c12 CLA and t10,c12 CLA + forskolin groups, mostly in CC where MUFA and an eicosatrienoic isomer decreased (P ≤ 0.043). Nevertheless, PUFA or FA and DMA total content were not affected. Arachidonic acid was reduced in t10,c12 CLA + forskolin CC and hexadecanal-DMA-16:0 in t10,c12 CLA CC. Forskolin alone increased (P ≤ 0.043) c9-18:1 in oocytes. In conclusion, maturation process clearly changed porcine COC FA and DMA profiles, mostly of CC, also more susceptible to modifications induced by t10,c12 CLA. This possibility of manipulating COC lipid composition during IVM could be used to improve oocyte quality/cryopreservation efficiency.
Sowińska, N; Frankowska, K; Filipczyk, A; Adamaszek, A; Nalik, K; Fic, K; Pietsch-Fulbiszewska, A
The aim of this study was to evaluate the effect of co-culture of denuded oocytes with cumulus cells (CC) or cumulus-oocyte complexes (COCs) on in vitro maturation (IVM) and in vitro fertilization (IVF). Immature oocytes were collected from ovaries of domestic cats following a routine ovariectomy. Oocytes were matured in vitro for 24 hr within four groups: (i) denuded oocytes (DO), (ii) DO co-cultured with CC, (iii) DO co-cultured with COC and (iv) COC as a control group. In further experiments, COCs were matured in vitro for 24 hr, and then, oocytes were randomly divided into four groups as previously described and fertilized in vitro. Embryos were cultured for up to 7 days. At the end of each experiment, oocytes/embryos were stained with Hoechst 33342 solution and observed under an inverted fluorescence microscope. The results of oocyte maturation showed that their meiotic competence decreased significantly in all experimental groups, compared to the control group. The maturation rates were approximately 45%, 24%, 43% and 76% in experiment 1, and 21%, 14%, 33% and 50% in experiment 2 in groups (i), (ii), (iii) and (iv), respectively. Examination of in vitro fertilization revealed that embryos developed up to the morula stage in all experimental groups. DO and oocytes cultured with COC during fertilization showed a lower cleavage rate-36% and 25% as opposed to those co-cultured with loose CC and the control group-43% and 42%, respectively. Results of this study indicate that cumulus cells connected with an oocyte into a cumulus-oocyte complex are irreplaceable for the maturation of domestic cat oocyte, but that the addition of loose CC may be beneficial for IVF. © 2016 Blackwell Verlag GmbH.
Full Text Available BACKGROUND: Oocytes are the female gametes which establish the program of life after fertilization. Interactions between oocyte and the surrounding cumulus cells at germinal vesicle (GV stage are considered essential for proper maturation or 'programming' of oocytes, which is crucial for normal fertilization and embryonic development. However, despite its importance, little is known about the molecular events and pathways involved in this bidirectional communication. METHODOLOGY/PRINCIPAL FINDINGS: We used differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT on bovine GV oocyte and cumulus cells and identified 811 and 1247 proteins in GV oocyte and cumulus cells, respectively; 371 proteins were significantly differentially expressed between each cell type. Systems biology modeling, which included Gene Ontology (GO and canonical genetic pathway analysis, showed that cumulus cells have higher expression of proteins involved in cell communication, generation of precursor metabolites and energy, as well as transport than GV oocytes. Our data also suggests a hypothesis that oocytes may depend on the presence of cumulus cells to generate specific cellular signals to coordinate their growth and maturation. CONCLUSIONS/SIGNIFICANCE: Systems biology modeling of bovine oocytes and cumulus cells in the context of GO and protein interaction networks identified the signaling pathways associated with the proteins involved in cell-to-cell signaling biological process that may have implications in oocyte competence and maturation. This first comprehensive systems biology modeling of bovine oocytes and cumulus cell proteomes not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level.
Macaulay, Angus D.; Gilbert, Isabelle; Scantland, Sara
, revealed transcripts common to all three fractions, suggesting the use of transferred transcripts for translation. Furthermore, the removal of potential RNA trafficking by stripping the cumulus cells caused a significant reduction in maturation rates, indicating the need for the cumulus cell RNA transfer...... that the somatic cells also contribute transcripts to the gamete. Here, we show that these transcripts can be visualized moving down the transzonal projections (TZPs) to the oocyte, and that a time course analysis revealed progressive RNA accumulation in the TZPs, indicating that RNA transfer occurs before......So far, the characteristics of a good quality egg have been elusive, similar to the nature of the physiological, cellular, and molecular cues leading to its production both in vivo and in vitro. Current understanding highlights a strong and complex interdependence between the follicular cells...
Influence of epidermal growth factor and insulin-like growth factor 1 on nuclear maturation and fertilization of buffalo cumulus oocyte complexes in serum free media and their subsequent development in vitro.
Purohit, G N; Brady, M S; Sharma, S S
The in vitro maturation, fertilization and development of Indian water buffalo (Bubalus sp.) cumulus oocyte complexes (COCs) to blastocysts were studied during culture, either in serum free tissue culture medium 199 (TCM 199) or Waymouth MB (WM). Based on different supplements added to these media, the experimental groups included: (a) no supplement (control); (b) hormones (FSH, LH and oestradiol) (c) Epidermal growth factor (EGF); (d) IGF-1; and (e) EGF + IGF-1. Experiments were conducted to note three end points: (1) nuclear maturation 24 h after culture (eight replicates); (2) fertilization 24 h after insemination (10 replicates); (3) development to blastocysts (nine replicates). The oocytes were cultured in groups of up to five per drop. Using a two-way (5 x 2) factorial model with interactions, the results were compared using generalized linear models with binomial errors and the logit link function. In experiment 1, the proportion of oocytes reaching metaphase II was higher for all the supplement treatments than the control treatment (t = 3.68, p hormone (chi2 = 17.23, p hormones (31.0%, chi2 = 12.5, p = 0.0004), IGF-1 (35.7%, chi2 = 20.53, p produce a significantly higher rate of progression to blastocysts compared to the control. Once again, media had no effect on outcome. It was concluded that maturation, fertilization and development of buffalo oocytes were enhanced by all supplements tested. Enhancement was maximal with the combination of EGF+IGF-1. In contrast, no significant differences were found between the two types of media used.
Li, H J; Sutton-McDowall, M L; Wang, X; Sugimura, S; Thompson, J G; Gilchrist, R B
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 benefits of c
Zhou, Ping; Wu, Yan-Guang; Wei, De-Li; Li, Qing; Wang, Gang; Zhang, Jie; Luo, Ming-Jiu; Tan, Jing-He
Our objectives were to study how cysteamine, cystine, and cumulus cells (CCs), as well as oocytes interact to increase oocyte intracellular glutathione (GSH) and thereby to establish an efficient in vitro maturation system for cumulus-denuded oocytes (DOs). Using M16 that contained no thiol as maturation medium, we showed that when supplemented alone, neither cystine nor cysteamine promoted GSH synthesis of mouse DOs, but they did when used together. Although goat CCs required either cysteamine or cystine to promote GSH synthesis, mouse CCs required both. In the presence of cystine, goat CCs produced cysteine but mouse CCs did not. Cysteamine reduced cystine to cysteine in cell-free M16. When TCM-199 that contained 83 microM cystine was used as maturation medium, supplementation with cysteamine alone had no effect, but supplementation with 100 microM cysteamine and 200 microM cystine increased blastulation of DOs matured with CC coculture to a level as high as achieved in cumulus-surrounded oocytes (COCs). Similar numbers of young were produced after two-cell embryos from mouse COCs or CC-cocultured DOs matured with optimal thiol supplementation were transferred to pseudopregnant recipients. It is concluded that 1) mouse CCs can use neither cysteamine nor cystine to promote GSH synthesis, but goat CCs can use either one; 2) goat CCs promote mouse oocyte GSH synthesis by reducing cystine to cysteine, but how they use cysteamine requires further investigation; and 3) mouse DOs can use neither cystine nor cysteamine for GSH synthesis, but they restore developmental capacity completely when matured in the presence of optimum supplementation of cysteamine, cystine, and CCs.
Racowsky, C; Baldwin, K V
Studies were carried out with the golden Syrian hamster to investigate the capacity of follicular fluid to maintain oocyte meiotic arrest and to determine the importance of cumulus-membrana granulosa cell contact in the regulation of meiotic status. The follicular fluid studies were conducted by cytological assessment of meiotic stage up to 6 hr after transferring cumulus-free oocytes into antra of explanted "host" follicles in vitro or into follicles of anesthetized animals prior to the gonadotropin surge at proestrus in vivo. The cumulus-membrana granulosa contact studies were undertaken with explanted follicles in which the oocyte-cumulus complex was dislodged from the underlying membrana granulosa, released into the antrum, and subsequently allowed to reestablish contact during 6 hr of incubation within the follicle. The extent of recontact of the dislodged complex with the underlying membrana granulosa was assessed visually at the end of incubation and was classified as close, moderate, or none. These various degrees of contact typically involved the following number of cumulus cells, as determined by serial sectioning of a representative sample of follicles after dislodgement and subsequent incubation: close, 32.7 +/- 1.78; moderate, 9.0 +/- 2.1; and no contact, 0. After 6 hr of incubation either in vitro or in vivo, few transferred oocytes remained at the germinal vesicle (GV) stage (18.8 +/- 8.7 and 17.3 +/- 4.0% GV, respectively). However, time course experiments revealed that meiotic resumption was significantly delayed in transferred oocytes compared with either liberated oocytes, spontaneously maturing oocytes, or follicle-enclosed oocytes induced to mature by luteinizing hormone in vitro (after 4 hr, transferred, 31.3 +/- 6.0% GV; liberated, 0% GV; follicle-enclosed, 0% GV; after 6 hr, 0% transferred oocytes exhibited a GV). In the dislodgement studies, after 6 hr of incubation, 26% of complexes reestablished close contact with the underlying membrana
Revelli, Alberto; Canosa, Stefano; Bergandi, Loredana; Skorokhod, Oleksii A; Biasoni, Valentina; Carosso, Andrea; Bertagna, Angela; Maule, Milena; Aldieri, Elisabetta; D'Eufemia, Maria Diletta; Evangelista, Francesca; Colacurci, Nicola; Benedetto, Chiara
The complex relationship between oocyte morphology, specific follicular fluid metabolites, gene expression in cumulus granulosa cells, and oocyte competence toward fertilization and embryo development still needs further clarification. Forty-six oocytes retrieved from the largest pre-ovulatory follicle of patients undergoing intra-cytoplasmic sperm injection (ICSI) were considered assessing: (a) oocyte morphological characteristics at polarized light microscopy (PLM), (b) specific follicular fluid (FF) metabolites previously suggested to influence oocyte competence (AMH, markers of redox status and of cytotoxicity), (c) transcription of AMH and AMH type II receptor genes in cumulus cells. Data were analyzed using mono-parametric tests and multivariable logistic analysis in order to correlate morphological and biochemical data with fertilization. Comparing normally fertilized oocytes (n = 29, F group) with unfertilized (n = 17, nF group) we observed that: (a) the meiotic spindle area and major axis were significantly higher in nF group and in fertilized oocytes undergoing an early embryo development arrest; (b) AMH level in FF was comparable in F and nF groups; (c) the FF of nF group contained significantly higher levels of cytotoxicity (lactate dehydrogenase) and oxidative stress (Cu,Zn-superoxide dismutase, catalase, 4-hydroxynonenal-protein conjugates) markers; (d) cumulus cells of nF group showed significantly higher AMH receptor type II gene expression. Taken together, these observations suggest that an excessive cytotoxicity level can alter AMH signal transduction within cumulus cells, in turn leading to partial inhibition of aromatase activity, altered cytoplasmic maturation and increased oxidative stress, factors able to impair oocyte fertilization competence and embryo growth.
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
Bilodeau-Goeseels, Sylvie; Magyara, Nora; Collignon, Coralie
The adenosine monophosphate-activated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET) inhibit resumption of meiosis in porcine cumulus-enclosed oocytes. The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine cumulus-enclosed oocytes; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells. The AMPK inhibitor compound C (CC; 1 μM) did not reverse the inhibitory effect of AICAR (1 mM) and MET (2 mM) on porcine oocyte meiosis. Additionally, CC had a significant inhibitory effect on its own. eNOS, c-Src and PI-3 kinase pathway inhibitors did not reverse the effect of metformin on porcine oocyte meiosis. The level of acetyl-CoA carboxylase (ACC) phosphorylation in oocytes and cumulus cells did not change in response to culture in the presence of MET, AICAR, CC, the CaMKK inhibitor STO-609 or the Ca2+ chelator BAPTA-AM for 3 h, but STO-609 increased the percentage of porcine cumulus-enclosed oocytes (CEO) that remained at the germinal vesicle (GV) stage after 24 h of culture. These results indicate that the inhibitory effect of MET and AICAR on porcine oocyte meiosis was probably not mediated through activation of AMPK.
Dimopoulou, M; Anifandis, G; Messini, C I; Dafopoulos, K; Kouris, S; Sotiriou, S; Satra, M; Vamvakopoulos, N; Messinis, I E
The present prospective study examined the follicular fluid oocyte/cumulus-free DNA concentrations (ff o/c-free DNA) during ovarian stimulation and the possible association between ff o/c-free DNA and embryological results such as embryo quality and pregnancy rate. Eighty-three women undergoing IV/ICSI-ET treatments were prospectively included in this study. ff o/c-free DNA was determined by conventional quantitative real time PCR-Sybr green detection approach. The 83 ff samples were categorized in two groups: group 1 (n = 62) with cumulus oocytes complexes (CoCs) ≥2 and group 2 (n = 21) with CoCs = 1. Group 1 revealed significant higher embryo quality in terms of mean score of embryo transfer (MSET), but lower ff o/c-free DNA concentrations compared to group 2. The two groups showed comparable pregnancy rates (positive hCG and clinical pregnancy). The higher the ff o/c-free DNA concentration, the lower the number of produced oocytes. ff o/c-free DNA did not seem to have any direct role in the IVF outcome. Further research is required to clarify whether ff o/c-free DNA is a biomolecular marker of embryo quality and IVF outcome.
van Tol, H T; van Eijk, M J; Mummery, C L; van den Hurk, R; Bevers, M M
Cumulus oocyte complexes (COCs) and cumulus oocyte complexes connected to a piece of the membrane granulosa (COCGs) were isolated from bovine antral follicles with a diameter of 2 to 8 mm. After culture of COCGs without gonadotrophic hormones for 22 hr approximately 50% of the oocytes were still in the germinal vesicle (GV) stage. Histology of the COCGs showed that the pieces of the membrana granulosa were free of thecal cells and parts of the basal membrane. This indicates that the membrana granulosa solely inhibits the progression of meiosis. To investigate the effect of gonadotropins on the resumption of meiosis of oocytes from small and medium sized antral follicles, COCs and COCGs were cultured with or without rec-hFSH or hCG. Addition of 0.05 IU rec-hFSH to the culture medium of COCGs resulted in germinal vesicle breakdown in 97.8% of the oocytes compared to 46% in the control group, and an increase of the diameter of the COCs (479 microns vs. 240 microns in the control group). Addition of 0.05 IU hCG to the culture medium had no effect on nuclear maturation (47.2% GV vs. 48.5% GV in the control group) nor on cumulus expansion (246 microns vs. 240 microns in the control group). RT-PCR on cDNA of the follicular wall, cumulus cells, granulosa cells, COCs, and oocytes revealed that mRNA for FSH receptor was present in all cell types except oocytes. mRNA of the LH receptor was detected exclusively in thecal cells. Nucleotide sequence analysis and alignment of the cloned PCR products showed the presence of two isoforms of the FSH receptor mRNA and two isoforms of the LH receptor mRNA. It is concluded that, in vitro, resumption of meiosis of oocytes, originating from small and medium sized antral follicles and meiotically arrested by the membrana granulosa, is triggered by FSH and not by LH. This is supported by the fact that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of these follicles.
van Tol, H T; Bevers, M M
The effect of follicular cells and their conditioned media on the FSH-induced oocyte maturation of oocytes surrounded by cumulus cells connected to the membrana granulosa (COCGs) was investigated. COCGs and cumulus oocyte complexes (COCs) were cultured for 22 hr in M199 supplemented with 0.05 IU FSH/ml in either the presence of pieces of theca cell layer or in the presence of pieces of membrana granulosa. COCGs and COCs were also cultured for 22 hr in either theca-cell conditioned medium (CMt) or in granulosa cell conditioned medium (CMg), both supplemented with 0.05 IU FSH/ml. To investigate the importance of cell-cell contacts between granulosa cells and cumulus cells, oocytes were cultured as COCs in CMt, as COCs in CMt supplemented with pieces of membrana granulosa, or as COCGs in CMt. In all groups the medium was supplemented with 0.05 IU FSH/ml. After culture the nuclear status of the oocytes was assessed using orcein staining. Culture of COCGs in the presence of theca cells as well as in CMt resulted in a significantly decreased proportion of oocytes that had undergone germinal vesicle breakdown (GVBD) at the end of the culture period as compared to the control. Of the oocytes that resumed meiosis in the presence of theca cells or in CMt, the proportion of oocytes that progressed up to the MII stage was significantly reduced. This indicates the production of a meiosis-inhibiting factor by theca cells. Culture with COCs instead of COCGs resulted in comparable results although the effect was less pronounced. The significant effect on the progression of meiosis of oocytes cultured as COCGs or as COCs, obtained in the presence of granulosa cells or in CMg, was much weaker than the effect of theca cells or culture in CMt. Culture of COCs in CMt supplemented with layers of membrana granulosa and 0.05 IU FSH/ml, resulted in significantly less oocytes that resumed meiosis as compared to culture of COCs in CMt. Of the oocytes that showed GVBD, the proportion that
Full Text Available ABSTRACT Background: Nitric oxide (NO have a dual action in mouse oocyte meiotic maturation which depends on its concentration, but the mechanisms by which it influences oocyte maturation has not been exactly clarified. In this study different signaling mechanisms which exist for in vitro maturation of meiosis was examined in cumulus cell-enclosed oocytes (CEOs after injection of pregnant mare's serum gonadotropin (PMSG to immature female mice. Methods: The CEOs were cultured in spontaneous maturation and hypoxanthine (HX arrested model. Results: Sodium nitroprusside (SNP, an NO donor, 10mM delayed germinal vesicle breakdown (GVBD significantly during the first 5 hrs of incubation and inhibited the formation of first polar body (PB1 at the end of 24 hrs of incubation. SNP (10-5M stimulated the meiotic maturation of oocytes significantly by overcoming the inhibition of HX. Sildenafil (a cGMP stimulator, 100 nM, had a significant inhibitory effects on both spontaneous meiotic maturation and HX-arrested meiotic maturation. Forskolin (an adenylate cyclase stimulator, 6µM and SNP (10mM had the same effects on GVBD. Forskolin reversed the SNP (10-5M stimulated meiotic maturation. Conclusion: These results suggest that differences in pathways are present between SNP-inhibited spontaneous meiotic maturation and SNP-stimulated meiotic maturation in mouse oocytes
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
Briggs, David C; Birchenough, Holly L; Ali, Tariq; Rugg, Marilyn S; Waltho, Jon P; Ievoli, Elena; Jowitt, Thomas A; Enghild, Jan J; Richter, Ralf P; Salustri, Antonietta; Milner, Caroline M; Day, Anthony J
The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.
Dietzel, E; Floehr, J; Van de Leur, E; Weiskirchen, R; Jahnen-Dechent, W
Does fetuin-B inhibit premature zona pellucida (ZP) hardening in mouse oocytes in vitro and thus increase IVF rate? Supplementation of oocyte in vitro maturation (IVM) media with recombinant mouse fetuin-B (rmFetuB) increased fertilization rate without affecting mouse embryo development into blastocysts. Mice deficient in fetuin-B are infertile owing to premature ZP hardening. Premature ZP hardening also occurs during oocyte IVM leading to decreased fertilization rate. We fertilized batches of 20-30 mouse metaphase II (Mll) stage oocytes from C57BL/6 mice with fresh sperm, and studied early embryo development until blastocyst hatching. Oocytes were maintained with or without rmFetuB during IVM and IVF. Exogenous rmFetuB was added to media prior to oocyte isolation. ZP hardening was quantified by chymotrypsin digestion timing and by counting attached sperm. In the absence of cumulus cells, rmFetuB dose-dependently inhibited ZP hardening and increased IVF rate (P = 0.039). Fetuin-B at ≥0.03 mg/ml also inhibited physiological, fertilization-triggered ZP hardening (indicated by increased sperm binding, P = 0.0002), without increasing embryo death. Exogenous rmFetuB increased IVF rate for up to 5 hours of IVM (P = 0.02 at 1 hour, P = 0.01 at 5 hours of IVM). Mll stage oocytes in this study were isolated from the ampullae of fetuin-B expressing mice. Thus, oocytes were protected against premature ZP hardening by endogenous fetuin-B. In humans and livestock, oocytes are usually isolated by follicle puncture before ovulation. In this situation, the deprivation of endogenous fetuin-B would occur earlier and the effect of exogenous fetuin-B in the IVF medium may be even more pronounced. Fertilization-triggered ZP hardening is essential for embryo development but in this study the effect of fetuin-B supplementation was only studied to blastocyst stage. Any influence of added fetuin-B on later embryo development after transplantation remains to be determined. The astacin
Moffatt, Odette; Drury, Sarah; Tomlinson, Matthew; Afnan, Masoud; Sakkas, Denny
To determine the expression of apoptosis-associated molecules on cumulus cells removed from individual oocytes of different maturity, inseminated oocytes and to investigate the possibility of an age-dependent expression. Analysis of apoptosis in cumulus cells isolated from oocytes of different stages of maturity. Assisted reproductive technology program of the Birmingham Women's Hospital, Birmingham, UK. Patients undergoing intracytoplasmic sperm injection or IVF cycles. Percentage of positive cumulus cells when assessed for nuclear DNA damage using the terminal deoxyuridine nucleotide end-labeling assay or stained with antibodies [Fas, Fas ligand, the antiapoptotic protein Bcl-xl, and the RNA-binding protein (TIAR)]. Cumulus cells collected from mature oocytes showed no significant difference in the percentage of apoptotic markers compared to those recovered from immature oocytes, whereas those from patients >/=38 years differed significantly. When cumulus cells were exposed to sperm the levels of apoptotic markers altered significantly from those not exposed to sperm. The results show that the cumulus cells of human oocytes are equipped with a mechanism to undergo apoptosis and that patient age and the exposure of cumulus cells to sperm can alter their profiles of apoptotic markers.
Pruksananonda, Kamthorn; Wasinarom, Artisa; Sereepapong, Wisan; Sirayapiwat, Porntip; Rattanatanyong, Prakasit; Mutirangura, Apiwat
The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls. The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1. The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (±2.66) vs. 75.40 (±4.92); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (±4.79) vs. 67.79 (±5.17); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients. These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS.
Grøndahl, M L; Nielsen, M Eilsø; Dal Canto, M B
, AMH receptor 2, FSH receptor, aromatase and androgen receptor were performed in CC in IVM patients where cumulus-oocyte-complex had expanded, CC in IVM patients where cumulus-oocyte-complex remained compacted, GC from immature follicles and CC and GC from IVF patients. Microarray data on corresponding...
Kiyosu, Chiyo; Tsuji, Takehito; Yamada, Kaoru; Kajita, Shimpei; Kunieda, Tetsuo
Natriuretic peptide type C (NPPC) and its high affinity receptor, natriuretic peptide receptor 2 (NPR2), have been assumed to be involved in female reproduction and have recently been shown to play an essential role in maintaining meiotic arrest of oocytes. However, the overall role of NPPC/NPR2 signaling in female reproduction and ovarian function is still less clear. Here we report the defects observed in oocytes and follicles of mice homozygous for Nppc(lbab) or Npr2(cn), mutant alleles of Nppc or Npr2 respectively to clarify the exact consequences of lack of NPPC/NPR2 signaling in female reproductive systems. We found that: i) Npr2(cn)/Npr2(cn) female mice ovulated a comparable number of oocytes as normal mice but never produced a litter; ii) all ovulated oocytes of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice exhibited abnormalities, such as fragmented or degenerated ooplasm and never developed to the two-cell stage after fertilization; iii) histological examination of the ovaries of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice showed that oocytes in antral follicles prematurely resumed meiosis and that immediately before ovulation, oocytes showed disorganized chromosomes or fragmented ooplasm; and iv) ovulated oocytes and oocytes in the periovulatory follicles of the mutant mice were devoid of cumulus cells. These findings demonstrate that NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation, which affects female fertility through the production of oocytes with developmental capacity.
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.
卵丘细胞与卵母细胞共处于同一个卵泡液微环境中,卵丘细胞与卵母细胞之间复杂的“对话机制”调控着卵母细胞的成熟和卵丘细胞的增殖延伸.在窦卵泡阶段,卵丘细胞由颗粒细胞分化而来,通过缝隙连接与卵母细胞共同形成一个结构和功能上的合胞体.卵泡发育不同时期,卵丘细胞对卵母细胞的代谢调控主要表现为:在窦卵泡期,卵丘细胞为卵母细胞发育提供必需的营养,而卵母细胞分泌的信号因子亦调控着卵丘细胞的增殖和延伸;在排卵前卵泡中,卵丘细胞主要通过调控卵母细胞中cAMP水平,促使卵母细胞恢复减数分裂;在排卵后卵泡中,卵丘细胞亦影响着精-卵结合及胚胎发育的过程.另外,伴随卵泡内微环境的变化,卵丘细胞与卵母细胞间发生着复杂的信号传递,从而对卵母细胞的发育实现分子水平的调控,其中部分基因可能作为卵母细胞发育成熟、胚胎发育及妊娠结局的分子标志物.%The cumulus cells (CCs) share the same follicular microenvironment with oocyte, and the bidirectional communication between CCs and oocyte is controlling the oocyte maturation and CCs proliferation. CCs originate from granulose cells (GCs) which differentiate into mural GCs and CCs during follicular antrum formation. The signaling between the CCs and oocyte via gap junctions and then they were made up of a structural and functional unit. During the different phases of folliculogenesis, CCs controlled the oocyte development as follows: during the antral phase, CCs provided necessary nourishment to support oocyte development and oocyte secreted signal factors to control the proliferation of CCs; during the pre-ovulatory phase, CCs promoted the oocyte to resume meiosis by controlling the cAMP; during the post-ovulation, the CCs also affected fertilization or embryos development. Furthermore, as the follicular microenvironment was changing, the complex
Sugiura, Koji; Su, You-Qiang; Li, Qinglei; Wigglesworth, Karen; Matzuk, Martin M.; Eppig, John J.
Mouse oocytes produce members of the transforming growth factor beta (TGFbeta) superfamily, including bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), as well as fibroblast growth factors (FGFs). These growth factors cooperate to regulate cumulus cell function. To identify potential mechanisms involved in these interactions, the ability of fully grown oocytes to regulate expression of BMP or FGF antagonists in cumulus cells was examined. Oocytes promoted cumulus cell expression of transcripts encoding antagonists to TGFbeta superfamily members, including Grem2, Htra1, Htra3, and Nog mRNAs. In contrast, oocytes suppressed cumulus cell expression of Spry2 mRNA, which encodes a regulator of receptor tyrosine kinase signals, such as FGF and epidermal growth factor (EGF) receptor signals. The regulation of Spry2 mRNA levels in cumulus cells was studied further as a model for analysis of potential mechanisms for cooperativity of FGF/EGF signaling with oocyte-derived members of the TGFbeta superfamily. Oocytes suppressed basal and FGF-stimulated Spry2 mRNA levels in cumulus cells but promoted EGF-stimulated levels. Furthermore, recombinant TGFbeta superfamily proteins, including BMP15 and GDF9, mimicked these effects of oocytes. Elevated expression of Spry2 mRNA in cumulus and mural granulosa cells correlated with human chorionic gonadotropin-induced expression of mRNAs encoding EGF-like peptides. Therefore, oocyte-derived members of the TGFbeta superfamily suppress FGF-stimulated Spry2 mRNA levels before the luteinizing hormone surge but promote Spry2 mRNA levels stimulated by EGF receptor-mediated signals after the surge. PMID:19553596
Pedersen, Hanne Skovsgaard; Callesen, Henrik; Løvendahl, Peter
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...
Full Text Available Polychlorinated biphenyls (PCBs are one of the most persistent and widespread groups of endocrine disrupting compounds in the ecosystem. These substances are present in sewage sludge that is spread in increasing amounts on arable land and pasture as fertilizer, and are ingested by farm animals with food and drinking water. This study investigated the effect of different PCB concentrations on pig oocyte in vitro maturation and developmental competence as well as examined the possible mechanisms involved. A concentration ranging from 0 to 1 ?g/mL of Aroclor 1254 (A1254, a pool of more than 60 PCB congeners, was added to the maturation medium, as its composition is considered environmentally relevant. A1254 had no effect on maturation of pig oocytes and on the number of oocytes that cleaved following parthenogenetic activation at any of the doses tested. By contrast, a significant decrease in the number of zygotes that developed to blastocyst stage became evident at a concentration of 10 ng/mL. The number of blastocysts obtained decreased significantly, and in a dose response manner with higher concentrations. Exposure to PCBs altered mitochondria relocation during maturation and this was associated with the lack of a cytoplasmic microtubule network. No effect on mitochondria activity was observed. A1254 exposure also perturbed gap-junction mediated communications between oocytes and cumulus cells. In conclusion, PCB exposure of pig oocytes during in vitro maturation significantly decreased oocyte developmental competence, altered both their cytoplasmic remodelling and the communication with the somatic compartment. These data indicated that accumulation of PCBs in the pig organism may have a detrimental effect on the reproductive efficiency in this species.
Full Text Available In in vitro fertilization (IVF cycles controlled ovarian hyperstimulation (COH is established by gonadotropins in combination with gonadotropin-releasing hormone (GnRH agonists or antagonists, to prevent premature luteinizing hormone (LH surge. The aim of our study was to improve the understanding of gene expression profile of cumulus cells (CC in terms of ovarian stimulation protocol and oocyte maturity. We applied Affymetrix gene expression profiling in CC of oocytes at different maturation stages using either GnRH agonists or GnRH antagonists. Two analyses were performed: the first involved CC of immature metaphase I (MI and mature metaphase II (MII oocytes where 359 genes were differentially expressed, and the second involved the two GnRH analogues where no differentially expressed genes were observed at the entire transcriptome level. A further analysis of 359 differentially genes was performed, focusing on anti-Müllerian hormone receptor 2 (AMHR2, follicle stimulating hormone receptor (FSHR, vascular endothelial growth factor C (VEGFC and serine protease inhibitor E2 (SERPINE2. Among other differentially expressed genes we observed a marked number of new genes connected to cell adhesion and neurotransmitters such as dopamine, glycine and γ-Aminobutyric acid (GABA. No differential expression in CC between the two GnRH analogues supports the findings of clinical studies where no significant difference in live birth rates between both GnRH analogues has been proven.
Gómez, E; Tarín, J J; Pellicer, A
To determine the effect of FSH/LH in vivo and epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) in vitro on human oocyte maturation. Oocyte-cumulus complexes were harvested from three different groups of patients:  unstimulated ovaries from women undergoing surgery;  multifollicular development achieved with a combination of FSH and LH in the absence of an ovulatory dose of hCG; and  oocyte-cumulus complexes retrieved after appropriate ovarian stimulation with FSH/LH and hCG for IVF purposes. In vitro fertilization program and patients undergoing surgery for benign disorders at the Instituto Valenciano de Infertilidad, Valencia, Spain. Oocyte-cumulus complexes from unstimulated ovaries collected at surgery by follicular puncture and washing. Oocyte-cumulus complexes from stimulated cycles obtained by ultrasound-guided transvaginal aspiration. Oocyte-cumulus complexes cultured in vitro in the absence or presence of different concentrations of EGF and IGF-I. Germinal vesicle breakdown and metaphase-II stage after 24 and 48 hours. Comparison of the spontaneous resumption of meiosis and metaphase II oocytes among groups showed significant differences between unstimulated and stimulated ovaries after 24 and 48 hours in culture. Administration of hCG accelerated the percentage of maturation by 24 hours. Further incubation of unstimulated oocyte-cumulus complexes with EGF and IGF-I significantly increased the percentage of metaphase-II oocytes after 24 and 48 hours in culture. Epidermal growth factor and IGF-I are able to augment spontaneous maturaion in immature human oocytes. Because spontaneous maturation is mainly observed when follicles have been exposed to pharmacological doses of hMG, it is suggested that increasing FSH levels within the follicle is coincident with the generation of a positive signal necessary to complete oocyte maturation in humans. This signal may be linked to the dynamics of growth factors within the follicle itself.
Munakata, Yasuhisa; Ichinose, Tomoya; Ogawa, Kaori; Itami, Nobuhiko; Tasaki, Hidetaka; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka
Lipid content, ATP content, and histone acetylation are thought to reflect the energy state of cells. In addition, the energy state closely associates with growth and developmental ability of oocytes. Oocyte growth is accompanied by active proliferation of the surrounding granulosa cells (GCs), and GCs play a key role in the provision of energy substrates to the oocytes. In the present study, we first examined the relationship among the average number of GCs per follicle, the average number of cumulus cells (CCs) per oocyte, and the average lipid content in oocytes that developed in vivo within individual donor gilts. Second, we validated the relationship between the number of cells surrounding oocytes and the energy states of oocytes by using an IVC system of oocyte granulosa cell complexes (OGCs) derived from early antral follicles. We collected cumulus cells and oocyte complexes (COCs) from antral follicles (3-5 mm in diameter) and found that average lipid content in oocytes significantly correlated with the average number of both GCs/follicle and CCs/oocyte (P cell number of OGCs, as well as the lipid content, ATP content, and acetylation level of H4K12 in oocytes grown in vitro. In addition, glucose consumption by OGCs was calculated from the sample media collected at Days 13 and 14. The lipid content of oocytes grown in vitro, significantly correlated with the number of cells surrounding the oocytes (P number of cells surrounding the oocytes (P number of cells surrounding the oocytes, and glucose uptake by OGCs is crucial for lipid content and ATP content, and H4K12 acetylation in oocytes.
Ritter, Lesley J; Sugimura, Satoshi; Gilchrist, Robert B
Oocytes progressively acquire the competence to support embryo development as oogenesis proceeds with ovarian folliculogenesis. The objectives of this study were to investigate oocyte-secreted factor (OSF) participation in the development of somatic cell epidermal growth factor (EGF) responsiveness associated with oocyte developmental competence. A well-established porcine model was employed using oocytes from small (4 mm) antral follicles, representing low vs moderate developmental competence, respectively. Cumulus-oocyte complexes (COCs) were treated in vitro with inducers of oocyte maturation, and cumulus cell functions and oocyte developmental competence were assessed. COCs from small follicles responded to FSH but, unlike COCs from larger follicles, were incapable of responding to EGF family growth factors known to mediate oocyte maturation in vivo, exhibiting perturbed cumulus expansion and expression of associated transcripts (HAS2 and TNFAIP6). Low and moderate competence COCs expressed equivalent levels of EGF receptor (EGFR) mRNA; however, the former had less total EGFR protein leading to failed activation of phospho-EGFR and phospho-ERK1/2, despite equivalent total ERK1/2 protein levels. Native OSFs from moderate, but not from low, competence oocytes established EGF responsiveness in low competence COCs. Four candidate recombinant OSFs failed to mimic the actions of native OSFs in regulating cumulus expansion. Treatment with OSFs and EGF enhanced oocyte competence but only of the low competence COCs. These data suggest that developmental acquisition by the oocyte of capacity to regulate EGF responsiveness in the oocyte's somatic cells is a major milestone in the oocyte's developmental program and contributes to coordinated oocyte and somatic cell development.
人卵丘细胞与卵母细胞之间存在密切联系,前者可以直接影响后者的发育、成熟以及所形成胚胎的质量.在辅助生殖技术中,通过对卵丘细胞的研究,可以为胚胎选择提供更加客观、准确且无创的方法,提高妊娠率,推动选择性单胚胎移植的发展,减少高序多胎妊娠及其所带来的不良妊娠结局.目前,从基因及转录水平探讨卵丘细胞与卵子质量的关系已成为研究热点.研究表明,卵丘细胞中卵母细胞发育相关基因表达上调能够预测优质胚胎；细胞周期检测点及DNA修复相关基因与卵子质量有关；细胞凋亡、葡萄糖生成、抗氧化应激相关基因及核转录因子I/B是预测卵子发育潜能和妊娠结局的生物学指标.%There is a close relationship between human cumulus granulosa cells and oocytes,because the former could influence the development and maturation of oocytes and therefore the embryos quality.In assisted reproductive technology,study on cumulus granulosa cells may offer us more objective,accurate and noninvasive criteria for embryo selection so that the pregnancy rates can be increased and the elective single embryo transfer protocol can be promoted.On the other hand,the high order multiple pregnancy rates and the poor outcomes can be also decreased considerably.Nowadays,it is a hot area to explore the relationship between cumulus cells and oocyte developmental potential at the genetic and transcriptional levels.It was reported that the up-regulation of oogenesis-related genes in cumulus cells can predict good quality embryos.Checkpoints and DNA repair-related genes are associated with oocyte quality.Cell apoptosis,glucogenesis,antioxidative stressrelated genes and transcription factor NFIB are the biological parameters to predict the oocyte developmental potential and pregnancy outcome.
Shi, Lin; Liu, Shan; Zhao, Wanqiu; Shi, Juanzi
The aim of this study was to compare the expression of microRNAs (miRNAs) in cumulus cells from polycystic ovary syndrome (PCOS) and non-PCOS women. In the present study, miRNA expression profiles of the cumulus cell samples were determined by miRNA microarrays. Quantification of selected miRNAs and predicted target genes was performed using quantitative real-time PCR (qRT-PCR). The results showed that miR-483-5p and miR-486-5p are significantly decreased in cumulus cells of PCOS patients PCOS (fold change >2, false discovery rate <0.001). qRT-PCR found that four predicted genes, SOCS3, SRF, PTEN and FOXO1, were significantly increased in PCOS cumulus cells (all P < 0.001), and IGF2 (host gene of miR-483-5p) was significantly decreased in PCOS cumulus cells (P < 0.001). These results indicated that miR-483-5p might play an important role in reducing insulin resistance, and that miR-486-5p might promote cumulus cell proliferation through activation of PI3K/Akt. The findings from this study provided new insights into the complex molecular mechanisms involved in PCOS by revealing pathways possibly regulated by miRNAs. The differences in miRNAs (miR-483-5p, miR-486-5p) and their target gene expression in cumulus cells may provide clues for future research and help to explain aberrant follicular development and subfertility in women with PCOS.
SAHOO, Lakshman; Singla, Suresh K.
Recovery rate and quality of oocytes were investigated from abattoir collected ovaries with and without corpora lutea (CL). The oocytes were categorized into four grades: grade-A, grade-B, grade-C, and grade-D based on the presence of the cumulus cells complex around the oocytes. The number of oocytes per ovary were 1.7 ±0.1 and 1.5 ±0.1, obtained from ovaries without CL and with CL, respectively. Recovery rate of different grades of oocytes from ovaries with CL and without CL were almost ide...
It was shown that expansion of porcine cumulus did not depend on oocyte-secreted factor(s), and it is therefore presumed that porcine CEEF may not be produced exclusively by the oocyte. In this experiment, we used mouse oocytectomized complexes (OOX), which were incapable of CEEF production, to assess the secretion of CEEF by evacuated zona, oocytes of different quality and somatic cells in the porcine follicles. The results showed that: (ⅰ) Evacuated zonae from both porcine and mouse oocytes did not produce CEEF. (ⅱ) Porcine oocytes of A, B and C types from 3 - 6 mm follicles were not significantly different in both production and activity of CEEF. (ⅲ) Both porcine OOX from 3 - 6 mm follicles and granulose cells from < 1 mm follicles secreted CEEF in a large quantity, independent of gonadotropins; mural granulose cells from 3-6 mm follicles, however, produced neglectable amount of CEEF. (ⅳ) The follicular fluid from 3-6 mm porcine follicles contained CEEF activity that was concentration-dependent, and thus it enabled cumulus expansion in 60% mouse OOX when used at 10% of concentration, but the expansion rate of mouse OOX decreased to 9% when the concentration was increased to 50%. (ⅴ) Mouse OOX cultured in porcine CEEF-containing M199 expanded only in the presence of gonadotropins, suggesting that the activity of porcine CEEF is hormone-de- pendent.
Partial characterization of the factor in theca-cell conditioned medium that inhibits the progression of FSH-induced meiosis of bovine oocytes surrounded by cumulus cells connected to the membrana granulosa.
van Tol, H T; Bevers, M M
A factor, secreted by theca cells, inhibits FSH induced resumption of meiosis in bovine oocytes that are surrounded by cumulus cells which are attached to a piece of the membrana granulosa (COCGs). In order to characterize this factor, theca cell conditioned medium (CMt) was heat-treated, filtered through a 5 kD spin off filter, charcoal treated, chloroform extracted and protease treated. To investigate whether the meiosis inhibiting factor produced by theca cells was also present in follicular fluid (FF), the same treatments were done with 50% bovine follicular fluid (bFF). COCGs, originating from 2 to 8 mm follicles of bovine ovaries collected at a slaughterhouse, were cultured in groups of 15 per 600 microl medium supplemented with 0.05 IU ml FSH for 22 hr at 39 degrees C in a humidified atmosphere of 5% CO(2). After culture the oocytes were denuded, stained with orcein, and the nuclear status assessed. Heat treatment did not affect the meiosis arresting capacity of CMt since a similar proportion of the oocytes remained at the GV stage after 22 hr of culture in heat treated CMt as compared to the proportion of oocytes in the GV stage after culture in untreated CMt. Filtering through a 5 kD spin-off filter revealed that the meiosis inhibiting action was maintained in the <5 kD fraction, although there was a significant (P < 0.05) loss of inhibiting activity compared to nonfiltered CMt. No significant decrease was observed in the meiosis arresting capacity of the <5 kD fraction after charcoal or protease treatment. Extraction of the <5 kD fraction with chloroform also did not affect the theca cell produced factor. The effect of the theca cell factor on the progression of meiosis of the oocytes that resumed meiosis, as demonstrated by a very low percentage of the oocytes that matured up to the M2 stage, was not affected following any of the treatments. With regard to bFF, the results show a lower percentage of the oocytes in the GV stage after culture in 50% bFF as
Cebral, E; Motta, A; de Gimeno, M F
Central and gonadal function can be affected by chronic consumption of high and moderate doses of ethanol. Few studies have been conducted to determine the effect of ethanol intake at ovarian and gamete level. Previously, we showed that fertilization rates of low chronic ethanol treated female mice were diminished. Also, our recent results indicated that moderate chronic intake of ethanol by immature females could alter the ovulatory quantity and produce morphological alterations in the superovulated oocytes. Furthermore, PGE production by oocyte cumulus complexes (OCCs) was reduced in the females treated with 10% (w/v) ethanol. In the present investigation, we studied the effects of 5% ethanol treatment given to immature mice for 30 days on the quality and quantity of oocytes superovulated at 16 h posthuman chronic gonadotrophin. Treated females had impaired ovulation rates (P < 0.05) as compared to the controls. The percentage of activated and morphologically abnormal oocytes was elevated in the ethanol-treated females (P < 0.05). PGE synthesis by the OCCs was higher than in the controls (P < 0.01). In summary, the administration of long-term ethanol at a relatively low dose to immature females produces decreased ovulation rates, abnormal oocyte morphology with high spontaneous activation and altered levels of PGE production by the oocytes' cumulus complexes. The relationship between the oocyte quality and abnormal synthesis of PGE is discussed.
Mazzoni, Gianluca; Razza, Eduardo; Pedersen, Hanne S.
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...
王兴玲; 蔡鹏飞; 张文娟; 肖雅琳
目的：探讨体外受精-胚胎移植（ IVF-ET）卵丘颗粒细胞的端粒长度与卵母细胞成熟度及妊娠结局的关系。方法：收集81例行体外受精-胚胎移植患者的卵丘颗粒细胞，根据患者的年龄，分为低龄（＜35岁）组42例和高龄（≥35岁）组39例。应用qRT-PCR测量卵丘颗粒细胞端粒长度。结果：卵丘颗粒细胞端粒长度与年龄呈负相关（r＝－0．267，P＝0．021），低、高龄组内成熟卵母细胞的卵丘颗粒细胞端粒长度明显比未成熟卵母细胞的长（P＜0．05）。高龄组妊娠者端粒长度较未妊娠者长（P＜0．05）。结论：在IVF-ET中，卵丘颗粒细胞的相对端粒长度随年龄增长逐渐缩短，可反映卵母细胞的成熟度；端粒长度缩短可能会影响妊娠结局。%Aim:To investigate the relationship of the relative telomere length in cumulus cells (CCs) with oocytes at different mature stages and the outcome of in vitro fertilization and embryo transfer (IVF-ET).Methods:Oocyte-cumulus complex samples were collected from 81 patients undergoing IVF-ET and CCs were manually separated .A total of 42 women (<35 years,the younger group) and 39 women(≥35 years, the older group) were collected.The oocyte maturation and the result of clinical pregnancy were recorded in different groups .DNA was extracted from CCs and assessed for telomere length by real-time quantitative PCR .Results: There were negative correlation between relative telomere length of CCs with the patients′age (r=-0.267, P=0.021).The relative telomere length of CCs was higher in mature oocytes than immature oocytes in both groups (P<0.05).There was a significantly higher telomere length of CCs in the pregnant sub-group than in the non-pregnant subgroup among the older group (P<0.05).Conclusion:In IVF-ET, the relative telomere length of CCs gradually shortened with age .The telomere length of CCs could reflect the degree of oocyte maturation and may be
Full Text Available Introduction: Superovulation produces complications in some patients, so invitro maturation of oocytes is used to decrease or eliminate these complications and improve IVF. Moreover, IVM is used for different aspects of reproductive researches. Slaughterhouse ovaries are the main source of oocytes for IVM and IVF studies. Different media has been introduced and experimented for in vitro maturation of oocytes. Animal's serum at estrus stage contains different hormones and proteins which are essential for oocyte maturation. The aim of this study was to compare three culture media for in vitro maturation (IVM of bovine oocytes; 1(controlTCM-199, 2HCG and follicular fluid (FF and 3 antibiotic. Methods: Rat estrus serum (RSS or fetal bovine serum (FBS was added to control medium. Total of 1789 compact cumulus oocyte complexes (COCs were aspirated from ovaries of slaughtered animals. Oocytes were randomly cultured in mentioned media and incubated in 38.5◦c, 5% CO2 and 95% humidity for 24 hours. The maturation of oocytes was judged according to cumulus cell expansion or randomly orcein stained oocytes and observation of polar bodies. Results: The results showed that maturation rate was significantly higher in second and third group (90.2%, 78.7% as compared to the control group (p<0.001. There was no significant difference between second and third groups (90.2 % vs. 86.6%. Conclusion: RSS is as effective as FBS for IVM of bovine oocytes and can be used as an alternative.
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.
Rodrigues-Cunha, Maria Carolina; Mesquita, Lígia G; Bressan, Fabiana; Collado, Maite Del; Balieiro, Júlio C C; Schwarz, Kátia R L; de Castro, Fernanda C; Watanabe, Osnir Y; Watanabe, Yeda F; de Alencar Coelho, Lia; Leal, Cláudia L V
Melatonin may have beneficial effects when used in oocyte maturation and embryo development culture. The effect of melatonin during IVM on meiosis resumption and progression in bovine oocytes and on expression of antioxidant enzymes, nuclear fragmentation and free radicals, as well as on embryo development were assessed. Cumulus-oocyte complexes were matured in vitro with melatonin (10(-9) and 10(-6) M), FSH (positive control), or without hormones (negative control) in defined medium. Maturation rates were evaluated at 6, 12, 18, and 24 hours. Transcripts for antioxidant enzymes (CuZnSOD, MnSOD, and glutathione peroxidase 4 (GPX4)) in oocytes and cumulus cells, nuclear fragmentation in cumulus cells (TUNEL) and reactive oxygen species levels in oocytes (carboxy-H2 difluorofluorescein diacetate) were determined at 24 hours IVM. Effect of treatments on embryo development was determined after in vitro fertilization and culture. At 12 hours, meiosis resumption rates in FSH and melatonin-treated groups were similar (69.6%-81.8%, P > 0.05). At 24 hours, most oocytes were in metaphase II, with FSH showing highest rates (90.0%, P 0.05). In cumulus cells, MnSOD expression was higher in FSH group (P 0.05). In conclusion, although melatonin during IVM in a defined medium does not stimulate nuclear maturation progression it does stimulate meiosis resumption and such treated oocytes support subsequent embryo development. Melatonin also shows cytoprotective effects on cumulus-oocyte complexes. Copyright © 2016 Elsevier Inc. All rights reserved.
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.
Ochsner, Scott A; Russell, Darryl L; Day, Anthony J; Breyer, Richard M; Richards, Joanne S
Ovulation, the release of fertilizable oocytes from mature follicles, involves tissue remodeling and increased prostaglandin (PG) signaling. Cyclooxygenase (COX)-2 is the rate-limiting enzyme during PG synthesis. Female mice null for either COX-2 or the PGE(2) receptor EP2 are infertile, show decreased ovulation, and exhibit abnormal cumulus expansion. Cumulus expansion is the production of a complex extracellular matrix surrounding the cumulus-oocyte complex (COC). Matrix components consist of hyaluronan, proteoglycans, and proteins with hyaluronan binding domains. One such hyaluronan binding protein is TNFalpha-stimulated gene 6 (TSG-6). By various methods, we show induction of TSG-6 and hyaluronan synthase-2 mRNA in ovaries of mice treated with pregnant mare serum gonadotropin and human chorionic gonadotropin. By in situ hybridization, we show that both genes are expressed in periantral mural granulosa cells and cumulus cells of the mouse ovary. Notably, RT-PCR and in situ hybridization show that TSG-6 mRNA but not hyaluronan synthase-2 mRNA expression is selectively reduced in cumulus cells of COX-2 and EP2 null mice. Western analysis further confirms that TSG-6 protein is reduced in isolated COCs but remains covalently associated with inter alpha-trypsin inhibitor in COX-2 null mice. These observations identify TSG-6 as a target of PG action and show that its production in ovulatory follicles is associated with proper formation of the cumulus-derived extracellular matrix.
Morais-de-Sá, Eurico; Mukherjee, Avik; Lowe, Nick; St Johnston, Daniel
The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. Here we show that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl.
Full Text Available Meiotic maturation of mammalian oocytes is a precisely orchestrated and complex process. Dimethyl sulfoxide (DMSO, a widely used solvent, drug, and cryoprotectant, is capable of disturbing asymmetric cytokinesis of oocyte meiosis in mice. However, in pigs, DMSO's effect on oocyte meiosis still remains unknown. We aimed to evaluate if DMSO treatment will affect porcine oocyte meiosis and the underlying molecular changes as well. Interestingly, we did not observe the formation of the large first polar body and symmetric division for porcine oocytes treated with DMSO, contrary to findings reported in mice. 3% DMSO treatment could inhibit cumulus expansion, increase nuclear abnormality, disturb spindle organization, decrease reactive oxygen species level, and elevate mitochondrial membrane potential of porcine oocytes. There was no effect on germinal vesicle breakdown rate regardless of DMSO concentration. 3% DMSO treatment did not affect expression of genes involved in spindle organization (Bub1 and Mad2 and apoptosis (NF-κB, Pten, Bcl2, Caspase3 and Caspase9, however, it significantly decreased expression levels of pluripotency genes (Oct4, Sox2 and Lin28 in mature oocytes. Therefore, we demonstrated that disturbed cumulus expansion, chromosome alignment, spindle organization and pluripotency gene expression could be responsible for DMSO-induced porcine oocyte meiotic arrest and the lower capacity of subsequent embryo development. Our results provide new insights on DMSO's effect on porcine oocyte meiosis and raise safety concerns over DMSO's usage on female reproduction in both farm animals and humans.
Payton, Rebecca R; Rispoli, Louisa A; Edwards, J Lannett
Results described herein provide insight regarding certain features of gamete RNA and how they compare to cumulus cell RNA. In particular, 28S/18S rRNA ratio and size distribution of RNA molecules differed in total RNA from oocytes versus surrounding cumulus cells. Specifically, oocyte total RNA had a lower rRNA ratio and an increased abundance of smaller RNA sizes compared to RNA from surrounding cumulus. Extensive efforts demonstrated that observed differences were repeatable whether oocyte maturation occurred in vitro or in vivo, and were similar between the nuclear stages examined. Features of oocyte RNA were conserved across six mammalian species, yet differed from surrounding cumulus. Profiles of sperm RNA were also examined but had no discernible ribosomal RNA peaks and were conserved across four mammalian species. Because the oocyte and spermatozoon are highly specialized cells representing unique molecular entities required for proper embryo development, dissimilarities described herein likely represent real gamete versus cumulus RNA differences.
Zhao, Gaoping; Wu, Kaifeng; Cui, Liang; Zhao, Lixia; Liu, Yiyi; Tan, Xiuwen; Zhou, Huanmin
Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 μl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glutamine, 0.11 mg/mL sodium pyruvate, and 25 mg/mL gentamycin. There were no significant differences among the three maturation media for oocyte maturation. Maturation rate of donkey oocytes in M1 was 53% for compact (Cp) cumulus-oocyte complexes and 75% for expanded (Ex) cumulus-oocyte complexes; in M2 these were 55 and 77%, respectively; and in M3, 58 and 75%. The percentage of cleaved parthenotes and 4- or 8-cell embryos were not significantly different for oocytes matured in the various media (61 and 24% for M1; 66 and 32% for M2; and 67 and 33% for M3). Oocytes matured in M3 tended to yield a higher rate of advanced embryo development (morula) than oocytes matured in M1 (22 vs 9%; P = 0.07). In conclusion, donkey oocytes were matured and parthenogenetically activated in vitro, using methods similar to those used in the horse.
Participation of Mitogen-activated Protein Kinase in Luteinizing Hormone-induced Differential Regulation of Steroidogenesis and Steroidogenic Gene Expression in Mural and Cumulus Granulosa Cells of Mouse Preovulatory Follicles
Su, You-Qiang; Nyegaard, Mette; Overgaard, Michael Toft
superovulation model for premature female mice was used to obtain MGCs and cumulus-oocyte complexes (COCs), and sensitive real-time reverse transcription polymerase chain reaction was used to simultaneously detect the expression levels of transcripts encoding key steroidogenic enzymes in the same sample. We...
Pedersen, Hanne Skovsgaard; Løvendahl, Peter; Larsen, Knud Erik
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......Oocyte competence has been related to mtDNA copy number, but a large variation in mtDNA copy number between oocytes has been observed, caused by, e.g. oocyte donor and oocyte size (Sato et al. 2014 PLOS ONE 9, e94488; Cotterill et al. 2013 Mol. Hum. Reprod. 19, 444–450; El Shourbagy et al. 2006...... 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...
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.
Waiz, Syma Ashraf; Raies-ul-Haq, Mohammad; Dhanda, Suman; Kumar, Anil; Goud, T. Sridhar; Chauhan, M. S.; Upadhyay, R. C.
In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly ( P peroxidase, and glutathione reductase were significantly ( P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.
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. Atte
Full Text Available Oocyte collection technique is important to obtain a maximum number of oocytes to be employed on in vitro production of embryos. In this study, immature bovine oocytes were collected from slaughterhouse ovaries by two techniques: aspiration of 2- to 6-mm follicles and slicing. Following collection, oocyte qualities were classified into four categories (A, B, C, and D on the basis of cumulus attachment. Oocytes of each category were matured in vitro in CO2 incubator for 22-24 hours and cumulus expansion and maturation rates were observed. The total number of oocytes (group A+B+C+D and yield of good quality oocytes (only group A and B recovered per ovary by aspiration were 12.02 and 8.21, and by slicing were 29.38 and 19.65 (P<0.01, respectively. The total cumulus cells expansion rates of A, B, C and D oocytes were 97.1%, 88.3%, 6.0% and 20.6% respectively. Maturation rates for A, B and C categories of oocytes were 91.4%, 82.3% and 35.0% respectively while no matured oocyte was observed for group D oocytes. Maturation rates were significantly different between group A and C and also between B and C but not between A and B (P<0.05. In conclusion, slicing technique recovered more oocytes per ovary (2.4 times than that of aspiration and the best maturation rate was observed from category A oocytes which surrounded by more than 3 layers of cumulus cells. However oocytes of category A and B can be considered as good quality oocytes.
Munakata, Yasuhisa; Kawahara-Miki, Ryoka; Shiratsuki, Shogo; Tasaki, Hidetaka; Itami, Nobuhiko; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka
Follicle development is accompanied by proliferation of granulosa cells and increasing oocyte size. To obtain high-quality oocytes in vitro, it is important to understand the processes that occur in oocytes and granulosa cells during follicle development and the differences between in vivo and in vitro follicle development. In the present study, oocytes and granulosa cells were collected from early antral follicles (EAFs, 0.5-0.7 mm in diameter), small antral follicles (SAFs, 1-3 mm in diameter), large antral follicles (LAFs, 3-7 mm in diameter), and in vitro grown oocyte-and-granulosa cell complexes (OGCs), which were cultured for 14 days after collection from EAFs. Gene expression was analyzed comprehensively using the next-generation sequencing technology. We found top upstream regulators during the in vivo follicle development and compared them with those in in vitro developed OGCs. The comparison revealed that HIF1 is among the top regulators during both in vivo and in vitro development of OGCs. In addition, we found that HIF1-mediated upregulation of glycolysis in granulosa cells is important for the growth of OGCs, but the cellular metabolism differs between in vitro and in vivo grown OGCs. Furthermore, on the basis of comparison of upstream regulators between in vivo and in vitro development of OGCs, we believe that low expression levels of FLT1 (VEGFA receptor), SPP1, and PCSK6 can be considered causal factors of the suboptimal development under in vitro culture conditions.
Rinaudo Paolo F
Full Text Available Abstract Background Ovarian stimulation for assisted reproductive technology (ART overcomes the physiologic process to develop a single dominant follicle. However, following stimulation, egg recovery rates are not 100%. The objective of this study is to determine if the follicular fluid hormonal environment is associated with oocyte recovery. Methods This is a prospective study involving patients undergoing ART by standard ovarian stimulation protocols at an urban academic medical center. A total of 143 follicular fluid aspirates were collected from 80 patients. Concentrations of FSH, hCG, estradiol, progesterone, testosterone and prolactin were determined. A multivariable regression analysis was used to investigate the relationship between the follicular fluid hormones and oocyte recovery. Results Intrafollicular FSH was significantly associated with oocyte recovery after adjustment for hCG (Adjusted odds ratio (AOR = 1.21, 95%CI 1.03–1.42. The hCG concentration alone, in the range tested, did not impact the odds of oocyte recovery (AOR = 0.99, 95%CI 0.93–1.07. Estradiol was significantly associated with oocyte recovery (AOR = 0.98, 95% CI 0.96–0.99. After adjustment for progesterone, the strength of association between FSH and oocyte recovery increased (AOR = 1.84, 95%CI 1.45–2.34. Conclusion The relationship between FSH and oocyte recovery is significant and appears to work through mechanisms independent of the sex hormones. FSH may be important for the physiologic event of separation of the cumulus-oocyte complex from the follicle wall, thereby influencing oocyte recovery. Current methods for inducing the final stages of oocyte maturation, with hCG administration alone, may not be optimal. Modifications of treatment protocols utilizing additional FSH may enhance oocyte recovery.
Al-aghbari, A M; Menino, A R
The objective of this work was to develop an effective vitrification technique for cryopreserving oocytes in sheep ovarian tissues. Ovaries were surgically recovered from 15 pubertal ewes and the ovarian cortex was cut into sections. Ovarian tissues were placed in equilibration medium consisting of 4% (v/v) ethylene glycol (EG) and 20% (v/v) FBS in TCM-199 on ice for 30 min and transferred to vitrification solution (35% EG, 5% polyvinylpyrrolidone, 0.4M trehalose and 20% FBS in TCM-199) for 5 min. Ovarian tissues were vitrified by dropping the tissue on the surface of a steel cube cooled by liquid nitrogen. Cumulus-enclosed oocyte complexes (COC) were also collected and vitrified following the procedure used for ovarian tissues. After 2-3 weeks of storage in liquid nitrogen, ovarian tissues and COC were thawed at 37 degrees C in 0.3M trehalose and COC in ovarian tissues were mechanically and enzymatically isolated. Vitrified COC and freshly collected COC were washed twice in maturation medium (TCM-199 supplemented with 0.255 mM pyruvate and 10% heat-treated estrus cow serum) and cultured in 50 microl drops of maturation medium under paraffin oil for 23-25h at 39 degrees C in a humidified atmosphere of 5% CO(2) in air. After culture, cumulus cells were removed by hyaluronidase treatment and vortexing and oocytes were fixed and stained. No significant differences were observed between vitrified oocytes, oocytes recovered from vitrified ovarian tissues and non-vitrified control oocytes in the percentage of oocytes with acceptable staining per total number of oocytes fixed or with visible chromatin per total number of oocytes with acceptable staining. However, fewer (P0.05) were observed due to treatment in the percentages of oocytes developing to metaphase II. These results demonstrate that sheep oocytes can be successfully cryopreserved by vitrification of ovarian tissues and exhibit in vitro maturation rates similar to that of vitrified and non-vitrified oocytes.
Full Text Available The aim of the study was to determine the influence of different concentrations of phytoestrogens biochanin A (BIO A; 20, 40, 50μg ml-1 and daidzein (DAI; 10, 20, 40, 50μg ml-1 on the course of meiotic maturation of pig oocytes. After a 24-hour cultivation, a stage of nuclear maturation was achieved and the areas of cumulus-oocyte complexes (COCs, as an indicator of cumulus expansion, were evaluated. The effects of both contaminants on oocytes were mani - fested from the lowest concentration used. Nuclear maturation was inhibited in a dose-dependent manner in the case of BIO A. Effects of DAI reached a plateau at a concentration of 20μg ml-1.Possible relationship to limited solubility of DAI was excluded because limits of DAI solubility in culture medium were confirmed at 50 μg ml-1.The cumulus expansion was also influenced in a different manner - reduction of the COC’s area by BIO A was dose-dependent, whereas DAI had the strongest effect on CCs in the lowest and highest concentrations used. Both phytoestrogens negatively influence the meiotic maturation of porcine oocytes but there are significant differences in their concrete effects which could relate to the diverse mechanisms of their acting on target cells.
Parks, Jason C; Patton, Alyssa L; McCallie, Blair R; Griffin, Darren K; Schoolcraft, William B; Katz-Jaffe, Mandy G
Corona cells surround the oocyte and maintain a close relationship through transzonal processes and gap junctions, and may be used to assess oocyte competence. In this study, the corona cell transcriptome of individual cumulus oocyte complexes (COCs) was investigated. Isolated corona cells were collected from COCs that developed into euploid blastocysts and were transferred in a subsequent frozen embryo transfer. Ten corona cell samples underwent RNA-sequencing to generate unique gene expression profiles. Live birth was compared with negative implantation after the transfer of a euploid blastocyst using bioinformatics and statistical analysis. Individual corona cell samples produced a mean of 21.2 million sequence reads, and 307 differentially expressed transcrpits (P corona cell transcriptome was successfully generated using RNA-sequencing. Key genes and signalling pathways were identified in association with implantation outcome after the transfer of a euploid blastocyst in a frozen embryo transfer. These data could provide novel biomarkers for the non-invasive assessment of embryo viability.
Full Text Available Impaired oocyte quality has been demonstrated in diabetic mice; however, the potential pathways by which maternal diabetes exerts its effects on the oocyte are poorly understood. Cumulus cells are in direct contact with the oocyte via gap junctions and provide essential nutrients to support oocyte development. In this study, we investigated the effects of maternal diabetes on the mitochondrial status in cumulus cells. We found an increased frequency of fragmented mitochondria, a decreased transmembrane potential and an aggregated distribution of mitochondria in cumulus cells from diabetic mice. Furthermore, while mitochondrial biogenesis in cumulus cells was induced by maternal diabetes, their metabolic function was disrupted as evidenced by lower ATP and citrate levels. Moreover, we present evidence suggesting that the mitochondrial impairments induced by maternal diabetes, at least in part, lead to cumulus cell apoptosis through the release of cytochrome c. Together the deleterious effects on cumulus cells may disrupt trophic and signaling interactions with the oocyte, contributing to oocyte incompetence and thus poor pregnancy outcomes in diabetic females.
Barnes, Stephen J.; Fisher, Louise A.; Godel, Bélinda; Pearce, Mark A.; Maier, Wolfgang D.; Paterson, David; Howard, Daryl L.; Ryan, Christopher G.; Laird, Jamie S.
An unusual occurrence of Pt-enriched pyroxenites in the Monts de Cristal igneous complex is characterized by unusually high ratios of Pt to other platinum-group elements (PGEs) and very low Cu and sulfide contents. Synchrotron X-ray fluorescence microscopy was used to identify over a hundred discrete grains of platinum minerals and relate their occurrence to textural associations in the host heteradcumulate orthopyroxenites. Element associations, backed up by FIB-SEM and PIXE probe observations, indicate that most of the Pt is associated with either As- or trace Cu-Ni-rich sulfides, or both. Some of the Pt-As grains can be identified as sperrylite, and most are likely to be Pt-Fe alloy. The relative abundances and volumes of Pt minerals to sulfide minerals are very large compared with typical magmatic sulfides. Almost all of the grains observed lie at or within a few tens of μm of cumulus orthopyroxene grain boundaries, and there is no significant difference between the populations of grains located inside or outside plagioclase oikocrysts. These oikocrysts are inferred to have crystallized either at the cumulus stage or very shortly thereafter, on the basis of their relationship to Ti enrichment in the margins of pyroxene grains not enclosed in oikocrysts. This relationship precludes a significant role of trapped intercumulus liquid in Pt deposition or mobilization and also allows a confident inference that Pt-rich and Pt-As-enriched phases precipitated directly from the magma at the cumulus stage. These observations lead to the conclusion that fractionation of Pt from other PGEs in this magmatic system is a consequence of a solubility limit for solid Pt metal and/or Pt arsenide.
Complete maturation of oocyte includes both nuclear and cytoplasmic maturation. At present, expulsion of the first polar body is known as the marker of oocyte nuclear maturation. Expansions of granulose cells and oocyte-corona-cumulus complex synchronize with the maturation of oocyte nuclei following the maturation of oocyte in the natural cycle. However, the expansion of oocyte-corona-cumulus complex and nuclear maturation are not synchronized due to disturbance of exogenous gonadotropins in controlled ovarian hyperstimulation (COH) cycles. The status of expansion of oocyte-corona-cumulus complex can not be used to estimate the maturation of oocyte maturation in COH cycles. The morphology characteristics of oocyte, migration of cortical granules and relative factors such as GDF9 , BMP15 etc. can not monitor cytoplasmic maturation fast and directly. In COH cycles, fertilization rate, cleavage rate, pregnancy rate and implantation rate display a rising trend along with the increased proportion of matured oocytes. The rate of available embryos from the oocytes with large follicular diameter is higher than that from the oocytes with small follicular diameter. Oocyte maturation rate and fertilization rate decrease but the fragments in embryos increase along with the reduction of follicular diameter. However, the diameter of dominant follicle does not reflect overall situation of follicular development in ovary. A group of follicles on behalf of the development synchronization of total follicles are named as "object follicles" in progress of COH. The "object follicles" determine the trigger time of hCG. In vitro investigations of mouse embryos also confirm that cultured oocyte in vitro for some time after hCG injection plays an important role in the maturation of oocyte nuclei and cytoplasm, where higher proportion and quicker speed of pronuclei development can be acquired after fertilization or artificial activation. High-quality embryo rate and pregnancy rate increase
Asada, M; Tetsuka, M; Ishikawa, H; Ohsumi, S; Fukui, Y
The aims of the present study were to improve in vitro maturation, fertilization and subsequent development of minke whale oocytes. We investigated the effects of different concentrations (0, 10 and 20%) of fetal whale serum (FWS) in maturation medium on nuclear maturation, morphological grade (A or B) of cumulus-oocyte complexes (COC) obtained from prepubertal and adult minke whales. Grade A (> or = 5 layers of cumulus cells) COC collected from the adult whales and cultured in the medium with 20% FWS had a higher (P whales and COC grades were not significantly affected by M-II oocytes. When in vitro fertilization of matured oocytes was performed in the presence of 20% FWS or 0.6% BSA in the fertilization medium, the proportions of sperm penetration and two-pronuclei formation in matured oocytes were not significantly different. Grade A COC cultured in a culture medium supplemented with 10% FWS cleaved at a higher rate (15.4%, P develop to morula (4.2%) compared with that of the oocytes from Grade B COC (2.5% and 0%). Coculture with granulosa cells during in vitro culture did not significantly affect cleavage and development to the morula stage. These results indicate that FWS addition in the maturation medium improved the rate of in vitro maturation and cleavage after insemination of minke whale oocytes. The BSA supplementation in fertilization medium was as effective as FWS supplementation for in vitro fertilization of matured oocytes. In vitro embryo production beyond the morula stage of minke whale oocytes could be possible, if Grade A COC was selected and cultured in the maturation medium supplemented with 10% or 20% FWS.
da Luz, Caroline M; da Broi, Michele G; Donabela, Flávia Cappello; Paro de Paz, Cláudia Cristina; Meola, Juliana; Navarro, Paula A
A deleterious effect of endometriosis on oocyte quality has been proposed. Evidence suggests that cumulus cells could be used as indirect biomarkers of oocyte quality. The PTGS2 gene, which encodes cyclooxygenase 2 (COX-2), is deregulated in endometriotic lesions and plays a crucial role in the acquisition of oocyte competence. To date, research evaluating PTGS2 expression in cumulus cells of infertile patients with endometriosis has not been conducted. The aim this study was to compare the expression levels of PTGS2 in cumulus cells of infertile women, with and without endometriosis, undergoing ovarian stimulation for intracytoplasmic sperm injection (ICSI). Therefore, a case-control study compared PTGS2 gene expression in the cumulus cells of 38 infertile patients with endometriosis and 40 without, using real-time polymerase chain reaction. For the first time, decreased expression of PTGS2 was found in cumulus cells of infertile women with endometriosis compared with controls (7.2 ± 10.5 versus 12.4 ± 15.7), which might be related to reduced levels of COX-2 in the cumulus cells of women with the disease. Consequently, we hypothesize that lower transcript levels of PTGS2 in cumulus cells may be involved in the impairment of oocyte quality, suggesting a possible mechanism involved in disease-related infertility. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
H. Jamil*, H. A. Samad, N. Rehman, Z. I. Qureshi and L. A. Lodhi
Full Text Available This study was designed to evaluate the efficacy of different somatic cell types and media in supporting in vitro maturation (IVM, in vitro fertilization (IVF and early embryonic development competence of buffalo follicular oocytes. Cumulus oocyte complexes were collected for maturation from follicles (>6mm of buffalo ovaries collected at the local abattoir. Oocytes were co-cultured in tissue culture medium (TCM-199 with either granulosa cells, cumulus cells, or buffalo oviductal epithelial cells (BOEC @ 3x106 cells/ml or in TCM-199 without helper cells (control at 39°C and 5%CO2 in humidified air. Fresh semen was prepared in modified Ca++ free Tyrode medium. Fertilization was carried out in four types of media: i Tyrode lactate albumin pyruvate (TALP, ii TALP+BOEC, iii modified Ca++ free Tyrode and iv modified Ca++ free Tyrode+BOEC. Fertilized oocytes were cultured for early embryonic development in TCM-199 with and without BOEC. Higher maturation rates were observed in the granulosa (84.24% and cumulus cells (83.44% than BOEC co culture system (73.37%. Highest fertilization rate was obtained in modified Ca++ free Tyrode with BOEC co culture (70.42%, followed by modified Ca++ free Tyrode alone (63.77%, TALP with BOEC (36.92% and TALP alone (10.94%. Development of early embryos (8-cell stage improved in TCM-199 with BOEC co culture than TCM-199 alone. From the results of this study, it can be concluded that addition of somatic cells (granulosa cells, cumulus cells results in higher maturation rates of buffalo follicular oocytes than BOEC co culture system, while fertilization rate improved in modified Ca++ free Tyrode with and without BOEC. Addition of BOEC to TCM-199 improved the developmental capacity of early embryo.
Michael J Bertoldo
Full Text Available Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK, an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues. Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed.
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.
Chaffin, Charles L; Lee, Young S; VandeVoort, Catherine A; Patel, Bela G; Latham, Keith E
Follicular somatic cells (mural granulosa cells and cumulus cells) and the oocyte communicate through paracrine interactions and through direct gap junctions between oocyte and cumulus cells. Considering that mural and cumulus cells arise through a common developmental pathway and that their differentiation is essential to reproductive success, understanding how these cells differ is a key aspect to understanding their critical functions. Changes in global gene expression before and after an ovulatory stimulus were compared between cumulus and mural granulosa cells to test the hypothesis that mural and cumulus cells are highly differentiated at the time of an ovulatory stimulus and further differentiate during the periovulatory interval. The transcriptomes of the two cell types were markedly different (>1500 genes) before an ovulatory hCG bolus but converged after ovulation to become completely overlapping. The predominant transition was for the cumulus cells to become more like mural cells after hCG. This indicates that the differentiated phenotype of the cumulus cell is not stable and irreversibly established but may rather be an ongoing physiological response to the oocyte.
Racowsky, C; McGaughey, R W
Liberated cumulus-enclosed pig oocytes were cultured either alone in follicular fluid or with membrana granulosa cells in a complex serum based medium. After 24 h, oocytes were air-dried for cytogenetic analysis, meiotic stage was scored, and viability of granulosa cells was determined. Neither the release from meiotic arrest nor the progression of maturation to metaphase II was significantly inhibited by either of these follicular components. Co-culture of membrana granulosa cells and oocytes significantly stimulated maturation in one experimental series, while viability of the somatic cells was maintained in all experiments. These results do not support the concept of a stable oocyte maturation inhibitor of granulosa cell origin in follicular fluid.
O'Doherty, Alan M; O'Gorman, Aoife; al Naib, Abdullah; Brennan, Lorraine; Daly, Edward; Duffy, Pat; Fair, Trudee
Ovarian follicle development in post-partum, high-producing dairy cows, occurs in a compromised endogenous metabolic environment (referred to as negative energy balance, NEB). Key events that occur during oocyte/follicle growth, such as the vital process of genomic imprinting, may be detrimentally affected by this altered ovarian environment. Imprinting is crucial for placental function and regulation of fetal growth, therefore failure to establish and maintain imprints during oocyte growth may contribute to early embryonic loss. Using ovum pick-up (OPU), oocytes and follicular fluid samples were recovered from cows between days 20 and 115 post-calving, encompassing the NEB period. In a complimentary study, cumulus oocyte complexes were in vitro matured under high non-esterified fatty acid (NEFA) concentrations and in the presence of the methyl-donor S-adenosylmethionine (SAM). Pyrosequencing revealed the loss of methylation at several imprinted loci in the OPU derived oocytes. The loss of DNA methylation was observed at the PLAGL1 locus in oocytes, following in vitro maturation (IVM) in the presence of elevated NEFAs and SAM. Finally, metabolomic analysis of postpartum follicular fluid samples revealed significant differences in several branched chain amino acids, with fatty acid profiles bearing similarities to those characteristic of lactating dairy cows. These results provide the first evidence that (1) the postpartum ovarian environment may affect maternal imprint acquisition and (2) elevated NEFAs during IVM can lead to the loss of imprinted gene methylation in bovine oocytes.
Mohammad H. Bahadori
Full Text Available Background: Oocyte invitro maturation is an enormously promising technology for the treatment of infertility, yet its clinical application remains limited owing to poor success rates. Therefore, this study was devised to evaluate the effect of hepatocyte growth factor (HGF on in vitro maturation of immature mouse oocytes and resulting embryos development. Materials and Method: Cumulus – oocyte complex and germinal vesicle were obtained from eighteen 6-8 weeks-old female NMRI mice 46-48 hours after administration of an injection of 5 IU PMSG (Pregnant Mares’ Serum Gonadotrophin. Oocytes were culture in TCM199 (Tissue culture medium-199 supplemented with dosages of 0, 10, 20, 50 and 100 ng/ml of HGF. After 24 hours, metaphase ІІ oocytes were co-incubated with sperms for 4-6 hours in T6 medium. Following isolation of two pronucleus embryos, cleavage of embryos was assessed in the same medium till blastocyst stage. The number of oocytes and embryos was recorded under an invert microscope and the rate of oocyte maturation, fertilization and embryos cleavage until blastocyst stage compared using of student χ2 test. Results: In all compared groups, oocytes growth and embryos development rate in the 20 ng/ml of HGF treatment group was significantly higher (p<0.05 than the control group (p<0.05.Conclusion: 20 ng/ml of HGF improved the nuclear maturation and embryo development up to blastocyst stage during culture condition
Full Text Available The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.
Dyce, Paul W; Shen, Wei; Huynh, Evanna; Shao, Hua; Villagómez, Daniel A F; Kidder, Gerald M; King, W Allan; Li, Julang
We previously reported the differentiation of cells derived from porcine female fetal skin into cells resembling germ cells and oocytes. A subpopulation of these cells expressed germ cell markers and formed aggregates resembling cumulus-oocyte complexes. Some of these aggregates extruded large oocyte-like cells (OLCs) that expressed markers consistent with those of oocytes. The objective of the current study was to further characterize OLCs differentiated from porcine skin-derived stem cells. Reverse transcriptase (RT)-polymerase chain reaction and Western blot revealed the expression of connexin37 and connexin43, both of which are characteristic of ovarian follicles. The expression of meiosis markers DMC1 and synaptonemal complex protein, but not STRA8 and REC8, was detected in the OLC cultures. Immunofluorescence with an antibody against synaptonemal complex protein on chromosome spreads revealed a very small subpopulation of stained OLCs that had a similar pattern to leptotene, zytotene, or pachytene nuclei during prophase I of meiosis. Sodium bisulfite sequencing of the differentially methylated region of H19 indicated that this region is almost completely demethylated in OLCs, similar to in vivo-derived oocytes. We also investigated the differentiation potential of male skin-derived stem cells in the same differentiation medium. Large cells with oocyte morphology were generated in the male stem cell differentiation cultures. These OLCs expressed oocyte genes such as octamer-binding transcription factor 4 (OCT4), growth differentiation factor-9b (GDF9B), deleted in azoospermia-like (DAZL), VASA, zona pellucida B (ZPB), and zona pellucida C (ZPC). It was concluded that skin-derived stem cells from both male and female porcine fetuses are capable of entering an oocyte differentiation pathway, but the culture system currently in place is inadequate to support the complete development of competent oocytes.
Sessions-Bresnahan, D R; Graham, J K; Carnevale, E M
IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine sperm capacitated with dilauroyl phosphatidylcholine would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In experiment 1, bovine oocytes were used for (1) IVF with bovine sperm, (2) IVF with equine sperm, and (3) intracytoplasmic sperm injections (ICSIs) with equine sperm. Presumptive zygotes were either stained with 4',6-diamidino-2-phenylindole from 18 to 26 hours at 2-hour intervals or evaluated for cleavage at 56 hours after addition of sperm. Equine sperm fertilized bovine oocytes; however, pronuclei formation was delayed compared with bovine sperm after IVF. The delayed pronuclear formation was not seen after ICSI. In experiment 2, bovine oocytes were assigned to the following five groups: (1) cumulus oocyte complexes (COCs) coincubated with bovine sperm; (2) COC exposed to sucrose then coincubated with bovine sperm; (3) COC coincubated with equine sperm; (4) COC exposed to sucrose, and coincubated with equine sperm; and (5) oocytes exposed to sucrose, and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P = 0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In experiment 3, oocytes were assigned to the following four groups: (1) IVF, equine, and bovine COC coincubated with equine sperm; (2) PVI of equine and bovine oocytes; (3) PVI with equine oocytes
Ríos, G L; Buschiazzo, J; Mucci, N C; Kaiser, G G; Cesari, A; Alberio, R H
The conditions for in vitro oocyte maturation impact on cytoplasmic and nuclear processes in the oocyte. These events are differentially influenced by the nature of the maturation inducer and the presence of intact cumulus in cumulus-oocyte complexes. Epidermal growth factor is the main growth factor promoting oocyte maturation. Also, hyaluronic acid (HA) produced by cumulus cells is known to be responsible for the correct structural and functional organization of the cumulus during oocyte maturation. Therefore, we evaluated the developmental competence of bovine oocytes matured in vitro in a maturation medium supplemented with both EGF and HA, compared to FSH and fetal bovine serum (FBS). In addition, the impact of IVM conditions on the proteomic profile of metaphase II bovine oocytes was analyzed by two-dimensional electrophoresis. Cumulus-oocyte complexes were matured in two media: (1) 10 ng/mL EGF, 15 μg/mL HA, and 100-μM cysteamine and (2) 0.01 UI/mL rh-FSH and 10% FBS. The percentages of first polar body and embryo production and the kinetics of embryo development and oocyte proteomic profiles were analyzed. Oocytes matured in the presence of EGF-HA showed an increase (6%, P < 0.05) in the percentage of polar body extrusion. The blastocyst rate was 3% (P < 0.05) higher in the FSH-FBS group, but no differences were found in the rate of expanded blastocyst neither in total embryo production between IVM conditions. Cleavage rate of oocytes matured with FSH-FBS was 5% higher (P < 0.05) with respect to EGF-HA-matured oocytes when evaluated 30 hours after fertilization. However, at Day 7, those inseminated oocytes that underwent division at a correct timing showed that although there are still early blastocysts in the FSH-FBS condition, EGF-HA embryos have developed completely into blastocysts. Still, the production rate of those embryos that achieved expansion was similar between both maturation conditions. On the other hand, noncleaved presumptive
Connolly, Amy A; Sugioka, Kenji; Chuang, Chien-Hui; Lowry, Joshua B; Bowerman, Bruce
During oocyte meiotic cell division in many animals, bipolar spindles assemble in the absence of centrosomes, but the mechanisms that restrict pole assembly to a bipolar state are unknown. We show that KLP-7, the single mitotic centromere-associated kinesin (MCAK)/kinesin-13 in Caenorhabditis elegans, is required for bipolar oocyte meiotic spindle assembly. In klp-7(-) mutants, extra microtubules accumulated, extra functional spindle poles assembled, and chromosomes frequently segregated as three distinct masses during meiosis I anaphase. Moreover, reducing KLP-7 function in monopolar klp-18(-) mutants often restored spindle bipolarity and chromosome segregation. MCAKs act at kinetochores to correct improper kinetochore-microtubule (k-MT) attachments, and depletion of the Ndc-80 kinetochore complex, which binds microtubules to mediate kinetochore attachment, restored bipolarity in klp-7(-) mutant oocytes. We propose a model in which KLP-7/MCAK regulates k-MT attachment and spindle tension to promote the coalescence of early spindle pole foci that produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly.
Full Text Available We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM, fertilization (IVF, and subsequent culture (IVC. Adjusting warmplate temperature to 42 °C during warming prevented temperature drops in a medium below 34.0 °C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38 °C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively. Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF of the surviving oocytes after warming at 38 °C and 42 °C was not different but lower (P<0.05 than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively. However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF from IVM medium (POM did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.
Liqin Wang; Jiapeng Lin; Juncheng Huang; Jing Wang; Yuncheng Zhao; Tong Chen
Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research on in vitro embryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving the in vitro culture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable exper...
Saber Mohammed Abd-Allah
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.
Yamamuro, Tadashi; Kano, Kiyoshi; Naito, Kunihiko
Cell division cycle 20 (CDC20) and fizzy/cell division cycle 20 related 1 (FZR1) are activators of the anaphase-promoting complex (APC), which ubiquitinates M-phase regulating proteins, such as cyclin B and securin, and induces their degradation. In the present study, porcine CDC20 and FZR1 were cloned by reverse transcriptase-polymerase chain reaction, and their functions in the meiotic maturation of porcine oocytes were analyzed. FZR1 was readily detected in porcine immature oocytes by immunoblotting, but its levels decreased substantially during maturation. In contrast, CDC20 levels rose during oocyte maturation and were highest by the second meiotic metaphase. The inhibition of CDC20 expression by the injection of CDC20 antisense RNA induced the meiotic arrest at the first meiotic metaphase (M1) and the accumulation of a large amount of cyclin B. On the other hand, the inhibition of FZR1 expression accelerated cyclin B accumulation and the start of germinal vesicle breakdown (GVBD), but did not affect the exit from M1. Conversely, the overexpression of FZR1 by the injection of FZR1 mRNA suppressed the cyclin B accumulation and retarded GVBD. Surprisingly, the injection of CDC20 mRNA into the immature oocytes could not increase CDC20 expression, but increased cyclin B accumulation and accelerated the meiotic progression. As CDC20 is a substrate of APC (FZR1), CDC20 might have competed with cyclin B and inhibited the FZR1 function. These results suggest that porcine FZR1 and CDC20 work on the maintenance of meiotic arrest at the first meiotic prophase and on the exit from M1, respectively, and that their functional phases are strictly distinguished during porcine oocyte maturation.
Cervantes, Miriam P; Palomino, J Manuel; Anzar, Muhammad; Mapletoft, Reuben J; Mastromonaco, Gabriela F; Adams, Gregg P
Two experiments were done to test the hypothesis that morphologic characteristics of wood bison cumulus-oocyte complexes (COC) are reflective of the ability of the oocyte to develop to an advanced embryonic stage after in vitro maturation, fertilization and culture, and to determine the effect of prolonging the interval from the end of superstimulation treatment to oocyte collection (FSH starvation period). Experiments were done during the anovulatory season. In Experiment 1, ovarian superstimulation was induced in 10 bison with two doses of FSH given at 48 h intervals beginning at the time of follicular wave emergence. COC were collected 3 days (72 h) after the last dose of FSH by follicular aspiration and classified as compact, expanded or denuded. The COC were matured in vitro for 24 h before fertilization in vitro (Day 0). Embryo development was assessed on Days 3, 7 and 8. The blastocyst rate was 7/34, 2/10 and 0/3 in COC classified as compact, expanded and denuded, respectively; however, only compact COC resulted in embryos that reached the expanded blastocyst stage. In Experiment 2, COC were collected at either 3 or 4 days (72 or 96 h) after the last dose of FSH (n = 16 bison/group) to determine the effect of the duration of FSH starvation on oocyte competence. The COC were classified as compact good (>3 layers of cumulus cells), compact regular (1-3 layers of cumulus cells), expanded or denuded, and then matured, fertilized and cultured in vitro. Although follicles were larger (P vitro. Importantly, oocytes collected from superstimulated bison during the anovulatory season were competent to develop to the blastocyst stage following in vitro maturation, fertilization and culture. Copyright © 2017 Elsevier Inc. All rights reserved.
Somfai, Tamás; Yoshioka, Koji; Tanihara, Fuminori; Kaneko, Hiroyuki; Noguchi, Junko; Kashiwazaki, Naomi; Nagai, Takashi; Kikuchi, Kazuhiro
We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42 °C during warming prevented temperature drops in a medium below 34.0 °C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38 °C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38 °C and 42 °C was not different but lower (Pvitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.
Olgan, Safak; Mumusoglu, Sezcan; Bozdag, Gurkan
Objective. To investigate in vitro fertilization (IVF) treatment outcomes of unilateral oocyte retrieval in patients with transvaginally inaccessible ovaries. Study Design. Ninety-two women who underwent unilateral oocyte retrieval were retrospectively matched for age, antral follicle count, and body mass index with 184 women who underwent bilateral oocyte retrieval. Each patient in bilateral oocyte retrieval group had the same number of cumulus oophorus complexes (COCs) from single ovary and had comparable number of follicles (±2) on contralateral site where follicular aspiration was performed. Results. The number of COCs, metaphase-2 oocytes, 2-pronuclei, and top-quality embryos was significantly lower in unilateral oocyte retrieval group. However, proportion of patients with an embryo transfer of at least one top-quality embryo was found to be comparable between unilateral and bilateral oocyte retrieval. Subsequently, clinical pregnancy and live birth rates were found to be similar between the groups. The ROC curve analysis revealed (AUC = 0.74, 95% CI 0.63–0.86, p = 0.001) that retrieved COCs ≥ 5 from single ovary had sensitivity of 76.0% and specificity of 64.2% for occurrence of a clinical pregnancy. Conclusion. The patients with unilateral oocyte retrieval have reasonable chance of success with IVF. The retrieval of ≥5 COCs from accessible ovary might result in better treatment outcomes among these patients. PMID:27123444
Full Text Available Objective. To investigate in vitro fertilization (IVF treatment outcomes of unilateral oocyte retrieval in patients with transvaginally inaccessible ovaries. Study Design. Ninety-two women who underwent unilateral oocyte retrieval were retrospectively matched for age, antral follicle count, and body mass index with 184 women who underwent bilateral oocyte retrieval. Each patient in bilateral oocyte retrieval group had the same number of cumulus oophorus complexes (COCs from single ovary and had comparable number of follicles (±2 on contralateral site where follicular aspiration was performed. Results. The number of COCs, metaphase-2 oocytes, 2-pronuclei, and top-quality embryos was significantly lower in unilateral oocyte retrieval group. However, proportion of patients with an embryo transfer of at least one top-quality embryo was found to be comparable between unilateral and bilateral oocyte retrieval. Subsequently, clinical pregnancy and live birth rates were found to be similar between the groups. The ROC curve analysis revealed (AUC = 0.74, 95% CI 0.63–0.86, p=0.001 that retrieved COCs ≥ 5 from single ovary had sensitivity of 76.0% and specificity of 64.2% for occurrence of a clinical pregnancy. Conclusion. The patients with unilateral oocyte retrieval have reasonable chance of success with IVF. The retrieval of ≥5 COCs from accessible ovary might result in better treatment outcomes among these patients.
This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.
Full Text Available The effect of microphysical and environmental factors on the development of precipitation in warm idealised cloud is explored using a kinematic modelling framework. A simple one-dimensional column model is used to drive a suite of microphysics schemes including a flexible multi-moment bulk scheme (including both single and dual moment cloud liquid water and a state-of-the-art bin-resolved scheme with explicit treatments of liquid and aerosol. The Factorial Method is employed to quantify and compare the sensitivities of each scheme under a set of controlled conditions, in order to isolate the effect of additional microphysical complexity in terms of the impact on surface precipitation. At relatively low updraught speeds, the sensitivity of the bulk schemes was found to depend on the assumptions made with regards the treatment of droplet activation. It was possible to achieve a much closer agreement between the single and dual moment bulk schemes by tuning the specified droplet number concentration in the single moment scheme, suggesting that a diagnostic representation of droplet number may be an acceptable alternative to the more expensive prognostic option. However the effect of changes in CCN concentration were found to produce a relatively stronger effect on precipitation in the bulk schemes compared to the bin scheme; this is believed to be a consequence of differences in the treatment of drop growth by collision and coalescence. Collectively, these results demonstrate the usefulness of the Factorial Method as a model development tool for quantitatively comparing and contrasting the behaviour of microphysics schemes of differing levels of complexity within a specified parameter space.
Full Text Available The selection of good quality oocytes is crucial for in vitro fertilization and somatic cloning. Brilliant cresyl blue (BCB staining has been used for selection of oocytes from several mammalian species. However, the effects of differential oocyte selection by BCB staining on nuclear reprogramming and in vivo development of SCNT embryos are not well understood. Immature compact cumulus-oocyte complexes (COCs were divided into control (not exposed to BCB, BCB+ (blue cytoplasm and BCB- (colorless cytoplasm groups. We found that BCB+ oocytes yielded a significantly higher somatic cell nuclear transfer (SCNT blastocyst rate and full term development rate of bovine SCNT embryos than the BCB- and control oocytes. BCB+ embryos (embryos developed from BCB+ oocytes showed increased acetylation levels of histone H3 at K9 and K18 (AcH3K9, AcH3K18, and methylation levels of histone H3 at K4 (H3K4me2 than BCB- embryos (embryos developed from BCB- oocytes at the two-cell stage. Furthermore, BCB+ embryos generated more total cells, trophectoderm (TE cells, and inner cell mass (ICM cells, and fewer apoptotic cells than BCB- embryos. The expression of SOX2, CDX2, and anti-apoptotic microRNA-21 were up-regulated in the BCB+ blastocysts compared with BCB- blastocysts, whereas the expression of pro-apoptotic gene Bax was down-regulated in BCB+ blastocysts. These results strongly suggest that BCB+ oocytes have a higher nuclear reprogramming capacity, and that BCB staining can be used to select developmentally competent oocytes for nuclear transfer.
Full Text Available Abstract Background In the past few years, cryopreservation of ovarian tissue has become an established procedure proposed in many centers around the world and transplantation has successfully resulted in full-term pregnancies and deliveries in human. This prospective study aims to evaluate the feasibility of vitrifying in vitro matured oocytes (IVM isolated at the time of ovarian tissue cryopreservation to improve the efficiency of fertility preservation programs. Methods Oocyte-cumulus complexes were retrieved from freshly collected ovarian cortex by aspirating antral follicular fluid, and were matured in vitro for 24-48 h prior to vitrification. Oocytes were matured in an IVM commercial medium (Copper Surgical, USA supplemented with 75 mIU/ml FSH and 75 mIU/ml LH and vitrified using a commercial vitrification kit (Irvine Scientific, California in high security vitrification straws (CryoBioSystem, France. Oocyte collection and IVM rates were evaluated according to the age, the cycle period and the amount of tissue collected. Results Immature oocyte retrieval from ovarian tissue was carried out in 57 patients between 8 and 35 years of age, undergoing ovarian tissue cryopreservation. A total of 266 oocytes were isolated, 28 of them were degenerated, 200 were at germinal vesicle stage (GV, 35 were in metaphase I (MI and 3 displayed a visible polar body (MII. The number of oocytes collected was positively correlated with the amount of tissue cryopreserved (p p = 0.005. Oocytes were obtained regardless of menstrual cycle period or contraception. A total maturation rate of 31% was achieved, leading to the vitrification of at least one mature oocyte for half of the cohort. Conclusions The study showed that a significant number of immature oocytes can be collected from excised ovarian tissue whatever the menstrual cycle phases and the age of the patients, even for prepubertal girls.
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
Inforzato, Antonio; Rivieccio, Vincenzo; Morreale, Antonio P; Bastone, Antonio; Salustri, Antonietta; Scarchilli, Laura; Verdoliva, Antonio; Vincenti, Silvia; Gallo, Grazia; Chiapparino, Caterina; Pacello, Lucrezia; Nucera, Eleonora; Serlupi-Crescenzi, Ottaviano; Day, Anthony J; Bottazzi, Barbara; Mantovani, Alberto; De Santis, Rita; Salvatori, Giovanni
PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.
Wani, N A
Experiments were conducted to study the efficiency of sequential treatments of ionomycine and ethanol combined with phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine) in inducing artificial activation in dromedary M-II oocytes. Cumulus oocyte complexes (COCs), collected from slaughterhouse ovaries were cultured at 38.5 degrees C in an atmosphere of 5% CO2 in air for 24-48 h. In experiment 1, the COCs were either fertilized in vitro or activated with 5 microM ionomycine for 5 min or 7% ethanol for 7 min, both followed by exposure to 6-diethylaminopurine or roscovitine for 4h. After 14-15 h of in vitro culture, the oocytes were fixed and stained with 1% aceto-orcein to evaluate their nuclear status. In experiment 2, the oocytes were activated in the same manner as in experiment 1 but were cultured for 7 days to evaluate their post-parthenogenetic development. In experiment 3, oocytes were exposed to the ionomycine for 2, 3, 4 or 5 min to evaluate the better exposure time while as in experiment 4, the oocytes matured for 28-48 h were activated to see the effect of aging on post-parthenogenetic development. Higher proportion (P0.05) in the proportion of oocytes activated with ethanol when compared with in vitro fertilized group. No significant difference was seen on the proportion of morula on day 7 of culture, however the development to blastocyst stage was higher (P0.05). The proportion of blastocysts obtained was higher (Pdromedary camel oocytes with ionomycine/6-DMAP is demonstrated and optimized in the present study for further use in the development of assisted reproductive techniques in this species.
Somfai, Tamás; Nakai, Michiko; Tanihara, Fuminori; Noguchi, Junko; Kaneko, Hiroyuki; Kashiwazaki, Naomi; Egerszegi, István; Nagai, Takashi; Kikuchi, Kazuhiro
Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.
Full Text Available Background: Bone morphogenetic protein-15 (BMP15 is a maternal gene necessary for mammalian reproduction. BMP15 expression increased in oocytes accompanied by follicle growth and development. The function and regulation mechanism of BMP15 in porcine cumulus cell apoptosis process is still unclear now. Methods: In this study, flow cytometry (FCM was used to analyze the effects of BMP15 with different concentrations to cumulus cell apoptosis. High-throughput sequencing technology was carried out to screen regulatory genes linked closely with BMP15. In order to confirm the function of (MCP-1/CCL2 and FBN1 in cumulus cell apoptosis, RNA interference (RNAi method was used to inhibit the expression of (MCP-1/CCL2 and FBN1. Apoptosis and proliferation of cumulus cell treated with siRNA transfection technology were measured by FCM, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide, quantitative real time-PCR (RT-qPCR and western blotting. Results: The results showed that the apoptosis levels of cumulus cell treated by BMP15 decreased significantly in a dose-dependent manner. The expression of related genes protein 1 (MCP-1/CCL2 and fibrillin1 (FBN1 were both regulated by BMP15. After transfection, the proliferation of porcine cumulus cells increased significantly and apoptosis of cumulus cells was prevented while FBN1 was silenced after BMP15 treatment. The proliferation of cumulus cells decreased significantly and apoptosis rate of cumulus cells increased significantly while CCL2 was silenced. Conclusion: The results obtained in this study firstly demonstrated that CCL2 and FBN1 are important regulatory factors of BMP15 in preventing cumulus cell apoptosis in porcine ovaries.
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.
Cervantes, Miriam P; Palomino, J Manuel; Anzar, Muhammad; Mapletoft, Reuben J; Adams, Gregg P
Experiments were done to compare the in vivo and in vitro maturational characteristics of cumulus-oocyte complexes (COC) collected from live wood bison. In Experiment 1 (anovulatory season), follicular ablation was done to synchronize follicle wave emergence among bison on Day -1, and FSH was given on Days 0 and 2. Bison were then assigned to 5 groups (n=5/group) in which COC were collected by transvaginal follicle aspiration on Day 4 and either fixed immediately with no maturation (control), matured in vitro for 24 or 30h, or collected on Day 5 after in vivo maturation for 24 or 30h (i.e., after hCG treatment). In Experiment 2 (ovulatory season), bison were treated as described for Experiment 1, but PGF2α (cloprostenol) was given to control the luteal phase on Days -9 and 3. In both experiments, cumulus cell expansion was more extensive following in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30h groups. Nuclear maturation occurred more rapidly in vitro; 60-70% of oocytes were at the MII stage 24h after in vitro maturation while only 25-27% of oocytes had reached the MII stage after 24h of in vivo maturation. In conclusion, nuclear maturation occurred more rapidly during in vitro vs. in vivo maturation, but was associated with less cumulus expansion than in vivo maturation. In vivo oocyte maturation was more complete at 30 vs. 24h after hCG treatment. Season had no effect on the maturational capacity of wood bison oocytes.
Full Text Available Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research on in vitro embryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving the in vitro culture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable experience to better identify oocyte quality because of the intense subjectivity involved (Gordon (2003, Madison et al. (1992 and De Loos et al. (1992. BCB staining is a function of glucose-6-phosphate dehydrogenase (G6PD activity, an enzyme synthesized in developing oocytes, which decreases in activity with maturation. Therefore, unstained oocytes (BCB− are high in G6PD activity, while the less mature oocytes stains are deep blue (BCB+ due to insuffcient G6PD activity to decolorize the BCB dye.
Full Text Available Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl phthalate (DEHP is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM, CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL and intracellular reactive oxygen species (ROS levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05. This effect was related to increased CC apoptosis (P<0.001 and reduced ROS levels (P<0.0001. At higher doses (12 and 1200 µM, DEHP induced apoptosis (P<0.0001 and ROS increase (P<0.0001 in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity, intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05, possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into
Crocomo, L F; Ariu, F; Bogliolo, L; Bebbere, D; Ledda, S; Bicudo, S D
The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 μm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos. © 2016 Blackwell Verlag GmbH.
Alvarez, G M; Ferretti, E L; Gutnisky, C; Dalvit, G C; Cetica, P D
Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose-dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6-AN) and the physiological (NADPH) inhibitors of PPP induced a dose-dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.
Kee Sang Park; Gun Ho Song; Hang Jin Kim; Hai Bum Song; Taek Hoo Lee; Sang Sik Chun
Objective To establish and evaluate the efficacy of the simple assessment system of oocyte maturity.Methods A total of 251 couples were enrolled in this study and female patients were divided into two groups. In group Ⅰ, oocytes were inseminated at 6 h after ovum pickup. In group Ⅱ, oocyte maturity was rapidly categorized by simple assessment system.Mature oocytes were inseminated at 3-4 h after ovum pick-up when oocyte-corona complexes (OCC) exhibited clear thick ring-like halo (RLH) and expanded cumulus cells (CC) or 5-6 h when OCC exhibited RLH and a few clumped and/or dark CC,respectively. Intermediate oocytes were inseminated at 7-8 h when RLH was not found around the OCC and CC were compacted and clumped and/or dark.Results Normal fertilization rate was higher in group Ⅱ (76.5%) than that in group Ⅰ(58.0%) (P＜0. 001). However, abnormal fertilization rate was higher in group Ⅰ(11.3%) than that in group Ⅱ (3.6%) (P＜0. 001). The cleavage (82.6% vs 90.0%),chemical pregnancy (4. 8% vs 3.9%), twin pregnancy (6. 7% vs 3. 9%) and implantation rate (8.4% vs 10. 6%) had no statistically differences between group Ⅰ and Ⅱ. Rate of clinical and singleton pregnancy was higher in group Ⅱ (35.3% and 31.4%) than those in group Ⅰ (24.8% and 18.2%) (P＜0. 05).Conclusion This simple assessment system is useful and effective for evaluation and categorization of the oocyte maturity with better reproductive outcomes.
Beker van Woudenberg, A.R.C.L. (Anna Rita Costa Lage)
Chapter 1 presents an overview on the aspects of oocyte maturation. Growth hormone (GH), released from the pituitary by the stimulus of GHRH, increases cumulus expansion and improves cytoplasmic maturation in bovine oocytes. GHRH is also expressed in extraneural tissues suggesting that GHRH also
Sun, Xing-Shen; Liu, Yong; Yue, Kui-Zhong; Ma, Suo-Feng; Tan, Jing-He
Changes in germinal vesicle (GV) chromatin configurations during growth and maturation of porcine oocytes were studied using a new method that allows a clearer visualization of both nucleolus and chromatin after Hoechst staining. The GV chromatin of porcine oocytes was classified into five configurations, based on the degree of chromatin condensation, and on nucleolus and nuclear membrane disappearance. While the GV1 to 4 configurations were similar to those reported by previous studies, the GV0 configuration was distinct by the diffuse, filamentous pattern of chromatin in the whole nuclear area. Most of the oocytes were at the GV0 stage in the layers of cumulus cells and those with less than one layer or no cumulus cells. Overall, our results suggested that (i) the GV0 configuration in porcine oocytes corresponded to the "nonsurrounded nucleolus" pattern in mice and other species; (ii) all the oocytes were synchronized at the GV1 stage before GVBD and this pattern might, therefore, represent a nonatretic state; (iii) the GV3 and GV4 configurations might represent stages toward atresia, or transient events prior to GVBD that could be switched toward either ovulation or atresia, depending upon circumstances; (iv) the in vitro systems currently used were not favorable for oocytes to switch toward ovulation (or final maturation); (v) the number of cumulus cells was not correlated with the chromatin configuration of oocytes, indicating that the beneficial effect of cumulus cells on oocyte maturation and development may simply be attributed to their presence during in vitro culture.
Jiang, Han; Wang, Ce; Guan, Jiyu; Wang, Lingyan; Li, Ziyi
The golden hamster is an excellent animal experimental model for oocyte research. The hamster oocytes are very useful in clinical examination of human spermatozoan activity. Non-fertile oocytes can lead to time-dependent processes of aging, which will affect the results of human spermatozoa examination. As a consequence there is a need to investigate the aging and anti-aging processes of golden hamster oocytes. In order to study the aging processes and parthenogenetic activation of golden hamster oocytes, in vivo oocytes, oocytes cultured with or without cumulus cells, and oocytes treated with Trichostatin A (TSA) or caffeine were collected and investigated. We found that: (1) spontaneous parthenogenetic activation, developmental potential (cleavage rate), and zona pellucida (ZP) hardening undergo age-dependent changes in in vivo, in vitro, and after TSA or caffeine treatment; (2) in vivo, oocytes became spontaneously parthenogenetic 25 h post-hCG treatment; (3) in vitro, cumulus cells did not significantly increase the parthenogenetic activation rate of cultured hamster oocytes; and (4) TSA or caffeine could delay spontaneous oocyte parthenogenetic activation and the aging processes by at least 5h, but also accelerated the hardening of the ZP. These results define the conditions for the aging and anti-aging processes in golden hamster oocytes. TSA and caffeine play roles in controlling spontaneous activation, which could facilitate the storage and use of golden hamster oocytes for studying processes relevant to human reproduction.
Wissing, Marie Louise; Sonne, Si Brask; Westergaard, David;
Corona radiata cells (CRCs) refer to the fraction of cumulus cells just adjacent to the oocyte. The CRCs are closely connected to the oocyte throughout maturation and their gene expression profiles might reflect oocyte quality. Polycystic ovary syndrome (PCOS) is a common cause of infertility...
Warzych, E; Peippo, J; Szydlowski, M; Lechniak, D
The objective of this study was to compare the effect of different supplements to the basic IVM medium (TCM199) on the efficiency of cattle oocyte maturation and blastocyst production, and the incidence of apoptosis in both oocytes and blastocysts. Two protein supplements (FBS and fafBSA) and a macromolecule (PVP40) were compared in a 3 treatmentsx9 replicates design. Cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries were matured for 24h in TCM199 medium supplemented with 10% FBS, 6% fafBSA or 4% PVP40 (50-70 COCs in each treatment/replicate), then inseminated and cultured in vitro for 8 days. Immature and mature oocytes as well as Day 8 blastocysts were subjected to TUNEL analysis. Cleavage rate was monitored on Day 2 post-insemination (pi), whereas blastocyst yield on Day 8 pi. The composition of maturation media did not affect zygotic cleavage rate on Day 2 (on average 71.0%), however the blastocyst rate on Day 8 pi was significantly lower (P<0.001) for embryos derived from oocytes matured with PVP40 (16.0%) than for those matured with FBS (22.4%) or fafBSA (22.1%). The rate of TUNEL positive oocytes differed significantly between immature (1.4%) and mature (11.2%) oocytes (P<0.01). Supplements to maturation medium were not related to the incidence of apoptosis in mature oocytes (11.2%) and the rate of oocytes at the second metaphase stage (71.5%). Cumulus cell expansion was reduced by maturation in medium supplemented with PVP40. This macromolecule was also correlated with higher apoptotic index in blastocysts (5.8%) when compared to FBS (3.2%) and fafBSA (3.1%; P<0.001). In conclusion, lower blastocyst rate and elevated apoptotic index in embryos derived from oocytes matured with PVP40 may suggest that synthetic macromolecule provides less balanced environment for oocyte maturation and therefore should be treated with caution.
Abdelkhalek, A E; Gabr, Sh A; Khalil, W A; Shamiah, Sh M; Pan, L; Qin, G; Farouk, M H
Application of assisted reproductive technology in camelidea, such as artificial insemination (AI) and embryo transfer, has been slow in comparison to that for other livestock species. In Egypt, there are few attempts to establish in vitro maturation (IVM) and fertilization (IVF) techniques in dromedary camel. The present study was carried out to produce Sudanese camel embryos using in vitro matured oocytes and epididymal spermatozoa. Dromedary camel ovaries were collected from abattoirs and then, the oocytes were aspirated from all the visible follicles on the ovarian surface (~2-8 mm in a diameter). Meanwhile, Fetal Dromedary Camel Serum (FDCS) was obtained from camel fetuses after slaughtering. Thereafter, only Cumulus Oocyte Complexes (COCs) were matured in vitro in the Tissue Culture Medium (TCM-199) complemented with 10% FDCS. Spermatozoa required for in vitro fertilization were collected from testes (epididymal cauda) of the slaughtered camel bulls. The results clearly showed that the maturation rate of oocytes at metaphase II was about 59.5% while the fertilization rate was around 70.4%. Intriguingly, the embryo rates determined were 13.1%, in 2-cell; 0.0%, in 4-cell; 34.7%, in 8-16% cell; 39.1%, in morula and 13.1% in a blastocyst stage. This study represented a successful in vitro production of Sudanese dromedary camel embryos from epididymal sperm cells and in vitro matured oocytes recovered from slaughtered camels.
Full Text Available This study evaluated the effect of melatonin supplementation of in vitro maturation media on in vitro maturation (IVM and in vitro fertilization (IVF rate of buffalo oocytes. Cumulus oocytes complexes (COCs were aspirated from follicles of 2-8 mm diameter. In experiment I, COCs were matured in IVM medium supplemented with 0 (control, 250, 500, and 1000 μM melatonin for 22-24 hours in CO2 incubator at 38.5°C with 5% CO2 and at 95% relative humidity. The maturation rate did not differ in media supplemented with melatonin at 250 μM, 500 μM, 1000 μM and control (0 μM. In experiment II, the matured oocytes were fertilized in 50 μl droplets of Tyrode’s Albumin Lactate Pyruvate (TALP medium having 10 ug/ml heparin for sperm (2 million/ml capacitation. The fertilization droplets were then kept for incubation at 5% CO2, 39°C and at 95% relative humidity for 18 hours. The fertilization rate was assessed by sperm penetration and pronuclear formation. Fertilization rate was improved when maturation medium was supplemented with 250 μM melatonin compared to control. In conclusion, melatonin supplementation to serum free maturation media at 250 μM improved the fertilization rate of buffalo oocytes.
Patel, Hiren; Chougule, Shruti; Chohan, Parul; Shah, Naval; Bhartiya, Deepa
Pluripotent stem cells are possibly the best candidates for regenerative medicine, and somatic cell nuclear transfer (SCNT) is one of the viable options to make patient-specific embryonic stem cells. Till date efficacy of SCNT embryos is very low and requires further improvement like ideal oocyte activation and in vitro culture system. The aim of the present study was to evaluate ideal oocyte activation using different stimulation protocols and to study the effect of cumulus co-culture conditions on embryo development. Results demonstrate that between electric stimulation and chemical stimulation using calcium ionomycin and ionophore, best oocyte activation was obtained using calcium ionomycin (5 microM for 5 min) which resulted in 83% cleavage followed by 7% of early blastocyst which further increased to 15% when a cumulus bed was also introduced during embryo culture. Sequential modified Charles Rosenkrans 2 (mCR2) medium was used for embryo culture in which glucose levels were increased from 1 mM to 5 mM from Day 3 onwards. SCNT using cumulus cells as donor somatic cell, calcium ionomycin to activate the reconstructed oocyte and embryo culture on a cumulus bed in sequential mCR2 medium, resulted in the development of 6% embryos to early blastocyst stage. Such technological advances will make SCNT a viable option to make patient-specific pluripotent stem cell lines in near future.
Bhuiyan, M M U; Suzuki, Y; Watanabe, H; Matsuoka, K; Fujise, Y; Ishikawa, H; Ohsumi, S; Fukui, Y
The cumulus-oocyte-complexes (COCs) recovery rates with respect to reproductive status per sei (Balaenoptera borealis) and Bryde's (B. edeni) whales were determined in Experiment 1. The number of COCs recovered ranged from 16.0 to 30.6 and from 6.7 to 26.8 per sei and Bryde's whales, respectively. The effects of COCs grades and protein supplementation in embryo culture medium on development of in vitro fertilized (IVF) embryos were evaluated in sei and Bryde's whales in Experiment 2. The COCs were classified into either Grade A (COCs with five or more layers of compact cumulus cells) or Grade B (COCs with less than five layers of compact or expanded cumulus cells) before being cultured for IVM. The cleavage (12.0 to 19.5%), 4-cell (8.0 to 12.0%) and 8-cell (4.0 to 8.0%) formation rates in sei whales did not vary significantly between embryos derived from either grade A or B oocytes and between embryos cultured in either fetal whale serum (FWS)- or bovine serum albumin (BSA)-supplemented medium. The cleavage (4.0 to 14.8%), 4-cell (0.0 to 7.5%) and 8-cell (0.0 to 2.6%) formation rates in Bryde's whales did not vary significantly between embryos derived from either grade A or B oocytes and between embryos cultured in either FWS- or BSA-supplemented medium. The grade B oocytes cultured in FWS-supplemented medium developed to morula stage (1.1%) in sei whales. In conclusion, the present study indicates that IVF in sei whales is possible to achieve cleaved embryos developing to morula stage. This is the first in vitro embryo production attempt in sei and Bryde's whales.
Coticchio, Giovanni; Dal-Canto, Mariabeatrice; Guglielmo, Maria-Cristina; Mignini-Renzini, Mario; Fadini, Rubens
Oocytes from medium-sized antral follicles have already completed their growth phase and, if released from the follicular environment and cultured in vitro, are able to resume the meiotic process and mature. However, in vitro maturation (IVM) does not entirely support all the nuclear and cytoplasmic changes that occur physiologically as an effect of the ovulatory stimulus. Regardless, oocyte IVM is widely applied for the breeding of agriculturally important species. In assisted reproduction technology, IVM has been proposed as an alternative treatment to circumvent the drawbacks of standard ovarian stimulation regimens. Initially introduced to eliminate the risks of ovarian hyperstimulation syndrome afflicting women presenting with polycystic ovaries, subsequently IVM has been suggested to represent an additional approach suitable also for normovulatory patients. So far, in children born from IVM cycles, no doubts of an increased incidence of congenital abnormalities have been raised. Many more births would be achieved if novel IVM systems, currently dominated by empiricism, could be conceived according to more physiological criteria. Recent findings shedding new light on the control of meiotic progression, the support of cumulus cells to the oocyte cellular reorganization occurring during maturation, and the modulation of the stimulus that promotes oocyte maturation downstream the mid-cycle gonadotropin signal are likely to provide crucial hints for the development of more efficient IVM systems.
Pitangui-Molina, Caroline P; Vireque, Alessandra A; Tata, Alessandra; Belaz, Katia Roberta A; Santos, Vanessa G; Ferreira, Christina R; Eberlin, Marcos N; Silva-de-Sá, Marcos Felipe; Ferriani, Rui A; Rosa-E-Silva, Ana Carolina J S
The phospholipid (PL) composition of embryo and oocyte membranes affects thermal phase behavior and several physicochemical properties such as fluidity and permeability. The characterization of PL profiles and the development of suitable in vitro maturation (IVM) protocols, that are able to modify membrane's composition, may result in significant improvements in oocyte developmental potential and cryotolerance. Using soybean phosphatidylcholine (PC) as a model supplement, we evaluated the effect of PL supplementation during IVM on bovine cumulus-oocyte-complex (COC). Substantial changes in the lipid profiles of oocyte membrane were observed and associated with pre-implantation data. The propensity of the PC supplement to become soluble in the maturation medium and/or diffuse into mineral oil was also assessed. Oocytes were matured in TCM without supplementation, i.e. control, (n=922) or supplemented with 50 or 100μM PC (n=994). The maturation media and mineral oil pre- and post- IVM, along with control and PC-treated oocytes were then analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and the lipid profiles were compared via principal component analysis (PCA). Soybean PCs are bioavailable and stable in IVM medium; further, PCs did not diffuse to the mineral oil, which also remained unaltered by the metabolism of treated oocytes. PC supplementation at 100μM resulted in substantially greater relative abundances of polyunsatured PL, namely PC (32:1), PC (34:2), PC (36:6), PC (36:4), and PC (38:6), in oocyte membrane. These differences indicated that short-term exposure to the PC supplement could indeed modify the lipid composition of IVM-oocytes in a dose-dependent manner. Membrane incorporation of polyunsaturated molecular species of PC was favored, and does so without compromising the viability of the subsequent embryo in regards to cleavage, blastocyst development and hatching rate. The reported approach will allow for the
Full Text Available Objective: This study examines the effects of hydrostatic pressure on in vitro maturation (IVM of oocytes derived from in vitro grown follicles.Materials and Methods: In this experimental study, preantral follicles were isolated from 12-day-old female NMRI mice. Each follicle was cultured individually in Alpha Minimal Essential Medium (α-MEM under mineral oil for 12 days. Then, follicles were induced for IVM and divided into two groups, control and experiment. In the experiment group follicles were subjected to 20 mmHg pressure for 30 minutes and cultured for 24-48 hours. We assessed for viability and IVM of the oocytes. The percentage of apoptosis in cumulus cells was determined by the TUNEL assay. A comparison between groups was made using the student’s t test.Results: The percentage of metaphase II oocytes (MII increased in hydrostatic pressure-treated follicles compared to controls (p<0.05. Cumulus cell viability reduced in hydrostatic pressure-treated follicles compared to controls (p<0.05. Exposure of follicles to pressure increased apoptosis in cumulus cells compared to controls (p<0.05.Conclusion: Hydrostatic pressure, by inducing apoptosis in cumulus cells, participates in the cumulus oocyte coupled relationship with oocyte maturation.
Lin, Li-Te; Wang, Peng-Hui; Chen, San-Nung; Li, Chia-Jung; Wen, Zhi-Hong; Cheng, Jiin-Tsuey; Tsui, Kuan-Hao
Growing studies have demonstrated that dehydroepiandrosterone (DHEA) may improve fertility outcomes in poor ovarian responders (PORs). The aim of this study was to compare clinical outcomes and cumulus cell (CC) expression before and after DHEA treatment in PORs undergoing in vitro fertilization (IVF) cycles. Six patients with poor ovarian response were enrolled in the study according to Bologna criteria. DHEA was supplied at least 2 months before patients entered into the next IVF cycle. Expression of apoptosis-related genes in CCs was determined by quantitative real-time PCR. Mitochondrial dehydrogenase activity of CCs was assessed by cell counting kit-8 assay. Metaphase II oocytes, maturation rate, embryos at Day 3, and fertilization rate significantly increased following DHEA treatment. Expression of cytochrome c, caspase 9, and caspase 3 genes in CCs were significantly reduced after DHEA therapy. Additionally, increased mitochondrial activity of CCs was observed following DHEA supplementation. DHEA supplementation may protect CCs via improved mitochondrial activity and decreased apoptosis, leading to better clinical outcomes in PORs.
Full Text Available Objective: Lower pregnancy rates of in vitro matured oocytes compared to those of invivo stimulated cycles indicate that optimization of in vitro maturation (IVM remains achallenge. Reduced developmental competence of in vitro matured oocytes shows thatcurrent culture systems for oocyte maturation do not adequately support nuclear and/orcytoplasmic maturation. Therefore this study evaluates the effects of different concentrationsof saffron (Crocus sativus L. aqueous extract (SAE, as an antioxidant agent on IVMof immature mouse oocytes.Materials and Methods: In this experimental study ,cumulus-oocyte complexes (COCswere collected from 6-8 weeks old novel medical research institute (NMRI female miceovaries. COCs were cultured in IVM medium supplemented with 0 (control, 5, 10, 20 and40 μg/ml of SAE in 5% CO2 at 37°C. The rates of maturation, fertilization and developmentwere recorded. ANOVA and Duncan’s protected least significant test, using the SASprogram was applied for all statistical analysis.Results: The maturation rate was significantly higher in all groups treated with differentconcentrations of SAE compared with the control group (p<0.05. However, the lowerconcentrations of SAE (10 and 5 μg/ml in maturation medium respectively increased thefertilization rate of oocytes and in vitro developmental competence when compared withthe control group (p<0.05.Conclusion: The results of this study indicate that lower concentrations of SAE are moreappropriate to be added to maturation medium when compared with other experimental andcontrol groups. Generally, we conclude that addition of appropriate amounts of natural extractssuch as SAE to maturation medium improves oocyte maturation and embryo development.
Full Text Available In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new cryoprotective agent (CPA, called as carboxylated ε-poly-L-lysine (COOH-PLL, has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90% of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20% (v/v and COOH-PLL 10% (w/v] or E15P15 group (EG 15% and COOH-PLL 15% were significantly higher than those with E10P20 (EG10% and COOH-PLL 20% or P30 group (PLL30%. The vitrified COCs in E20P10 group developed to term at a high success rate (46.2% and it was significantly higher than that in control (E30 group (34.8%. Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mouse oocytes.
Hammami, S; Morató, R; Romaguera, R; Roura, M; Catalá, M G; Paramio, M T; Mogas, T; Izquierdo, D
The aim of this study was to test the effect of insulin-transferrin-selenium (ITS) and L-ascorbic acid (AA) supplementation and the hormonal level during in vitro maturation (IVM) of small oocytes from pre-pubertal goat on the blastocyst yield and quality. Concretely, we used four maturation media: conventional IVM medium (CM), growth medium (GM: CM+ITS+AA and low level of hormones), modified CM (mCM: CM with low level of hormones) and modified GM (mGM: CM+ITS+AA and normal level of hormones). Cumulus-oocyte complexes (COCs) were classified into two categories according to oocyte diameter: <125 μm and ≥ 125 μm. Large oocytes were matured 24 h in CM (Treatment A). Small oocytes were matured randomly in six experimental groups: Treatment B: 24 h in CM; Treatment C: 12 h in GM and 12 h in CM; Treatment D: 24 h in mGM; Treatment E: 12 h in mGM and 12 h in CM; Treatment F: 12 h in mCM and 12 h in CM; and Treatment G: 12 h in GM and 12 h in mGM. After IVM, oocytes were fertilized and cultured for 8 days. The blastocyst quality was assessed by the survival following vitrification/warming and the mean cell number. When different maturation media were combined, the blastocyst rate did not improve. The large oocytes produced the highest blastocysts yield. However, the culture of small oocytes in GM (53.3%) enhanced the post-warming survival of blastocysts compared to large oocytes matured in CM (35.7%). In conclusion, IVM of pre-pubertal goat small oocytes in GM would be useful to improve the quality of in vitro-produced blastocysts.
Fernandez-Gonzalez, Lorena; Hribal, Romy; Stagegaard, Julia; Zahmel, Jennifer; Jewgenow, Katarina
Assisted reproductive techniques are becoming widely applied to the breeding of endangered species, but establishing reliable protocols for the production of embryos in vitro is challenging because of the scarcity of sample material. In our study, we applied an assisted reproductive technique protocol for IVM and intracytoplasmic sperm injection (ICSI), developed in the domestic cat, to oocytes retrieved from ovaries of four 2-year-old lionesses (Panthera leo) eight hours postmortem. In total, 68 cumulus-oocyte complexes of good quality were randomly distributed and cultured for 32 to 34 hours in two different maturation culture media, consisting of Medium 199 with Earle's salts, 3 mg/mL BSA, 0.1 mg/mL cysteine, 1.4 mg/mL sodium pyruvate, 0.6 mg/mL sodium lactate, 0.15 mg/mL l-glutamine, and 0.055 mg/mL gentamicin. Hormonal supplementation of IVM_1 was 0.02 IU/mL FSH and 0.05 IU/mL LH; IVM_2 consisted of 1.64 IU/mL FSH, 1.06 IU/mL LH, and 1 μg/mL 17ß-estradiol. Differences in hormonal supplementation did not produce significant differences in oocyte maturation rates, which were 39.4% in IVM_1 and 34.3% in IVM_2. Matured oocytes were microinjected with homologous frozen-thawed spermatozoa, and subsequent cleavage rates were 30.8% and 58.3%, respectively. Half of the embryos derived from oocytes matured in IVM_1 developed into blastocysts, whereas only 28.6% of embryos from oocytes matured in IVM_2 reached the blastocyst stage. Morula stages were present from Day 6 onward, and blastocyst stages from Day 9 on, indicating a slower developmental speed in comparison with domestic cats. This is the first report of in vitro-produced blastocysts using ICSI in the lion, and the results report that IVM and ICSI can be successfully performed with cumulus-oocyte complexes retrieved from ovaries after eight hours of shipping, obtaining competent embryos in culture. Copyright © 2015 Elsevier Inc. All rights reserved.
Germond, M; Nocera, D; Senn, A; Rink, K; Delacretaz, G; Pedrazzini, T; Hornung, J P
The safety of microdrilling the zona pellucida of mouse oocytes with a 1.48 microm diode laser has been investigated by determining the ability of mouse oocytes to fertilize in vitro and develop in vivo. Mice born after transfer of control and zona pellucida-microdrilled embryos into foster mothers were submitted to anatomical and immunohistochemical investigations, and their aptitude to breed was assessed in two subsequent generations. Decoronization of the oocytes with hyaluronidase induced a reduction of the fertilization and implantation rates, which was attributed to a zona hardening phenomenon. After laser zona pellucida microdrilling, these rates were restored to those obtained with embryos derived from untreated oocyte-cumulus complexes. Pups derived from zona pellucida microdrilled embryos were comparable with those obtained from control embryos, confirming the lack of deleterious effects of the laser treatment. In conclusion, the 1.48 microm diode laser allows safe microdrilling of the zona pellucida of mouse oocytes after decoronization with hyaluronidase. Based on the health of the F2 generation and the lack of neuroanatomical and neurochemical differences, we concluded that this technology may be investigated in the human, particularly when the zona pellucida represents the main impediment for fertilization or embryo hatching.
Full Text Available A Holstein cow of high genetic merit, in late lactation (205 days and diagnosed with salpingitis (after 4 infertile services and veterinary consultation, was subjected to 1 trans-vaginal oocyte collection attempt, prior to slaughter.Of an estimated 10 follicles punctured, a total of 4 cumulus-oocyte complexes were retrieved. These were matured in vitro in a maturation medium for 24 hours. After 24 hours maturation, the oocytes were fertilised in vitro with Percoll-processed frozen / thawed imported semen, of the owner's choice. Fertilisation was achieved in a modified Tyrode's medium. At 18 hours post-insemination, the presumptive zygotes were transferred into culture in vitro in Charles Rosenkran's aminoacid medium and supplemented on Day 4 post-insemination with 10 % foetal calf serum. All in vitro procedures were conducted in 50 mℓ medium droplets, under oil, in a humidified incubator at 38.5 oC in 5% CO2 in air. Three of the potential zygotes cleaved and, by Day 7 of culture, these had developed to the morula stage. The embryos were frozen in 1.5 M ethylene glycol and later transferred non-surgically to synchronised Holstein recipient heifers. One morula resulted in the only pregnancy and subsequent birth of a healthy heifer calf. An independent commercial company confirmed parentage through standard bloodtyping assays. The genetic salvage of oocytes, for in vitro production of embryos, has potential benefits to the producer.
Beek, J; Nauwynck, H; Maes, D; Van Soom, A
In this study, we report for the first time on a possible contribution of metalloproteases in sperm passage through the cumulus matrix in pigs. The presence of 20 μM 1,10-phenanthroline (1,10-PHEN), inhibitor of zinc-dependent metalloproteases, strongly inhibited the degree of sperm penetration in cumulus-intact (CI), but not in cumulus-free (CF), porcine oocytes during IVF. The inhibitory effect of 1,10-PHEN was due to the chelation of metal ions as a non-chelating analog (1,7-PHEN) did not affect IVF rates. Furthermore, incubation with 1,10-PHEN did not affect sperm binding to the zona pellucida nor sperm motility, membrane integrity, or acrosomal status. These findings led to the assumption that 1,10-PHEN interacts with a sperm- or cumulus-derived metalloprotease. Metalloproteases are key players in physiological processes involving degradation or remodeling of extracellular matrix. In vivo, their proteolytic activity is regulated by tissue inhibitors of metalloproteases (TIMP1-TIMP4). We tested the effect of TIMP3 on fertilization parameters after porcine IVF. Similar to 1,10-PHEN, TIMP3 inhibited total fertilization rate of CI but not CF oocytes and did not influence sperm quality parameters. Although the inhibitory effect was stronger in CI oocytes, TIMP3 also reduced the degree of sperm penetration in CF oocytes, suggesting the involvement of a metalloprotease in a subsequent step during fertilization. In conclusion, our results indicate the involvement of TIMP3-sensitive, zinc-dependent metalloprotease activity in sperm passage through the cumulus oophorus in pigs. The results should provide the basis for further biochemical research toward the localization and identification of the metalloprotease involved.
Pereira, G R; Lorenzo, P L; Carneiro, G F; Ball, B A; Pegoraro, L M C; Pimentel, C A; Liu, I K M
In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E2), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles <30 mm in diameter and matured for 30 h at 38.5°C in air with 5% CO2. In experiment 1, selected cumulus-oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 μg/ml E2, 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-α-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO3-iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E2, gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P < 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E2, gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.
Full Text Available In this report, we present a case of in vitro maturation (IVM with surgical retrieved testicular sperm in a normo-ovulatory female. Human chorionic gonadotropin-primed IVM, testicular biopsy for sperm retrieval and intracytoplasmic sperm injection with fresh sperm were performed. Fourteen cumulus-oocyte complexes were obtained in germinal vesicle or metaphase I stage, eight oocytes reached metaphase II, seven presumptive zygotes were obtained, and three cleavage stages embryos in day 2 were transferred producing a singleton pregnancy. A single healthy newborn was obtained. Our results suggest that IVM may be an alternative for in vitro fertilization in normo-ovulatory women even if surgical retrieval of sperm is needed. Further research is required to depict contributing factors to the success of IVM in indications different from polycystic ovaries syndrome and the role of male gamete.
González-Ortega, Claudia; Piña-Aguilar, Raul Eduardo; Cancino-Villareal, Patricia; Gutiérrez-Gutiérrez, Antonio Martin
In this report, we present a case of in vitro maturation (IVM) with surgical retrieved testicular sperm in a normo-ovulatory female. Human chorionic gonadotropin-primed IVM, testicular biopsy for sperm retrieval and intracytoplasmic sperm injection with fresh sperm were performed. Fourteen cumulus-oocyte complexes were obtained in germinal vesicle or metaphase I stage, eight oocytes reached metaphase II, seven presumptive zygotes were obtained, and three cleavage stages embryos in day 2 were transferred producing a singleton pregnancy. A single healthy newborn was obtained. Our results suggest that IVM may be an alternative for in vitro fertilization in normo-ovulatory women even if surgical retrieval of sperm is needed. Further research is required to depict contributing factors to the success of IVM in indications different from polycystic ovaries syndrome and the role of male gamete. PMID:27803591
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.
Semra Kahraman MD
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
Kahraman, Semra; Ulker, Kahraman; Pirkevi Çetinkaya, Caroline; Kumtepe Colakoglu, Yesim; Yelke, Hakan K.; Yilanlioglu, Cihangir
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 of the procedure
This thesis focuses on interactively visualizing, and ultimately simulating, cumulus clouds both in virtual reality (VR) and with a standard desktop computer. The cumulus clouds in question are found in data sets generated by Large-Eddy Simulations (LES), which are used to simulate a small section
This thesis focuses on interactively visualizing, and ultimately simulating, cumulus clouds both in virtual reality (VR) and with a standard desktop computer. The cumulus clouds in question are found in data sets generated by Large-Eddy Simulations (LES), which are used to simulate a small section o
Kwon, Jeong-Woo; Jin, Yong-Xun; Park, Shun-Ha; Wang, Hai-Yang; Sun, Tian-Yi; Zhang, Jia-Bao; Kim, Nam-Hyung
In the present study, we investigated the potential role of glucose and pyruvate in the cytoplasmic maturation of porcine oocytes by investigating the effect of glucose and/or pyruvate supplementation, in the presence or absence of 10% porcine follicular fluid (PFF), on meiotic maturation and subsequent embryo development. In the absence of 10% PFF, without exogenous addition of glucose and pyruvate, the medium seemed unable to support maturation. In the presence of 10% PFF, the addition of 5.6 mM glucose and/or 2 mM pyruvate during in vitro maturation of cumulus enclosed oocytes increased MII oocyte and blastocyst rates. In contrast, oocytes denuded of cumulus cells were not able to take full advantage of the glucose in the medium, as only pyruvate was able to increase the MII rate and the subsequent early embryo developmental ability. Treatment of cumulus enclosed oocytes undergoing maturation with 200 μM dehydroepiandrosterone (DHEA), a pentose phosphate pathway inhibitor, or 2 μM iodoacetate (IA), a glycolysis inhibitor, significantly reduced GHS, intra-oocyte ATP, maternal gene expression, and MPF activity levels. DHEA was also able to increase ROS and reduce the levels of NADPH. Moreover, blastocysts of the DHEA- or IA-treated groups presented higher apoptosis rates and markedly lower cell proliferation cell rates than those of the non-treated group. In conclusion, our results suggest that oocytes maturing in the presence of 10% PFF can make full use of energy sources through glucose metabolism only when they are accompanied by cumulus cells, and that pentose phosphate pathway (PPP) and glycolysis promote porcine oocyte cytoplasmic maturation by supplying energy, regulating maternal gene expression, and controlling MPF activity. PMID:27997591
Zhang, Meijia; Xia, Guoliang
Mammalian oocytes grow and undergo meiosis within ovarian follicles. Fully grown oocytes are arrested at the first meiotic prophase by a mural granulosa origin "arrester" until a surge of luteinizing hormone (LH) from the pituitary at the mid-cycle stimulates the immature oocyte to resume meiosis. Recent evidence indicates that natriuretic peptide precursor type C (NPPC) produced by mural granulosa cells stimulates the generation of cyclic guanosine 3',5'-monophosphate (cGMP) by cumulus cell natriuretic peptide receptor 2 (NPR2), which diffuses into oocyte via gap junctions and inhibits oocyte phosphodiesterase 3A (PDE3A) activity and cyclic adenosine 3',5'-monophosphate (cAMP) hydrolysis and maintains meiotic arrest with a high intraoocyte cAMP level. This cAMP is generated through the activity of the Gs G-protein by the G-protein-coupled receptor, GPR3 and GPR12, and adenylyl cyclases (ADCY) endogenous to the oocyte. Further studies suggest that endocrine hormones, such as follicle-stimulating hormone (FSH), LH, 17β-estradiol (E2) and oocyte-derived paracrine factors (ODPFs), participate in oocyte meiosis possibly by the regulation of NPPC and/or NPR2. A detailed investigation of NPPC and NPR2 expression in follicle cells will elucidate the precise molecular mechanisms of gonadotropins, and control the arrest as well as resumption of meiosis.
Ola, Safiriyu Idowu; Sun, Qing-Yuan
The need for accurate selection of the best oocytes for in vitro fertilization protocols and thus, production of embryos has driven the search for oocyte quality markers from morphological criteria to biochemical parameters. Current studies are focused on the biochemical constituents of the follicular fluid and gene expression profiling of the cumulus cells. These parameters are, however, affected by factors that must be considered before making a judgment of the oocyte's quality. These includes factors such as the type of hormonal stimulation protocol, age of oocyte donor and heat stress on the donor, all of which have been reported to influence the concentrations of many hormones, apolipoproteins, metabolites, fatty acids and growth factors in the follicular fluid and the expression of several genes in the cumulus cells. Another important point to note is species variation in the response to these extraneous influences, which thus calls for species targeted investigations. As reports are still scanty and investigations assumed to be very keen, we employed this review paper to bring attention of researchers and clinicians to those factors that may come to bear on the outcome of their investigations on oocyte and embryo quality.
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
Khatir, H; Anouassi, A; Tibary, A
Parthenogenetic activation of the oocyte represents an important step in the somatic cloning. The aim of the present study was to evaluate the effectiveness (in term of in vitro development) of different methods of parthenogenetic activation of dromedary oocytes. Selected cumulus-oocytes-complexes (n=1264) collected by follicular aspiration from ovaries obtained postmortem were matured in vitro (IVM) for 30 h then divided randomly into seven groups and submitted to artificial activation. Two groups were preactivated with 25 microM of calcium ionophore (CaI) for 20 min then incubated for 4h with either 2mM 6-dimethylaminopurine (6-DMAP) (group 1, n=202) or with 10 microg/mL cycloheximide (CHX) (group 2, n=194). Group 3 (n=172) and group 4 (n=184), oocytes were pretreated with 5 microM ionomycin (Iono) for 5 min then incubated with either 2mM 6-DMAP or 10 microg/mL cycloheximide for 4h, respectively. Group 5 (n=161) and group 6 (n=155) oocytes were preactivated with electrical stimulation (ES) then activated with either 2mM 6-DMAP or 10 microg/mL cycloheximide for 4h, respectively. Group 7 (n=196) oocytes were submitted to in vitro fertilization (IVF) and served as a control. All groups containing oocytes were cultured in vitro following activation or IVF, at 38.5 degrees C under 5% CO(2) in air with >95% humidity. The in vitro development rates of dromedary oocytes exposed to 6-DMAP after CaI (61%), ES (74%) and the IVF group (71%) were similar and significantly greater (Pdromedary embryos was examined by transfer to synchronized recipients. An embryonic vesicle was seen by ultrasonography at 15 days post transfer in four females (CaI/6-DMAP: 1/5; 20%, IVF: 3/10; 30%). The only pseudopregnancy obtained with an activated embryo resorbed at 25 days. One of the females receiving the IVF produced embryos aborted at 2 months and the other two females carried to term and gave birth to healthy calves (one female and one male). This study shows that artificial activation of
Elham Mokhber Maleki
Full Text Available Background: Crocin is an active ingredient of saffron (Crocus sativus L. and its antioxidant properties have been previously investigated. This carotenoid scavenges free radicals and stimulates glutathione (GSH synthesis; consequently, it may protect cells against oxidative stress. The aim of this research is to protect oocytes from oxidative stress by the addition of a natural source antioxidant. Materials and Methods: In the present in vitro experimental study, we collected cumulus oocyte complexes (COCs from mouse ovaries of euthanized, 6-8 week-old female Naval Medical Research Institute (NMRI mice. Oocytes were subjected to in vitro maturation (IVM in the presence of either crocin (5 or 10 μg/ml, 5 mM buthionine-[S-R]- sulfoximine (BSO, or the combination of crocin plus BSO. Oocytes that matured in vitro in a medium without crocin or BSO supplements were considered as controls. Following 16-18 hours of IVM, matured oocytes (n=631 were fertilized by capacitated sperm from NMRI male mice, and cultured in vitro for up to 96 hours to assess preimplantation embryonic development. The levels of GSH in metaphase II (MII oocytes after IVM (n=240 were also assessed by the 5, 5-dithio-bis (2-nitrobenzoic acid (DTNB-GSH reductase recycling assay. Results: Supplementation of IVM media with 10 μg/ml crocin significantly (P<0.05 increased nuclear maturation, preimplantation development and GSH concentrations compared with the control group. Maturation of oocytes in IVM medium supplemented with BSO alone or the combination of 5 μg/ml crocin and BSO drastically decreased GSH concentrations and subsequently resulted in low rates of maturation, fertilization and blastocyst development. However, the combination of 10 μg/ml crocin with 5 mM BSO increased the level of nuclear maturation which was comparable to the control group. Conclusion: Supplementation of IVM media with crocin can improve nuclear maturation rates and subsequent developmental potential
Mokhber Maleki, Elham; Eimani, Hussein; Bigdeli, Mohammad Reza; Golkar Narenji, Afsane; Abedi, Reyhane
Background Crocin is an active ingredient of saffron (Crocus sativus L.) and its antioxidant properties have been previously investigated. This carotenoid scavenges free radicals and stimulates glutathione (GSH) synthesis; consequently, it may protect cells against oxidative stress. The aim of this research is to protect oocytes from oxidative stress by the addition of a natural source antioxidant. Materials and Methods In the present in vitro experimental study, we collected cumulus oocyte complexes (COCs) from mouse ovaries of euthanized, 6-8 week-old female Naval Medical Research Institute (NMRI) mice. Oocytes were subjected to in vitro maturation (IVM) in the presence of either crocin (5 or 10 μg/ml), 5 mM buthionine-[S-R]- sulfoximine (BSO), or the combination of crocin plus BSO. Oocytes that matured in vitro in a medium without crocin or BSO supplements were considered as controls. Following 16-18 hours of IVM, matured oocytes (n=631) were fertilized by capacitated sperm from NMRI male mice, and cultured in vitro for up to 96 hours to assess preimplantation embryonic development. The levels of GSH in metaphase II (MII) oocytes after IVM (n=240) were also assessed by the 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay. Results Supplementation of IVM media with 10 µg/ml crocin significantly (P<0.05) increased nuclear maturation, preimplantation development and GSH concentrations compared with the control group. Maturation of oocytes in IVM medium supplemented with BSO alone or the combination of 5 µg/ml crocin and BSO drastically decreased GSH concentrations and subsequently resulted in low rates of maturation, fertilization and blastocyst development. However, the combination of 10 µg/ml crocin with 5 mM BSO increased the level of nuclear maturation which was comparable to the control group. Conclusion Supplementation of IVM media with crocin can improve nuclear maturation rates and subsequent developmental potential of mouse
Sambasiva Rao, Brahmasani; Uma Mahesh, Yelisetti; Lakshmikantan, Uthanda Raman; Suman, Komjeti; Venu Charan, Katari; Shivaji, Sisinthy
The ability to rescue gametes from endangered or wildlife species and to subsequently produce viable embryos holds tremendous potential as a means to increase the population size of endangered or wildlife species. The objective of this study was to assess the meiotic and developmental competence of oocytes recovered from postmortem ovaries of the Indian blackbuck. Oocytes collected from the ovaries of dead blackbucks were allowed to mature in vitro and then tested for developmental potential by activation with ionomycin followed by treatment with 6-dimethylaminopurine. The average number of oocytes recovered per ovary was 10.9, and recovery of the oocytes did not depend on the presence or absence of the corpus luteum, on the side, size and weight of the ovaries or on the type of oocytes recovered. The proportion of good quality oocytes showing cumulus expansion and extrusion of the first polar body were 79.3% and 46.1% when cultured with gonadotropins. In vitro maturation studies indicated that the proportion of oocytes that reached MII stage was significantly higher when good quality oocytes (68%) were used compared with fair quality oocytes (48%) when cultured in the presence of gonadotropins. Furthermore, fifty eight percent of the in vitro matured oocytes cleaved, and thirteen percent of the cleaved oocytes developed into blastocysts. These findings suggest that the oocytes recovered from postmortem ovaries of the blackbuck can be utilized for production of embryos.
Zhu, Jiang; Lin, Fei-Hu; Zhang, Jie; Lin, Juan; Li, Hong; Li, You-Wei; Tan, Xiu-Wen; Tan, Jing-He
In spite of great efforts, the mechanisms for postovulatory oocyte aging are not fully understood. Although our previous work showed that the FasL/Fas signaling facilitated oocyte aging, the intra-oocyte signaling pathways are unknown. Furthermore, the mechanisms by which oxidative stress facilitates oocyte aging and the causal relationship between Ca2+ rises and caspase-3 activation and between the cell cycle and apoptosis during oocyte aging need detailed investigations. Our aim was to address these issues by studying the intra-oocyte signaling pathways for Fas/FasL to accelerate oocyte aging. The results indicated that sFasL released by cumulus cells activated Fas on the oocyte by increasing reactive oxygen species via activating NADPH oxidase. The activated Fas triggered Ca2+ release from the endoplasmic reticulum by activating phospholipase C-γ pathway and cytochrome c pathway. The cytoplasmic Ca2+ rises activated calcium/calmodulin-dependent protein kinase II (CaMKII) and caspase-3. While activated CaMKII increased oocyte susceptibility to activation by inactivating maturation-promoting factor (MPF) through cyclin B degradation, the activated caspase-3 facilitated further Ca2+releasing that activates more caspase-3 leading to oocyte fragmentation. Furthermore, caspase-3 activation and fragmentation were prevented in oocytes with a high MPF activity, suggesting that an oocyte must be in interphase to undergo apoptosis.
Full Text Available UCP2 plays a physiological role by regulating mitochondrial biogenesis, maintaining energy balance, ROS elimination, and regulating cellular autophagy in numerous tissues. But the exact roles of UCP2 in cumulus cells are still not clear. Genipin, a special UCP2 inhibitor, was added into the cultural medium to explore the roles of UCP2 in human cumulus cells. There were no significant differences in ATP and mitochondrial membrane potential levels in cumulus cells from UCP2 inhibiting groups as compared with the control. The levels of ROS and Mn-SOD were markedly elevated after UCP2 inhibited Genipin. However, the ratio of reduced GSH to GSSG significantly declined after treatment with Genipin. UCP2 inhibition by Genipin also resulted in obvious increase in the active caspase-3, which accompanied the decline of caspase-3 mRNA. The level of progesterone in culture medium declined obviously after Genipin treatment. But there was no significant difference in estradiol concentrations. This study indicated that UCP2 is expressed in human cumulus cells and plays important roles on mediate ROS production, apoptotic process, and steroidogenesis, suggesting UCP2 may be involved in regulation of follicle development and oocyte maturation and quality.
Khatir, H; Anouassi, A; Tibary, A
The effect of source of cumulus-oocytes-complexes (COCs), maturation and fertilization conditions on developmental competence of dromedary embryos was examined. Thirty-six adult females were superovulated with equine Chorionic Gonadotropin (eCG) injection (3500 IU, IM) and divided in three groups of 12 females each. Group 1 provided 138 COC's collected from follicles >or= 5 mm 10 days after stimulation prior hCG treatment and matured in vitro for 30 h. Group 2 provided 120 in vivo matured oocytes which were aspirated from their follicles 20 h after hCG (3000 IU, IV) given on day 10 follow eCG injection. Group 3 provided 65 in vivo matured/fertilized oocytes. Females in Group 3 received hCG on day 10 following eCG treatment and then were mated 24 h later. Fertilized oocytes were collected from the oviducts of females 48-h post-mating. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of COCs. All cultures were performed in three replicates (n = 3) at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. Nuclear maturation rate for Groups 1 and 2 was determined by epifluorescence microscopy in a sample of COCs (n = 30) denuded, fixed and stained with Hoechst 33342. To study the viability of obtained embryos, hatched blastocysts from each group were transferred to recipients followed by pregnancy diagnosis using ultrasonography at 15, 60 and 90 days. The percentage of COCs reaching metaphase II (MII) after 30 h of maturation was slightly but not significantly higher for in vivo matured oocytes (28/30; 93%) than those in vitro matured (25/30; 84%). The total rate of cleavage (2 cells to blastocyst stage) was not different for the three groups. However, significantly (p dromedary embryos may be directly related to the intrinsic quality (cytoplasmic maturation) of oocytes.
Kalo, Dorit; Hadas, Ron; Furman, Ori; Ben-Ari, Julius; Maor, Yehoshua; Patterson, Donald G; Tomey, Cynthia; Roth, Zvi
We examined acute exposure of Holstein cows to di(2-ethylhexyl) phthalate (DEHP) and its carryover effects on ovarian function and oocyte developmental competence. Synchronized cows were tube-fed with water or 100 mg/kg DEHP per day for 3 days. Blood, urine and milk samples were collected before, during and after DEHP exposure to examine its clearance pattern. Ovarian follicular dynamics was monitored through an entire estrous cycle by ultrasonographic scanning. Follicular fluids were aspirated from the preovulatory follicles on days 0 and 29 of the experiment and analyzed for phthalate metabolites and estradiol concentration. The aspirated follicular fluid was used as maturation medium for in-vitro embryo production. Findings revealed that DEHP impairs the pattern of follicular development, with a prominent effect on dominant follicles. The diameter and growth rate of the first- and second-wave dominant follicles were lower (P 25 mm). The pattern of growth and regression of the corpus luteum differed between groups, with a lower volume in the DEHP-treated group (P < 0.05). The follicular fluid aspirated from the DEHP-treated group, but not the controls, contained 23 nM mono(2-ethylhexyl) phthalate. Culturing of cumulus oocyte complexes in the follicular fluid aspirated from DEHP-treated cows reduced the proportion of oocytes progressing to the MII stage, and the proportions of 2- to 4-cell-stage embryos (P < 0.04) and 7-day blastocysts (P < 0.06). The results describe the risk associated with acute exposure to DEHP and its deleterious carryover effects on ovarian function, nuclear maturation and oocyte developmental competence.
Kalo, Dorit; Hadas, Ron; Furman, Ori; Ben-Ari, Julius; Maor, Yehoshua; Patterson, Donald G.; Tomey, Cynthia; Roth, Zvi
We examined acute exposure of Holstein cows to di(2-ethylhexyl) phthalate (DEHP) and its carryover effects on ovarian function and oocyte developmental competence. Synchronized cows were tube-fed with water or 100 mg/kg DEHP per day for 3 days. Blood, urine and milk samples were collected before, during and after DEHP exposure to examine its clearance pattern. Ovarian follicular dynamics was monitored through an entire estrous cycle by ultrasonographic scanning. Follicular fluids were aspirated from the preovulatory follicles on days 0 and 29 of the experiment and analyzed for phthalate metabolites and estradiol concentration. The aspirated follicular fluid was used as maturation medium for in-vitro embryo production. Findings revealed that DEHP impairs the pattern of follicular development, with a prominent effect on dominant follicles. The diameter and growth rate of the first- and second-wave dominant follicles were lower (P 25 mm). The pattern of growth and regression of the corpus luteum differed between groups, with a lower volume in the DEHP-treated group (P < 0.05). The follicular fluid aspirated from the DEHP-treated group, but not the controls, contained 23 nM mono(2-ethylhexyl) phthalate. Culturing of cumulus oocyte complexes in the follicular fluid aspirated from DEHP-treated cows reduced the proportion of oocytes progressing to the MII stage, and the proportions of 2- to 4-cell-stage embryos (P < 0.04) and 7-day blastocysts (P < 0.06). The results describe the risk associated with acute exposure to DEHP and its deleterious carryover effects on ovarian function, nuclear maturation and oocyte developmental competence. PMID:26154164
Full Text Available We examined acute exposure of Holstein cows to di(2-ethylhexyl phthalate (DEHP and its carryover effects on ovarian function and oocyte developmental competence. Synchronized cows were tube-fed with water or 100 mg/kg DEHP per day for 3 days. Blood, urine and milk samples were collected before, during and after DEHP exposure to examine its clearance pattern. Ovarian follicular dynamics was monitored through an entire estrous cycle by ultrasonographic scanning. Follicular fluids were aspirated from the preovulatory follicles on days 0 and 29 of the experiment and analyzed for phthalate metabolites and estradiol concentration. The aspirated follicular fluid was used as maturation medium for in-vitro embryo production. Findings revealed that DEHP impairs the pattern of follicular development, with a prominent effect on dominant follicles. The diameter and growth rate of the first- and second-wave dominant follicles were lower (P 25 mm. The pattern of growth and regression of the corpus luteum differed between groups, with a lower volume in the DEHP-treated group (P < 0.05. The follicular fluid aspirated from the DEHP-treated group, but not the controls, contained 23 nM mono(2-ethylhexyl phthalate. Culturing of cumulus oocyte complexes in the follicular fluid aspirated from DEHP-treated cows reduced the proportion of oocytes progressing to the MII stage, and the proportions of 2- to 4-cell-stage embryos (P < 0.04 and 7-day blastocysts (P < 0.06. The results describe the risk associated with acute exposure to DEHP and its deleterious carryover effects on ovarian function, nuclear maturation and oocyte developmental competence.
Abe, Yasuyuki; Hara, Kenshiro; Matsumoto, Hiromichi; Kobayashi, Jin; Sasada, Hiroshi; Ekwall, Hans; Rodriguez-Martinez, Heriberto; Sato, Eimei
To improve the feasibility of nylon-mesh holder for vitrification of bovine cumulus-oocytes complexes (GV-COCs) having germinal vesicle, this study was conducted to demonstrate effects of sugars and protocol of exposure in vitrification on subsequent in vitro maturation, ultrastructural changes, and in vitro development in bovine immature oocytes after cryopreservation using nylon mesh. Before vitrification, GV-COCs were exposed to the cryoprotectant, which was composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose (EFS40) or 0.3 M trehalose (EFT40), either by single step or in a stepwise way. The maturation rates in the stepwise exposure with EFS40 or EFT40 were significantly higher (P < 0.05) compared with the corresponding rates in the single step. In the stepwise exposure, few abnormalities were observed compared with the single-step exposure, where most oocytes showed a highly vacuolated cytoplasm with many ruptured mitochondria. Cleavage rates in fertilized oocytes previously exposed stepwise to EFS40 or EFT40 were significantly higher than those exposed by the single-step procedure. The cleaved embryos derived from the stepwise exposure to EFS40 developed to blastocysts. After transfer of blastocysts derived from vitrified GV oocytes, a female calf was born. These results indicate that vitrification of large numbers of bovine GV-COCs using a nylon-mesh holder accompanied with stepwise exposure minimizes structural damage in organelles, resulting in yield of viable blastocysts following in vitro embryo production.
George W. Smith
Full Text Available Development of improved procedures for in vitro maturation of oocytes collected from prepubertal goats has applications for in vitro embryo production and accompanying strategies for genetic improvement. The objective of described studies was to determine the effects of oocyte grade, in vitro maturation time, antioxidant supplementation and concentrations of estradiol in the maturation medium on in vitro maturation of oocytes harvested from 1-6 mm follicles present on the ovaries (obtained from an abattoir of 1-6 month-old prepubertal Boer goats. Rates of progression to metaphase II were greater for grade 1 oocytes (>3 compact layers of cumulus cells and evenly granulated cytoplasm than grade 2 oocytes (in vitro maturation in the presence of high concentrations of estradiol (10 and 100 mg/mL on progression to metaphase II was observed, and no effect was observed in response to 1 mg/mL estradiol treatment as compared with control. Results suggest that oocyte selection and beta-mercaptoethanol supplementation can positively influence progression to metaphase II of oocytes harvested from ovaries of prepubertal goats, whereas high concentrations of estradiol are inhibitory to in vitro maturation.
Dadashpour Davachi, Navid; Kohram, Hamid; Zare Shahneh, Ahmad; Zhandi, Mahdi; Goudarzi, Abbas; Fallahi, Roozbeh; Masoudi, Reza; Yousefi, Ali Reza; Bartlewski, Pawel M
The acquisition of fertilization ability by oocytes is one of the prerequisites for successful in vitro embryo production. In the present study, we examined the influence of conspecific ampulla oviductal epithelial cells incubated with cumulus-oocyte complexes (COCs) throughout the IVM phase on the developmental competence and maturation-promoting factor (MPF) activity of sheep oocytes. There were six experimental groups in this study, namely four groups with and two groups without oviductal epithelial cells added to IVM media: adult COCs matured in vitro with the ampulla oviductal epithelial cells obtained from adult (AAE; G1) or prepubertal donors (prepubertal sheep ampulla oviductal epithelial cells [PAE]; G4), COCs obtained from prepubertal animals cocultured with AAE (G2) or PAE (G3), and adult (C1) and prepubertal sheep COCs (C2) matured without oviductal epithelial cells. Coincubation of oocytes retrieved from both adult and sexually immature donors with AAE (G1 and G2) resulted in significantly improved rates of metaphase-II (M-II) attainment (G1: 85.1 ± 2.0 and G2: 40.2 ± 1.3) and blastocyst formation (G1: 42.2 ± 1.1 and G2: 21.2 ± 1.0) as well as blastocyst development (total cell count; G1: 130.3 ± 7.8, G2: 70.2 ± 3.5) compared with their respective controls (C1: 94.3 ± 4.1 and C2: 49.7 ± 2.0). Prior to IVM, the activity of MPF was greater (P vitro embryo production efficiency. A significant increase in MPF activity following IVM of G2 oocytes could be responsible, at least partly, for the improved rate of blastocyst formation after IVF of prepubertal sheep oocytes. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.
Hashem, N; Secher, J O; Pryor, J H; Long, C R; Looney, C R; Avery, B; Hyttel, P; Stroebech, L
Laboratory ware for the in vitro-produced embryos is generally made from embryo-tested plastic instead of glass. The quality of the plastic is crucial for the outcome because plastic is often toxic to gametes (Nijs et al. 2009 Fertil. Steril. 92, 527-535). In addition, gas molecules permeate through the plastic at a rate that depends on a variety of factors, such as diffusion coefficient and thickness of the plastic. In an incubator with appropriate concentration of CO2 and vented culture vessels, the gas permeability of the plastic is not important. When oocytes are transported outside a controlled atmosphere, gas permeability, toxicity, and oocyte cumulus cell CO2 metabolism could perturb the outcome. Medium containing bicarbonate buffer increases pH outside of a controlled atmosphere within minutes, whereas medium buffered with HEPES maintains suitable pH for hours. Previously, we tested that gas permeability differs among plastic vials and glass vials with no cumulus-oocyte complexes (COC) by measuring pH after 2, 5, and 24h at the same temperature. The objective of this study was to compare pH post-maturation, blastocyst development rates on Day 8 post-IVF (Day 0=IVF) between 2 different 1.2-mL polypropylene cryovials (A: VWR DK, 479-1219; B: Sigma-Aldrich, St. Louis, MO, USA, CLS430289), glass vial (VWR DK, NSCAC4015-96), and 4-well plate (4WP) as control (Thermo Fisher Scientific, Waltham, MA, USA, 144444). A total of 1135 abattoir-derived COC in Exp. 1 and 133 in Exp. 2 were divided equally between the treatments (20-25 COC per vessel). Vials/4WP contained 0.8/0.5mL of BO-IVM HEPES, a HEPES/bicarbonate medium (IVF Bioscience; BO-HEPES-IVM, UK). Maturation lasted 22 to 24h at 38.8°C in an incubator with either a humidified atmosphere of 5.5% CO2 in air (Exp. 1) or with no CO2 contact (Exp. 2). In Exp. 1, oocyte vials were matured without a vial lid while in Exp. 2 vial lids were closed. Statistical analysis was performed with chi-square and mean±SD. In Exp
Lee, Eunsong; Song, Kilyoung
The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts.
Solé, M; Santaló, J; Boada, M; Clua, E; Rodríguez, I; Martínez, F; Coroleu, B; Barri, P N; Veiga, A
How does vitrification affect oocyte viability? Vitrification does not affect oocyte viability in oocyte donation cycles. Oocyte vitrification is performed routinely and successfully in IVF and oocyte donation programs. This is a prospective study performed between June 2009 and February 2012 to compare ongoing pregnancy rates and other indices of viability between fresh and vitrified oocytes. A total of 99 donations with more than 16 oocytes (MII) in which oocytes were allocated both to a synchronous recipient (fresh oocytes) and to an asynchronous recipient (vitrified oocytes) were included. The participants were consenting couples (donors and recipients) from the oocyte donation program. On the day of retrieval, the oocytes allocated to the synchronous recipient were inseminated and those allocated for banking were denuded of cumulus and vitrified. Vitrified oocytes were microinjected with spermatozoa 2 h after warming. Embryo transfer was performed on Day 2 of development in both groups, and the remaining embryos were cryopreserved on Day 3. Clinical pregnancy was defined by a positive fetal heartbeat at 6 weeks. A total of 989 oocytes were warmed and 85.6% survived. No significant differences were observed between fresh and vitrified oocytes: fertilization rate (80.7 versus 78.2%), ongoing embryo rate (71.0 versus 68.2%) or good-quality embryo rate (54.1 versus 49.8%). The mean number of embryos transferred was similar in both groups (1.82 ± 0.44 versus 1.90 ± 0.34). The implantation rate (33.3 versus 34.0%) and the multiple pregnancy rate (27.7 versus 20.8) were also similar between both groups (P > 0.05). The live birth rate per cycle was 38.4% in the recipients of fresh oocytes and 43.4% in the recipients of vitrified oocytes (P > 0.05). Eighty five frozen embryo transfers were also evaluated. Comparing embryos from fresh and vitrified oocytes there were no significant differences in the embryo survival rate (70.1 versus 65.8%), clinical pregnancy rate
AN; Xiaorong(安晓荣); GOU; Kemian(苟克勉); ZHU; Shien(朱士恩); GUAN; Hong(关宏); HOU; Jian(侯健); LIN; Aixing(林爱星); ZENG; Shenming(曾申明); TIAN; Jianhui(田见辉); CHEN; Yongfu(陈永福)
Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2-3 h, was significantly lower than that of the 3-6 h groups (31.0%), while not significantly different among 3-4 h (P < 0.05), 4-5 h, and 5-6 h groups (P ≥ 0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.
Gutnisky, Cynthia; Dalvit, Gabriel C; Thompson, Jeremy G; Cetica, Pablo D
The relationship between pentose phosphate pathway (PPP) activity in cumulus-oocyte complexes (COCs) and oxidative and mitochondrial activity in bovine oocytes was evaluated with the aim of analysing the impact of two inhibitors (NADPH and 6-aminonicotinamide (6-AN)) and a stimulator (NADP) of the key enzymes of the PPP on the maturation rate, oxidative and mitochondrial activity and the mitochondrial distribution in oocytes. The proportion of COCs with measurable PPP activity (assessed using brilliant cresyl blue staining), glucose uptake, lactate production and meiotic maturation rate diminished when 6-AN (0.1, 1, 5 and 10mM for 22h) was added to the maturation medium (P<0.05). The addition of NADPH did not modify glucose uptake or lactate production, but reduced PPP activity in COCs and meiotic maturation rates (P<0.05). The presence of NADP (0.0125, 0.125, 1.25 and 12.5mM for 22h of culture) in the maturation medium had no effect on PPP activity in COCs, glucose uptake, lactate production and meiotic maturation rate. However, in the absence of gonadotropin supplementation, NADP stimulated both glucose uptake and lactate production at 12.5mM (the highest concentration tested; P<0.05). NADP did not modify cleavage rate, but decreased blastocyst production (P<0.05). During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22h maturation, which was also related to progressive mitochondrial migration. Inhibiting the PPP with 6-AN or NADPH led to reduced oxidative and mitochondrial activity compared with the respective control groups and inhibition of mitochondrial migration (P<0.05). Stimulation of the PPP with NADP increased oxidative and mitochondrial activity at 9h maturation (P<0.05) and delayed mitochondrial migration. The present study shows the significance of altering PPP activity during bovine oocyte IVM, revealing that there is a link between the activity of the PPP and the oxidative status of the oocyte.
李志凌; 林虹; 肖婉芬; 王玉莲
The significance of the performance of conventional in vitro fertilization and intracytoplasmic sperm injection (IVF/ICSI) using sibling oocytes from couples with subfertile male or unexplained infertility was evaluated. A total of 410 sibling oocyte cumulus-corona complexes (OCCC)from 21 couples with subfertile male (group A) and 11 unexplained infertile couples (group B) were randomly divided, in order of retrieval, into two groups inseminated either by conventional IVF or by ICSI. The treatment outcomes and the influence of infertility factors on fertilization in each group were compared. The results showed that although the two pronuclear (2PN) fertilization rate per injected sibling oocytes was significantly higher after ICSI (group A: 68.2 % ±28.8 %; group B: 66.2 %±24.9 %) than after conventional IVF (group A: 41.8 %±32.7 %; group B: 40. 1 %±22.1 %), the other variables studied included: the fertilization rates of per allocated sibling oocytes IVF/ICSI, the fertilization rates of sibling oocytes IVF/ICSI after excluding failed IVF fertilization cycles, as well as the cleavage rates of normal fertilization were not statistically significant (P＞0.05). Similarly, though the total fertilization failure rate in the IVF group (group A: 42.9 %;group B: 36.4 %) was significantly higher than in the ICSI group (group A: 4.8 %; group B: 0),we did not cancel cycles due to the normal fertilization of sibling oocytes. Embryo transfer was possible in all 32 couples. There were 10 clinical pregnancies in the two groups. We also discovered a possible association between some semen parameters and sperm functions of group A, and women age and duration of infertility of group B and fertilization. It is suggested that adoption of the split IVF/ICSI technology in the above cases may help eliminate fertilization failures. This is also a useful method to investigate the effect of single factor on the employment of assisted reproductive tech nology.
Khatir, H; Anouassi, A; Tibary, A
The aim of this work was to determine the effect of follicle size on camel oocyte quality as measured by developmental competence in vitro and in vivo. Ovaries from a local slaughterhouse were dissected to obtain two classes of follicle size: small (3-6 mm) and large (>6 mm) follicles. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of cumulus oocyte complexes (COCs). All cultures were done in four replicates at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. The COCs were matured for 28 h in TCM-199 medium supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/mL EGF, and 250 microM cysteamine. Nuclear maturation rate for each class of follicle size was determined by contrast phase microscopy in a sample of COCs (n=30) denuded, fixed and stained with aceto-orcein. In vitro fertilization was performed using fresh semen (0.5 x 10(6)spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 5% O(2) and 90% N(2). The percentage of COCs reaching metaphase II (MII) after 28 h of maturation was 87% (26/30) and 73% (22/30) for oocytes originating from large and small follicles, respectively (P>0.1). The rate of total cleavage (two cells to blastocyst stage) was greater (P6 mm) was assessed by transfer to synchronized recipients. None of the hatched blastocysts from small follicles resulted in a pregnancy whereas 68% (15/22) of the transferred hatched embryos from large follicles developed into a 25-day pregnancy. Of the resulting 15 pregnancies, 53% (n=8) aborted (five between 2 and 4 months and three between 5 and 7 months of pregnancy). The remaining seven pregnant females gave birth to normal healthy offsprings (four females and three males). The present study shows that dromedary oocytes developmental competence is acquired late during the final phase of follicular
Full Text Available Objective: This study aimed to evaluate the effect of seasonal variability on the assisted reproductive technique (ART success rate.Materials and methods: This study was a descriptive – analytical survey performed on 91 infertile women undergoing intracytoplasmic sperm injection – embryo transfer (ICSI-ET in different seasons. The patients aged less than 35 years old and had normal LH/FSH ratio. All patients entered long protocol down regulation treatment cycle and the picked up oocytes were transferred to GIII medium in the infertility laboratory. The cumulus characteristics, oocyte parameters including number of the retrieved oocytes, morphological characteristics, fertilization and degeneration rate and number of cleaved embryos were recorded. Data were analyzed by SPSS software. Results: The number of embryos was significantly higher in autumn. Abnormal morphological parameters (color, size, zona thickness and the degeneration rate were significantly higher in spring. The number of retrieved oocytes, MI, MII oocytes and fertilization rate had no significant seasonal variations.Conclusion: The results of this study showed a significant seasonal variation in morphological parameters of the oocytes, degeneration rate and the number of formed embryos.
Xie, Juan-Ke; Wang, Qian; Zhang, Ting-Ting; Yin, Shen; Zhang, Cui-Lian; Ge, Zhao-Jia
Controlled ovarian stimulation by exogenous gonadotrophins is a key procedure during the in vitro fertilization cycle to obtain a sufficient number of oocytes in humans. Previous studies demonstrated that repeated superovulation had deleterious effects on the ovaries. However, whether repeated superovulation adversely affects the mitochondrial functions of cumulus cells remains unclear. In this study, mice were divided into three groups: superovulation once (R1); superovulation three times (R3), and superovulation five times (R5). We evaluated the effects of repeated superovulation on mitochondrial DNA copies (mtDNA) and observed decreased mtDNA copies per cell with increasing number of superovulation cycles. Further, we investigated the DNA methylation status in exon 2 and the mRNA expression level of nuclear-encoded DNA polymerase gamma A (PolgA). The results showed that the DNA methylation levels of PolgA in R1 and R5 were slightly lower than in R3. Additionally, the altered DNA methylation in PolgA coincided with the changes in PolgA expression in cumulus cells. We also found that the mRNA expression of COX1, CYTB, ND2, and ND4 was altered by repeated superovulation in cumulus cells. Thus, repeated superovulation had adverse effects on mitochondrial function.
Wani, Na; Wernery, U
The present experiment was aimed to compare the effect of different protein supplementation sources, foetal calf serum (FCS), oestrous dromedary serum (EDS) and BSA, in experiment 1, and the effect of different concentrations of epidermal growth factor (EGF), in experiment 2, on in vitro nuclear maturation of the dromedary oocytes. Cumulus oocyte complexes (COCs) were harvested from the ovaries collected from a local slaughterhouse by aspirating the visible follicles in PBS supplemented with 5% FCS. Pooled COCs were randomly distributed to 4-well culture plates containing 500 μl of the maturation medium and cultured at 38.5 °C in an atmosphere of 5% CO(2) in air for 32-36 h. The basic maturation medium consisted of TCM-199 supplemented with 0.1 mg/ml L-glutamine, 0.8 mg/ml sodium bicarbonate, 0.25 mg/ml pyruvate, 50 μg/ml gentamicin, 10 μg/ml bFSH, 10 μg/ml bLH and 1 μg/ml estradiol. In experiment 1, this medium was supplemented with 10% FCS, 10% EDS or 0.4% BSA, whereas in experiment 2, it was supplemented with 0.4% BSA and 0, 10, 20 or 50 ng/ml of EGF. The oocytes were fixed, stained with 1% aceto-orcein stain and their nuclear status was evaluated. Oocytes were classified as germinal vesicle, diakinesis, metaphase-I, anaphase-I (A-I), metaphase-II (M-II) and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. There was no difference (p > 0.05) observed in the proportion of oocytes reaching M-II stage between the media supplemented with FCS (71.5 ± 4.8), EDS (72.8 ± 2.9) and BSA (72.7 ± 6.2). In experiment 2, a higher proportion (p dromedary camel oocytes can be supplemented with any of the three protein sources, i.e. FCS, EDS and BSA without any significant differences on the maturation rates. Also, a supplementation of 20 ng/ml of EGF in the maturation medium seems to be optimal and improves the nuclear maturation of dromedary camel oocytes.
Objective To assess the role of mitochondria in the early embryonic development of ageing mice.Methods Mitochondria isolated from cumulus granular cells of aged mice were microinjected into oocytes or zygotes of aged mice. In the setting of oocyte injection, mitochondria were transferred via intracytoplasmic sperm injection (ICSI+MIT), and ICSI without mitochondrial transfer. In the setting of zygote injection, mitochondria were directly microinjected into fertilized oocytes (MIT), and those injected with buffer alone (mock injection) or not injected (uninjected) served as controls.Results Although the rates of oocyte cleavage between ICSI and ICSI+MIT groups were not statistically different (P>0.05), the rate of blastocyst in the ICSI+MIT group was significantly higher than that in ICSI group (P<0.05). Although both the cleavage and blastocyst rates of mock injection group were significantly lower than those of uninjected group (P<0.05), likely due to mechanical damages of the cells by microinjection, the decrease of these rates was prevented by mitochondrial transfer. After mitochondrial transfer, the rates of both cleavage and blastocyst were significantly improved over the mock-injection group (P<0.05).Conclusion Mitochondrial transplantation can improve the developmental potential of early embryos of aged mice.
Co-culture of buffalo (Bubalus bubalis) preantral follicles with antral follicles: a comparative study of developmental competence of oocytes derived from in vivo developed and in vitro cultured antral follicles.
Sharma, G Taru; Dubey, Pawan K; Nath, Amar; Saikumar, G
The present study was undertaken to examine whether the presence of antral follicles (AFs) affects the survival, growth and steroidogenesis of preantral follicles (PFs) and compare the maturation and developmental competence of buffalo oocytes derived from in vivo developed and in vitro cultured AFs. Two experiments were carried out. In experiment I, PFs (200-250 μm) were isolated and cultured with or without AFs (3-5 mm) in TCM-199 medium that contained 10% fetal bovine serum (FBS), 1% insulin transferin selenium (ITS), 20 ng/ml epidermal growth factor (EGF), 0.5 μg/ml follicle-stimulating hormone (FSH) and 100 ng/ml insulin-like growth factor (IGF)-I. In experiment II, in vitro developmental competence was compared for the cumulus-oocyte complexes (COCs) recovered from in vivo developed and in vitro cultured AFs. Survival, growth, development of antrum, accumulation of estradiol and progesterone was (P cultured with AFs. Developmental competence of both types of follicular oocytes did not differ significantly in terms of maturation and cleavage rate, but morula and blastocyst production rate were (P vitro cultured antral follicular oocytes. In conclusion, co-culture of PFs with AFs supports long-term survival and growth of buffalo PFs and this co-culture system plays a dual role for in vitro production of embryos as well as understanding the relationship between developing PFs and AFs.
Cinar, Ozgur; Semiz, Olcay; Can, Alp
Although many countries banned of its usage, carbofuran (CF) is still one of the most commonly used carbamate derivative insecticides against insects and nematodes in agriculture and household, threatening the human and animal health by contaminating air, water, and food. Our goal was to evaluate the potential toxic effects of CF on mammalian oocytes besides mitotic cells. Caspase-dependent apoptotic pathway was assessed by immunofluorescence and western blot techniques. Alterations in the meiotic spindle formation after CF exposure throughout the in vitro maturation of mice oocyte-cumulus complexes (COCs) were analyzed by using a 3D confocal laser microscope. Maturation efficiency and kinetics were assessed by direct observation of the COCs. Results indicated that the number of TUNEL-positive cells increased in CF-exposed groups, particularly higher doses (>250 µM) in a dose-dependent fashion. The ratio of anticleaved caspase-3 labeled cells in those groups positively correlated with TUNEL-positivity. Western blot analysis confirmed a significant increase in active caspase-3 activity. CF caused a dose-dependent accumulation of oocytes at prometaphase-I (PM-I) of meiosis. Partial loss of spindle microtubules (MTs) was noted, which consequently gave rise to a diamond shape spindle. Aberrant pericentrin foci were noted particularly in PM-I and metaphase-I (M-I) stages. Conclusively, CF (1) induces programmed cell death in a dose-dependent manner, and (2) alters spindle morphology most likely through a mechanism that interacts with MT assembly and/or disorientation of pericentriolar proteins. Overall, data suggest that CF could give rise to aneuploidy or cell death in higher doses, therefore reduce fertilization and implantation rates. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: email@example.com.
Schoenfelder, Martin; Einspanier, Ralf
In response to the gonadotropin surge, the compact cumulus-oocyte complex (COC) undergoes expansion by synthesis of the mucopolysaccharide hyaluronan (HA) accompanying oocyte maturation. The objective of the present study was to quantify mRNA transcripts of the HA synthase (HAS) 1, HAS2, and HAS3 and the HA-receptors CD44 and RHAMM (receptor for HA-mediated motility). Additionally, we determined the histological localization of HA and its receptor, CD44, in maturing bovine COCs and cultured granulosa cells (GCs). Full-length transcript of bovine HAS2 and a part of the bovine RHAMM sequence has been made available. Real-time reverse transcriptase-polymerase chain reaction was used for individual mRNA expressions of bovine COCs in comparison to follicular GC gonadotropin treatment. Localization of CD44 and HA were done by immunohistochemistry and biotinylated HA-binding protein, respectively. Gonadotropins caused a rapid, 120-fold increase of HAS2 mRNA, whereas a delayed, 2-fold up-regulation of HAS3 mRNA was observed. The HAS1 transcripts were barely detected. Expression of CD44 mRNA greatly increased during in vitro maturation of COCs, indicating an important role when compared to an unchanged, steady-state RHAMM expression. As a consequence, HA was locally enriched after COC expansion, but only limited change was observed in the GCs. In cultured GCs, HAS2 expression was stimulated through FSH application, followed by the effective treatments of FSH+LH and LH. Treatment with LH induced the highest increase of the CD44 receptor, followed by FSH and FSH+LH treatments. These results suggest that HAS2 is mainly responsible for rapid HA synthesis in bovine COCs and GCs. In bovine COCs, the transcriptional up-regulation of both HAS2 and the receptor CD44 appear to be important prerequisites for initiating HA-mediated effects during final oocyte development and sperm-egg interaction.
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.
Sohel, Md Mahmodul Hasan; Hoelker, Michael; Noferesti, Sina Seifi; Salilew-Wondim, Dessie; Tholen, Ernst; Looft, Christian; Rings, Franca; Uddin, Muhammad Jasim; Spencer, Thomas E; Schellander, Karl; Tesfaye, Dawit
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.
Goudet, Ghylène; Douet, Cécile; Kaabouba-Escurier, Aurore; Couty, Isabelle; Moros-Nicolás, Carla; Barrière, Philippe; Blard, Thierry; Reigner, Fabrice; Deleuze, Stefan; Magistrini, Michèle
Most wild and domestic donkey breeds are currently endangered or threatened. Their preservation includes the creation of a Genome Resource Bank. Embryos cryopreservation allows the preservation of genetics from both male and female and is the fastest method to restore a breed. Because embryo production in vivo is limited in equids, our objective was to establish conditions for in vitro production of embryos in donkey using ovum pick up (OPU), IVM, IVF, and in vitro culture of zygotes. Donkey cumulus-oocyte complexes (COCs) were collected by transvaginal ultrasound-guided aspirations OPU in adult cyclic jennies and in vitro matured in tissue culture medium 199 supplemented with fetal calf serum and epidermal growth factor for 24, 30, 34, or 38 hours. They were preincubated with oviductal fluid for 30 minutes, coincubated with frozen-thawed donkey semen treated with procaine for 18 hours, and cultured for 30 hours in Dulbecco's Modified Eagle Medium-F12 supplemented with NaHCO3, fetal calf serum, and gentamycin. From the five OPU sessions, we collected 92 COCs in 193 follicles (48%) with an average of 4.2 COCs per jenny. All COCs were expanded after more than 24-hour IVM. At collection, jennies oocytes contained a germinal vesicle. Metaphase 1 oocytes were observed after 30-hour IVM and 44% were in metaphase 2 after 34-hour IVM. In our conditions, IVM of donkey oocytes was slower than IVM of equine oocytes and optimal duration for donkey oocytes IVM may be 34 hours. Only 15% of jennies oocytes contained two pronuclei after coincubation with donkey spermatozoa and none of them developed further after 48 hours post-IVF. Moreover, some parthenogenetic activation occurred. Thus, the treatment of donkey sperm with procaine may not be efficient for IVF. In conclusion, we established for the first time conditions for OPU in jennies with high recovery rates. We reported that IVM of jennies oocytes can produce 44% of metaphase 2 oocytes after 34 hours in culture
Liu, Yong; Wang, Huili; Lu, Jinhua; Miao, Yiliang; Cao, Xinyan; Zhang, Ling; Wu, Xiaoqing; Wu, Fengrui; Ding, Biao; Wang, Rong; Luo, Mingjiu; Li, Wenyong; Tan, Jinghe
Somatic cell nuclear transfer (SCNT) requires large numbers of matured oocytes. In vitro-matured (IVM) oocytes have been used in SCNT in many animals. We investigated the use of IVM oocytes in Rex rabbit SCNT using Rex rabbit ovaries obtained from a local abattoir. The meiotic ability of oocytes isolated from follicles of different diameters was studied. Rex rabbit SCNT was optimized for denucleation, activation, and donor cell synchronization. Rex rabbit oocytes grew to the largest diameter (110 μm) when the follicle diameter was 1.0 mm. Oocytes isolated from 0.7-mm follicles acquired maturation ability. More than 90% of these oocytes matured after IVC for 18 h. The developmental potential of oocytes isolated from >1-mm follicles was greater than that of oocytes isolated from 0.7- to 1.0-mm follicles. The highest activation rates for IVM Rex rabbit oocytes were seen after treatment with 2.5 μM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) and 5 μg/mL cycloheximide (CHX) for 1 h. Ionomycin induced the chromatin of IVM oocytes to protrude from the oocyte surface, promoting denucleation. Fetal fibroblast cells (FFCs) and cumulus cells (CCs) were more suitable for Rex rabbit SCNT than skin fibroblast cells (SFCs) (blastocyst rate was 35.6 ± 2.2% and 38.0 ± 6.0% vs. 19.7 ± 3.1%). The best fusion condition was a 2DC interval for 1 sec, 1.6 kV/cm voltages, and 40 μsec duration in 0.28 M mannitol. In conclusion, the in vitro maturation of Rex rabbit oocytes and SCNT procedures were studied systematically and optimized in this study.
A Dehghan Manshadi
Full Text Available Background & aim: In vitro maturation of oocytes is a promising technique for reducing the costs and complications of ovarian stimulation by gonadotropins. The aim of this study was to investigate the effects of combination of insulin-like growth factor-1 and antioxidant cysteamine and &beta-Mercaptoethanol on maturation and fertilization of immature oocytes. Methods: in this experimental study, following 48 hrs injection of 7.5 IU PMSG to immature female mice, the germinal vesicle oocytes from ovaries were removed and transferred to TCM199 culture medium containing 50 ng /ml insulin-like growth factor-1 and 100 &mumol Cysteamine and &beta -Mercaptoethanol. After 24 hrs of culture, the oocytes of MII in IVF were fertilized and embryonic development to the two cells was studied under an inverted microscope. Data analysis was performed by using ANOVA and Post hoc Tukey test. Results: The results showed that the rate of maturation, fertilization and 2-cell embryo formation in GV oocytes with cumulus cells in TCM199 medium containing insulin-like growth factor-1, Cysteamine and BME were 92.10, 93.30, 80.60% and in the GV oocytes without Cumulus cells were cultured in the same medium were 65.80, 64.00, 58.60% respectively which were statistically significant (P <0.001. Conclusion: In the present study, the simultaneous combination of insulin-like growth factor-1, &beta-Mercaptoethanol and CYS increased maturation, fertilization and developmental rate to 2-cells stage with cumulus cells more than the oocyte without cumulus cells to a greater extent. This represented the need of adding supplemental growth factors and antioxidants to the medium and is associated with cumulus cells.
Breininger, E; Vecchi Galenda, B E; Alvarez, G M; Gutnisky, C; Cetica, P D
Oocyte maturation depends on the metabolic activity of cumulus-oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2-oxoglutarate (5, 10 and 20 mm) or hydroxymalonate (30, 60 and 100 mm) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10(-5) and (2.54 ± 0.32) 10(-5) , and for MDH, the U were (4.72 ± 0.42) 10(-5) and (4.38 ± 0.25) 10(-5) for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10(-3) and (0.94 ± 0.12) 10(-3) , and for MDH (9.08 ± 0.93) 10(-3) and (1.89 ± 0.10) 10(-3) for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2-oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.
Full Text Available Myeloperoxidase (MPO, an abundant heme-containing enzyme present in neutrophils, monocytes, and macrophages, is produced in high levels during inflammation, and associated with poor reproductive outcomes. MPO is known to generate hypochlorous acid (HOCl, a damaging reactive oxygen species (ROS utilizing hydrogen peroxide (H2O2 and chloride (Cl-. Here we investigate the effect of activated immune cells and MPO on oocyte quality. Mouse metaphase II oocytes were divided into the following groups: 1 Incubation with a catalytic amount of MPO (40 nM for different incubation periods in the presence of 100 mM Cl- with and without H2O2 and with and without melatonin (100 μM, at 37°C (n = 648/648 total number of oocytes in each group for oocytes with and without cumulus cells; 2 Co-cultured with activated mouse peritoneal macrophage and neutrophils cells (1.0 x 106 cells/ml in the absence and presence of melatonin (200 μM, an MPO inhibitor/ROS scavenger, for different incubation periods in HTF media, at 37°C (n = 200/200; 3 Untreated oocytes incubated for 4 hrs as controls (n = 73/64. Oocytes were then fixed, stained and scored based on the microtubule morphology and chromosomal alignment. All treatments were found to negatively affect oocyte quality in a time dependent fashion as compared to controls. In all cases the presence of cumulus cells offered no protection; however significant protection was offered by melatonin. Similar results were obtained with oocytes treated with neutrophils. This work provides a direct link between MPO and decreased oocyte quality. Therefore, strategies to decrease MPO mediated inflammation may influence reproductive outcomes.
Donner, Leo J.
Cumulus convection plays a significant role in determining the structure of the terrestrial water vapor field. Cumulus convection acts directly on the moisture field by condensing and precipitating water vapor and by redistributing water vapor through cumulus induced eddy circulations. The mechanisms by which cumulus convection influences the terrestrial water vapor distribution is outlined. Calculations using a theory due to Kuo is used to illustrate the mechanisms by which cumulus convection works. Understanding of these processes greatly aids the ability of researchers to interpret the seasonal and spatial distribution of atmospheric water vapor by providing information on the nature of sources and sinks and the global circulation.
Lamer, K.; Kollias, P.; Ghate, V. P.; Luke, E. P.
Cumulus clouds play a critical role in modulating the radiative and hydrological budget of the lower troposphere. These clouds, which are ubiquitous in regions of large-scale subsidence over the oceans, tend to be misrepresented in global climate models. Island-based, long-term, high-resolution ground-based observations can provide valuable insights on the factors controlling their macroscopic and microphysical properties and subsequenlty assist in model evaluation and guidance. Previous studies, limited to fair-weather cumuli over land, revealed that their fractional coverage is only weakly correlated with several parameters; the best ones being complex dynamical characteristics of the subcloud layer (vertical velocity skewness and eddy coherence). Other studies noted a relationship between cumuli depth and their propensity to precipitate. The current study will expand on such analysis by performing detail characterization of the full spectrum of shallow cumulus fields from non-precipitating to precipitating in the context of the large-scale forcing (i.e. thermodynamic structure and subsidence rates). Two years of ground-based remote sensing observations collected at the Atmospheric Radiation Measurement (ARM) Eastern North Atlantic (ENA) site are used to document macroscopic (cloud depth, cord length, cover), microphysical (liquid water path, cloud base rain rate) and dynamical (cloud base mass flux, eddy dissipation rate) cumuli properties. The observed variability in shallow cumulus is examined in relation to the variability of the large-scale environment as captured by the humidity profile, the magnitude of the low-level horizontal winds and near-surface aerosol conditions.
Wigglesworth, Karen; Lee, Kyung-Bon; O’Brien, Marilyn J.; Peng, Jia; Matzuk, Martin M.; Eppig, John J.
Coordinated regulation of oocyte and ovarian follicular development is essential for fertility. In particular, the progression of meiosis, a germ cell-specific cell division that reduces the number of chromosomes from diploid to haploid, must be arrested until just before ovulation. Follicular somatic cells are well-known to impose this arrest, which is essential for oocyte–follicle developmental synchrony. Follicular somatic cells sustain meiotic arrest via the natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system, and possibly also via high levels of the purine hypoxanthine in the follicular fluid. Upon activation by the ligand NPPC, NPR2, the predominant guanylyl cyclase in follicular somatic cells, produces cyclic guanosine monophosphate (cGMP), which maintains meiotic arrest after transfer to the oocyte via gap junctions. Here we report that both the NPPC/NPR2 system and hypoxanthine require the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme required for the production of guanylyl metabolites and cGMP. Furthermore, oocyte-derived paracrine factors, particularly the growth differentiation factor 9–bone morphogenetic protein 15 heterodimer, promote expression of Impdh and Npr2 and elevate cGMP levels in cumulus cells. Thus, although the somatic compartment of ovarian follicles plays an essential role in the maintenance of oocyte meiotic arrest, as has been known for many years, this function of the somatic cells is surprisingly regulated by signals from the oocyte itself. PMID:23980176
Yan-Guang Wu; Yong Liu; Ping Zhou; Guo-Cheng Lan; Dong Han; De-Qiang Miao; Jing-He Tan
Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue (BCB) staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained (BCB+) and those unstained (BCB-) according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB+ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotro-pin stimulation on the competence of BCB+ oocytes were also analyzed. In the large- and medium-size groups, BCB+ oocytes were larger and showed more surrounded nucleoli (SN) chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity (determined as the intracellular GSH level and pattern of mitochondrial distribution) and higher developmental potential after in vitro maturation (IVM) than the BCB- oocytes. Adult mice produced more BCB+ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB+ oocytes. The BCB+ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB+ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.
Nogueira, D; Ron-El, R; Friedler, S; Schachter, M; Raziel, A; Cortvrindt, R; Smitz, J
Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6-12 mm) were retrieved 34-36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro matured (67% vs. 46%, P = 0.01). In controls, immature CEOs retrieved with moderate expansion reached higher maturation rates compared to fully compacted CEOs, but in PMC groups, high values of maturation were achieved for both morphological classes of CEOs. No effect of PMC on fertilization was observed. A 24-h PMC period proved to be the most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in vitro groups. In summary, PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs. This resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intraoocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.
Ko, Duck Sung; Lee, Sun-Hee; Park, Dong-Wook; Yang, Kwang Moon; Lim, Chun Kyu
The goal of this study was to evaluate the pregnancy potential of immature (metaphase I or germinal vesicle stage) oocytes retrieved in intracytoplasmic sperm injection (ICSI) cycles. A total of 1,871 couples with infertility underwent 2,984 ICSI cycles. Cycles in which three or fewer oocytes were retrieved were included in this study in order to evaluate the pregnancy potential of immature oocytes. Cycles were divided into five groups (group I-V), according to the maturation status of the oocytes at the time of cumulus cell removal and ICSI. The fertilization and pregnancy rates after ICSI were analyzed and compared among the study groups based on the maturation status of the retrieved oocytes. The retrieval of only immature oocytes was associated with a significant decrease in the fertilization rate (76.1%±37.3% vs. 49.0%±49.1%, 66.7%±48.7%; group I vs. group II, group III, respectively) and the average number of transferred embryos (1.5±0.7 vs. 1.1±0.4, 1.1±0.6). The cycle cancellation rate was significantly higher when only immature oocytes were retrieved. The clinical pregnancy rate decreased significantly when the transferred embryos had originated from immature oocytes (16.9% vs. 10.3%, 1.2%). In ICSI cycles, the fertilization potential and pregnancy potential of the immature oocytes retrieved in ICSI cycles were inferior to those of mature oocytes. Therefore, increasing the number of injectable oocytes and transferrable embryos by using immature oocytes after their spontaneous in vitro maturation does not necessarily improve pregnancy outcomes.
Zhang, Yue; Han, Jun; Zhu, Cheng-Cheng; Tang, Feng; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen
T-2 toxin is a main type A trichothecene mycotoxin which is the most toxic trichothecence. T-2 toxin has posed various toxic effects on human and animals in vigorous cell proliferation tissues like lymphoid, hematopoietic and gastrointestinal tissues, while HT-2 toxin is the major metabolite which is deacetylated by T-2 toxin. In this study, we focused on the toxic effects of HT-2 on porcine oocyte maturation. We treated the porcine oocyte with HT-2 toxin in vitro, and we first found that HT-2 treatment inhibited porcine oocyte polar body extrusion and cumulus cell expansion. We observed the disrupted meiotic spindle morphology after treatment, which might be due to the reduced p-MAPK protein level. Actin distribution was also disturbed, indicating that HT-2 affects cytoskeleton of porcine oocytes. We next explored the causes for the failure of oocyte maturation after HT-2 treatment. We found that HT-2 treated oocytes showed the increased ROS level, which indicated that oxidative stress had occurred. We also detected autophagy as well as early apoptosis in the treatment oocytes. Due to the fact that oxidative stress could induced apoptosis, our results indicated that HT-2 toxin caused oxidative stress induced apoptosis and autophagy, which further affected porcine oocyte maturation. PMID:27658477
Deng, Wei-Ping; Ren, Zhao-Rui
Oocyte development and maturation is a complicated process. The nuclear maturation and cytoplasmic maturation must synchronize which can ensure normal oocyte fertilization and following development. Mitochondrial is the most important cellular organell in cytoplasm, and the variation of its distribution during oocyte maturation, the capacity of OXPHOS generating ATP as well as the content or copy number or transcription level of mitochondrial DNA play an important role in oocyte development and maturation. Therefore, the studies on the variation of mitochondrial distribution, function and mitochondrial DNA could enhance our understanding of the physiology of reproduction and provide new insight to solve the difficulties of assisted reproduction as well as cloning embryo technology.
Full Text Available Abstract Background RUNX2 is a transcription factor, whose expression has been recently identified in the mouse ovary. Regulation of RUNX2 expression and its function in the human ovary have not been determined yet. The aim of the present study is the investigation of the possible correlation between RUNX2 gene expression in cumulus cells and controlled ovarian stimulation and pregnancy outcomes after ART treatment. Methods A total of 41 patients undergoing ICSI treatment for male factor infertility were enrolled into a specific ART program, during which cumulus cells were collected. The expression of RUNX2 gene in cumulus cells was examined by real-time PCR. Results Concerning RUNX2 gene expression, 12 out of 41 women were detected with RUNX2 expression, with ratios ranging from 0.84 to 1.00, while 28 out of 41 women had no expression (ratio = 0. Only 1 woman presented a weak RUNX2 gene expression (ratio = 0.52. From 8 women that proceeded to pregnancy, 7 of them did not express RUNX2 gene in cumulus cells, while one was the woman with weak gene expression that also achieved pregnancy. The group of women without RUNX2 expression presented higher number of follicles (p = 0.013, higher number of retrieved oocytes (p = 0.016, higher basal LH serum levels (p = 0.016 and higher peak estradiol levels (p = 0.013, while the number of fertilized oocytes differed marginally between the two groups (p = 0.089. Moreover, RUNX2 expression was negatively associated with LH levels (OR = 0.22, p = 0.021 and E2 levels (OR = 0.25, p = 0.026. Conclusions Consequently, based on the preliminary findings of the present pilot study a potential inhibitory mechanism of RUNX2 gene is observed in the ovary when high mRNA levels are detected, suggesting that RUNX2 could possibly be used as a candidate genetic marker in the monitoring of the outcome of an ART treatment.
Full Text Available The diffuse all-sky surface irradiances measured at two nearby wavelengths in the visible spectral range and their model clear-sky counterparts are two main components of a new method for estimating the fractional sky cover of different cloud types, including cumulus clouds. The performance of this method is illustrated using 1-min resolution data from ground-based Multi-Filter Rotating Shadowband Radiometer (MFRSR. The MFRSR data are collected at the US Department of Energy Atmospheric Radiation Measurement (ARM Climate Research Facility (ACRF Southern Great Plains (SGP site during the summer of 2007 and represent 13 days with cumulus clouds. Good agreement is obtained between estimated values of the fractional sky cover and those provided by a well-established independent method based on broadband observations.
LING Jian; LI Chongyin; JIA Xiaolong
Vertical cumulus momentum transport is an important physical process in the tropical atmosphere and plays a key role in the evolution of the tropical atmospheric system.This paper focuses on the impact of the vertical cumulus momentum transport on Madden-Julian Oscillation (MJO) simulation in two global climate models (GCMs).The Tiedtke cumulus parameterization scheme is applied to both GCMs [CAM2 and Spectral Atmospheric general circulation Model of LASG/IAP (SAMIL)].It is found that the MJO simulation ability might be influenced by the vertical cumulus momentum transport through the cumulus parameterization scheme.However,the use of vertical momentum transport in different models provides different results.In order to improve model's MJO simulation ability,we must introduce vertical cumulus momentum transport in a more reasonable way into models.Furthermore,the coherence of the parameterization and the underlying model also need to be considered.
Full Text Available Since the introduction of in vitro fertilization (IVF in clinical practice of infertility treatment, the indicators for high quality embryos were investigated. Cumulus cells (CC have a specific gene expression profile according to the developmental potential of the oocyte they are surrounding, and therefore, specific gene expression could be used as a biomarker. The aim of our study was to combine more than one biomarker to observe improvement in prediction value of embryo development. In this study, 58 CC samples from 17 IVF patients were analyzed. This study was approved by the Republic of Slovenia National Medical Ethics Committee. Gene expression analysis [quantitative real time polymerase chain reaction (qPCR] for five genes, analyzed according to embryo quality level, was performed. Two prediction models were tested for embryo quality prediction: a binary logistic and a decision tree model. As the main outcome, gene expression levels for five genes were taken and the area under the curve (AUC for two prediction models were calculated. Among tested genes, AMHR2 and LIF showed significant expression difference between high quality and low quality embryos. These two genes were used for the construction of two prediction models: the binary logistic model yielded an AUC of 0.72 ± 0.08 and the decision tree model yielded an AUC of 0.73 ± 0.03. Two different prediction models yielded similar predictive power to differentiate high and low quality embryos. In terms of eventual clinical decision making, the decision tree model resulted in easy-to-interpret rules that are highly applicable in clinical practice.
Susor, Andrej; Ellederova, Zdenka; Jelinkova, Lucie; Halada, Petr; Kavan, Daniel; Kubelka, Michal; Kovarova, Hana
In this study, we performed proteomic analysis of porcine oocytes during in vitro maturation. Comparison of oocytes at the initial and final stages of meiotic division characterized candidate proteins that were differentially synthesized during in vitro maturation. While the biosynthesis of many of these proteins was significantly decreased, we found four proteins with increased biosynthetic rate, which are supposed to play an essential role in meiosis. Among them, the ubiquitin C-terminal hydrolase-L1 (UCH-L1) was identified by mass spectrometry. To study the regulatory role of UCH-L1 in the process of meiosis in pig model, we used a specific inhibitor of this enzyme, marked C30, belonging to the class of isatin O-acyl oximes. When germinal vesicle (GV) stage cumulus-enclosed oocytes were treated with C30, GV breakdown was inhibited after 28 h of culture, and most of the oocytes were arrested at the first meiosis after 44 h. The block of metaphase I-anaphase transition was not completely reversible. In addition, the inhibition of UCH-L1 resulted in elevated histone H1 kinase activity, corresponding to cyclin-dependent kinase(CDK1)-cyclin B1 complex, and a low level of monoubiquitin. These results supported the hypothesis that UCH-L1 might play a role in metaphase I-anaphase transition by regulating ubiquitin-dependent proteasome mechanisms. In summary, a proteomic approach coupled with protein verification study revealed an essential role of UCH-L1 in the completion of the first meiosis and its transition to anaphase.
Hanna, Carol B.; Yao, Shan; Wu, Xuemei; Jensen, Jeffrey T.
Objective To identify a cGMP-specific phosphodiesterase (PDE) in non-human primate germinal vesicle (GV) oocytes and establish a proposed effect on oocyte maturation through preliminary experiments in mouse GV oocytes. Design Controlled non-human primate and rodent experiments. Setting Academic research institution. Animals Rhesus macaques and B6/129F1 mice. Interventions Rhesus macaques were stimulated with FSH to collect GV oocytes and cumulus for gene expression analysis. Female mice were stimulated with PMSG to collect GV oocytes. Main Outcome Measures PDE transcript expression in primate GV oocytes and cumulus cells. Fluorescence polarization (FP) measurements of PDE3A activity. Spontaneous resumption of meiosis in mouse GV oocytes. Results Five PDE transcripts were detected in Rhesus GV oocytes, only PDE9A was cGMP-specific. FP assays indicated cGMP has an inhibitory effect on PDE3A while the PDE9 inhibitor, BAY73-6691, did not. Similarly, BAY73-6691, had little effect on preventing spontaneous maturation in oocytes, but did augment the inhibitory effects of cGMP. Inclusion of 0µM (control), 10µM, 100µM, and 1 mM BAY73-6691 significantly increased the proportion of mouse oocytes maintaining GV arrest in the presence of the cGMP analog 8-Br-cGMP at: 100µM (8.8%, 11.4%, 18.8%, and 28%), 500µM (21.1%, 38.1%, 74.5%,and 66.5%), and 1 mM (57.8%, 74.5%, 93.9%, and 94.0%) respectively, when P<0.05. Conclusions PDE9 is a cGMP-specific hydrolyzing enzyme present in primate oocytes, and PDE9 antagonists augment the inhibitory effect of cGMP during spontaneous in vitro maturation of GV mouse oocytes. PMID:22704629
Full Text Available Introduction: Standard concentrations of antibiotics in culture media are thought to have no detectable toxic effects on the cultured cells. However, since antibiotics are biologically active substances, the possibility that they interfere to some extent with cellular processes occurring in the cultured cells can not always be totally excluded. This study, therefore, was conducted to assess whether the presence of penicllin-streptomycin (pen-strep during in vitro maturation (IVM of bovine cumulus oocyte complexes (COCs affect nuclear and cytoplasmic maturation and subsequent embryo development. Materials and Methods: Bovine COCs were matured at 39oC in a humidified atmosphere with 5 % CO2 in air for 24 h in: 1- culture medium M 199 supplemented with 10 % FCS (Fetal calf serum, 0.05 IU/ml rhFSH (recombinant human FSH and 100 units penicillin and 100 ?g streptomycin/ ml. 2- culture medium M 199 without FCS and rhFSH in the presence of pen-strep. Cultures without antibiotics served as control. Six series of experiments, each consisted of at least 3 replicates, were performed. Results: In vitro maturation in the presence of pen-strep in culture medium supplemented with FCS and rhFSH significantly (P<0.05 increased the percentage of MII oocytes, however, when the COCs were divided, on the basis of appearance of the cumulus investment, into bright and dark groups, this effect was less obvious in both types of COCs, 76% vs 72% in bright COCs (P= 0.149 or 83% vs 80% in dark COCs (P=0.296 in treated and control groups respectively. The percentage of oocytes with type III of cortical granules (CGs distribution was not affected in the presence of pen-strep. The COCs expansion after IVM was not affected by the presence of antibiotics in culture medium. The subsequent embryo development of IVM/IVF produced ova, which were exposed to pen-strep during IVM, was significantly (P<0.05 decreased with respect to blastocyst formation by day 9. In vitro maturation in
Guilherme Fazan Rossi
Full Text Available The objective of this study was to evaluate the effects of feeding primiparous Nellore cows supplements rich in protected or unprotected linoleic (n-6 and linolenic (n-3 acids before and after calving on the number of follicles, total number of cumulus-oocyte complexes (COCs collected by aspiration (OPU and oocytes suitable for culture (grades I, II and III, and in vitro embryo production (IVEP. The donor cows were randomly divided into three groups: control (n=7, Megalac-E® (n=8; 100 g/donor/day, and flaxseed (n=7; 1.0 kg/donor/day. The diets were offered at least 30 days precalving and 75 days postcalving. The animals were submitted to OPU on postcalving days 30, 45, 60 and 75. Recovered oocytes were selected and those considered suitable for culture were submitted to the IVEP procedures. The data were analyzed using a completely randomized design with repeated measures over time. There was no effect of supplementation on the number of follicles, the number of recovered COCs and those suitable for culture, or IVEP. However, the rate of oocytes suitable for culture recovered by OPU was higher on postcalving days 60 and 75 when compared to days 30 and 45. Additionally, the rate of embryos produced in vitro increased after postcalving day 45. In conclusion, pre- and postcalving supplementation with 100 g/day Megalac-E® or 1.0 kg/day flaxseed does not alter the number of oocytes obtained in vivo or IVEP rates of lactating primiparous Nellore donor cows. However, IVEP improves when the programs are carried out 45 days after calving.
Full Text Available Zuo Gui Wan (ZGW and You Gui Wan (YGW are two classic formulas used in clinical treatment of infertility in traditional Chinese medicine (TCM. However, the actions of the formulas remain to be proven at the cellular and molecular levels. In this study, we investigate whether the two formulas have any effect on germ cell formation and differentiation by culturing rat medicated serums containing YGW or ZGW with stem cells derived from human first trimester umbilical cord. Our results showed that while the normal rat serums had no significant effects, the rat medicated serums had significant effects on the differentiation of the stem cells into oocyte-like cells (OLCs based on (1 cell morphological changes that resembled purative cumulus-oocyte complexes (COCs; (2 expressions of specific markers that were indicative of germ cell formation and oocyte development; and (3 estradiol production by the COC-like cells. Furthermore, ZGW medicated serums exhibited more obvious effects on specific gene expressions of germ cells, whereas YGW medicated serums showed stronger effects on estradiol production. Accordingly, our study provides evidence demonstrating for the first time that one of molecular and cellular actions of YGW or ZGW in treating human reproductive dysfunctions may be through an enhancement of neooogenesis.
Hu, Xiang; Lu, Hua; Deng, Yan-Li; Wan, Qian; Yie, Shang-Mian
Zuo Gui Wan (ZGW) and You Gui Wan (YGW) are two classic formulas used in clinical treatment of infertility in traditional Chinese medicine (TCM). However, the actions of the formulas remain to be proven at the cellular and molecular levels. In this study, we investigate whether the two formulas have any effect on germ cell formation and differentiation by culturing rat medicated serums containing YGW or ZGW with stem cells derived from human first trimester umbilical cord. Our results showed that while the normal rat serums had no significant effects, the rat medicated serums had significant effects on the differentiation of the stem cells into oocyte-like cells (OLCs) based on (1) cell morphological changes that resembled purative cumulus-oocyte complexes (COCs); (2) expressions of specific markers that were indicative of germ cell formation and oocyte development; and (3) estradiol production by the COC-like cells. Furthermore, ZGW medicated serums exhibited more obvious effects on specific gene expressions of germ cells, whereas YGW medicated serums showed stronger effects on estradiol production. Accordingly, our study provides evidence demonstrating for the first time that one of molecular and cellular actions of YGW or ZGW in treating human reproductive dysfunctions may be through an enhancement of neooogenesis.
Barton, B R; Hertig, A T
Quiescent oocytes of the monkey Cebus albifrons were examined with the electron microscope. In many respects the ultrastructure of these cells was similar to that of other mammalian species. Elongate and oval mitochondria, lamellar Golgi complexes, small profiles of smooth endoplasmic reticulum, and vacuolar organelles were randomly distributed around a round nucleus which usually contained a nucleolus and clumps of heterochromatin. Among the unusual morphological characteristics of these oocytes are 'membranous aggregates', membrane-bound organelles containing a complex of convoluted membranes, some very dense rod-like structures and a droplet of moderate density which resembles lipid. A similar droplet is frequently found in mitochondria. Rough endoplasmic reticulum is abundant in many of these oocytes, forming parallel arrays and concentric rings around the nucleus. Folded membrane complexes, apparent elaborations of smooth endoplasmic reticulum, are frequently found in the cytoplasm in continuity with cisternae of smooth and rough endoplasmic reticulum and associated with vesicles which often contain flocculent material. The morphology of Cebus oocytes suggests a greater rate of steroid and protein synthesis, transport, and storage than is usually indicated by the ultrastructure of other mammalian oocytes.
Lai, Fang-Nong; Ma, Jun-Yu; Liu, Jing-Cai; Wang, Jun-Jie; Cheng, Shun-Feng; Sun, Xiao-Feng; Li, Lan; Li, Bo; Nyachoti, Charles Martin; Shen, Wei
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. Copyright © 2015 Elsevier Inc. All rights reserved.
Full Text Available One of the biggest prerequisites for pregnancy is the fertilization step, where a human haploid spermatozoon interacts and penetrates one haploid oocyte in order to produce the diploid zygote. Although fertilization is defined by the presence of two pronuclei and the extraction of the second polar body the process itself requires preparation of both gametes for fertilization to take place at a specific time. These preparations include a number of consecutive biochemical and molecular events with the help of specific molecules and with the consequential interaction between the two gametes. These events take place at three different levels and in a precise order, where the moving spermatozoon penetrates (a the outer vestments of the oocyte, known as the cumulus cell layer; (b the zona pellucida (ZP; where exocytosis of the acrosome contents take place and (c direct interaction of the spermatozoon with the plasma membrane of the oocyte, which involves a firm adhesion of the head of the spermatozoon with the oocyte plasma membrane that culminates with the fusion of both sperm and oocyte membranes (Part I. After the above interactions, a cascade of molecular signal transductions is initiated which results in oocyte activation. Soon after the entry of the first spermatozoon into the oocyte and oocyte activation, the oocyte’s coat (the ZP and the oocyte’s plasma membrane seem to change quickly in order to initiate a fast block to a second spermatozoon (Part II. Sometimes, two spermatozoa fuse with one oocyte, an incidence of 1%–2%, resulting in polyploid fetuses that account for up to 10%–20% of spontaneously aborted human conceptuses. The present review aims to focus on the first part of the human sperm and oocyte interactions, emphasizing the latest molecular and cellular mechanisms controlling this process.
May-Panloup, Pascale; Boucret, Lisa; Chao de la Barca, Juan-Manuel; Desquiret-Dumas, Valérie; Ferré-L'Hotellier, Véronique; Morinière, Catherine; Descamps, Philippe; Procaccio, Vincent; Reynier, Pascal
There is a great inter-individual variability of ovarian ageing, and almost 20% of patients consulting for infertility show signs of premature ovarian ageing. This feature, taken together with delayed childbearing in modern society, leads to the emergence of age-related ovarian dysfunction concomitantly with the desire for pregnancy. Assisted reproductive technology is frequently inefficacious in cases of ovarian ageing, thus raising the economic, medical and societal costs of the procedures. Ovarian ageing is characterized by quantitative and qualitative alteration of the ovarian oocyte reserve. Mitochondria play a central role in follicular atresia and could be the main target of the ooplasmic factors determining oocyte quality adversely affected by ageing. Indeed, the oocyte is the richest cell of the body in mitochondria and depends largely on these organelles to acquire competence for fertilization and early embryonic development. Moreover, the oocyte ensures the uniparental transmission and stability of the mitochondrial genome across the generations. This review focuses on the role played by mitochondria in ovarian ageing and on the possible consequences over the generations. PubMed was used to search the MEDLINE database for peer-reviewed original articles and reviews concerning mitochondria and ovarian ageing, in animal and human species. Searches were performed using keywords belonging to three groups: 'mitochondria' or 'mitochondrial DNA'; 'ovarian reserve', 'oocyte', 'ovary' or 'cumulus cells'; and 'ageing' or 'ovarian ageing'. These keywords were combined with other search phrases relevant to the topic. References from these articles were used to obtain additional articles. There is a close relationship, in mammalian models and humans, between mitochondria and the decline of oocyte quality with ageing. Qualitatively, ageing-related mitochondrial (mt) DNA instability, which leads to the accumulation of mtDNA mutations in the oocyte, plays a key role in
Anamaria Jeni Pernes
It is known that L-ascorbic acid (vitamin C can modulate many biochemical processes intracellularly or extracellularly as antioxidant. The aim of the present study was to examine the effects of media supplementation with ascorbic acid on canine oocyte meiotic maturation, viability and the cumulus cell expansion. Various concentrations of ascorbic acid supplemented in in vitro maturation (IVM media were tested. Canine oocyte was exposed to different levels of ascorbic acid (0, 50, 150, 250, 500, 750µM. Cumulus expansion, meiotic maturation and degeneration of oocytes were assessed 72 h after in vitro culture. As results, on the group treated with 250µM ascorbic acid was a significant difference compared to the control group on nuclear maturation in stages metaphase I (MI and metaphase II (MII (26.98% vs. 6.00%. The groups treated with 50, 150, 250, 500µM had an increase in stage (GVBD, and a significant decrease of degenerate-undefined oocytes compared with the control (23.31%, 18.85%, 13.41% vs 40.80. Concentration 750µM had similar effect to that in the control group. The groups treated with 50, 150, 250, 500µM had an increase in meiosis resumption(GVBD, metaphase I (MI and metaphase II (MII with the best result in the group treated with 250 µM ascorbic acid.
Solak, Kamila A; Santos, Regiane R; van den Berg, Martin; Blaauboer, Bas J; Roelen, Bernard A J; van Duursen, Majorie B M
Flavanones such as naringenin (NAR) and 8-prenylnaringenin (8-PN) are increasingly used as dietary supplements despite scientific concern regarding adverse effects on female reproduction upon excessive intake. In the present study, NAR and 8-PN (0.3-1μM) significantly affected porcine oocyte maturation in vitro by decreasing cumulus expansion. In addition, NAR and 8-PN decreased percentages of meiotic spindle formation, oocyte cleavage and blastocyst formation. The effects of NAR and 8-PN were different from estradiol (3.12μM)-induced effects. Still, the flavanone-induced effects were observed at concentrations that can be found in human plasma upon supplement intake and that resemble physiological estrogen equivalence levels in follicular fluids. Considering that abnormal oocyte maturation can cause subfertility, our study warrants that precautions are in place and excessive intake of NAR and 8-PN e.g. via dietary supplements should be avoided by women.
Bogolyubov, Dmitry S; Batalova, Florina M; Kiselyov, Artyom M; Stepanova, Irina S
The first ultrastructural and immunomorphological characteristics of the karyosphere (karyosome) and extrachromosomal nuclear bodies in the red flour beetle, Tribolium castaneum, are presented. The karyosphere forms early in the diplotene stage of meiotic prophase by the gathering of all oocyte chromosomes in a limited nuclear volume. Using the BrUTP assay, T. castaneum oocyte chromosomes united in the karyosphere maintain their transcriptional activity until the end of oocyte growth. Hyperphosphorylated RNA polymerase II and basal transcription factors (TFIID and TFIIH) were detected in the perichromatin region of the karyosphere. The T. castaneum karyosphere has an extrachromosomal capsule that separates chromosomes from the rest of the nucleoplasm. Certain structural proteins (F-actin, lamin B) were found in the capsule. Unexpectedly, the karyosphere capsule in T. castaneum oocytes was found to be enriched in TMG-capped snRNAs, which suggests that the capsule is not only a structural support for the karyosphere, but may be involved in biogenesis of snRNPs. We also identified the counterparts of 'universal' extrachromosomal nuclear domains, Cajal bodies (CBs) and interchromatin granule clusters (IGCs). Nuclear bodies containing IGC marker protein SC35 display some features unusual for typical IGCs. SC35 domains in T. castaneum oocytes are predominantly fibrillar complex bodies that do not contain trimethyl guanosine (TMG)-capped small nuclear (sn) RNAs. Microinjections of 2'-O-methyl (U)22 probes into the oocytes allowed revealing poly(A)+ RNAs in these nuclear domains. Several proteins related to mRNA export (heterogeneous ribonucleoprotein core protein A1, export adapters Y14 and Aly and export receptor NXF1) were also detected there. We believe that unusual SC35 nuclear domains of T. castaneum oocytes are possibly involved in mRNP but not snRNP biogenesis.
Huang, Weiping; Nagano, Masashi; Kang, Sung-Sik; Yanagawa, Yojiro; Takahashi, Yoshiyuki
Bovine ovaries offer a large pool of oocytes that could be used for in vitro production of embryos of genetically valuable animals. The effects of in vitro growth (IVG) culture duration (10, 12, and 14 days) on the viability and growth of bovine oocytes derived from early antral follicles (0.5-1 mm diameter) in this study. In addition, the effect of pre-IVM culture with phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) on nuclear maturation of IVG oocytes was also evaluated. In experiment 1, oocyte viability observed after 10 or 12 days of IVG culture was greater (P culture. Oocyte diameters and proportions of oocytes at metaphase II stage were greater (P culture where used when compared with 10 days culture. In addition, the proportion of oocytes at metaphase II stage was greater (P culture was performed for oocytes derived from 12 and 14 days of IVG culture. When 12 and 14 days of IVG culture followed by pre-IVM culture were compared in experiment 2, cumulus cell membrane integrity was greater (P culture (24.5%) was greater (P culture was considered the optimal processing system for bovine oocytes derived from early antral follicles when oocyte viability, diameter, maturation, and development competences were considered.
Aydos, Alp; Gurel, Aykut; Oztemur Islakoglu, Yasemin; Noyan, Senem; Gokce, Bagdagul; Ecemis, Tolga; Kaya, Cemil; Aksu, Arif Tarik
Polycystic ovary syndrome (PCOS) is a metabolic and endocrine disorder which affects women of reproductive age with prevalence of 8–18%. The oocyte within the follicle is surrounded by cumulus cells (CCs), which connect with mural granulosa cells (MGCs) that are responsible for secreting steroid hormones. The main aim of this study is comparing gene expression profiles of MGCs and CCs in PCOS and control samples to identify PCOS-specific differentially expressed genes (DEGs). In this study, two microarray databases were searched for mRNA expression microarray studies performed with CCs and MGCs obtained from PCOS patients and control samples. Three independent studies were selected to be integrated with naive meta-analysis since raw meta-data from these studies were found to be highly correlated. DEGs in these somatic cells were identified for PCOS and control groups. This study enabled us to reveal dysregulation in MAPK (mitogen activated protein kinase), insulin and Wnt signaling pathways between CCs and MGCs in PCOS. The meta-analysis results together with qRT-PCR validations provide evidence that molecular signaling is dysregulated through MGCs and CCs in PCOS, which is important for follicle and oocyte maturation and may contribute to the pathogenesis of the syndrome. PMID:27997581
Full Text Available Mesenchymal stem cells (MSCs secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF. Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM, α-minimum essential medium (α-MEM or human tubal fluid (HTF. Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2% than DMEM (54.7%, α-MEM (63.5% and HTF (27.1%. Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively but there was no significant difference compared with α-MEM (85 vs 80.3%. The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.
Protein kinase C (PKC) is a superfamily of Ser/Thr protein kinases that is distributed widely in eukaryotes. It plays key regulatory roles at multiple steps of oocyte meiotic maturation and fertilization. During the process of meiotic maturation, the activation of PKC in cumulus cells stimulates meiotic maturation, whereas the activation of PKC in oocytes results in the inhibition of germinal vesicle breakdown. PKC activity increases following the meiotic maturation, and decreases at the transition of metaphase/anaphase in meiosis I, so as to facilitate the release of the first polar body and the entry of meiosis II. In fertilization of mammalian oocytes, PKC may act as one of the downstream targets of Ca2+ to stimulate the cortical granule exocytosis, release the oocytes from MII arrest and to induce pronucleus formation. PKC is also involved in the regulation of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Several PKC isoforms have been identified in mammalian oocytes, and there is evidence showing that classical PKCs may be the principal mediator of oocyte cortical reaction.
Full Text Available Abstract During maturation, the last phase of oogenesis, the oocyte undergoes several changes which prepare it to be ovulated and fertilized. Immature oocytes are arrested in the first meiotic process prophase, that is morphologically identified by a germinal vesicle. The removal of the first meiotic block marks the initiation of maturation. Although a large number of molecules are involved in complex sequences of events, there is evidence that a calcium increase plays a pivotal role in meiosis re-initiation. It is well established that, during this process, calcium is released from the intracellular stores, whereas less is known on the role of external calcium entering the cell through the plasma membrane ion channels. This review is focused on the functional role of calcium currents during oocyte maturation in all the species, from invertebrates to mammals. The emerging role of specific L-type calcium channels will be discussed.
Ching, J. K. S.; Shipley, S. T.; Browell, E. V.; Brewer, D. A.
Observations are presented which substantiate the hypothesis that significant vertical exchange of ozone and aerosols occurs between the mixed layer and the free troposphere during cumulus cloud convective activity. The experiments utilized the airborne Ultra-Violet Differential Absorption Lidar (UV-DIAL) system. This system provides simultaneous range resolved ozone concentration and aerosol backscatter profiles with high spatial resolution. Evening transects were obtained in the downwind area where the air mass had been advected. Space-height analyses for the evening flight show the cloud debris as patterns of ozone typically in excess of the ambient free tropospheric background. This ozone excess was approximately the value of the concentration difference between the mixed layer and free troposphere determined from independent vertical soundings made by another aircraft in the afternoon.
Zuev, V.E.; Titov, G.A. [Institute of Atmospheric Optics, Tomsk (Russian Federation)
This work presents a 3-D model of radiative transfer which is used to study the relationship between the spatial distribution of cumulus clouds and fluxes (albedo and transmittance) of visible solar radiation.
Full Text Available Abstract Background Oocyte developmental competence is highly affected by the phase of ovarian follicular wave. Previous studies have shown that oocytes from subordinate follicles recovered at growth phase (day 3 after estrus are developmentally more competent than those recovered at dominance phase (day 7 after estrus. However, the molecular mechanisms associated with these differences are not well elucidated. Therefore, the objective of this study was to investigate transcript abundance of bovine oocytes retrieved from small follicles at growth and dominance phases of the first follicular wave and to identify candidate genes related to oocyte developmental competence using cDNA microarray. Results Comparative gene expression analysis of oocytes from growth and dominance phases and subsequent data analysis using Significant Analysis of Microarray (SAM revealed a total of 51 differentially regulated genes, including 36 with known function, 6 with unknown function and 9 novel transcripts. Real-time PCR has validated 10 transcripts revealed by microarray analysis and quantified 5 genes in cumulus cells derived from oocytes of both phases. The expression profile of 8 (80% transcripts (ANAXA2, FL396, S100A10, RPL24, PP, PTTG1, MSX1 and BMP15 was in agreement with microarray data. Transcript abundance of five candidate genes in relation to oocyte developmental competence was validated using Brilliant Cresyl Blue (BCB staining as an independent model. Furthermore, localization of mRNA and protein product of the candidate gene MSX1 in sections of ovarian follicles at days 0, 1, 3 and 7 of estrous cycle showed a clear fluorescent signal in both oocytes and cumulus cells with higher intensity in the former. Moreover, the protein product was detected in bovine oocytes and early cleavage embryos after fertilization with higher intensity around the nucleus. Conclusion This study has identified distinct sets of differentially regulated transcripts between
Paynter, S J; Cooper, A; Gregory, L; Fuller, B J; Shaw, R W
Equilibration of oocytes with cryoprotectants is a prerequisite of low temperature storage. However, cryoprotectant exposure may induce damage via osmotic stress. Knowledge of cell membrane permeability characteristics and their temperature dependence would facilitate the design of cryopreservation protocols in which osmotic stress is minimized and the incidence of intracellular freezing is reduced. To obtain such data, the volume change of donated human oocytes following exposure to cryoprotectant was measured at a variety of temperatures. After removal of cumulus cells, each oocyte was placed in a 5 microl droplet of phosphate-buffered medium. The oocyte was held in position by suction generated using a fine pipette and perfused with 1 ml 1.5 mol/l dimethylsulphoxide (DMSO) at 30, 24 or 10 degrees C. The volume of the oocyte before, during and after perfusion was recorded by videomicroscopy. Oocyte volume was calculated from radius measurements and the Kedem-Katchalsky (K-K) passive coupled transport coefficients, namely L(p) (hydraulic permeability), P(DMSO) (permeability to DMSO) and sigma (reflection coefficient) were derived. The resulting coefficients were L(p) = 1. 65 +/- 0.15, 0.70 +/- 0.06 and 0.28 +/- 0.04 microm/min.atm; P(DMSO) = 0.79 +/- 0.10, 0.25 +/- 0.04 and 0.06 +/- 0.01 microm/s and sigma = 0.97 +/- 0.01, 0.94 +/- 0.03 and 0.96 +/- 0.01 at 30, 24 and 10 degrees C respectively. The activation energy for L(p) was 14.70 and for P(DMSO) was 20.82 kcal/mol. The permeability parameters of human oocytes are higher than those of murine oocytes, suggesting that they require a shorter period of exposure to DMSO with concomitantly reduced toxic effects.
Lin, Tao; Zhang, Jin Yu; Diao, Yun Fei; Kang, Jung Won; Jin, Dong-Il
In the present study, a porcine system was supplemented with sorbitol during in vitro maturation (IVM) or in vitro culture (IVC), and the effects of sorbitol on oocyte maturation and embryonic development following parthenogenetic activation were assessed. Porcine immature oocytes were treated with different concentrations of sorbitol during IVM, and the resultant metaphase II stage oocytes were activated and cultured in porcine zygote medium-3 (PZM-3) for 7 days. No significant difference was observed in cumulus expansion and the nuclear maturation between the control and sorbitol-treated groups, with the exception of the 100 mM group, which showed significantly decreased nuclear maturation and cumulus expansion. There was no significant difference in the intracellular reactive oxygen species (ROS) levels between oocytes matured with 10 or 20 mM sorbitol and control groups, but 50 and 100 mM groups had significantly higher ROS levels than other groups. The 20 mM group showed significant increases in intracellular glutathione and subsequent blastocyst formation rates following parthenogenetic activation compared with the other groups. During IVC, supplementation with sorbitol significantly reduced blastocyst formation and increased the apoptotic index compared with the control. The apoptotic index of blastocysts from the sorbitol-treated group for entire culture period was significantly higher than those of the partially sorbitol-exposed groups. Based on these findings, it can be concluded that the addition of a low concentration of sorbitol (20 mM) during IVM of porcine oocytes benefits subsequent blastocyst development and improves embryo quality, whereas sorbitol supplement during IVC has a negative effect on blastocyst formation.
When a sperm and oocyte unite into one cell upon fertilization, membranous fusion between the sperm and oocyte occurs. In mice, Izumo1 and a tetraspanin molecule CD9 are required for sperm-oocyte fusion as one of the oocyte factors, and another tetraspanin molecule CD81 is also thought to involve in this process. Since these two tetraspanins often form a complex upon cell-cell interaction, it is probable that such a complex is also formed in sperm-oocyte interaction; however, this possibility is still under debate among researchers. Here we assessed this problem using mouse oocytes. Immunocytochemical analysis demonstrated that both CD9 and CD81 were widely distributed outside the oocyte cell membrane, but these molecules were separate, forming bilayers, confirmed by immunobiochemical analysis. Electron-microscopic analysis revealed the presence of CD9- or CD81-incorporated extracellular structures in those bilayers. Finally, microinjection of in vitro-synthesized RNA showed that CD9 reversed a fusion defect in CD81-deficient oocytes in addition to CD9-deficient oocytes, but CD81 failed in both oocytes. These results suggest that both CD9 and CD81 independently work upon sperm-oocyte fusion as extracellular components.
Jennifer D. Small
Full Text Available We analyse the cloud microphysical response to entrainment mixing in warm cumulus clouds observed from the CIRPAS Twin Otter during the GoMACCS field campaign near Houston, Texas, in summer 2006. Cloud drop size distributions and cloud liquid water contents from the Artium Flight phase-Doppler interferometer in conjunction with meteorological observations are used to investigate the degree to which inhomogeneous versus homogeneous mixing is preferred as a function of height above cloud base, distance from cloud edge and aerosol concentration. Using four complete days of data with 101 cloud penetrations (minimum 300 m in length, we find that inhomogeneous mixing primarily explains liquid water variability in these clouds. Furthermore, we show that there is a tendency for mixing to be more homogeneous towards the cloud top, which we attribute to the combination of increased turbulent kinetic energy and cloud drop size with altitude which together cause the Damköhler number to increase by a factor of between 10 and 30 from cloud base to cloud top. We also find that cloud edges appear to be air from cloud centres that have been diluted solely through inhomogeneous mixing. Theory predicts the potential for aerosol to affect mixing type via changes in drop size over the range of aerosol concentrations experienced (moderately polluted rural sites to highly polluted urban sites. However, the observations, while consistent with this hypothesis, do not show a statistically significant effect of aerosol on mixing type.
Li, Qinglei; McKenzie, Laurie J.; Matzuk, Martin M.
Prediction and improvement of oocyte competence are two critical issues in assisted reproductive technology to improve infertility therapy. The lack of reliable and objective predictors of oocyte developmental competence for oocyte/embryo selection during in vitro fertilization hampers the effectiveness of this technology. Likewise, the low pregnancy rate resulting from in vitro maturation of human oocytes represents a major obstacle for its clinical application. Oocyte competence is progressively acquired during follicular development, and the oocyte plays a dominant role in regulating granulosa cell functions and maintaining the microenvironment appropriate for the development of its competence. Hence, granulosa cell functions are reflective of oocyte competence, and molecular markers of granulosa cells are potentially reliable predictors of oocyte quality. With the advent of the functional genomics era, the transcriptome of granulosa cells has been extensively characterized. Experimental data supporting granulosa cell markers as predictors of oocyte competence are now emerging in both animal models and humans. Future efforts should focus on integrating granulosa cell genetic markers as parameters for oocyte/embryo selection. Moreover, novel in vitro evidence highlights the effectiveness of exogenous oocyte-secreted factors in promoting oocyte developmental competence in animal models. The challenge in evaluating the effect of oocyte-secreted factors on oocyte quality in a clinical setting is to standardize the various preparations of these recombinant proteins and decipher their complex interactions/cooperativity within the germline-somatic cell regulatory loop. PMID:18996952
Ruiz, Jaime; Paulo Santayana, R; José Mendoza, M; Leandra Landeo, J; Huamán, Elizabeth; Ticllacuri, Flamel; Fidel Mujica, L; Silva, Mauricio; Ratto, Marcelo H
We evaluated the effect of in vitro maturation time, sperm selection and oxygen tension on alpaca embryo development. In Experiment I, Cumulus Oocyte- Complexes (COCs) were obtained from abattoir ovaries and in vitro matured in TCM-199 for 24 (n = 217), 28 (215), or 32 h (223) at 38.5 °C, high humidity and 5% CO2 in air. Oocytes from 24 (n = 392), 28 (n = 456) or 32 (n = 368) h groups were in vitro fertilized with epididymal sperm and cultured in SOFaa at 38.5 °C, high humidity and 5% CO2, 5% O2 and 90% N2 for 7 days. Embryo development was evaluated on Day 2, 5 and Day 7 of in vitro culture (Day 0 = in vitro fertilization). In Experiment II, a 2 by 2-factorial design was used to determine the effect of sperm selection (Swim-up vs Percoll) and oxygen tension (20% vs 5%) during embryo culture and their interaction on embryo development. COCs were in vitro matured for 32 h at 38.5 °C and 5% CO2 in air and then in vitro inseminated with epididymal sperm processed by swim-up or Percoll. Zygotes were cultured in SOFaa + cumulus cells at 38.5 °C under 20 or 5% of O2 tension and high humidity for 7 days. A total of 235, 235, 253 and 240 oocytes were assigned to: swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, groups respectively. The proportion of oocytes reaching MII stage was highest after 32 h of in vitro maturation (P < 0.05). Blastocyst rate (29.1 ± 2.7%) was also highest for COCs matured for 32 h (Exp I). In Experiment II, Blastocysts rate (26.03 ± 4.7; 27.7 ± 4.3; 29.7 ± 3.8 and 27.6 ± 4.2% for swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, respectively) was not affected by sperm selection method (P = 0.8), oxygen tension (P = 0.9) or their interaction (P = 0.5). Copyright © 2017 Elsevier Inc. All rights reserved.
Makita, Miho; Ueda, Mayuko; Miyano, Takashi
In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4-0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts.
Lin, L; Kragh, P M; Purup, S
cumulus cells were removed with 1 mg mL-1 hyaluronidase and pipetting, and oocytes were used as recipients for somatic nuclear transfer with handmade cloning (HMC) method. Porcine fetal fibroblasts were used as nuclear donor cells. Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol...... cell number in blastocysts after somatic cell nuclear transfer (SCNT). In conclusion, a simple NaCl pre-treatment of oocytes has improved the in vitro efficiency of porcine SCNT......Modified environmental stress was reported to improve the developmental competence and cryotolerance of porcine oocytes, such as high hydrostatic pressure (HHP; Du et al. 2008 Cloning Stem Cells, Epub ahead of print) and osmotic stress (Lin et al. 2008 Reprod. Biomed. Online, in press). HHP also...
Porcu, Eleonora; Fabbri, Raffaella; Damiano, Giuseppe; Fratto, Rosita; Giunchi, Susanna; Venturoli, Stefano
The use of chemotherapy and radiotherapy in oncological patients may reduce their reproductive potential. Sperm cryopreservation has been already used in men affected by neoplastic disease. Oocyte cryopreservation might be an important solution for these patients at risk of losing ovarian function. A program of oocyte cryopreservation for oncological patients is also present in our center. From June 1996 to January 2000, 18 patients awaiting chemotherapy and radiotherapy for neoplastic disease were included in our oocyte cryopreservation program. Our experience documents that oocyte storage may be a concrete and pragmatic alternative for oncological patients. The duration of oocyte storage does not seem to interfere with oocyte survival as pregnancies occurred even after several years of gamete cryopreservation in liquid nitrogen.
JIA Xiao-long; LI Chong-yin; LING Jian
Madden-Julian Oscillations (MJO) in six integrations using an AGCM with different cumulus parameterization schemes and resolutions are examined to investigate their impacts on the MJO simulation.Results suggest that the MJO simulation can be affected by both resolution and cumulus parameterization,though the latter,which determines the fundamental ability of the AGCM in simulating the MJO and the characteristics of the simulated MJO,is more crucial than the former. Model resolution can substantially affect the simulated MJO in certain aspects. Increasing resolution cannot improve the simulated MJO substantially,but can significantly modulate the detailed character of the simulated MJO; meanwhile,the impacts of resolution are dependent on the cumulus parameterization,determining the basic features of the MJO. Changes in the resolution do not alter the nature of the simulated MJO but rather regulate the simulation itself,which is constrained by cumulus parameterization schemes. Theretbre,the vertical resolution needs to be increased simultaneously. The vertical profile of diabatic heating may be a crucial factor that is responsible for these different modeling results. To a large extent,it is determined by the cumulus parameterization scheme used.
Kristensen, Stine G; Pors, Susanne E; Yding Andersen, Claus
PURPOSE OF REVIEW: The ovarian reserve comprises an enormous surplus of follicles. Despite this, some women produce insufficient numbers of oocytes by conventional fertility treatments. However, recent technical accomplishments may transform assisted reproductive technology (ART) in such a way...... 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....
Graham C. Gilchrist
Full Text Available Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV oocytes, metaphase II (MII oocytes, and presumptive zygotes (PZ. Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR. Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05. To determine whether changes in specific primary miRNA (pri-miRNA transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo.
Gettelman, Andrew [University Corporation for Atmospheric Research (NCAR), Boulder, CO (United States)
This project was successfully able to meet its’ goals, but faced some serious challenges due to personnel issues. Nonetheless, it was largely successful. The Project Objectives were as follows: 1. Develop a unified representation of stratifom and cumulus cloud microphysics for NCAR/DOE global community models. 2. Examine the effects of aerosols on clouds and their impact on precipitation in stratiform and cumulus clouds. We will also explore the effects of clouds and precipitation on aerosols. 3. Test these new formulations using advanced evaluation techniques and observations and release
Kalous, J; Sutovsky, P; Rimkevicova, Z; Shioya, Y; Lie, B L; Motlik, J
Membrana granulosa was isolated from healthy large antral follicles of prepubertal or cyclic gilts stimulated with PMSG or PMSG and hCG. Ultrastructural observations revealed that pieces of pig membrana granulosa were associated with the basement membrane. The cattle cumulus-enclosed oocytes (COC) were placed in the rolled pieces of the pig membrana granulosa (PMG). After 8 and 24 hr of coculture with PMG from prepubertal gilts, only 16% and 21% of oocytes underwent GVBD, respectively. PMG from PMSG-stimulated cyclic gilts blocked the resumption of meiosis in all COC. The inhibitory effect of heterologous granulosa cells was fully reversible. When COC were initially incubated for 2 and 4 hr, subsequent culture in PMG prevented GVBD in 100% and 36% of oocytes, respectively. This suggests that functional contact between COC and PMG was established during the first 2 hr of coculture. To follow metabolic cooperation between PMG and COC, PMG was prelabeled with 3H-uridine and cocultured with COC. Autoradiography on semithin sections revealed the intensive passage of 3H-uridine from PMG into the cumulus layer and an oocyte. COC placed in PMG after GVBD (8 and 12 hr of an initial incubation) did not extrude the first polar body. PMG isolated from cyclic gilts after PMSG and hCG stimulation also inhibited GVBD of COC. Since nearly all COC placed in PMG isolated 10 and 12 hr after hCG remained in the GV stage after 24 hr of coculture, the hCG stimulation did not substantially diminish the meiosis inhibiting activity of PMG.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhang, Yuan-Wei; Liu, Yu-Sheng; Ma, Chun-Hong
During folliculogenesis, cumulus cells surrounding the oocyte differentiate into corona radiata cells (CRCs) and cumulus oophorus cells (COCs), which are involved in gonadal steroidogenesis and the development of germ cells. Several studies suggested that microRNAs (miRNAs) play an important regulatory role at the post-transcriptional level in cumulus cells. However, comparative miRNA profiles and associated processes in human CRCs and COCs have not been reported before. In this study, miRNA profiles were obtained from CRCs and COCs using next generation sequencing in women undergoing controlled ovarian stimulation for IVF. A total of 785 and 799 annotated miRNAs were identified in CRCs and COCs, while high expression levels of six novel miRNAs were detected both in CRCs and in COCs. In addition, different expression patterns in CRCs and COCs were detected in 72 annotated miRNAs. To confirm the miRNA profile in COCs and CRCs, quantitative real-time PCR was used to validate the expression of annotated miRNAs, differentially expressed miRNAs, and novel miRNAs. The miRNAs in the let-7 family were found to be involved in the regulation of a broad range of biological processes in both cumulus cell populations, which was accompanied by a large amount of miRNA editing. Bioinformatics analysis showed that amino acid and energy metabolism were targeted significantly by miRNAs that were differentially expressed between CRCs and COCs. Our work extends the current knowledge of the regulatory role of miRNAs and their targeted pathways in folliculogenesis, and provides novel candidates for molecular biomarkers in the research of female infertility. PMID:25188034
Full Text Available During folliculogenesis, cumulus cells surrounding the oocyte differentiate into corona radiata cells (CRCs and cumulus oophorus cells (COCs, which are involved in gonadal steroidogenesis and the development of germ cells. Several studies suggested that microRNAs (miRNAs play an important regulatory role at the post-transcriptional level in cumulus cells. However, comparative miRNA profiles and associated processes in human CRCs and COCs have not been reported before. In this study, miRNA profiles were obtained from CRCs and COCs using next generation sequencing in women undergoing controlled ovarian stimulation for IVF. A total of 785 and 799 annotated miRNAs were identified in CRCs and COCs, while high expression levels of six novel miRNAs were detected both in CRCs and in COCs. In addition, different expression patterns in CRCs and COCs were detected in 72 annotated miRNAs. To confirm the miRNA profile in COCs and CRCs, quantitative real-time PCR was used to validate the expression of annotated miRNAs, differentially expressed miRNAs, and novel miRNAs. The miRNAs in the let-7 family were found to be involved in the regulation of a broad range of biological processes in both cumulus cell populations, which was accompanied by a large amount of miRNA editing. Bioinformatics analysis showed that amino acid and energy metabolism were targeted significantly by miRNAs that were differentially expressed between CRCs and COCs. Our work extends the current knowledge of the regulatory role of miRNAs and their targeted pathways in folliculogenesis, and provides novel candidates for molecular biomarkers in the research of female infertility.
Khatir, Hadj; Anouassi, AbdelHaq
Dromedary offspring have never been produced fully in vitro. We have previously demonstrated that embryos obtained by culture in semi-defined medium (mKSOMaa) have better in vitro development ability than those cultured with oviductal epithelial cells. The aim of the present experiment was to study the pregnancy rate after embryo transfer of in vitro-produced (IVP) dromedary embryos cultured in semi-defined modified medium (mKSOMaa). IVM/IVF procedures were conducted on six hundred and sixty four (664) cumulus oocytes complexes (COCs) aspirated from ovaries collected at a local slaughterhouse and cultured in vitro (38.5 degrees C; 5% CO2, and maximum humidity >95%). Maturation was completed by incubation in TCM-199 medium supplemented with 10% heat-treated Fetal Calf Serum (FCS), 10 ng/mL EGF, 1 microg/mL FSH, 1 microg/mL E2 and 500 microM cysteamine for 30 h. In vitro fertilization was performed using fresh semen (0.5 x 10(6) spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 38.5 degrees C, 5% CO2 and 90% N2 with maximum humidity (>95%). All IVC steps were done in seven replicates. The cleavage rate (two cells to blastocyst stage) was 64% (425/664) and the percentage of oocytes reaching the blastocyst stage was 23% (155/664). The hatching rate of blastocyst obtained after culture was 46% (71/155). Good quality hatched blastocysts (n = 66) were transferred individually to synchronized recipients. Pregnancy rates, determined by ultrasonography at 15, 60 and 90 days after embryo transfer (ET), were 38%, 32% and 27%, respectively. Out of 18 pregnant females 5 aborted between the fifth and seventh month of pregnancy and 13 females (20%) remained pregnant. After 385 days of pregnancy, the first healthy and normal male-dromedary offspring produced fully in vitro was born at a birth weight of 38 kg. More dromedary calves (n = 4) were born later on. The remaining pregnant females (n = 8) are due to calf within the next months. In
朱淑娟; 李威; 吴效科
Human follicle development requires the recruitment of primordial follicles into a cohort of growing follicles from which one follicle is selected to ovulate a mature oocyte. During this developmental process, complex endocrine and intraovarian paracrine signals create a changing intrafollicular hormonal. With this microenvironment, appropriate cumulus cell-oocyte signaling governs oocyte developmental competence, defined as the ability of the oocyte to complete meiosis and undergo fertilization, embryogenesis. Many of these mechanisms are perturbed in polycystic ovary syndrome (PCOS), which is characterized by ovarian hyperandrogenism, hyperinsulinemia from insulin resistance and reduced fecundity. In addition to these endocrinopathies, pcos also is characterized by paracrine dysregulation of follicle development by intraovarian proteins of the transforming growth factor p (TGF- P) family.%人卵泡发育始于始基卵泡募集到形成大量生长卵泡,随之从中选择一个发育成熟进行排卵.在生长发育过程中,复杂的内分泌和卵巢旁分泌信号通路使卵泡内激素发生变化.大量卵丘细胞—卵母细胞在这个微环境中调控卵母细胞的生长发育能力,卵母细胞的发育能力表现在完成减数分裂、受精、着床等.多囊卵巢综合征( PCOS)的甾体激素合成异常,糖、脂代谢机制受损,特征性表现为高雄激素血症、胰岛素抵抗致高胰岛素血症及不孕.除内分泌异常外,还表现为卵巢内转化生长因子-β (TGF-β)蛋白家族对卵泡发育的异常调节.
Xu, Kuan-Man; Arakawa, Akio
Semiprognostic tests are performed against data simulated by a cumulus ensemble model to evaluate the Arakawa-Schubert (A-S) cumulus parametrization. It is found that the A-S cumulus parametrization is generally valid despite the existence of mesoscale organization in cumulus convection. The nondiagnostic and nondeterministic aspects of the A-S cumulus parametrization are examined by testing the sensitivity of the parametrization to the horizontal grid resolution. It is also shown that the inclusion of convective-scale downdrafts improves the results of semiprognostic tests.
Burkhardt, Sabrina; Borsos, Máté; Szydlowska, Anna; Godwin, Jonathan; Williams, Suzannah A; Cohen, Paula E; Hirota, Takayuki; Saitou, Mitinori; Tachibana-Konwalski, Kikuë
Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis . Rec8-containing cohesin, bound to Smc3/Smc1α or Smc3/Smc1β, maintains bivalent cohesion in mammalian meiosis [2-6]. In females, meiotic DNA replication and recombination occur in fetal oocytes. After birth, oocytes arrest at the prolonged dictyate stage until recruited to grow into mature oocytes that divide at ovulation. How cohesion is maintained in arrested oocytes remains a pivotal question relevant to maternal age-related aneuploidy. Hypothetically, cohesin turnover regenerates cohesion in oocytes. Evidence for post-replicative cohesion establishment mechanism exists, in yeast and invertebrates [7, 8]. In mouse fetal oocytes, cohesin loading factor Nipbl/Scc2 localizes to chromosome axes during recombination [9, 10]. Alternatively, cohesion is maintained without turnover. Consistent with this, cohesion maintenance does not require Smc1β transcription, but unlike Rec8, Smc1β is not required for establishing bivalent cohesion [11, 12]. Rec8 maintains cohesion without turnover during weeks of oocyte growth . Whether the same applies to months or decades of arrest is unknown. Here, we test whether Rec8 activated in arrested mouse oocytes builds cohesion revealed by TEV cleavage and live-cell imaging. Rec8 establishes cohesion when activated during DNA replication in fetal oocytes using tamoxifen-inducible Cre. In contrast, no new cohesion is detected when Rec8 is activated in arrested oocytes by tamoxifen despite cohesin synthesis. We conclude that cohesion established in fetal oocytes is maintained for months without detectable turnover in dictyate-arrested oocytes. This implies that women's fertility depends on the longevity of cohesin proteins that established cohesion in utero.
Stachecki, James J; Cohen, Jacques; Garrisi, John; Munné, Santiago; Burgess, Colleen; Willadsen, Steen M
Previous investigations revealed that choline-based freezing media developed in our laboratory were superior to conventional sodium-based media for storing mouse oocytes. This paper examines the ability of the choline-based medium CJ2 and a modified form of this medium, CJ3, to cryopreserve unfertilized human oocytes. Oocytes that were consented for research and matured overnight, as well as freshly collected, donor, mature metaphase II (MII) oocytes, were cryopreserved using choline-based media and an optimized slow-cooling protocol. The results showed higher survival and fertilization rates when CJ3 supplemented with 0.2 mmol/l sucrose was used as compared with CJ2 supplemented with either 0.1 mmol/l or 0.2 mmol/l sucrose. Freshly collected oocytes were more difficult to cryopreserve than those matured in vitro. Modification of the base medium proved to be one of the key factors in obtaining survival rates over 90%. Fertilization rates, embryo development, and genetic analysis of embryos resulting from control and frozen-thawed oocytes are provided. There appears to be a high correlation between chromosomal anomalies and abnormal morphology in embryos from thawed oocytes.
Van der Elst Josiane
Full Text Available Abstract Background We studied the benefit of using in vitro matured metaphase I (MI oocytes for ICSI in patients with a maximum of 6 mature metaphase II (MII oocytes at retrieval. Methods In 2004, 187 ICSI cycles were selected in which maximum 6 MII oocytes and at least one MI oocyte were retrieved. MI oocytes were put in culture to mature until the moment of ICSI, which was performed between 2 to 11 hours after oocyte retrieval (day 0. In exceptional cases, when the patient did not have any mature oocyte at the scheduled time of ICSI, MI oocytes were left to mature overnight and were injected between 19 to 26 hours after retrieval (day 1. Embryos from MI oocytes were chosen for transfer only when no other good quality embryos from MII oocytes were available. Outcome parameters were time period of in vitro maturation (IVM, IVM and fertilization rates, embryo development, clinical pregnancy rates, implantation rates and total MI oocyte utilization rate. Results The overall IVM rate was 43%. IVM oocytes had lower fertilization rates compared to in vivo matured sibling oocytes (52% versus 68%, P Conclusion Fertilization of in vitro matured MI oocytes can result in normal embryos and pregnancy, making IVM worthwhile, particularly when few MII oocytes are obtained at retrieval.
MacLennan, Marie; Crichton, James H; Playfoot, Christopher J; Adams, Ian R
Meiosis is one of the defining events in gametogenesis. Male and female germ cells both undergo one round of meiotic cell division during their development in order to reduce the ploidy of the gametes, and thereby maintain the ploidy of the species after fertilisation. However, there are some aspects of meiosis in the female germline, such as the prolonged arrest in dictyate, that appear to predispose oocytes to missegregate their chromosomes and transmit aneuploidies to the next generation. These maternally-derived aneuploidies are particularly problematic in humans where they are major contributors to miscarriage, age-related infertility, and the high incidence of Down's syndrome in human conceptions. This review will discuss how events that occur in foetal oocyte development and during the oocytes' prolonged dictyate arrest can influence meiotic chromosome segregation and the incidence of aneuploidy in adult oocytes.
Full Text Available We propose an approach to stochastic parameterization of shallow cumulus clouds to represent the convective variability and its dependence on the model resolution. To collect the information about the individual cloud lifecycles and the cloud ensemble as a whole, we employ a Large-Eddy Simulation model (LES and a cloud tracking algorithm, followed by conditional sampling of clouds at the cloud-base level. In the case of a shallow cumulus ensemble, the cloud-base mass flux distribution is bimodal due to the different shallow cloud subtypes. Each distribution mode can be approximated with a Weibull distribution, explaining the deviation from a single-parameter exponential shape through the diversity in cloud lifecycles. The exponential distribution of cloud mass flux previously suggested for deep convection parameterization is a special case of the Weibull distribution, which opens a way towards unification of the statistical convective ensemble formalism of shallow and deep cumulus clouds. Based on the empirical and theoretical findings, a stochastic model has been developed to simulate a shallow convective cloud ensemble. It is formulated as a compound random process, with the number of convective elements drawn from a Poisson distribution, and the cloud mass flux sampled from a mixed Weibull distribution. Convective memory is accounted for through the explicit cloud lifecycles, making the model formulation consistent with the choice of the Weibull cloud mass flux distribution function. The memory of individual shallow clouds is required to capture the correct convective variability. The resulting distribution of the subgrid convective states in the considered shallow cumulus case is scale-adaptive – the smaller the grid size, the broader the distribution.
Full Text Available We propose an approach to stochastic parameterisation of shallow cumulus clouds to represent the convective variability and its dependence on the model resolution. To collect information about the individual cloud lifecycles and the cloud ensemble as a whole, we employ a large eddy simulation (LES model and a cloud tracking algorithm, followed by conditional sampling of clouds at the cloud-base level. In the case of a shallow cumulus ensemble, the cloud-base mass flux distribution is bimodal, due to the different shallow cloud subtypes, active and passive clouds. Each distribution mode can be approximated using a Weibull distribution, which is a generalisation of exponential distribution by accounting for the change in distribution shape due to the diversity of cloud lifecycles. The exponential distribution of cloud mass flux previously suggested for deep convection parameterisation is a special case of the Weibull distribution, which opens a way towards unification of the statistical convective ensemble formalism of shallow and deep cumulus clouds. Based on the empirical and theoretical findings, a stochastic model has been developed to simulate a shallow convective cloud ensemble. It is formulated as a compound random process, with the number of convective elements drawn from a Poisson distribution, and the cloud mass flux sampled from a mixed Weibull distribution. Convective memory is accounted for through the explicit cloud lifecycles, making the model formulation consistent with the choice of the Weibull cloud mass flux distribution function. The memory of individual shallow clouds is required to capture the correct convective variability. The resulting distribution of the subgrid convective states in the considered shallow cumulus case is scale-adaptive – the smaller the grid size, the broader the distribution.
Full Text Available Implantation failure and early pregnancy loss have been reported to be closely related to the quality of mammalian oocytes; however, the pregnant outcome of embryos from in-vitro matured (IVM oocytes remains unknown. In this study we examined spindle assembly and chromosome segregation during differentiation, and the duration of IVM of mouse oocytes. The resulting implantation and pregnancy outcomes were analyzed to clarify the relationship between the spindle and chromosomes of IVM oocytes and implantation and early pregnancy. Cumulus-enclosed germinal vesicle oocytes were collected and randomly cultured in IVM medium with different IVM durations. One part of IVM oocytes were analyzed the spindle and chromosome morphology by immunofluorescence method, and the other part of them were fertilized by intracytoplasmic sperm injection. The resulting embryos were transferred into pseudo-pregnant female mice, and the post-implantation and full term development was observed. The chromosome aberrations and incorrect spindle assembly seems not affect the early development and blastocyst cell number derived from IVM oocytes, however the development potential of the resulting embryos after implantation were significant decreased with the ratio increasing of chromosome aberrations and incorrect spindle assembly. Accordingly, the full-term development was also decreased. In conclusion, the present study showed the spindle assembly of in vitro-matured oocytes was one of the most important factors that affected the implantation and ongoing pregnancy rates of IVM oocytes, and the improvement by an appropriate duration of maturation in vitro will enhance the post-implantation development potential of the resulting embryos, and decrease implantation failure and early pregnancy loss.
Li, Min; Zhao, Hong-Cui; Li, Rong; Yu, Yang; Qiao, Jie
Implantation failure and early pregnancy loss have been reported to be closely related to the quality of mammalian oocytes; however, the pregnant outcome of embryos from in-vitro matured (IVM) oocytes remains unknown. In this study we examined spindle assembly and chromosome segregation during differentiation, and the duration of IVM of mouse oocytes. The resulting implantation and pregnancy outcomes were analyzed to clarify the relationship between the spindle and chromosomes of IVM oocytes and implantation and early pregnancy. Cumulus-enclosed germinal vesicle oocytes were collected and randomly cultured in IVM medium with different IVM durations. One part of IVM oocytes were analyzed the spindle and chromosome morphology by immunofluorescence method, and the other part of them were fertilized by intracytoplasmic sperm injection. The resulting embryos were transferred into pseudo-pregnant female mice, and the post-implantation and full term development was observed. The chromosome aberrations and incorrect spindle assembly seems not affect the early development and blastocyst cell number derived from IVM oocytes, however the development potential of the resulting embryos after implantation were significant decreased with the ratio increasing of chromosome aberrations and incorrect spindle assembly. Accordingly, the full-term development was also decreased. In conclusion, the present study showed the spindle assembly of in vitro-matured oocytes was one of the most important factors that affected the implantation and ongoing pregnancy rates of IVM oocytes, and the improvement by an appropriate duration of maturation in vitro will enhance the post-implantation development potential of the resulting embryos, and decrease implantation failure and early pregnancy loss.
Ernst, E H; Grøndahl, M L; Grund, S; Hardy, K; Heuck, A; Sunde, L; Franks, S; Andersen, C Y; Villesen, P; Lykke-Hartmann, K
Do specific transcriptome dynamics in human oocytes from primordial and primary follicles identify novel pathways in oocyte activation? The transcriptomic profiles in oocytes from primordial and primary follicles, respectively, revealed several new canonical pathways as putative mediators of oocyte dormancy and activation. Cellular signaling pathways including PI3K/AKT and AKT/mTOR as well as TGF-β and IGF signaling are known to regulate the primordial-to-primary transition in mammalian follicle development. We performed a class comparison study on human oocytes from primordial (n = 436) and primary (n = 182) follicles donated by three women having ovarian tissue cryopreserved before chemotherapy. RNA was extracted from oocytes from primordial and primary follicles isolated by Laser Capture Microdissection, and submitted to the HiSeq Illumina platform. Data mapping, quality control, filtering and expression analysis were performed using Tophat (2.0.4), Cufflinks (2.0.2), BWA (0.6.2) and software R. Modeling of complex biological systems was performed using the IPA® software. Finally, qPCR and immunohistochemistry were employed to explore expression and localization of selected genes and products in human ovarian tissue. We found 223 and 268 genes down-regulated and up-regulated, respectively, in the oocytes during the human primordial-to-primary follicle transition (P 2). IPA® enrichment analysis revealed known pathways ('mTOR Signaling', 'PI3K/AKT Signaling' and 'PTEN Signaling') as well as enriched canonical pathways not previously associated with human ovarian follicle development such as 'ErB Signaling' and 'NGF Signaling' in the down-regulated category and 'Regulation of eIF4 and P70S6K Signaling' and 'HER-2 Signaling in Breast Cancer' in the up-regulated group. Additionally, immunohistochemistry on human ovarian tissue explored the intraovarian localization of VASA, FOXO1 and eIF4E. http://users-birc.au.dk/biopv/published_data/ernst_2017/. This is a
Jennifer R. Prentice
Full Text Available The preservation of the female portion of livestock genetics has become an international priority; however, in situ conservation strategies are extremely expensive. Therefore, efforts are increasingly focusing on the development of a reliable cryopreservation method for oocytes, in order to establish ova banks. Slow freezing, a common method for cryopreservation of oocytes, causes osmotic shock (solution effect and intracellular ice crystallization leading to cell damage. Vitrification is an alternative method for cryopreservation in which cells are exposed to a higher concentration of cryoprotectants and frozen with an ultra rapid freezing velocity, resulting in an ice crystal free, solid glass-like structure. Presently, vitrification is a popular method for cryopreservation of embryos. However, vitrification of oocytes is still challenging due to their complex structure and sensitivity to chilling.
Prentice, Jennifer R; Anzar, Muhammad
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.
Mitogen-activated protein kinase (MAPK) is a family of Ser/Thr protein kinases expressed widely in eukaryotic cells. MAPK is activated by a cascade of protein kinase phosphorylation and plays pivotal roles in regulating meiosis process in oocytes. As an important physical substrate of MAPK, p90rsk mediates numerous MAPK functions. MAPK was activated at G2/M transition during meiosis. Its activity reached the peak at MⅠ stage and maintained at this level until the time before the pronuclear formation after fertilization. There is complex interplay between MAPK and MPF in the meiosis regulation. Furthermore, other intracellular signal transducers, such as cAMP, protein kinase C and protein phosphotase, ect., also regulated the activity of MAPK at different stages during meiosis in oocytes. In the present article, the roles of MAPK signaling pathway in oocyte meiosis are reviewed and discussed.
Full Text Available The objective of this study was to compare the effectiveness of the protocols for superstimulation of follicular growth in Thai native heifers. Heifers (n = 20 were randomly divided into four groups of five heifers/group. Heifers were given a single dose by i.m. administration of 100 mg Follicle Stimulating Hormone dissolved in polyvinylpyrrolidone (FSHp at 24 h. Ovum pick up (OPU occurred at 72 h (F24O72 protocol; Group 1 or 96 h (F24O96 protocol; Group 2, and at 36 h and OPU at 72 h (F36O72 protocol; Group 3 or 96 h (F36O96 protocol; Group 4 after follicular ablation. The dynamics of ovarian follicular growth were monitored by twice-daily ultrasonographic examinations. Blood sample collections were performed every 12 h after initiation of treatment for assessment of FSH, E2 and P4 profiles. All heifers were subjected to eight repeated sequential sessions of OPU. The follicular deviation commenced 24±5.32 h after follicular ablation in all groups. The circulatory FSH surged quickly from 24 to 36 h (>0.8 ng/ml after follicular ablation and circulatory estrogen levels steadily increased from 36 h until OPU in all groups. At the end of the OPU sessions, the mean number of aspirated follicles/heifer/session in F36O72 protocol (Group 3 and F36O96 protocol (Group 4 were higher than in the two other groups (p<0.05. The number of cumulus-oocyte complexes (COCs, cleaved and day 8 blastocysts rates in the F36O72 protocol (Group 3 were higher than in the other groups (p<0.05. It can be concluded that a single dose i.m. administration of 100 mg FSHp at 36 h and OPU at 72 h after follicular ablation (F36O72 protocol; Group 3 was the most effective protocol for superstimulation of follicular growth for repeated OPU and subsequent in vitro embryo production in Thai native heifers.
Significance: The current study provides the first proteomic profile of an oocyte of a cnidarian organism the starlet sea anemone N. vectensis and gives new insights on the ancient origin of an oocyte proteome template. The comparative analysis with a chordate oocyte suggests that the oocyte proteome predates the divergence of the cnidarian and bilaterian lineages. In addition, the data generated in the study will serve as a valuable resource for further developmental and evolutional studies.
Wang, Yijing; Teng, Zhen; Li, Ge; Mu, Xinyi; Wang, Zhengpin; Feng, Lizhao; Niu, Wanbao; Huang, Kun; Xiang, Xi; Wang, Chao; Zhang, Hua; Xia, Guoliang
In mammalian ovaries, a fixed population of primordial follicles forms during the perinatal stage and the oocytes contained within are arrested at the dictyate stage of meiotic prophase I. In the current study, we provide evidence that the level of cyclic AMP (cAMP) in oocytes regulates oocyte meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary. Our results show that the early meiotic development of oocytes is closely correlated with increased levels of intra-oocyte cAMP. Inhibiting cAMP synthesis in fetal ovaries delayed oocyte meiotic progression and inhibited the disassembly and degradation of synaptonemal complex protein 1. In addition, inhibiting cAMP synthesis in in vitro cultured fetal ovaries prevented primordial follicle formation. Finally, using an in situ oocyte chromosome analysis approach, we found that the dictyate arrest of oocytes is essential for primordial follicle formation under physiological conditions. Taken together, these results suggest a role for cAMP in early meiotic development and primordial follicle formation in the mouse ovary. © 2015. Published by The Company of Biologists Ltd.
Sugiyama, Miyako; Kawahara-Miki, Ryoka; Kawana, Hirosuke; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka
Mitochondrial numbers increase during oocyte growth. In this study, we collected oocytes and granulosa cell complexes (OGCs) from early antral follicles (EAFs) of aged cows (> 120 months of age) and examined the effects of resveratrol on mitochondrial generation, degradation, and quality in oocytes grown in vitro. We also examined the effects of resveratrol on gene expression of the granulosa cells. Resveratrol (20 µM) enhanced the expression of SIRT1 and induced autophagy in both granulosa cells and oocytes derived from aged cows. Culturing the OGCs with resveratrol increased mitochondrial DNA copy numbers in oocytes grown in vitro. Furthermore, resveratrol increased the ATP content in oocytes and improved the developmental ability of the oocytes to the blastocyst stage. Gene expression profiles in granulosa cells, as evaluated by next-generation sequencing technology, revealed that resveratrol enhanced the expression of EIF2-related genes but downregulated the expression of mammalian target of rapamycin (mTOR)-, inflammation-, and cholesterol homeostasis-related genes in granulosa cells. In conclusion, resveratrol affected both oocytes and granulosa cells derived from aged cows and improved the quality of oocytes grown in vitro through upregulation of mitochondrial biogenesis and degradation in growing oocytes and conditioning of granulosa cells.
Full Text Available An in vitro zona-free hamster oocyte penetration test was utilized in 24 trials in order to evaluate the capacitation media used for ram sperm. A pool of fresh semen was collected from three crossed breed rams. Two semen drops were washed by centrifugation and incubated in high ionic strength treatment (HIS or in a defined medium with HEPES, on heat ewe serum and heparin. After the incubation to promote capacitation, simplified triple-stain technique was used to evaluate the spontaneous acrosome reaction of the capacitated sperm. Superovulation in 96 golden hamsters was induced by PMSG and hCG. The oocytes were treated with hyaluronidase and trypsin to remove, respectively, the cumulus cells and the zona pellucida. Oocytes and capacitated sperm were incubated during 3 hours for further penetration. Then, oocytes were fixed and stained, being evaluated under phase contrast microscope. No significant statistical difference (p >; 0.05 was found between media, concerning the penetration rate of the capacitated sperm and between number of sperm viable with acrosome reaction after the capacitation treatment using two different media. It was concluded that both media utilized were effective in capacitating ram sperm.
Iwayama, Hiroshi; Hochi, Shinichi; Kato, Megumi; Hirabayashi, Masumi; Kuwayama, Masashige; Ishikawa, Hajime; Ohsumi, Seiji; Fukui, Yutaka
Germinal-vesicle-stage oocytes enclosed with compact cumulus cell layers (COCs) were recovered from adult or prepubertal minke whale ovaries, and were vitrified in a solution containing 15% ethylene glycol, 15% DMSO and 0.5 M sucrose using either a Cryotop or an open-pulled straw (OPS) as the cryodevice. The post-warm COCs with normal morphology were cultured for 40 h in a 390 mosmol in vitro maturation medium, and oocytes extruding the first polar body were considered to be matured. The proportion of morphologically normal COCs after vitrification and warming was higher when the COCs were cryopreserved by Cryotop (adult origin, 88.4%; prepubertal origin, 80.8%) compared with the OPS (adult origin, 67.7%; prepubertal origin, 64.2%). The oocyte maturation rate was higher in the adult/Cryotop group (29.1%) compared with those of the prepubertal/Cryotop group (14.4%), the adult/OPS group (14.3%) and the prepubertal/OPS group (10.6%). These results indicate that the Cryotop is a better device than the OPS for vitrification of immature oocytes from adult minke whales.
JIA Xiaolong; LI Chongyin
The sensitivity of the simulated tropical intraseasonal oscillation or MJO (Madden and Julian oscilla tion)to different cumulus parameterizations is studied by using an atmospheric general circulation model (GCM)-SAMIL(Spectral Atmospheric Model of IAP LASG).Results show that performance of the model in simulating the MJO alters widely when using two different cumulus parameterization schemes-the moist convective adjustment scheme(MCA)and the Zhang-McFarlane(ZM)scheme.MJO simulated by the MCA scheme was found to be more realistic than that simulated by the ZM scheme.MJO produced by the ZM scheme is too weak and shows little propagation characteristics.Weak moisture convergence at low levels simulated by the ZM scheme is not enough to maintain the structure and the eastward propagation of the oscillation.These two cumulus schemes produced different vertical structures of the heating profile.The heating profile produced by the ZM scheme is nearly uniform with height and the heating is too weak compared to that produced by the MCA,which maybe contributes greatly to the failure of simulating a reasonable MJO.Comparing the simulated MJO by these two schemes indicate that the MJO simulated by the GCM is highly sensitive to cumulus parameterizations implanted in.The diabatic heating profile plays an important role in the performance of the GCM.Three sensitivity experiments with different heating profiles are designed in which modified heating profiles peak respectively in the upper troposphere(UH), middle troposphere(MH),and lower troposphere(LH).Both the LH run and the MH run produce eastward propagating signals on the intraseasonal timescale,while it is interesting that the intraseasonal timescale signals produced by the UH run propagate westward.It indicates that a realistic intraseasonal oscillation is more prone to be excited when the maximum heating concentrates in the middle-low levels,especially in the middle levels,while westward propagating disturbances axe more
Kumar, Dinesh; Mohanty, U. C.; Kumar, Krishan
The cloud processes play an important role in all forms of precipitation. Its proper representation is one of the challenging tasks in mesoscale numerical simulation. Studies have revealed that mesoscale feature require proper initialization which may likely to improve the convective system rainfall forecasts. Understanding the precipitation process, model initial condition accuracy and resolved/sub grid-scale precipitation processes representation, are the important areas which needed to improve in order to represent the mesoscale features properly. Various attempts have been done in order to improve the model performance through grid resolution, physical parameterizations, etc. But it is the physical parameterizations which provide a convective atmosphere for the development and intensification of convective events. Further, physical parameterizations consist of cumulus convection, surface fluxes of heat, moisture, momentum, and vertical mixing in the planetary boundary layer (PBL). How PBL and Cumulus schemes capture the evolution of thunderstorm have been analysed by taking thunderstorm cases occurred over Kolkata, India in the year 2011. PBL and cumulus schemes were customized for WSM-6 microphysics because WSM series has been widely used in operational forecast. Results have shown that KF (PBL scheme) and WSM-6 (Cumulus Scheme) have reproduced the evolution of surface variable such as CAPE, temperature and rainfall very much like observation. Further, KF and WSM-6 scheme also provided the increased moisture availability in the lower atmosphere which was taken to higher level by strong vertical velocities providing a platform to initiate a thunderstorm much better. Overestimation of rain in WSM-6 occurs primarily because of occurrence of melting and freezing process within a deeper layer in WSM-6 scheme. These Schemes have reproduced the spatial pattern and peak rainfall coverage closer to TRMM observation. It is the the combination of WSM-6, and KF schemes
Opinnäytetyön tavoitteena oli tutkia ja löytää hyviä ergonomiaratkaisuja Cumulus Hotel Lappeenrannan vastaanottoon ennen syksyllä 2017 alkavaa remonttia. Ergonomia tarkoittaa ihmisen ja työn yhteensovittamista. Sen tehtävä on tutkia ihmisen ja tekniikan sekä ympäristön vuorovaikutusta ja saada aikaan ihmiselle mieluisa ja toimiva toimintaympäristö työntekoa varten. Teoriaosuudessa käsitellään ergonomiaa käsitteenä ja avataan sen alalajeja fyysistä sekä kognitiivista ergonomiaa. Lisäksi e...
Full Text Available The cloud processes play an important role in all forms of precipitation. Its proper representation is one of the challenging tasks in mesoscale numerical simulation. Studies have revealed that mesoscale feature require proper initialization which may likely to improve the convective system rainfall forecasts. Understanding the precipitation process, model initial condition accuracy and resolved/sub grid-scale precipitation processes representation, are the important areas which needed to improve in order to represent the mesoscale features properly. Various attempts have been done in order to improve the model performance through grid resolution, physical parameterizations, etc. But it is the physical parameterizations which provide a convective atmosphere for the development and intensification of convective events. Further, physical parameterizations consist of cumulus convection, surface fluxes of heat, moisture, momentum, and vertical mixing in the planetary boundary layer (PBL. How PBL and Cumulus schemes capture the evolution of thunderstorm have been analysed by taking thunderstorm cases occurred over Kolkata, India in the year 2011. PBL and cumulus schemes were customized for WSM-6 microphysics because WSM series has been widely used in operational forecast. Results have shown that KF (PBL scheme and WSM-6 (Cumulus Scheme have reproduced the evolution of surface variable such as CAPE, temperature and rainfall very much like observation. Further, KF and WSM-6 scheme also provided the increased moisture availability in the lower atmosphere which was taken to higher level by strong vertical velocities providing a platform to initiate a thunderstorm much better. Overestimation of rain in WSM-6 occurs primarily because of occurrence of melting and freezing process within a deeper layer in WSM-6 scheme. These Schemes have reproduced the spatial pattern and peak rainfall coverage closer to TRMM observation. It is the the combination of WSM-6
Lord, S. J.
The verification of the Arakawa and Schubert (1974) cumulus parameterization is continued using a semiprognostic approach. Observed data from Phase III of GATE are used to provide estimates of the large-scale forcing of a cumulus ensemble at each observation time. Instantaneous values of the precipitation and the warming and drying due to cumulus convection are calculated using the parameterization. The results show that the calculated precipitation agrees very well with estimates from the observed large-scale moisture budget and from radar observations. The calculated vertical profiles of cumulus warming and drying also are quite similar to the observed. It is shown that the closure assumption adopted in the parameterization (the cloud-work function quasi-equilibrium) results in errors of generally less than 10% in the calculated precipitation. The sensitivity of the parameterization to some assumptions of the cloud ensemble model and the solution method for the cloud-base mass flux is investigated.
Full Text Available Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1 a decrease of the fertilization rate and index; and (2 a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.
Full Text Available Principle of oocyte cryoinjury is first overviewed and then research history of cryopreservation using bovine oocytes is summarized for the last two decades with a few special references to recent progresses. Various types of cryodevices have been developed to accelerate the cooling rate and applied to the oocytes from large domestic species enriched with cytoplasmic lipid droplets. Two recent approaches include the qualitative improvement of IVM oocytes prior to the vitrification and the short-term recovery culture of vitrified-warmed oocytes prior to the subsequent IVF. Supplementation of L-carnitine to IVM medium of bovine oocytes has been reported to reduce the amount of cytoplasmic lipid droplets and improve the cryotolerance of the oocytes, but it is still controversial whether the positive effect of L-carnitine is reproducible. Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes, was inhibited by a short-term culture of the postwarm oocytes in the presence of Rho-associated coiled-coil kinase (ROCK inhibitor. Use of an antioxidant α-tocopherol, instead of the ROCK inhibitor, also supported the revivability of the postwarm bovine oocytes. Further improvements of the vitrification procedure, combined with pre- and postvitrification chemical treatment, would overcome the high sensitivity of bovine oocytes to cryopreservation.
Angelier, N.; Moreau, N.A.; N' Da, E.A.; Lautredou, N.F. (Centre de Biologie Cellulaire, Ivry-sur-Seine (France))
Female Pleurodeles waltl newts (Amphibia, urodele), usually raised at 20 degrees C, were submitted to low temperatures; oocytes responded to this cold stress by drastic changes both in lampbrush chromosome structure and in protein pattern. Preexisting lateral loops of lampbrush chromosomes were reduced in size and number, while cold-induced loops which were tremendously developed, occurred on defined bivalents of the oocyte at constant, reproducible sites. A comparison of protein patterns in control and stressed oocytes showed two main differences: in stressed oocytes, overall protein synthesis was reduced, except for a set of polypeptides, the cold-stress proteins; second, there was a striking inversion of the relative amount of beta- and gamma-actin found in the oocyte nucleus before and after cold stress. Whereas beta-actin was the predominant form in control oocytes, gamma-actin became the major form in stressed oocytes.
Lisková, Lucie; Susor, Andrej; Pivonková, Katerina; Sasková, Adéla; Karabínová, Pavla; Kubelka, Michal
The multiprotein complexes known as condensins (I and II) are major players in chromosome dynamics in mitotic and meiotic cells. Here, we report for the first time the detection of different condensin subunits from both complexes in mammalian oocytes. Using immunoblotting analysis we examined expression levels of condensin subunits during meiotic maturation of porcine oocytes. The expression of the core subunit structural maintenance of chromosomes 2 (SMC2), identical in both condensin complexes, did not change significantly during maturation. Similarly, there was no significant change in the expression of the chromosome associated protein (CAP)-H and CAP-H2 subunits, components of condensin I and II, respectively. Conversely, the expression profiles of CAP-G, CAP-D2 (condensin I) and CAP-D3 (condensin II) were more interesting. At least two isoforms of the CAP-D2 subunit were detected, along with three isoforms of the CAP-D3 and CAP-G subunits. We suggest that this diverse migration of subunit isoforms is due to post-translational modification. Earlier, it was reported that non-SMC proteins are phosphorylated by cyclin-dependent kinase 1. In the present study, we analysed the phosphorylation status of the three subunits in oocyte extracts using alkaline phosphatase treatment and we found that at least the fastest migrating form of CAP-D3 was likely to be phosphorylated in maturing porcine oocytes. In addition, the localisation of CAP-H and CAP-H2 subunits was examined using immunofluorescence staining with specific antibodies, as well as following microinjection of their enhanced green fluorescent protein-tagged mRNA into germinal vesicle-stage oocytes. CAP-H was found in the cytoplasm, whereas CAP-H2 was localised within the nucleus.
Zhang, Xia; Liu, Xiaoyan; Chen, Li; Wu, Dan-Ya; Nie, Zheng-Wen; Gao, Ying-Ying; Miao, Yi-Liang
Caffeine, as an oocyte aging inhibitor, was used in many different species to control or delay oocyte aging. However, the safety of caffeine and developmental competence of aged oocytes inhibited by caffeine has not been studied systematically. So we detected the spindle morphology, distribution of cortical granules, zona pellucida hardening and pronucleus formation to assess oocyte quality of caffeine treated oocytes. We found that aged oocytes treated by caffeine maintained weak susceptibility to activating stimuli and regained normal competent after aged further 6 hr. Caffeine maintained the spindle morphology, changed cortical granules distribution of aged oocytes and could not prevent zona pellucida hardening. Furthermore, caffeine increased pronucleus formation of aged oocytes and decreased fragmentation after fertilization. These results suggested that caffeine could maintain the quality of aged oocytes safely in mouse.
Bogolyubov, D S; Batalova, F M; Ogorzałek, A
An oocyte nucleus contains different extrachromosomal nuclear domains collectively called nuclear bodies (NBs). In the present work we revealed, using immunogold labeling electron microscopy, some marker components of interchromatin granule clusters (IGCs) and Cajal bodies (CBs) in morphologically heterogeneous oocyte NBs studied in three hemipteran species: Notostira elongata, Capsodes gothicus (Miridae) and Velia caprai (Veliidae). Both IGC and CB counterparts were revealed in oocyte nuclei of the studied species but morphological and biochemical criteria were found to be not sufficient to determine carefully the define type of oocyte NBs. We found that the molecular markers of the CBs (coilin and non-phosphorylated RNA polymerase II) and IGCs (SC35 protein) may be localized in the same NB. Anti-SC35 antibody may decorate not only a granular material representing "true" interchromatin granules but also masks some fibrillar parts of complex NBs. Our first observations on the hemipteran oocyte NBs confirm the high complexity and heterogeneity of insect oocyte IGCs and CBs in comparison with those in mammalian somatic cells and amphibian oocytes.
Swain, J E; Smith, G D
The mammalian oocyte is notorious for high rates of chromosomal abnormalities. This results in subsequent embryonic aneuploidy, resulting in infertility and congenital defects. Therefore, understanding regulatory mechanisms involved in chromatin remodeling and chromosome segregation during oocyte meiotic maturation is imperative to fully understand the complex process and establish potential therapies. This review will focus on major events occurring during oocyte meiosis, critical to ensure proper cellular ploidy. Mechanistic and cellular events such as chromosome condensation, meiotic spindle formation, as well as cohesion of homologues and sister chromatids will be discussed, focusing on the role of reversible phosphorylation in control of these processes.
Jin, J X; Lee, S; Khoirinaya, C; Oh, A; Kim, G A; Lee, B C
Spermine plays an important role in protection from reactive oxygen species (ROS) in bacteria, yeast, and mammalian cells, but there are few studies on the effects of spermine on porcine oocyte maturation and subsequent embryo development. The aim of this study was to determine the effects of spermine on in vitro maturation (IVM) of porcine oocytes and their developmental competence after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). We evaluated nuclear maturation, intracellular glutathione (GSH), and ROS levels in oocytes, and their subsequent embryonic development, as well as gene expression in mature oocytes, cumulus cells, and PA blastocysts. After treatment with various concentrations of spermine in IVM culture medium, there was no significant difference in nuclear maturation rate. However, spermine treatment groups (10- 500 µM) showed significantly increased intracellular GSH levels and decreased ROS levels compared to the control ( cells ( < 0.05). was increased in spermine-treated oocytes. Levels of transcription for and were significantly increased in PA blastocysts. In conclusion, 10 µM spermine supplementation during IVM improved the development of porcine PA and SCNT embryos by increasing intracellular GSH, scavenging ROS levels, and regulating gene expression.
Since the beginning of IVF, cryopreservation concern spermatozoa or embryos due to the poor efficiency of oocyte freezing. To date, oocyte vitrification allows changing our practice privileging female gamete vitrification instead of human embryo freezing.
Shao, Hua; Ma, Chunqi; Zhang, Xuan; Li, Ruizhen; Miller, Ann L; Bement, William M; Liu, X Johné
Aurora B (Aur-B) plays multiple roles in mitosis, of which the best known are to ensure bi-orientation of sister chromatids by destabilizing incorrectly attached kinetochore microtubules and to participate in cytokinesis. Studies in Xenopus egg extracts, however, have indicated that Aur-B and the chromosome passenger complex play an important role in stabilizing chromosome-associated spindle microtubules. Aur-B stabilizes spindle microtubules in the egg extracts by inhibiting the catastrophe kinesin MCAK. Whether or not Aur-B plays a similar role in intact oocytes remains unknown. Here we have employed a dominant-negative Aur-B mutant (Aur-B122R, in which the ATP-binding lysine(122) is replaced with arginine) to investigate the function of Aur-B in spindle assembly in Xenopus oocytes undergoing meiosis. Overexpression of Aur-B122R results in short bipolar spindles or monopolar spindles, with higher concentrations of Aur-B122R producing mostly the latter. Simultaneous inhibition of MCAK translation in oocytes overexpressing Aur-B122R results in suppression of monopolar phenotype, suggesting that Aur-B regulates spindle bipolarity by inhibiting MCAK. Furthermore, recombinant MCAK-4A protein, which lacks all four Aur-B phosphoryaltion sites and is therefore insensitive to Aur-B inhibition but not wild-type MCAK, recapitulated the monopolar phenotype in the oocytes. These results suggest that in vertebrate oocytes that lack centrosomes, one major function of Aur-B is to stabilize chromosome-associated spindle microtubules to ensure spindle bipolarity.
Wissing, Marie Louise; Sonne, Si Brask; Westergaard, David;
individual oocytes developing into embryos selected for transfer. CRCs were isolated in a two-step denudation procedure, separating outer cumulus cells from the inner CRCs. Extracted RNA was amplified and transcriptome profiling was performed with Human Agilent® arrays. The transcriptomes of CRCs showed......-related genes and cell cycle pathways in PCOS CRCs could indicate a disturbed or delayed final maturation and differentiation of the CRCs in response to the human chorionic gonadotropin (hCG) surge. However, this had no effect on the in vitro development of the corresponding embryos. Future studies are needed....... It is controversial whether PCOS associate with diminished oocyte quality. The purpose of this study was to compare individual human CRC samples between PCOS patients and controls. All patients were stimulated by the long gonadotropin-releasing hormone (GnRH) agonist protocol. The CRC samples originated from...
Wissing, Marie Louise; Sonne, Si Brask; Westergaard, David;
. It is controversial whether PCOS associate with diminished oocyte quality. The purpose of this study was to compare individual human CRC samples between PCOS patients and controls. All patients were stimulated by the long gonadotropin-releasing hormone (GnRH) agonist protocol. The CRC samples originated from...... individual oocytes developing into embryos selected for transfer. CRCs were isolated in a two-step denudation procedure, separating outer cumulus cells from the inner CRCs. Extracted RNA was amplified and transcriptome profiling was performed with Human Agilent® arrays. The transcriptomes of CRCs showed...... no individual genes with significant differential expression between PCOS and controls, but gene set enrichment analysis identified several cell cycle- and DNA replication pathways overexpressed in PCOS CRCs (FDR
Peter, J. R.; S. T. Siems; Jensen, J.B.; Gras, J. L.; Ishizaka, Y.; Hacker, J. M.
International audience; Airborne measurements, obtained during the Asian Aerosol Characterisation Experiment (ACE-Asia), of SO2 and condensation nuclei (CN) concentrations were made in the local environment of a cumulus cloud band. Conserved quantities, wet equivalent potential temperature ?q, and total water content Q, were used to identify the sources of air detrained on the downwind side of the cumulus band. It was found that ~65% of the detrained air originated from below cloud base and t...
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 foll
Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K
Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.
Montserrat, Pallas Seijas
Cryopreservation ofhuman oocytes to delay fertility also be an option for women who are going to be subjected to a cancer/autoimmune treatment. It allows for creating a bank of oocytes for donation in assisted reproduction centers. The legislation allows the use of cryopreserved oocytes throughout the reproductive life of women with what conservation could last up to 48-50 years. Oocyte vitrification is a ultrafast freezing method in which cryoprotectants are used to prevent the formation of ice crystals within the cell. Treatment for oocyte vitrification process is similar to IVF treatment, ending at the time of obtaining the ova. The eggs obtained in the laboratory are classified according to maturity and quality. The apartments will be cryopreserved by vitrification technique tanks and maintained in liquid nitrogen until used for reproductive purposes.
Motlík, J; Sutovský, P; Kalous, J; Kubelka, M; Moos, J; Schultz, R M
Bovine cumulus-enclosed oocytes, initially cultured up to diakinesis (8 h of initial culture) or metaphase I (12 h of initial culture), were subsequently co-cultured for 6 h in contact with pig membrana granulosa (PMG) cells and then assayed for histone H1 and MAP kinase activities. In addition, the phosphorylation state of ERK 1,2 proteins was determined by Western blotting. The alterations in nuclear envelope breakdown, meiotic spindle formation and the patterns of chromosome condensation were analysed by immunofluorescence and transmission electron microscopy. The diakinesis-stage oocytes (initially cultured for 8 h) already possessed high histone H1 kinase and MAP kinase activities that were correlated with condensed and partially individualised chromosomes. The ERK 1 and most ERK 2 proteins were partly phosphorylated. Following the 6 h co-culture of these oocytes with PMG a rapid decrease in MAP kinase activity and a slower decrease in histone H1 kinase occurred, as well as ERK 1 and ERK 2 dephosphorylation. Both kinase activities and ERK 1,2 phosphorylation were fully restored following the release of the oocytes from co-culture and a subsequent culture in the absence of PMG. Moreover, the clumped bivalents were reindividualised and 56% of these oocytes reached metaphase II after 20 h of culture without PMG. The metaphase I oocytes, initially cultured for 12 h, displayed a fusiform meiotic spindle and a metaphase array of chromosomal bivalents, accompanied by high levels of both histone H1 and MAP kinase activity. Co-culture of MI oocytes with PMG abolished the activity of both kinases and caused the dephosphorylation of ERK 1 and ERK 2. Furthermore, the spindle microtubules were depolymerised and the chromosomal bivalents clumped into a single mass. Neither of the protein kinase activities nor the meiotic spindle were restored following subsequent culture in the absence of PMG for up to 20 h. These observations indicate that under in vitro conditions
Zahra Khodabandeh Jahromi
Full Text Available Objective: The aim of this study was to investigate the effects of two vitrification protocolson mouse metaphase stage II (MII oocytes and evaluate their effects on the expressionof heat shock protein A1A (HSP A1A and MnSOD genes.Materials and Methods: Groups of approximately 15 oocytes without cumulus complexeswere collected and vitrified with 10% (v/v ethylene glycol (EG + 10% (v/v dimethylsulphoxide(DMSO + 0.5M sucrose in group A (VSI and 14.5% (v/v EG + 14.5% PROH+ 0.5M sucrose in group B (VSII, respectively. Thawing after vitrification was performdby placing the oocytes into 1M sucrose for 1 minute and two diluted solutions, each for3 minutes. After thawing, the oocytes were fertilized and cultured in vitro to develop intothe pronuclear stage. The survival rate of vitrified-warmed oocytes and rate of fertilizationwere evaluated. In addition, gene expressions (HSP A1A, MnSOD and β actin of vitrifiedwarmedoocytes were also examined by reverse transcription polymerase chain reaction(RT-PCR.Results: Survival rates of each group were separately compared to the control. The resultshowed significant differences between each experimental group compared to the control(p≤ 0.001. The survival rate of oocytes after warming was higher in VSI (91.2% ± 1.7compared to VSII (89.2% ± 1.5 but there were no significant differences between the twogroups. The rate of fertilization was significantly (p≤0.05 reduced in vitrified-warmed (VSI:39% ± 5.8; VSII: 34% ± 5.7 oocytes compared to the control (88.36% ± 2.3. The expressionof MnSOD increased in the vitrified-warmed oocytes when compared to controloocytes. We also detected HSP A1A only in the control and VSI group.Conclusion: Vitrification of oocytes by cryotop resulted in high survival rate; low developmentalcompetence and fertilization rate of vitrified-warmed oocytes which may be a resultof changing expression of important genes after thawing.
Full Text Available Background & objectives: The major cause of fertilisation failure after ICSI is failure of the oocyte to initiate the biochemical processes necessary for activation. This inability could be ascribed to cytoplasmic immaturity of those gametes even if they had reached nuclear maturity. The activation of a mature oocyte is characterised by release from metaphase II (MII arrest and extrusion of the second polar body, followed by pro-nuclear formation. The aim of this study was to evaluate the fate of in vitro matured (IVM metaphase I (MI oocytes subjected to intracytoplasmic sperm injection (ICSI at different time intervals after extrusion of the first polar body (1PB in in vitro fertilization (IVF cycles. Methods: A total of 8030 oocytes were collected from 1400 ICSI cycles, 5504 MII at the time of cumulus retrieval. Four hundred eight metaphase II (MII (27.1% matured to MII after in vitro culture for 2-26 h and 5389 sibling MII in the moment of oocyte denudation were injected. On the other hand, 49 ICSI cycles containing only MI oocytes at retrieval were injected at three different time intervals after reaching the MII. The intervals were as follows: 2-6 h (n=10, 8-11 h (n=4 and 23-26 h (n=10. Fertilization and development potential were evaluated in both studies. Results: Fertilization, embryo cleavage and quality were significantly lower in IVM MI compared to MII at time of denudation. Pregnancy rate was higher in group MII. Pregnancy was achieved in three embryo transfers when ICSI was performed within 2-6 h (group I and 8-11 h (group II after PB extrusion. One pregnancy was obtained in group I and a healthy neonate was born. Interpretation & conclusions: Immature oocytes from women whose ovaries have been stimulated could be matured, fertilized by ICSI, cleaved in vitro and to give rise to a live birth. However, the developmental competence of embryos derived from immature oocytes is reduced, compared with sibling in vivo matured oocytes
Sheffield, A. M.; van den Heever, S. C.; Saleeby, S. M.
Tropical convection has been observed to contain three modes of convection, the middle mode of which is cumulus congestus clouds. This study investigates the impacts of aerosol, specifically those aerosols that can serve as cloud condensation nuclei, on the growth and development of congestus clouds observed within idealized cloud-resolving model (CRM) simulations conducted under a state of radiative-convective equilibrium (RCE). The model employed here is the Regional Atmospheric Modeling System (RAMS). RAMS CRM simulations were completed using a large two-dimensional domain (7200 km) at fine resolution (1 km) and long duration (100 days). Results indicate that congestus in more polluted conditions extend to greater heights more frequently than those developing in clean cases. Greater cloud water mass and ice mass forms in more polluted conditions, though ice forms at a fraction of the rate of the cloud mass. The importance of vapor diffusional growth of cloud droplets in the more polluted conditions is highlighted as one such process contributing to congestus development through latent heat release.
Sana N Khan
Full Text Available Hydrogen peroxide (H2O2 is a relatively long-lived signaling molecule that plays an essential role in oocyte maturation, implantation, as well as early embryonic development. Exposure to relatively high levels of H2O2 functions efficiently to accelerate oocyte aging and deteriorate oocyte quality. However, little precise information exists regarding intra-oocyte H2O2 concentrations, and its diffusion to the oocyte milieu. In this work, we utilized an L-shaped amperometric integrated H2O2-selective probe to directly and quantitatively measure the real-time intra-oocyte H2O2 concentration. This investigation provides an exact measurement of H2O2 in situ by reducing the possible loss of H2O2 caused by diffusion or reactivity with other biological systems. This experiment suggests that the intra-oocyte H2O2 levels of oocytes obtained from young animals are reasonably high and remained constant during the procedure measurements. However, the intra-oocyte H2O2 concentration dropped significantly (40-50% reduction in response to catalase pre-incubation, suggesting that the measurements are truly H2O2 based. To further confirm the extracellular diffusion of H2O2, oocytes were incubated with myeloperoxidase (MPO, and the diffused H2O2 triggered MPO chlorinating activity. Our results show that the generated hypochlorous acid (HOCl facilitated the deterioration in oocyte quality, a process that could be prevented by pre-incubating the oocytes with melatonin, which was experimentally proven to be oxidized utilizing HPLC methods. This study is the first to demonstrate direct quantitative measurement of intracellular H2O2, and its extracellular diffusion and activation of MPO as well as its impact on oocyte quality. These results may help in designing more accurate treatment plans in assisted reproduction under inflammatory conditions.
Pirro, Valentina; Oliveri, Paolo; Ferreira, Christina Ramires; González-Serrano, Andrés Felipe; Machaty, Zoltan; Cooks, Robert Graham
The development of sensitive measurements to analyze individual cells is of relevance to elucidate specialized roles or metabolic functions of each cell under physiological and pathological conditions. Lipids play multiple and critical roles in cellular functions and the application of analytical methods in the lipidomics area is of increasing interest. In this work, in vitro maturation of porcine oocytes was studied. Two independent sources of chemical information (represented by mass spectra in the positive and negative ion modes) from single oocytes (immature oocytes, 24-h and 44-h in vitro matured oocytes) were acquired by using desorption electrospray ionization-mass spectrometry (DESI-MS). Low and mid-level data fusion strategies are presented with the aim of better exploring the large amount of chemical information contained in the two mass spectrometric lipid profiles. Data were explored by principal component analysis (PCA) within the two multi-block approaches to include information on free fatty acids, phospholipids, cholesterol-related molecules, di- and triacylglycerols. After data fusion, clearer differences among immature and in vitro matured porcine oocytes were observed, which provide novel information regarding lipid metabolism throughout oocyte maturation. In particular, changes in TAG composition, as well as increase in fatty acid metabolism and membrane complexity were evidenced during the in vitro maturation process. This information can assist the improvement of in vitro embryo production for porcine species.
Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei
Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.
Motohashi, Hideyuki H; Taniguchi, Ryoma; Sakamoto, Junpei; Sankai, Tadashi; Kada, Hidemi
For in vitro growth and maturation of mouse oocytes (IVG-IVM), serum is added to media up to and including the stage of oocyte maturation; this subsequently supports oocytes through fertilization and early embryo development. However, problems may occur with sera, such as batch differences and issues of biosafety. The purpose of the present study was to determine the capacity for fertilization and pre- and post-implantation development of oocytes that underwent IVG-IVM with a serum substitute. Oocyte-granulosa cell complexes from preantral follicles were cultured in medium with either fetal bovine serum (FBS), Serum Substitute Supplement™ (SSS), or Knockout™ Serum Replacement (KSR) for 10days, and were then allowed to mature for 17 h. Subsequently, more than 90% of oocytes underwent germinal vesicle breakdown (GVBD) and more than 70% reached metaphase II, with no significant difference between the groups. A lower fertilization rate, presumably due to zona hardening, was found in the serum substitute groups. Nevertheless, more than 50% of the inseminated oocytes were fertilized and 35%-45% of them underwent first cleavage and developed to the blastocyst stage. Following embryo transfer, one and four live offspring were produced from the SSS and KSR groups, respectively. The present study demonstrated that murine IVG-IVM oocytes cultured in media with a serum substitute, achieved fertilization in vitro, pre- and post-implantation development, and the delivery of live pups, although the efficiency of the process is reduced compared to FBS supplementation. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
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.
Full Text Available Polo-like kinase 1 (PLK1 orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1's functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC. Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.
鲍晓; 徐家伟; 孙莹璞
缺乏准确、客观、非侵入性的胚胎评估标准是目前体外受精-胚胎移植技术所面临的主要挑战之一。本文对卵丘细胞内基因表达与胚胎及卵子发育潜能之间的相关研究进行了总结，发现卵丘细胞内特定基因的表达与胚胎发育、成熟及发育潜能之间存在紧密的联系。近年来，通过实时定量逆转录聚合酶链反应（qRT-PCR）及微阵列技术筛选出一系列颗粒细胞中能够预测受精结局、胚胎形态学评分、妊娠结局等胚胎发育潜能的候选标记基因，这些基因主要涉及卵丘扩展、脂类代谢、细胞凋亡等过程。然而，卵母细胞及胚胎的发育受卵泡内、外多种因素的影响。欲确立一种全面、准确、非侵入性的胚胎发育潜能评估方法仍需更深入的研究。%One of the major challenges in in-vitro fertilization-embryo transfer (IVF-ET) technology is lack of an objective, accurate, noninvasive criteria for evaluating the developmental potential of embryo. We reviewed here the expressions of those genes in cumulus cells related to the developmental potential of oocyte, suggesting that there was a close relationship between the expression of some specific genes in human cumulus cells and oocyte development, maturation, and embryo developmental potential. Recently, using qRT-PCR and microarray technologies, a series of candidate genes in cumulus and granulosa cells were found to be possible markers to predict the developmental potential of embryo, such as those genes related to cumulus expansion, lipid metabolism, cell apoptosis and other aspects, which could be useful to predict fertilization outcomes, embryo morphology and pregnant outcomes. However, the development of oocyte and embryo could be affected by various factors. It is necessary to do more detailed study to develop a accurate, comprehensive and non-invasive test for evaluating the developmental potential of oocyte and embryo.
Itami, Nobuhiko; Munakata, Yasuhisa; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka
In vitro culture of the oocyte granulosa cell complexes (OGCs) from early antral follicles (EAFs) shows granulosa cell (GC) proliferation, but to a lesser extent than that observed in vivo during follicle development. As the number of GCs closely relates to energy sufficiency of the oocytes, enhancement of GC proliferation influences oocyte development. GC proliferation depends on glycolysis and insulin-mediated AKT/mTOR signaling pathway; therefore, addition of culture medium containing insulin and glucose may potentially promote GC proliferation and hence improve oocyte development. In the present study, we assessed the effect of exogenous insulin and glucose concentration on GC proliferation and oocyte energy status as well as developmental abilities of porcine oocytes grown in vitro. In the presence of 5.5 mM of glucose (Low), a comparison of 10 versus 20 μg/ml insulin showed that high insulin enhanced GC proliferation but exhausted glucose from the medium, which resulted in low energy status including lipid and adenosine triphosphate of the oocyte. Whereas, in the presence of 20 μg/ml insulin, medium with 11 mM glucose (High) enhanced GC proliferation and oocyte energy status as well as developmental ability up to the blastocyst stage. Considering that there was no difference in OGCs development observed with medium (10 μg/ml insulin) containing 5.5 versus 11 mM glucose, we concluded that the combination of high insulin and glucose enhanced GC proliferation and energy status of oocytes as well as the developmental ability of the oocytes grown in vitro.
Flemr, Matyas; Ma, Jun; Schultz, Richard M.; Svoboda, Petr
In mammalian somatic cells, several pathways that converge on deadenylation, decapping, and 5'-3' degradation are found in cytoplasmic foci known as P-bodies. Because controlled mRNA stability is essential for oocyte-to-zygote transition, we examined the dynamics of P-body components in mouse oocytes. We report that oocyte growth is accompanied by loss of P-bodies and a subcortical accumulation of several RNA-binding proteins, including DDX6, CPEB, YBX2 (MSY2), and the exon junction complex. ...
Laird, John L.; Harshvardhan
Clouds are extremely important with regard to the transfer of solar radiation at Earth's surface. This study investigates Cumulus Solar Irradiance Reflection (CSIR) using ground-based pyranometers. CSIR events are short-term increases in solar radiation observed at the surface as a result of reflection off the sides of convective clouds. When Sun-cloud observer geometry is favorable, these occurrences produce characteristic spikes in the pyranometer traces and solar irradiance values may exceed expected clear-sky values. Ultraviolet CSIR events were investigated during the summer of 1995 using UVA and UVB pyranometers. Observed data were compared to clear-sky curves which were generated using a third degree polynomial best-fit line technique. Periods during which the observed data exceeded this clear-sky curve were identified as CSIR events. The magnitude of a CSIR event was determined by two different quantitative calculations. The MAC (magnitude above clear-sky) is an absolute measure of the difference between the observed and clear-sky irradiances. Maximum MAC values of 3.4 Win -2 and 0.0169 Wm -2 were observed at the UV-A and UV-B wavelengths, respectively. The second calculation determined the percentage above clear-sky (PAC) which indicated the relative magnitude of a CSIR event. Maximum UV-A and UV-B PAC magnitudes of 10.1% and 7.8%, respectively, were observed during the study. Also of interest was the duration of the CSIR events which is a function of Sun-cloud-sensor geometry and the speed of cloud propagation over the measuring site. In both the UV-A and UV-B wavelengths, significant CSIR durations of up to 30 minutes were observed. C 1997 Elsevier Science B.V.
Pirro, Valentina, E-mail: firstname.lastname@example.org [Department of Chemistry, University of Turin, Via Pietro Giuria 7, Turin 10125 (Italy); Oliveri, Paolo [Department of Pharmacy, University of Genoa, Via Brigata Salerno 13, Genoa 16147 (Italy); Ferreira, Christina Ramires [Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907 (United States); González-Serrano, Andrés Felipe [Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Hoeltystrasse 10, 31535 Neustadt, Mariensee (Germany); Machaty, Zoltan [Department of Animal Sciences, Purdue University, 915 W. State St., West Lafayette, IN 47907 (United States); Cooks, Robert Graham [Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907 (United States)
Highlights: • Repeated analysis by DESI(±)-MS of intact single oocytes for lipid characterization. • Deployment of a data fusion strategy to merge positive and negative ion mode data. • Enhanced interpretation of metabolic changes by more efficient analysis of spectral data. • Discovery of increased fatty acid metabolism and membrane complexity during maturation. • Assistance in the improvement of in vitro embryo production for porcine species. - Abstract: The development of sensitive measurements to analyze individual cells is of relevance to elucidate specialized roles or metabolic functions of each cell under physiological and pathological conditions. Lipids play multiple and critical roles in cellular functions and the application of analytical methods in the lipidomics area is of increasing interest. In this work, in vitro maturation of porcine oocytes was studied. Two independent sources of chemical information (represented by mass spectra in the positive and negative ion modes) from single oocytes (immature oocytes, 24-h and 44-h in vitro matured oocytes) were acquired by using desorption electrospray ionization-mass spectrometry (DESI-MS). Low and mid-level data fusion strategies are presented with the aim of better exploring the large amount of chemical information contained in the two mass spectrometric lipid profiles. Data were explored by principal component analysis (PCA) within the two multi-block approaches to include information on free fatty acids, phospholipids, cholesterol-related molecules, di- and triacylglycerols. After data fusion, clearer differences among immature and in vitro matured porcine oocytes were observed, which provide novel information regarding lipid metabolism throughout oocyte maturation. In particular, changes in TAG composition, as well as increase in fatty acid metabolism and membrane complexity were evidenced during the in vitro maturation process. This information can assist the improvement of in vitro embryo
De Vico, G; Peretti, V; Losa, G A
The present study aimed at verifying whether immature cat oocytes with morphologic irregular cytoplasm display self-similar features which can be analytically described by fractal analysis. Original images of oocytes collected by ovariectomy were acquired at a final magnification of 400x with a CCD video camera connected to an optic microscope. After greyscale thresholding segmentation of cytoplasm, image profiles were submitted to fractal analysis using FANAL++, a program which provided an analytical standard procedure for determining the fractal dimension (FD). The presentation of the oocyte influenced the magnitude of the fractal dimension with the highest FD of 1.91 measured on grey-dark cytoplasm characterized by a highly connected network of lipid droplets and intracellular membranes. Fractal analysis provides an effective quantitative descriptor of the real cytoplasm morphology, which can influence the acquirement of in vitro developmental competence, without introducing any bias or shape approximation and thus contributes to an objective and reliable classification of feline oocytes.
G De Vico
Full Text Available The present study aimed at verifying whether immature cat oocytes with morphologic irregular cytoplasm display selfsimilar features which can be analytically described by fractal analysis. Original images of oocytes collected by ovariectomy were acquired at a final magnification of 400 X with a CCD video camera connected to an optic microscope. After greyscale thresholding segmentation of cytoplasm, image profiles were submitted to fractal analysis using FANAL++, a program which provided an analytical standard procedure for determining the fractal dimension (FD. The presentation of the oocyte influenced the magnitude of the fractal dimension with the highest FD of 1.91 measured on grey-dark cytoplasm characterized by a highly connected network of lipid droplets and intracellular membranes. Fractal analysis provides an effective quantitative descriptor of the real cytoplasm morphology, which can influence the acquirement of in vitro developmental competence, without introducing any bias or shape approximation and thus contributes to an objective and reliable classification of feline oocytes.
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). (auth)
Full Text Available Normal female fertility depends on normally occuring oogenesis and maturation progress. Oogenesis and folliculogenesis are different progresses but occure in a harmony and at the same time. Oogenesis includes the events that take place matur ovum produced from primordial germ cells. Although folliculogenesis includes the stages primordial, primary, secondary, matur (Graaf follicules in the influece of gonadotropines and local growth factors. During oocyte maturation meiosis is distrupted till the puberty. Under LH influence it starts again and first meiosis completes before ovulation. Oocyte maturation can be regarded as the process of coming metaphase II from prophase I of oocyte at the puberty and can be studied as nuclear and cytoplasmic maturation. Meiosis is completed when fertilization occures and zygot is formed. In this article oogenesis, folliculogenesis and oocyte maturation process are summerized with related studies and reiews are revised. [Archives Medical Review Journal 2009; 18(4.000: 227-240
J Konc; S Cseh; E Varga; R Kriston; K Kanyó
Embryo cryopreservation(CP) has became a very important part of the clinical use of in vitro fertilization. Oocyte CP offers more advantages compared with embryo freezing with regard to less ethical, legal and moral problems. However, the efficiency of this procedure is still low, which prevents its clinical application in wide range. The aim of our paper is to review the basic principles, technical and safety aspects and current status of oocyte cryopreservation in human assisted reproduction.
Seidler, Emily A; Moley, Kelle H
Mitochondrial production of cellular energy is essential to oocyte function, zygote development and successful continuation of pregnancy. This review focuses on several key functions of healthy oocyte mitochondria and the effect of pathologic states such as aging, oxidative stress and apoptosis on these functions. The effect of these abnormal conditions is presented in terms of clinical presentations, specifically maternal obesity, diminished ovarian reserve and assisted reproductive technologies.
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.
Sud, Y. C.; Chao, Winston C.; Walker, G. K.
The roles of the Critical Cloud Work Function (CCWF) data set and the upper and lower bounds on entrainment by cumulus plumes in the Arakawa-Schubert cumulus parameterization (ASCP) in the GLA GCM (Geller et al., 1988) were investigated in two sets of experiments. It was found that the horizontal and vertical distribution of cumulus heating can be altered in ASCP by adjusting these parameters. These changes can have a strong influence on the vertical structure of condensation heating, water vapor distribution, temperature, and rainfall. The CCWF is an important limiting parameter that controls the onset of different cloud types; increasing the threshold values of CCWF for all clouds tends to concentrate the rainfall into a narrower ITCZ and affects the rainfall during the initial adjustment period.
Alvarez Sedó, Cristian; Schatten, Heide; Combelles, Catherine M; Rawe, Vanesa Y
The oocyte's meiotic spindle is a dynamic structure that relies on microtubule organization and regulation by centrosomes. Disorganization of centrosomal proteins, including the nuclear mitotic apparatus (NuMA) protein and the molecular motor complex dynein/dynactin, can lead to chromosomal instability and developmental abnormalities. The present study reports the distribution and function of these proteins in human oocytes, zygotes and early embryos. A total of 239 oocytes, 90 zygotes and discarded embryos were fixed and analyzed with confocal microscopy for NuMA and dynactin distribution together with microtubules and chromatin. Microtubule-associated dynein-dependent transport functions were explored by inhibiting phosphatase and ATPase activity with sodium-orthovanadate (SOV). At germinal vesicle (GV) stages, NuMA was dispersed across the nucleoplasm. After GV breaks down, NuMA became cytoplasmic before localizing at the spindle poles in metaphase I and II oocytes. Aberrant NuMA localization patterns were found during oocyte in vitro maturation. After fertilization, normal and abnormal pronuclear stage zygotes and embryos displayed translocation of NuMA to interphase nuclei. SOV treatment for up to 2 h induced lower maturation rates with chromosomal scattering and ectopic localization of NuMA. Accurate distribution of NuMA is important for oocyte maturation, zygote and embryo development in humans. Proper assembly of NuMA is likely necessary for bipolar spindle organization and human oocyte developmental competence.
Flemr, Matyas; Ma, Jun; Schultz, Richard M; Svoboda, Petr
In mammalian somatic cells, several pathways that converge on deadenylation, decapping, and 5'-3' degradation are found in cytoplasmic foci known as P-bodies. Because controlled mRNA stability is essential for oocyte-to-zygote transition, we examined the dynamics of P-body components in mouse oocytes. We report that oocyte growth is accompanied by loss of P-bodies and a subcortical accumulation of several RNA-binding proteins, including DDX6, CPEB, YBX2 (MSY2), and the exon junction complex. These proteins form transient RNA-containing aggregates in fully grown oocytes with a surrounded nucleolus chromatin configuration. These aggregates disperse during oocyte maturation, consistent with recruitment of maternal mRNAs that occurs during this time. In contrast, levels of DCP1A are low during oocyte growth, and DCP1A does not colocalize with DDX6 in the subcortical aggregates. The amount of DCP1A markedly increases during meiosis, which correlates with the first wave of destabilization of maternal mRNAs. We propose that the cortex of growing oocytes serves as an mRNA storage compartment, which contains a novel type of RNA granule related to P-bodies.
Camargos, Maria das Graças R S; Lobach, Veronica N M; Pereira, Francisco A N; Lemos, Cláudia N C D; Reis, Fernando M; Camargos, Aroldo F
The present study aimed to correlate morphometric parameters of the oocytes with the occurrence of fertilization following intracytoplasmic sperm injection (ICSI). In a prospective, controlled cohort design, women (n = 32) who were candidates for ICSI had oocytes (n = 258) collected and submitted to morphometric evaluation using the Cronus3 software program. The morphometric parameters obtained were oocyte diameter, perivitelline space width, zona pellucida thickness, and first polar body diameter. The median oocyte diameter was similar in cases in which fertilization occurred compared with those in which fertilization failed (75.2 and 75.9 μm, respectively; P = .218). The 2 groups also had similar measurements of perivitelline space, zona pellucida, and first polar body. However, the best quality zygotes identified by a morphological score resulted from oocytes with larger diameter (75.6 vs 74.0 μm; P < .01) and narrow perivitelline space (5.3 vs 7.1 μm; P < .01). Embryo development, as assessed by cleavage at second day of culture, was not significantly associated with oocyte morphometric parameters. These findings suggest that morphometric parameters of the oocytes do not correlate with the occurrence of fertilization following ICSI but may assist in selecting oocytes more likely to originate high-quality zygotes.
Lindsey, L.; Randall, D. A.; Dazlich, D.
The horizontal resolution of cumulus cloud simulations not only affects the computational cost of running a cloud resolving model (CRM), but it affects the results of the model as well. It is necessary to find the coarsest resolution that can be used without compromising the accuracy of the results. This study was carried out using the System for Atmospheric Modeling (SAM), a three-dimensional cloud resolving model. The forcing data used for the model came from two different field campaigns, the GARP Atlantic Tropical Experiment (GATE) and Atmospheric Radiation Measurement Program (ARM) at the Southern Great Plains site. GATE took place during the summer of 1974 over the tropical Atlantic Ocean. The ARM field campaign took place during the summer of 1997. A constant large scale forcing was applied to the temperature and moisture fields for the GATE case. The model was run until the atmospheric water content stabilized. With the ARM forcing data, a diurnal cycle of solar insolation was applied. The model was run until the soil moisture adjusted. For both GATE and ARM, a 20-day period was analyzed. The effects of horizontal grid spacing on rainfall rates, updraft and downdraft velocities, and cloud cover are presented in this study. Grid spacings of 8 km, 4 km, 2 km, and 500 m were studied for the GATE runs, and 4 km, 2 km, 1 km, and 500 m were studied for the ARM runs. For the GATE runs, resolution does not have a great impact on rainfall rates. A more radical effect is seen on updrafts and downdrafts, which intensify with higher resolutions. The overall cloud cover for the GATE runs decrease with increased resolution. The ARM runs are more sensitive to horizontal resolution than the GATE runs. Domain averaged rainfall rates decrease with increased resolution. Local rainfall rates, on the contrary, increase with increased resolution. In the ARM runs updrafts and downdrafts intensify with increased resolution. Cloud cover decreases with increased resolution. Grid
Sobinoff, A.P. [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308 (Australia); Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia); Beckett, E.L.; Jarnicki, A.G. [Centre for Asthma and Respiratory Disease, The University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308 (Australia); Sutherland, J.M. [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308 (Australia); Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia); McCluskey, A. [Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia); Hansbro, P.M. [Centre for Asthma and Respiratory Disease, The University of Newcastle and Hunter Medical Research Institute, Callaghan, NSW 2308 (Australia); McLaughlin, E.A., E-mail: email@example.com [Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308 (Australia); Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308 (Australia)
Cigarette smoke is a reproductive hazard associated with pre-mature reproductive senescence and reduced clinical pregnancy rates in female smokers. Despite an increased awareness of the adverse effects of cigarette smoke exposure on systemic health, many women remain unaware of the adverse effects of cigarette smoke on female fertility. This issue is compounded by our limited understanding of the molecular mechanisms behind cigarette smoke induced infertility. In this study we used a direct nasal exposure mouse model of cigarette smoke-induced chronic obstructive pulmonary disease to characterise mechanisms of cigarette-smoke induced ovotoxicity. Cigarette smoke exposure caused increased levels of primordial follicle depletion, antral follicle oocyte apoptosis and oxidative stress in exposed ovaries, resulting in fewer follicles available for ovulation. Evidence of oxidative stress also persisted in ovulated oocytes which escaped destruction, with increased levels of mitochondrial ROS and lipid peroxidation resulting in reduced fertilisation potential. Microarray analysis of ovarian tissue correlated these insults with a complex mechanism of ovotoxicity involving genes associated with detoxification, inflammation, follicular activation, immune cell mediated apoptosis and membrane organisation. In particular, the phase I detoxifying enzyme cyp2e1 was found to be significantly up-regulated in developing oocytes; an enzyme known to cause molecular bioactivation resulting in oxidative stress. Our results provide a preliminary model of cigarette smoke induced sub-fertility through cyp2e1 bioactivation and oxidative stress, resulting in developing follicle depletion and oocyte dysfunction. - Highlights: • Cigarette smoke exposure targets developing follicle oocytes. • The antral follicle oocyte is a primary site of ovarian cigarette smoke metabolism. • Cyp2e1 is a major enzyme involved in ameliorating smoke-induced ovotoxicity. • Cigarette smoke causes oocyte
Krarup, T.; Pedersen, T.; Faber, M.
MICE are born with a finite number of oocytes which develop in foetal life from primordial oogonia and their direct mitotic progeny. After birth no new oocytes are formed, and the total number of oocytes decreases with advancing age. During the first 2 weeks of life this decrease is due to degene......MICE are born with a finite number of oocytes which develop in foetal life from primordial oogonia and their direct mitotic progeny. After birth no new oocytes are formed, and the total number of oocytes decreases with advancing age. During the first 2 weeks of life this decrease is due...
[Cajal bodies in insect oocytes. II. New data on the molecular composition of cajal bodies in oocytes of the house cricket Acheta domesticus with special reference to interactions between cajal bodies and interchromatin granule clusters].
Stepanova, I S; Bogoliubov, D S; Parfenov, V N
Cajal bodies (CBs) in oocytes of the house cricket Acheta domesticus are large, perfectly spherical nuclear organelles with a complex internal structure. These consist of a fibrillar coilin-containing matrix and a central cavity with a prominent fibrogranular body inside; the latter has been referred to as an "internal" interchromatin granule cluster (IGC). Within the matrix of CBs we detected transcriptional co-activators CBP/p300 and TATA-box binding protein (TBP). No RNA polymerase II was revealed in CBs of both normal and actynomycin D treated oocytes. In the nucleoplasm of A. domesticus oocytes, besides CBs, free IGCs were observed. In oocytes treated with actynomycin D, the amount of "free" IGCs in the nucleoplasm increase significantly, granular and fibrillar components of IGCs were seen segregated, and RNA polymerase II and CBP/p300 were found to be accumulated in fibrillar zones of IGCs.
Cobo, Ana; Remohí, José; Chang, Ching-Chien; Nagy, Zsolt Peter
Oocyte donation is an efficient alternative to using own oocytes in IVF treatment for different indications. Unfortunately, 'traditional' (fresh) egg donations are challenged with inefficiency, difficulties of synchronization, very long waiting periods and lack of quarantine measures. Given the recent improvements in the efficiency of oocyte cryopreservation, it is reasonable to examine if egg donation through oocyte cryopreservation has merits. The objective of the current manuscript is to review existing literature on this topic and to report on the most recent outcomes from two established donor cryobank centres. Reports on egg donation using slow freezing are scarce and though results are encouraging, outcomes are not yet comparable to a fresh egg donation treatment. Vitrification on the other hand appears to provide high survival rates (90%) of donor oocytes and comparable fertilization, embryo development, implantation and pregnancy rates to traditional (fresh) egg donation. Besides the excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg cryobank and makes it likely that this approach becomes the future standard of care. Oocyte donation is one of the last resorts in IVF treatment for couples challenged with infertility problems. However, traditional (fresh) egg donation, as it is performed today, is not very efficient, as typically all eggs from one donor are given to only one recipient, it is arduous as it requires an excellent synchronization between the donor and recipient and there are months or years of waiting time. Because of the development of an efficient oocyte cryopreservation technique, it is now possible to cryo-store donor (as well as non-donor) eggs, maintaining their viability and allowing their use whenever there is demand. Therefore, creating a donor oocyte cryobank would carry many advantages
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.
Caamaño, J N; Muñoz, M; Diez, C; Gómez, E
The meiotic spindle structure plays a key role in normal chromosome alignment and segregation during meiosis. Polarized light microscopy (PLM) allows non-invasive evaluation of the meiotic spindle of metaphase oocytes from different animal species. The purpose of this article is to review the use of PLM in animal reproduction, mainly in the assessment of the meiotic spindle in oocytes. A brief overview of the methods to assess the meiotic spindle is presented as well as the principles behind the PLM. The use of PLM to evaluate oocyte quality and spindle morphology is discussed and the results on the viability of the oocytes after being exposed to PLM are presented. Several researchers showed that PLM could be successfully implemented on cryopreservation, nuclear transfer and intracytoplasmic sperm injection procedures as a tool to improve the outcome of these procedures. In addition, PLM can be used to develop studies on oocyte maturation and spindle dynamics. However, the information on the practical use of this technology in farm animals is very limited and further studies are needed to assess the importance of PLM in animal reproduction.
Almonacid, Maria; Terret, Marie-Emilie; Verlhac, Marie-Hélène
The position of the nucleus in a cell can instruct morphogenesis in some cases, conveying spatial and temporal information and abnormal nuclear positioning can lead to disease. In oocytes from worm, sea urchin, frog and some fish, nucleus position regulates embryo development, it marks the animal pole and in Drosophila it defines the future dorso-ventral axis of the embryo and of the adult body plan. However, in mammals, the oocyte nucleus is centrally located and does not instruct any future embryo axis. Yet an off-center nucleus correlates with poor outcome for mouse and human oocyte development. This is surprising since oocytes further undergo two extremely asymmetric divisions in terms of the size of the daughter cells (enabling polar body extrusion), requiring an off-centering of their chromosomes. In this review we address not only the bio-physical mechanism controlling nucleus positioning via an actin-mediated pressure gradient, but we also speculate on potential biological relevance of nuclear positioning in mammalian oocytes and early embryos. Copyright © 2017. Published by Elsevier Ltd.
Davenport, Richard; Davenport, Janice C.
The cytoplasm of young oocytes of the ascidians contains high concentrations of proteins which are stainable with alkaline fast green at pH 8.1 and above. These proteins cannot be stained even with acid dyes at low pH unless RNA is removed. Deamination and formalin blockage of amino groups is incapable of destroying the net positive charge on these protein molecules in the presence of RNA, but these treatments destroy the charge if RNA is removed. It is therefore concluded that basic proteins and RNA exist as a nucleoprotein complex in the ribosomes of these young oocytes. The detectable RNA of the mature oocytes and unfertilized eggs shows no evidence of being associated with basic proteins. PMID:14287183
Full Text Available Changes in oocyte quality can have great impact on the developmental potential of early embryos. Here we test whether nuclear genome transfer from a developmentally incompetent to a developmentally competent oocyte can restore developmental potential. Using in vitro oocyte aging as a model system we performed nuclear transfer in mouse oocytes at metaphase II or at the first interphase, and observed that development to the blastocyst stage and to term was as efficient as in control embryos. The increased developmental potential is explained primarily by correction of abnormal cytokinesis at anaphase of meiosis and mitosis, by a reduction in chromosome segregation errors, and by normalization of the localization of chromosome passenger complex components survivin and cyclin B1. These observations demonstrate that developmental decline is primarily due to abnormal function of cytoplasmic factors involved in cytokinesis, while the genome remains developmentally fully competent.
Neggers, R.A.J.; Siebesma, A.P.; Lenderink, G.; Holtslag, A.A.M.
Three closure methods for the mass flux at cloud base in shallow cumulus convection are critically examined for the difficult case of a diurnal cycle over land. The closure methods are first evaluated against large-eddy simulations (LESs) by diagnosing all parameters appearing in the closure equatio
Fahad, Abdullah Al
Tropical Cyclone Viyaru, formerly known as Cyclonic Storm Mahasen was a rapidly intensifying, category 01B storm that made landfall in Chittagong, Bangladesh on the 16th of May, 2013. In this study, the sensitivity of numerical simulations of tropical cyclone to cumulus physics parametrization is carried out with a view to determine the best cumulus physics option for prediction of the cyclones track, timing, and central pressure evolution in the Bay of Bengal. For this purpose, the tropical cyclone Viyaru has been simulated by WRF ARW in a nested domain with NCEP Global Final Analysis(FNL) data as initial and boundary conditions. The model domain consists of one parent domain and one nested domain. The resolution of the parent domain is 36 km while the nested domain has a resolution of 12 km. Five numerical simulations have been done with the same micro-physics scheme (WSM3), planetary boundary layer scheme,NOAH land surface scheme but different Cumulus Parametrization scheme. Four cumulus Parametrization sc...
Apr 26, 2010 ... Department of Animal Science, College of Agriculture and Natural ... After culture for 24 h, over 80% of COCs had full cumulus cells expansion. ..... Agawal A, Tamer MS, Mohamed AB, Jashoman B, Juan GA (2006). Oxidative ...
Tsai, Sujune; Jhuang, Yating; Spikings, Emma; Sung, Ping-Jyun; Lin, Chiahsin
The developmental oogenesis of gorgonian coral was investigated at the histological level. The objective of this study was to examine and improve the understanding of Junceella juncea oogenesis using ultrastructural methods, such as histological sectioning and transmission electron microscopy. At least three types of yolk materials were observed in this study: yolk body, lipid granules and cortical alveoli. Some of the complex yolk materials were encompassed by concentric or arched layers of smooth and rough endoplasmic reticulum and the Golgi complex in early stage oocytes. Different types of vesicles were found in both early and late stage oocytes and some granules could be seen inside the empty vesicles. This may be a possible method for elaborating complex yolk materials. Homogeneous yolks from different types of inclusions were abundant and the autosynthesis of yolk may be a major mechanism in J. juncea oocytes. This is the first report of the ultrastructural observation of oogenesis in gorgonian coral species using transmission electron microscopy. Our study obtained relatively detailed information at the ultrastructural level, and it provides an overview of the oocyte ultrastucture of the gorgonian coral J. juncea.
Moniruzzaman, Mohammad; Miyano, Takashi
Mammalian ovaries are endowed with a huge number of small oocytes (primordial oocytes) in primordial follicles. A small number of primordial oocytes start to grow, while others remain quiescent. Little is known about the mechanism regulating the activation of primordial oocytes. Recently, we found that primordial follicles in mature cows and prepubertal pigs took longer to initiate growth in xenografts compared with those in neonatal animals. We think that primordial oocytes in adult mammals are different from those in neonatal mammals. In this review, we summarize the results regarding the activation of primordial oocytes in neonatal and adult ovaries of different species and propose a model in which ovaries of neonatal mammals contain a mixed population of both quiescent and activated primordial oocytes, while almost all primordial oocytes are quiescent in adult females. The dormancy of primordial oocytes may be required to reserve the non-growing oocyte pool for the long reproductive life in mammals. FOXO3 is considered one of the molecules responsible for the dormancy of primordial oocytes in adult ovaries. These quiescent primordial oocytes are activated, perhaps by certain mechanisms involving the interaction between stimulatory and inhibitory factors, to enter the growth phase.
In this experiment, the bovine follicular oocytes were aspirated from the ovaries of Chinese Holsteins with laparoscope made in China. The results were as following: for identifying the suitable negative aspiration pressure, six different pressures (50, 100, 150, 200, 250 and 300mmHg)were tested. The aspiration pressure of 100mmHg was the best. Its oocyte recovery rate was 37. 2%, and G I , G Ⅱ oocyte rate was 89. 5%. The heifers were picked up by laparoscope once or twice a week. Each heifer was collected with 2. 4 oocytes once a week or 4. 4 oocytes twice a week.Its oocyte recovery rate was 48. 0% and the G Ⅰ ,G Ⅱ oocyte rate was 93. 5%. In addition, 1.9 oocytes were collected from each cow once a week or 5.4 oocytes from each cow twice a week. Its oocyte recovery rate was 51.7% and the G Ⅰ , G Ⅱ oocyte rate was 85. 1%. It showed that it was possible to pick up bovine oocyte twice a week. Two cows were picked up twice a week for several weeks(53 times). 268 follicles were aspirated(5.1 follicles per cow per time), and 141 oocytes were recovered(2.7 oocytes per cow per time). The oocyte recovery rate was 52.5%, and the G Ⅰ , G Ⅱ oocyte rate was 85. 1%. It was advisable to pick up oocytes twice a week continuously. Some cows in estrous cycles were superovulated with PMSG(500IU). Each of them could be recovered 2.3 follicles and 1.1 oocytes, the others were superovulated with FSH(0. 7mg) , each of them could be aspirated with 4.4 follicles and 2.3 oocytes. It was obvious that the effect of OPU(oocyte pick up) by superovulation with FSH was much better than that with PMSG. The best time for OPU with laparoscope was at the beginning of cow's estrous cycles. At the first day of their estrus, each of them could be averagely aspirated with 8 follicles and 5.7 oocytes.
Critser, J K; Arneson, B W; Aaker, D V; Ball, G D
Three experiments were conducted for evaluation of the efficacy of conventional freezing or vitrification of hamster oocytes for use in a human sperm penetration assay (hSPA). In experiment 1, oocytes were cryopreserved and evaluated for survival on the basis of morphologic criteria. Survival of vitrified oocytes and that of frozen oocytes were not different, whereas all cryopreserved groups had lower survival than noncryopreserved controls. In experiment 2, oocytes were conventionally frozen or vitrified and evaluated in an hSPA. Vitrified oocytes had a lower frequency of sperm penetration than frozen oocytes, and all cryopreserved groups had lower penetration rates than untreated controls. In experiment 3, oocytes were exposed to the cryoprotectant used to vitrify (VS1) or freeze (DMSO) but not cooled prior to evaluation in an hSPA. Exposure to DMSO but not VS1 reduced hSPA values. It is concluded from these experiments that while all cryopreserved oocytes do not survive, at current stages of development conventionally frozen oocytes perform better than vitrified oocytes in the hSPA and losses associated with conventional freezing procedures may be related to cryoprotectant exposure, whereas vitrification losses are more probably due to events associated with rapid cooling and/or warming of the oocytes.
Park, Chan Woo; Lee, Sun Hee; Yang, Kwang Moon; Lee, In Ho; Lim, Kyung Teak; Lee, Ki Heon; Kim, Tae Jin
The aim of this study was to report a case series of in vitro matured (IVM) oocyte freezing in gynecologic cancer patients undergoing radical surgery under time constraints as an option for fertility preservation (FP). Case series report. University-based in vitro fertilization center. Six gynecologic cancer patients who were scheduled to undergo radical surgery the next day were referred for FP. The patients had endometrial (n=2), ovarian (n=3), and double primary endometrial and ovarian (n=1) cancer. Ex vivo retrieval of immature oocytes from macroscopically normal ovarian tissue was followed by mature oocyte freezing after IVM or embryo freezing with intracytoplasmic sperm injection. A total of 53 oocytes were retrieved from five patients, with a mean of 10.6 oocytes per patient. After IVM, a total of 36 mature oocytes were obtained, demonstrating a 67.9% maturation rate. With regard to the ovarian cancer patients, seven IVM oocytes were frozen from patient 3, who had stage IC cancer, whereas one IVM oocyte was frozen from patient 4, who had stage IV cancer despite being of a similar age. With regard to the endometrial cancer patients, 15 IVM oocytes from patient 1 were frozen. Five embryos were frozen after the fertilization of IVM oocytes from patient 6. Immature oocytes can be successfully retrieved ex vivo from macroscopically normal ovarian tissue before radical surgery. IVM oocyte freezing provides a possible FP option in patients with advanced-stage endometrial or ovarian cancer without the risk of cancer cell spillage or time delays.
Kristensen, Stine Gry; Pors, Susanne Elisabeth; Andersen, Claus Yding
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....
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.
Konc J; Kanyo K; Varga E; Kriston R; Cseh S
Objective To evaluate the value of oocyte cryopreservation (CP) in our clinical ICSIprogram.Methods Freezing procedure with medium containing 1.5 mol/L propanediol (PrOH)+ 0.3 mol/L succrose and traditional slow-freezingprotocol were used. Thawed oocytes were fertilized with ICSI (4-6 h after thawing), and fertilization was assessed 12-16 h later. Laser assisted hatching was performed on all transferred embryos and embryo transfer was carried out 48-72 h after ICSI.Results Eighty-five eggs were thawed and survival rate of 75.3% (64/85) was obtained.Sixty-four oocytes were inseminated with ICSI, 47fertilized (47/64; 73.4%) and a cleavage rate of 85% (40/47) was obtained. Embryo transfers were performed in 18patients, and 4 (19%) resulted in clinical pregnancies. One of the pregnancies encountered first trimester abortion. Implantation rate were 17.2% (5/29) per embryo and 5. 8% (5/85) per egg thawed. In all cases, chorion biopsy was performed resulting46 XY kariotype.Conclusion Our results provide further evidence of that although egg freezing cannot currently claim to be a routine procedure in human IVF, there will certainly be a place for oocyte CP in reproductive medicine in the future.
Batalova, F M; Bogoliubov, D S
Structure and composition of the karyosphere (karyosome) capsule were studied in the oocytes of a laboratory insect, Tribolium castaneum, with the use of electron microscopy and immunoelectron cytochemistry. Basing on the study of nuclear structure dynamics, we distinguished 8 stages that characterize the period of oocyte growth. At the diplotene stage, T. castaneum oocyte chromosomes conjoin early into a compact karyosphere, but a significant chromatin condensation does not occur. The process of karyosphere formation is accompanied by the development of an extensive extrachromosome capsule surrounding chromatin. The capsule consists of a material of different morphological types. Significant molecular components of the T. castaneum karyosphere capsule are represented by the proteins of nuclear matrix including F-actin and lamin B. Besides the structural proteins, the Sm proteins of small nuclear (sn) RNPs and mature 2,2,7-trimethyl guanosine (TMG) 5'-capped snRNAs are revealed immunocytochemically in the karyosphere capsule. The obtained data can form a basis for further expansion of ideas on the functions of the karyosphere capsule as a specialized extrachromosomal nuclear domain of the oocytes. We believe that the T. castaneum karyosphere capsule plays not only a structural role, but may be involved directly in the processes related to gene expression.
Gui, Liming; Homer, Hayden
The spindle assembly checkpoint (SAC) averts aneuploidy by coordinating proper bipolar chromosomal attachment with anaphase-promoting complex/cyclosome (APC/C)-mediated securin and cyclin B1 destruction required for anaphase onset. The generation of a Mad2-based signal at kinetochores is central to current models of SAC-based APC/C inhibition. During mitosis, kinetochores of polar-displaced chromosomes, which are at greatest risk of mis-segregating, recruit the highest levels of Mad2, thereby ensuring that SAC activation is proportionate to aneuploidy risk. Paradoxically, although an SAC operates in mammalian oocytes, meiosis I (MI) is notoriously error prone and polar-displaced chromosomes do not prevent anaphase onset. Here we find that Mad2 is not preferentially recruited to the kinetochores of polar chromosomes of wild-type mouse oocytes, in which polar chromosomes are rare, or of oocytes depleted of the kinesin-7 motor CENP-E, in which polar chromosomes are more abundant. Furthermore, in CENP-E-depleted oocytes, although polar chromosomal displacement intensified during MI and the capacity to form stable end-on attachments was severely compromised, all kinetochores nevertheless became devoid of Mad2. Thus, it is possible that the ability of the SAC to robustly discriminate chromosomal position might be compromised by the propensity of oocyte kinetochores to become saturated with unproductive attachments, thereby predisposing to aneuploidy. Our data also reveal novel functions for CENP-E in oocytes: first, CENP-E stabilises BubR1, thereby impacting MI progression; and second, CENP-E mediates bi-orientation by promoting kinetochore reorientation and preventing chromosomal drift towards the poles.
Objective: To report two sister-cases of primary infertility for oocyte developmental disorders. Methods: A retrospective clinical study was performed to analyze two sister-cases of oocyte developmental disorders. Results: Two sister-cases underwent outpatient ovulation induction by conventional GnRHa protocol. There were only cumulus granulosa cell clusters without oocytes through microscopic examination on retrieval day. Oocytes were also not obtained by remedial hCG treatment. Thus oocyte dys-plasia could be diagnosed. Subsequently it was acquired that their grandparents were cousin consanguineous marriages. Younger sister-patient with donor oocyte cycle surgery got successful pregnancy of intrauterine single fetal. Conclusion: The family and medical history should be asked in detail for IVF treatment. Donor scheme for oocyte developmental disorders is a feasible and effective option.%目的:报道2例IVF治疗中卵子发育障碍原发性不孕姐妹患者.方法:对本院收治的2例卵子发育障碍原发性不孕患者姐妹进行回顾性总结与分析.结果:均采用常规GnRHa长方案促排卵,取卵日卵泡穿刺镜检仅见卵丘颗粒细胞团,均未见到卵子,补救性hCG治疗亦未取得卵子,诊断为卵子发育异常.追问病史得知其祖父母为表兄妹近亲婚配.患者(妹妹)行受卵治疗,宫内单胎成功妊娠.结论:门诊IVF治疗建病历时应详细询问患者家族史,供卵方案是目前IVF治疗卵子发育障碍原发性不孕较常用且有效的方法.
Yu, X.; Berg, L.; Berkowitz, C.; Alexander, L.; Lee, Y.; Ogren, J.; Andrews, B.
Clouds play an active role in the processing and cycling of atmospheric constituents. Gases and particles can partition to cloud droplets by absorption and condensation as well as activation and pact scavenging. The Cumulus Humilis Aerosol Processing Study (CHAPS) aimed at characterizing freshly emitted aerosols above, within and below fields of cumulus humilis (or fair-weather cumulus) in the vicinity of Oklahoma City. The experiment took place in June 2007. Evolution of aerosol and cloud properties downwind of the Oklahoma City is of particular interest in this project. These observations of a mid-size and mid-latitude city can be used in the development and evaluation of regional-scale and global climate model cumulus parameterizations that describes the transport and transformations of these aerosols by fair-weather cumulus. The Department of Energy (DOE) G-1 aircraft was one of the main platforms used in CHAPS. It carried a suite of instruments to measure properties of interstitial aerosols behind an isokinetic inlet and a set of duplicate instruments to determine properties of activated particles behind a counter-flow virtual impactor (CVI). The sampling line to the Aerodyne Aerosol Mass Spectrometer was switched between the isokinetic inlet and the CVI to allow characterization of interstitial particles out of clouds in contrast to particles activated in clouds. Trace gases including ozone, carbon monoxide, sulfur dioxide, and a series of volatile organic compounds (VOCs) were also measured as were key meteorological state parameters including liquid water content, cloud drop size, and dew point temperature were measured. This presentation will focus on results related to the transformation and transport of aerosols and trace gases observed in fair-weather cumulus and compare these results with concurrent observations made outside these clouds. Our interest will focus on the differences in particle size and composition under varying conditions. The role of
Yu, X.; Berg, L. K.; Berkowitz, C. M.; Lee, Y.; Alexander, L.; Ogren, J. A.; Andrews, B.
The Cumulus Humilis Aerosol Processing Study (CHAPS) provided a unique opportunity to study cloud processing of aerosols. Clouds play an active role in the processing and cycling of atmospheric constituents. Within in a cloud, gases and particles can partition to cloud droplets by absorption and condensation as well as activation and impact scavenging. The Department of Energy (DOE) G-1 aircraft was used as one of the main platforms in CHAPS. G-1 flight tracks were designed to characterize aerosols at cloud top and cloud base as well as within individual cumulus humilis (or fair-weather cumulus), in the vicinity of Oklahoma City. Measurements of interstitial aerosols and residuals of activated condensation cloud nuclei were conducted simultaneously. The interstitial aerosols were measured downstream of an isokinetic inlet, and the activated particles downstream of a counter-flow virtual impactor (CVI). The sampling line to the Aerodyne Aerosol Mass Spectrometer (AMS) was switched between the isokinetic inlet and the CVI to allow characterization of non-activated particles outside of clouds in contrast to particles activated in clouds. Trace gases including ozone, carbon monoxide, sulfur dioxide, and a series of volatile organic compounds (VOCs) were measured. Key meteorological state parameters included liquid water content, cloud drop size, and dew point. In this presentation, we will focus on case studies of CCN properties in cumulus humilis. The first analysis summarizes three case studies of measurements made at cloud bottom and in-cloud by the AMS. The size-resolved composition is different between background and activated particles. The second analysis links in situ measurements of aerosol, trace gas, and VOCs to look into the sources of CCN. For instance, by comparing the characteristic m/z ratios by AMS and tracers like CO or isoprene, one can gain more insight into the role of primary and secondary organic aerosols in CCN and background aerosols. The third
Payne, D; Flaherty, S P; Barry, M F; Matthews, C D
In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.
Adona, Paulo R; Leal, Cláudia L V; Biase, Fernando H; De Bem, Tiago H; Mesquita, Lígia G; Meirelles, Flávio V; Ferraz, André L; Furlan, Luiz R; Monzani, Paulo S; Guemra, Samuel
Gene expression profiling of in vivo- and in vitro-matured bovine oocytes can identify transcripts related to the developmental potential of oocytes. Nonetheless, the effects of in vitro culturing oocytes are yet to be fully understood. We tested the effects of in vitro maturation on the transcript profile of oocytes collected from Bos taurus indicus cows. We quantified the expression of 1488 genes in in vivo- and in vitro-matured oocytes. Of these, 51 genes were up-regulated, whereas 56 were down-regulated (≥2-fold) in in vivo-matured oocytes in comparison with in vitro-matured oocytes. Quantitative real-time polymerase chain reaction (PCR) of nine genes confirmed the microarray results of differential expression between in vivo- and in vitro-matured oocytes (EZR, EPN1, PSEN2, FST, IGFBP3, RBBP4, STAT3, FDPS and IRS1). We interrogated the results for enrichment of Gene Ontology categories and overlap with protein-protein interactions. The results revealed that the genes altered by in vitro maturation are mostly related to the regulation of oocyte metabolism. Additionally, analysis of protein-protein interactions uncovered two regulatory networks affected by the in vitro culture system. We propose that the differentially expressed genes are candidates for biomarkers of oocyte competence. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence.
Bernd Rosenbusch; Michael Schneider; Hans Wilhelm Michelmann
Human oocytes that remained unfertilized in programmes of assisted reproduction have been analysed cytogenetically for more than 20 years to assess the incidence of aneuploidy in female gametes. However, the results obtained so far are not indisputable as a consequence of difficulties in evaluating oocyte chromosome preparations. Because of the lack of guidelines, we decided to summarize for the first time, the possible pitfalls in human oocyte chromosome analysis. Therefore, we screened the material from our previous studies and compiled representative, complicated cases with recommendations for their cytogenetic classification. We point out that maturity and size of the oocyte are important parameters and that fixation artefacts, as well as the particular structure of oocyte chromosomes, may predispose one to misinterpretations. Moreover, phenomena related to oocyte activation and fertilization are illustrated and explained. This compilation may help to avoid major problems in future studies and contribute to a more precise, and uniform assessment of human oocyte chromosomes.
Ahmed Z Balboula
Full Text Available Aurora B kinase (AURKB is the catalytic subunit of the chromosomal passenger complex (CPC, an essential regulator of chromosome segregation. In mitosis, the CPC is required to regulate kinetochore microtubule (K-MT attachments, the spindle assembly checkpoint, and cytokinesis. Germ cells express an AURKB homolog, AURKC, which can also function in the CPC. Separation of AURKB and AURKC function during meiosis in oocytes by conventional approaches has not been successful. Therefore, the meiotic function of AURKC is still not fully understood. Here, we describe an ATP-binding-pocket-AURKC mutant, that when expressed in mouse oocytes specifically perturbs AURKC-CPC and not AURKB-CPC function. Using this mutant we show for the first time that AURKC has functions that do not overlap with AURKB. These functions include regulating localized CPC activity and regulating chromosome alignment and K-MT attachments at metaphase of meiosis I (Met I. We find that AURKC-CPC is not the sole CPC complex that regulates the spindle assembly checkpoint in meiosis, and as a result most AURKC-perturbed oocytes arrest at Met I. A small subset of oocytes do proceed through cytokinesis normally, suggesting that AURKC-CPC is not the sole CPC complex during telophase I. But, the resulting eggs are aneuploid, indicating that AURKC is a critical regulator of meiotic chromosome segregation in female gametes. Taken together, these data suggest that mammalian oocytes contain AURKC to efficiently execute meiosis I and ensure high-quality eggs necessary for sexual reproduction.
Kim, Sung Han; Chung, Ee-Yung; Lee, Ki-Young
Ultrastructural studies of oocyte degeneration in the oocyte, and the functions of follicle cells during oocyte degeneration are described to clarify the reproductive mechanism on oocyte degeneration of Mactra chinensis using cytological methods. Commonly, the follicle cells are attached to the oocyte. Follicle cells play an important role in oocyte degeneration. In particular, the functions of follicle cells during oocyte degeneration are associated with phagocytosis and the intracellular di...
Yao, Mao-Sung; Del Genio, Anthony D.
An improved version of the GISS Model II cumulus parameterization designed for long-term climate integrations is used to study the effects of entrainment and multiple cloud types on the January climate simulation. Instead of prescribing convective mass as a fixed fraction of the cloud base grid-box mass, it is calculated based on the closure assumption that the cumulus convection restores the atmosphere to a neutral moist convective state at cloud base. This change alone significantly improves the distribution of precipitation, convective mass exchanges, and frequencies in the January climate. The vertical structure of the tropical atmosphere exhibits quasi-equilibrium behavior when this closure is used, even though there is no explicit constraint applied above cloud base.
Del Genio, Anthony D
Moist convection plays a leading role in the dynamics and energy budget of Earth's tropics and influences the sensitivity of Earth's climate to greenhouse gas increases. Because individual convective cells are much smaller than the gridboxes of 3-dimensional global climate models (GCMs), these models parameterize the effects of an ensemble of moist convective updrafts and downdrafts on the environment. Cumulus parameterization has been a focus of the terrestrial meteorology community for half a century. Only in past decade, however, have GCMs with moist convective physics been applied to other planets. Given our lack of detailed knowledge about convective clouds except on Earth, planetary GCMs are often designed with very simple approaches to cumulus parameterization, adopted from the earliest generations of terrestrial GCMs. These parameterizations were based on breakthroughs in understanding of convection in their time. However, at the same time that planetary GCMs have begun to emerge, a quiet revolution i...
Shallow-cumulus clouds constitute the most prominent cloud type on Earth, and their response to changing environmental conditions is critical for climate sensitivity. Research over the last decade has pointed out the importance of the interplay between clouds, convection, turbulence and circulation in controlling this response. Unfortunately, numerical models represent this interplay in diverse ways, which translates into different shallow-cumulus cloud feedbacks in climate change. Climate models predict that the cloud-base cloud fraction is very sensitive to changes in environmental conditions, while process models suggest that it is very resilient to such changes. To understand and solve this contradiction, a field campaign named EUREC4A (Elucidating the role of clouds-circulation coupling in climate) will be organized in the lower Atlantic trades in Jan-Fev 2020. The scientific objectives of this campaign will be presented, and the experimental strategy envisioned to reach these objectives will be discussed.
Eck, T. F.; Holben, B. N.; Reid, J. S.; Arola, A.; Ferrare, R. A.; Hostetler, C. A.; Crumeyrolle, S. N.; Berkoff, T. A.; Welton, E. J.; Lolli, S.; Lyapustin, A.; Wang, Y.; Schafer, J. S.; Giles, D. M.; Anderson, B. E.; Thornhill, K. L.; Minnis, P.; Pickering, K. E.; Loughner, C. P.; Smirnov, A.; Sinyuk, A.
During the July 2011 Deriving Information on Surface conditions from Column and Vertically Resolved Observations Relevant to Air Quality (DISCOVER-AQ) field experiment in Maryland, significant enhancements in Aerosol Robotic Network (AERONET) sun-sky radiometer measured aerosol optical depth (AOD) were observed in the immediate vicinity of non-precipitating cumulus clouds on some days. Both measured Ångström exponents and aerosol size distribution retrievals made before, during and after cumulus development often suggest little change in fine mode particle size; therefore, implying possible new particle formation in addition to cloud processing and humidification of existing particles. In addition to sun-sky radiometer measurements of large enhancements of fine mode AOD, lidar measurements made from both ground-based and aircraft-based instruments during the experiment also measured large increases in aerosol signal at altitudes associated with the presence of fair weather cumulus clouds. These data show modifications of the aerosol vertical profile as a result of the aerosol enhancements at and below cloud altitudes. The airborne lidar data were utilized to estimate the spatial extent of these aerosol enhancements, finding increased AOD, backscatter and extinction out to 2.5 km distance from the cloud edge. Furthermore, in situ measurements made from aircraft vertical profiles over an AERONET site during the experiment also showed large increases in aerosol scattering and aerosol volume after cloud formation as compared to before. The 15-year AERONET database of AOD measurements at the Goddard Space Flight Center (GSFC), Maryland site, was investigated in order to obtain a climatological perspective of this phenomenon of AOD enhancement. Analysis of the diurnal cycle of AOD in summer showed significant increases in AOD from morning to late afternoon, corresponding to the diurnal cycle of cumulus development.
Vial, Jessica; Bony, Sandrine; Stevens, Bjorn; Vogel, Raphaela
Shallow cumulus clouds in the trade-wind regions are at the heart of the long standing uncertainty in climate sensitivity estimates. In current climate models, cloud feedbacks are strongly influenced by cloud-base cloud amount in the trades. Therefore, understanding the key factors controlling cloudiness near cloud-base in shallow convective regimes has emerged as an important topic of investigation. We review physical understanding of these key controlling factors and discuss the value of the different approaches that have been developed so far, based on global and high-resolution model experimentations and process-oriented analyses across a range of models and for observations. The trade-wind cloud feedbacks appear to depend on two important aspects: (1) how cloudiness near cloud-base is controlled by the local interplay between turbulent, convective and radiative processes; (2) how these processes interact with their surrounding environment and are influenced by mesoscale organization. Our synthesis of studies that have explored these aspects suggests that the large diversity of model responses is related to fundamental differences in how the processes controlling trade cumulus operate in models, notably, whether they are parameterized or resolved. In models with parameterized convection, cloudiness near cloud-base is very sensitive to the vigor of convective mixing in response to changes in environmental conditions. This is in contrast with results from high-resolution models, which suggest that cloudiness near cloud-base is nearly invariant with warming and independent of large-scale environmental changes. Uncertainties are difficult to narrow using current observations, as the trade cumulus variability and its relation to large-scale environmental factors strongly depend on the time and/or spatial scales at which the mechanisms are evaluated. New opportunities for testing physical understanding of the factors controlling shallow cumulus cloud responses using
Gohin, Maella; Bodinier, Pascal; Fostier, Alexis; Chesnel, Franck; Bobe, Julien
While it is generally well accepted that the ovarian follicular sites of estradiol-17β (E2) synthesis are restricted to somatic cells, the possible contribution of the germinal compartment has received little or no attention in teleosts. In order to demonstrate the expression of ovarian aromatase in the oocyte, cyp19a1a mRNA was studied in ovarian follicles by in situ hybridization. In addition, the expression of cyp19a1a was studied in both somatic and germinal compartments of the ovarian follicle in rainbow trout (Oncorhynchus mykiss) during final oocyte maturation (i.e., maturational competence acquisition and subsequent meiosis resumption) by real-time PCR. The enzymatic activity of ovarian aromatase was also studied in both somatic and germinal compartments of the ovarian follicle. Finally, E2 levels were monitored in follicle-enclosed oocytes throughout the pre-ovulatory period. We were able to demonstrate a significant ovarian aromatase expression and activity in the late vitellogenic oocyte. Furthermore, a dramatic decrease in aromatase expression and activity occurs in the oocyte during late oogenesis, concomitantly with the trend observed in surrounding follicular layers. We also report an unexpected increase of E2 levels in the oocyte during the pre-ovulatory period. To our knowledge, these observations are reported for the first time in any teleost species. Together, our data support the hypothesis of the participation of the germinal compartment in follicular estrogen synthesis and a biological role of E2 during oocyte and/or early embryo development.
Collado-Fernandez, Esther; Picton, Helen M; Dumollard, Rémi
Metabolic studies of mammalian embryos started with the development of in vitro culture systems more than 40 years ago. More recently, metabolic studies have begun to shed light on the requirements of growing oocytes/follicles from the earliest stages of folliculogenesis. While growing oocytes preferentially metabolise pyruvate over glucose, the somatic compartment of ovarian follicles is more glycolytic. The metabolic preferences of the oocyte are reflected in the early zygote, which becomes increasingly dependent on glycolytic energy production as development progresses to the blastocyst stage. Furthermore, the intricate metabolic relationship between each oocyte and its somatic surroundings is critical for oocyte growth and developmental competence. Measurements of amino acid turnover in bovine oocytes indicate that glutamine, arginine and leucine are consistently depleted, while alanine is produced, showing similarities with amino acid turnover in preimplantation embryos. Amino acid profiling is a good predictor of embryo quality and might also turn out to be a predictor of oocyte developmental competence. Finally, recent studies have uncovered lipid metabolism in oocytes and early embryos, suggesting that endogenous fatty acids might be used for energy production. Together, metabolic studies have revealed the multiplicity of energetic substrates used by oocytes and early embryos, and suggest that the versatility of the metabolic pathways available for energy production is key for high developmental potential. Metabolic studies of early embryos are now being applied to follicle culture, and the goal of describing the metabolome of the growing oocyte in its follicle is now very attainable.
Moniruzzaman, Mohammad; Lee, Jibak; Zengyo, Mai; Miyano, Takashi
Mammalian ovaries are endowed with a large number of primordial follicles, each containing a nongrowing oocyte. Only a small population of primordial oocytes (oocytes in primordial follicles) is activated to enter the growth phase throughout a female's reproductive life. Little is known about the mechanism regulating the activation of primordial oocytes. Here, we found that the primordial oocytes from infant pigs (10- to 20-day-old) grew to full size at 2 months after xenografting to immunodeficient mice, whereas those from prepubertal pigs (6-month-old) survived without initiation of their growth even after 4 months; thereafter, they started to grow and reached full size after 6 months. These results suggest that the mechanism regulating the activation of primordial oocytes in prepubertal pigs is different from that in infant pigs. In this regard, the involvement of FOXO3, a forkhead transcription factor, was studied. In prepubertal pigs, FOXO3 was detected in almost all (94+/-2%) primordial oocyte nuclei, and in infant pigs, 42+/-7% primordial oocytes were FOXO3 positive. At 4 months after xenografting, the percentage of FOXO3-positive primordial oocytes from prepubertal pigs had decreased to the infant level. Further, siRNA was designed to knock down porcine FOXO3. FOXO3-knockdown primordial follicles from prepubertal pigs developed to the antral stage accompanied by oocyte growth at 2 months after xenografting. These results suggest that primordial oocytes are dormant in prepubertal pigs by a FOXO3-related mechanism to establish a nongrowing oocyte pool in the ovary, and that a transient knockdown of the FOXO3 activates the primordial oocytes to enter the growth phase.
Robert F. Casper
Full Text Available Background: Oocyte cryopreservation is potentially the best way to preserve female fertility forunmarried women or young girls at risk of losing ovarian function. The aim of this study was tocompare fertilization and embryo development in frozen-thawed oocytes to their fresh siblings inwomen undergoing in vitro fertilization (IVF and embryo transfer (ET.Materials and Methods: Eleven infertile women undergoing infertility treatment, between theages of 24 to 37 years (mean ± SD = 31.6 ± 3.5, were included in this study. Mature oocytesfrom each patient were randomized into cryopreserved and fresh groups prior to intracytoplasmicsperm injection (ICSI. One hundred and thirty nine oocytes were retrieved, of which 105 were atmetaphase II (MII. Forty- five fresh MII oocytes were kept in culture whereas their sibling 60 MIIoocytes were cryopreserved using a slow cooling protocol. The frozen oocytes remained in LN2for 2 hours before thawing. ICSI was performed 1-2 hours after thawing for frozen oocytes and 4-5hours after retrieval for fresh oocytes. Fertilization and embryo development were compared.Results: Following thawing, 31 oocytes (51.6 % survived and 22 fertilized (79% while 32 freshoocytes fertilized upon ICSI (71%. The mean ± SE scores for embryos developing from frozenthawedoocytes were significantly lower at 48 and 72 hours post-ICSI than for embryos resultingfrom fresh oocytes (p<0.05.Conclusion: Our data demonstrated that oocyte freezing resulted in acceptable survival ratesfollowing cryopreservation, and similar fertilization rates following ICSI as compared to the freshsibling oocytes. However the number of blastomeres and the embryo quality on day three wassuperior in embryos from fresh oocytes when compared to the frozen oocytes.
Carpintero, Nayara López; Suárez, Onica Armijo; Mangas, Carmen Cuadrado; Varea, Carolina González; Rioja, Rubén Gómez
Various components of follicular fluid are suggested as biochemical predictors of oocyte quality. Previous studies of follicular steroid hormone levels have shown disparate results when related with fertilization outcomes. The objective of the study was to relate the levels of steroid hormones of each individual follicle with oocyte maturation, fertilization results, embryo quality, and pregnancy rates. Prospective cohort study in a university hospital. In 31 patients, who underwent intracytoplasmic sperm injection, it was performed an ultrasound guided aspiration of follicular fluid of the first two mature follicles from each ovary. Follicular levels of estradiol, progesterone, testosterone, and dehydroepiandrosterone sulfate were measured by chemiluminescent immunoassay. Generalized estimating equation model. In follicular fluids with mature oocyte presence, in normal as well as in failed fertilization, there was a positive correlation between follicular testosterone and progesterone (r = 0.794, P = 0.0001 and r = 0.829, P = 0.0001). Progesterone levels were higher in cases of normal fertilization compared to failed fertilization (P = 0.003). B quality embryos came from oocytes immersed in follicular fluids with higher estradiol values and higher estradiol/progesterone and estradiol/testosterone ratios than those of C quality (P = 0.01; P = 0.0009; P = 0.001). Estradiol levels were higher in patients who achieved pregnancy (P = 0.02). The analysis of follicular hormone composition could be considered as an additional tool in oocyte selection.
Quinlan, Margot E
Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.
Balboula, Ahmed Z; Blengini, Cecilia S; Gentilello, Amanda S; Takahashi, Masashi; Schindler, Karen
During oocyte meiotic maturation, Aurora kinase C (AURKC) is required to accomplish many critical functions including destabilizing erroneous kinetochore-microtubule (K-MT)attachments and regulating bipolar spindle assembly. How localized activity of AURKC is regulated in mammalian oocytes, however, is not fully understood. Female gametes from many species, including mouse, contain stores of maternal transcripts that are required for downstream developmental events. We show here that depletion of maternal RNA in mouse oocytes resulted in impaired meiotic progression, increased incidence of chromosome misalignment and abnormal spindle formation at metaphase I (Met I), and cytokinesis defects. Importantly, depletion of maternal RNA perturbed the localization and activity of AURKC within the chromosomal passenger complex (CPC). These perturbations were not observed when translation was inhibited by cycloheximide (CHX) treatment. These results demonstrate a translation-independent function of maternal RNA to regulate AURKC-CPC function in mouse oocytes. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: firstname.lastname@example.org.
Das, Arunika; Shah, Shital J.; Fan, Bensen; Paik, Daniel; DiSanto, Daniel J.; Hinman, Anna Maria; Cesario, Jeffry M.; Battaglia, Rachel A.; Demos, Nicole; McKim, Kim S.
Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule–chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation. PMID:26564158
Yurdun Kuyucu; Ozgul Tap
Normal female fertility depends on normally occuring oogenesis and maturation progress. Oogenesis and folliculogenesis are different progresses but occure in a harmony and at the same time. Oogenesis includes the events that take place matur ovum produced from primordial germ cells. Although folliculogenesis includes the stages primordial, primary, secondary, matur (Graaf) follicules in the influece of gonadotropines and local growth factors. During oocyte maturation meiosis is distrupted til...
Remohí, J; Gartner, B; Gallardo, E; Yalil, S; Simón, C; Pellicer, A
To determine accumulated conception and live birth rates in ovum donation. Retrospective study from a computer database. Pregnancies with one gestational sac observed by ultrasound have been included as conceptional cycles and pregnancies that resulted in one live child were recorded for the analysis of the live birth rates. Life table analysis was applied. Oocyte donation program at the Instituto Valenciano de Infertilidad. Three hundred ninety-seven recipients undergoing a total of 627 ETs were analyzed. Ovarian stimulation and ovum pick-up in donors. Uterine ET in recipients after appropriate exogenous steroid replacement. Accumulated and estimated (95% confidence intervals [CI]) conception and live birth rates in the oocyte donation program as well as considering age and cause of infertility of the recipients. Pregnancy rate after one cycle was 53.4% (CI 50.9% to 55.9%), with a delivery rate of 42.6% (CI 40.1% to 45.1%). Accumulated pregnancy rate increased up to 94.8% (CI 90.6% to 99.0%) after four transfers. Similarly, live birth rates reached 88.7% (CI 88.1% to 89.3%) after four attempts of ET by ovum donation. Cycle fecundity rates were maintained at approximately 50% after each attempt. Implantation rate was 18.3% (430/2,340 replaced embryos). Age and cause of entering the program did not influence the overall results of ovum donation. Oocyte donation is a successful treatment modality for infertile couples that offers even higher success rates than natural conception. No difference in cumulative pregnancy rate was observed regardless of recipient age, indication for oocyte donation, or number of cycles attempted.
Yu, X.; Berg, L. K.; Berkowitz, C. M.; Alexander, M. L.; Lee, Y.; Laskin, A.; Ogren, J. A.; Andrews, B.
The Cumulus Humilis Aerosol Processing Study (CHAPS) provided a unique opportunity to study aerosol and cloud processing. Clouds play an active role in the processing and cycling of atmospheric constituents. Gases and particles can partition to cloud droplets by absorption and condensation as well as activation and pact scavenging. The Department of Energy (DOE) G-1 aircraft was used as one of the main platforms in CHAPS. Flight tracks were designed and implemented to characterize freshly emitted aerosols on cloud top and cloud base as well as with cloud, i.e., cumulus humilis (or fair-weather cumulus), in the vicinity of Oklahoma City. Measurements of interstitial aerosols and residuals of activated condensation cloud nuclei were conducted simultaneously. The interstitial aerosols were determined downstream of an isokinetic inlet; and the activated particles downstream of a counter-flow virtual impactor (CVI). The sampling line to the Aerodyne Aerosol Mass Spectrometer was switched between the isokinetic inlet and the CVI to allow characterization of interstitial particles out of clouds in contrast to particles activated in clouds. Trace gases including ozone, carbon monoxide, sulfur dioxide, and a series of volatile organic compounds (VOCs) were also measured as were key meteorological state parameters including liquid water content, cloud drop size, and dew point temperature were measured. This work will focus on studying CCN properties in cumulus humilis. Several approaches will be taken. The first is single particle analysis of particles collected by the Time-Resolved Aerosol Sampler (TRAC) by SEM/TEM coupled with EDX. We will specifically look into differences in particle properties such as chemical composition and morphology between activated and interstitial ones. The second analysis will link in situ measurements with the snap shots observations by TRAC. For instance, by looking into the characteristic m/z obtained by AMS vs. CO or isoprene, one can gain
Hwang, Grace; Sun, Fengyun; O'Brien, Marilyn; Eppig, John J; Handel, Mary Ann; Jordan, Philip W
SMC complexes include three major classes: cohesin, condensin and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis have not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre-driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase IIα to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females. © 2017. Published by The Company of Biologists Ltd.
Full Text Available Objective: Artificial stimulation of mouse oocyte, in the absence of sperm contribution,can induce its parthenogenic activation of oocyte. Ultrasound is one of the newest methodsfor artificial activation of mammal oocytes, and its successful utilization in pig oocyteactivation has been recently reported. Our objective was to assess the effect of ultrasoundon mouse oocyte activation.Materials and Methods: Our groups included1 control group, 3 experimental groups consistingof 1, 2 and 3 repetitions of ultrasound exposure, and 3 sham groups handled similarto experimental groups but ultrasound system was off during treatments.In experimental groups, adult female NMRI mice at the interval between pregnant mareserum gonadotropin (PMSG and human corionic gonadotropin (hCG injections, wereexposed to continuous ultrasound with 3.28 MHz frequency and peak intensity (Ipk = 355mW/cm2.Sixteen hours after injection of hCG, the mice were euthanized and their oocytes werecollected; thereafter, parthenogenic oocytes were counted.Results: Data analysis using the ANOVA test shows a significant increase in the number ofparthenogenic oocytes in mice with 3 overall exposures to ovarian ultrasound (p<0.05.A significant decrease in the number of metaphase II (MII oocytes numbers was alsoseen in mice treated with ultrasound (p<0.05.Conclusion: Ultrasound is thought to induce pores generation in oocyte membranes andprovides an easier inward transport of Ca++ into oocytes. This phenomenon can inducemeiosis resumption in immature oocytes. With increased exposure repetitions from 1 to 3times and greater Ca++ arrival, oocytes can be parthenogenetically activated.
Hanna, R E B; Moffett, D; Forster, F I; Trudgett, A G; Brennan, G P; Fairweather, I
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.
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: email@example.com [Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160 (United States)
Highlights: • PTK2b is expressed in oocytes and is activated following fertilization. • PTK2b suppression in oocytes prevents fertilization, but not parthenogenetic activation. • PTK2b suppression prevents the oocyte from fusing with or incorporating bound sperm. • PTK2b suppressed oocytes that fail to fertilize do not exhibit calcium oscillations. - Abstract: Fertilization triggers rapid changes in intracellular free calcium that serve to activate multiple signaling events critical to the initiation of successful development. Among the pathways downstream of the fertilization-induced calcium transient is the calcium-calmodulin dependent protein tyrosine kinase PTK2b or PYK2 kinase. PTK2b plays an important role in fertilization of the zebrafish oocyte and the objective of the present study was to establish whether PTK2b also functions in mammalian fertilization. PTK2b was activated during the first few hours after fertilization of the mouse oocyte during the period when anaphase resumption was underway and prior to the pronuclear stage. Suppression of PTK2b kinase activity in oocytes blocked sperm incorporation and egg activation although sperm-oocyte binding was not affected. Oocytes that failed to incorporate sperm after inhibitor treatment showed no evidence of a calcium transient and no evidence of anaphase resumption suggesting that egg activation did not occur. The results indicate that PTK2b functions during the sperm-egg fusion process or during the physical incorporation of sperm into the egg cytoplasm and is therefore critical for successful development.
Wang, Qian; Wei, Haojie; Du, Juan; Cao, Yan; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Chen, Dandan; Ma, Wei
Haspin-catalyzed histone H3 threonine 3 (Thr3) phosphorylation facilitates chromosomal passenger complex (CPC) docking at centromeres, regulating indirectly chromosome behavior during somatic mitosis. It is not fully known about the expression and function of H3 with phosphorylated Thr3 (H3T3-P) during meiosis in oocytes. In this study, we investigated the expression and sub-cellular distribution of H3T3-P, as well as its function in mouse oocytes during meiotic division. Western blot analysis revealed that H3T3-P expression was only detected after germinal vesicle breakdown (GVBD), and gradually increased to peak level at metaphase I (MI), but sharply decreased at metaphase II (MII). Immunofluorescence showed H3T3-P was only brightly labeled on chromosomes after GVBD, with relatively high concentration across the whole chromosome axis from pro-metaphase I (pro-MI) to MI. Specially, H3T3-P distribution was exclusively limited to the local space between sister centromeres at MII stage. Haspin inhibitor, 5-iodotubercidin (5-ITu), dose- and time-dependently blocked H3T3-P expression in mouse oocytes. H3T3-P inhibition delayed the resumption of meiosis (GVBD) and chromatin condensation. Moreover, the loss of H3T3-P speeded up the meiotic transition to MII of pro-MI oocytes in spite of the presence of non-aligned chromosomes, even reversed MI-arrest induced with Nocodazole. The inhibition of H3T3-P expression distinguishably damaged MAD1 recruitment on centromeres, which indicates the spindle assembly checkpoint was impaired in function, logically explaining the premature onset of anaphase I. Therefore, Haspin-catalyzed histone H3 phosphorylation is essential for chromatin condensation and the following timely transition from meiosis I to meiosis II in mouse oocytes during meiotic division.
Full Text Available Final oocyte maturation (FOM is a process involving a complex set of genetical, biochemical, and morphological mechanisms. FOM involves the shift of a post-vitellogenic follicle to a pre-ovulated oocyte, which is necessary for fertilization by spermatozoan to occur. This process is regulated by a maturation-inducing steroid (MIS at the follicular level. In other species of scienids fish the MIS, a hydroxilated derivatives of progestagen 17, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S, was identified. Although Micropogonias furnieri is the second fishery resource of Uruguay, basic knowledge about its endocrine process is very scarce. The aim of this work was to investigate what steroids are synthesized in vitro by the oocyte follicle of M. furnieri during the maturation process. Fragments of ovary (1 g in three stages: post-vitellogenic (PV, maturing (Mtg, and mature (M were incubated with 1 mug.g-1 of tritiated progesterone (P at 30, 60, and 180 min. After extraction with ethanol and dichloromethane, steroid metabolites were purified by TLC and rpHPLC. Two progesterone derivatives with identical chromatographic properties of 20beta-S and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P were purified. In other Teleost fish these steroids are biologically activ as MIS. The 17,20beta-P was clearly detected in Mtg and M stages and confirmed by enzymatic oxidation with enzyme 20beta-HSD. The 20beta-S was strongly detected in all Mtg oocytes. The results do not corroborate 20beta-S as a major hormone synthesized in the ovary in FOM as occurs in other scienid fish. A differential steroid synthesis in the advanced oocyte stages suggests that the 20beta-S is acting as a MIS in M. furnieri.
Liu, Shuai; Nong, Suqun; Ma, Qingyan; Chen, Baojian; Liu, Mingjun; Pan, Tianbiao; Liao, D. Joshua
The maternal-to-embryonic transition (MET) is a complex process that occurs during early mammalian embryogenesis and is characterized by activation of the zygotic genome, initiation of embryonic transcription, and replacement of maternal mRNA with embryonic mRNA. The objective of this study was to reveal the temporal expression and localization patterns of PTTG1 during early porcine embryonic development and to establish a relationship between PTTG1 and the MET. To achieve this goal, reverse transcription-polymerase chain reaction (RT-PCR) was performed to clone porcine PTTG1. Subsequently, germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, zygotes, 2-, 4-, and 8-cell-stage embryos, morulas, and blastocysts were produced in vitro and their gene expression was analyzed. The results revealed that the coding sequence of porcine PTTG1 is 609-bp in length and that it encodes a 202-aa polypeptide. Using qRT-PCR, PTTG1 mRNA expression was observed to be maintained at high levels in GV- and MII-stage oocytes. The transcript levels in oocytes were also significantly higher than those in embryos from the zygote to blastocyst stages. Immunohistochemical analyses revealed that porcine PTTG1 was primarily localized to the cytoplasm and partially localized to the nucleus. Furthermore, the PTTG1 protein levels in MII-stage oocytes and zygotes were significantly higher than those in embryos from the 2-cell to blastocyst stage. After fertilization, the level of this protein began to decrease gradually until the blastocyst stage. The results of our study suggest that porcine PTTG1 is a new candidate maternal effect gene (MEG) that may participate in the processes of oocyte maturation and zygotic genome activation during porcine embryogenesis. PMID:27058238
Park, Chan Woo; Lee, Sun Hee; Yang, Kwang Moon; Lee, In Ho; Lim, Kyung Teak; Lee, Ki Heon
Objective The aim of this study was to report a case series of in vitro matured (IVM) oocyte freezing in gynecologic cancer patients undergoing radical surgery under time constraints as an option for fertility preservation (FP). Methods Case series report. University-based in vitro fertilization center. Six gynecologic cancer patients who were scheduled to undergo radical surgery the next day were referred for FP. The patients had endometrial (n=2), ovarian (n=3), and double primary endometrial and ovarian (n=1) cancer. Ex vivo retrieval of immature oocytes from macroscopically normal ovarian tissue was followed by mature oocyte freezing after IVM or embryo freezing with intracytoplasmic sperm injection. Results A total of 53 oocytes were retrieved from five patients, with a mean of 10.6 oocytes per patient. After IVM, a total of 36 mature oocytes were obtained, demonstrating a 67.9% maturation rate. With regard to the ovarian cancer patients, seven IVM oocytes were frozen from patient 3, who had stage IC cancer, whereas one IVM oocyte was frozen from patient 4, who had stage IV cancer despite being of a similar age. With regard to the endometrial cancer patients, 15 IVM oocytes from patient 1 were frozen. Five embryos were frozen after the fertilization of IVM oocytes from patient 6. Conclusion Immature oocytes can be successfully retrieved ex vivo from macroscopically normal ovarian tissue before radical surgery. IVM oocyte freezing provides a possible FP option in patients with advanced-stage endometrial or ovarian cancer without the risk of cancer cell spillage or time delays. PMID:27358831
A tangent linear and an adjoint of the large-scale precipitation and the cumulus convection processes in the National Meteorological Center's NMC/ETA regional forecast model are developed. The effects of discontinuities in the Betts Miller cumulus convection scheme are examined and applicability of derivative minimization methods in four-dimensional variational (4D VAR) data assimilation is considered. It is demonstrated that discontinuities present in the control Betts Miller cumulus convection scheme increase linearization errors to a large extent and have adverse effects on 4D VAR data assimilation. In the experiments performed, discontinuities in the cumulus convection scheme have the most serious effect in low layers. These problems can be reduced by modifying the scheme to make it more continuous in low layers. Positive effects of inclusion of cumulus convection in 4D VAR data assimilation are found in upper layers, especially in humidity fields. The "observations" used are optimal interpolation analyses of temperature, surface pressure, wind and specific humidity. By inclusion of other data, more closely related to the convective processes, such as precipitation and clouds, more benefits should be expected. Even with the difficulties caused by discontinuities, derivative minimization techniques appear to work for the data assimilation problems. In order to get more general conclusions, more experiments are needed with different synoptic situations. The inclusion of other important physical processes such as radiation, surface friction and turbulence in the forecast and the corresponding adjoint models could alter the results since they may reinforce the effects of discontinuities.
Guimarães, A C D; Quagio-Grassiotto, I
The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups.
Grøndahl, Marie Louise; Borup, Rehannah; Vikeså, Jonas
Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human...... 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...
Full Text Available Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H2S in the process of porcine oocyte aging. H2S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS, cystathionine-γ-lyase (CSE and 3-mercaptopyruvate sulfurtransferase (MPST. We demonstrated that H2S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H2S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H2S from a donor (Na2S.9H2O significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H2S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H2S on MPF and MAPK activities were detected and the intracellular mechanism underlying H2S activity remains unclear, our study clearly demonstrates the role of H2S in the regulation of porcine oocyte aging.
Tombes, R. M.; Simerly, C.; Borisy, G. G.; Schatten, G.
During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.
Fong, S. Lie; Baart, E. B.; Martini, E.; Schipper, I.; Visser, J. A.; Themmen, A. P. N.; de Jong, F. H.; Fauser, B. J. C. M.; Laven, J. S. E.
Serum anti-Mullerian hormone (AMH) concentrations decline with increasing age and constitute a Sensitive marker for ovarian ageing. In addition, basal serum AMH concentrations predict ovarian response during IVF cycles. Concomitantly, oocyte quantity and embryo quality decrease with advancing age. H
Qiao, Fengxue; Liang, Xin-Zhong
This study examines the effects of five cumulus closure assumptions on simulations of summer precipitation in the continental U.S. by utilizing an ensemble cumulus parameterization (ECP) that incorporates multiple alternate closure schemes into a single cloud model formulation. Results demonstrate that closure algorithms significantly affect the summer mean, daily frequency and intensity, and diurnal variation of precipitation, with strong regional dependence. Overall, the vertical velocity (W) closure produces the smallest summer mean biases, while the moisture convergence (MC) closure most realistically reproduces daily variability. Both closures have advantages over others in simulating U.S. daily rainfall frequency distribution, though both slightly overestimate intense rain events. The MC closure is superior at capturing summer rainfall amount, daily variability, and heavy rainfall frequency over the Central U.S., but systematically produces wet biases over the North American Monsoon (NAM) region and Southeast U.S., which can be reduced by using the W closure. The instability tendency (TD) and the total instability adjustment (KF) closures are better at capturing observed diurnal signals over the Central U.S. and the NAM, respectively. The results reasonably explain the systematic behaviors of several major cumulus parameterizations. A preliminary experiment combining two optimal closures (averaged moisture convergence and vertical velocity) in the ECP scheme significantly reduced the wet (dry) biases over the Southeast U.S. in the summer of 1993 (2003), and greatly improved daily rainfall correlations over the NAM. Further improved model simulation skills may be achieved in the future if optimal closures and their appropriate weights can be derived at different time scales based on specific climate regimes.
Litta, A. J.; Chakrapani, B.; Mohankumar, K.
Heavy rainfall events become significant in human affairs when they are combined with hydrological elements. The problem of forecasting heavy precipitation is especially difficult since it involves making a quantitative precipitation forecast, a problem well recognized as challenging. Chennai (13.04°N and 80.17°E) faced incessant and heavy rain about 27 cm in 24 hours up to 8.30 a.m on 27th October 2005 completely threw life out of gear. This torrential rain caused by deep depression which lay 150km east of Chennai city in Bay of Bengal intensified and moved west north-west direction and crossed north Tamil Nadu and south Andhra Pradesh coast on 28th morning. In the present study, we investigate the predictability of the MM5 mesoscale model using different cumulus parameterization schemes for the heavy rainfall event over Chennai. MM5 Version 3.7 (PSU/NCAR) is run with two-way triply nested grids using Lambert Conformal Coordinates (LCC) with a nest ratio of 3:1 and 23 vertical layers. Grid sizes of 45, 15 and 5 km are used for domains 1, 2 and 3 respectively. The cumulus parameterization schemes used in this study are Anthes-Kuo scheme (AK), the Betts-Miller scheme (BM), the Grell scheme (GR) and the Kain-Fritsch scheme (KF). The present study shows that the prediction of heavy rainfall is sensitive to cumulus parameterization schemes. In the time series of rainfall, Grell scheme is in good agreement with observation. The ideal combination of the nesting domains, horizontal resolution and cloud parameterization is able to simulate the heavy rainfall event both qualitatively and quantitatively.
Qiao, Fengxue; Liang, Xin-Zhong
This study examines the effects of five cumulus closure assumptions on simulations of summer precipitation in the continental U.S. by utilizing an ensemble cumulus parameterization (ECP) that incorporates multiple alternate closure schemes into a single cloud model formulation. Results demonstrate that closure algorithms significantly affect the summer mean, daily frequency and intensity, and diurnal variation of precipitation, with strong regional dependence. Overall, the vertical velocity (W) closure produces the smallest summer mean biases, while the moisture convergence (MC) closure most realistically reproduces daily variability. Both closures have advantages over others in simulating U.S. daily rainfall frequency distribution, though both slightly overestimate intense rain events. The MC closure is superior at capturing summer rainfall amount, daily variability, and heavy rainfall frequency over the Central U.S., but systematically produces wet biases over the North American Monsoon (NAM) region and Southeast U.S., which can be reduced by using the W closure. The instability tendency (TD) and the total instability adjustment (KF) closures are better at capturing observed diurnal signals over the Central U.S. and the NAM, respectively. The results reasonably explain the systematic behaviors of several major cumulus parameterizations. A preliminary experiment combining two optimal closures (averaged moisture convergence and vertical velocity) in the ECP scheme significantly reduced the wet (dry) biases over the Southeast U.S. in the summer of 1993 (2003), and greatly improved daily rainfall correlations over the NAM. Further improved model simulation skills may be achieved in the future if optimal closures and their appropriate weights can be derived at different time scales based on specific climate regimes.
Brast, Maren; Schemann, Vera; Neggers, Roel
In this study we investigate the scale-adaptivity of a new parameterization scheme for shallow cumulus clouds in the gray zone. The Eddy-Diffusivity Multiple Mass-Flux (or ED(MF)n ) scheme is a bin-macrophysics scheme, in which subgrid transport is formulated in terms of discretized size densities. While scale-adaptivity in the ED-component is achieved using a pragmatic blending approach, the MF-component is filtered such that only the transport by plumes smaller than the grid size is maintained. For testing, ED(MF)n is implemented in a large-eddy simulation (LES) model, replacing the original subgrid-scheme for turbulent transport. LES thus plays the role of a non-hydrostatic testing ground, which can be run at different resolutions to study the behavior of the parameterization scheme in the boundary-layer gray zone. In this range convective cumulus clouds are partially resolved. We find that at high resolutions the clouds and the turbulent transport are predominantly resolved by the LES, and the transport represented by ED(MF)n is small. This partitioning changes towards coarser resolutions, with the representation of shallow cumulus clouds becoming exclusively carried by the ED(MF)n. The way the partitioning changes with grid-spacing matches the results of previous LES studies, suggesting some scale-adaptivity is captured. Sensitivity studies show that a scale-inadaptive ED component stays too active at high resolutions, and that the results are fairly insensitive to the number of transporting updrafts in the ED(MF)n scheme. Other assumptions in the scheme, such as the distribution of updrafts across sizes and the value of the area fraction covered by updrafts, are found to affect the location of the gray zone.
Gatebe, C. K.; Varnai, T.; Poudyal, R.; Ichoku, C.; King, M. D.
Forest fires can burn large areas, but can also inject smoke into the upper troposphere/lower stratosphere (UT/LS), where stakes are even higher for climate, because emissions tend to have a longer lifetime, and can produce significant regional and even global climate effects, as is the case with some volcanoes. Large forest fires are now believed to be more common in summer, especially in the boreal regions, where pyrocumulus (pyroCu), and occasionally pyrocumuionimbus (pyroCb) clouds are formed, which can transport emissions into the UT/LS. A major difficulty in developing realistic fire plume models is the lack of observational data within fire plumes that resolves structure at a few 100 m scales, which can be used to validate these models. Here, we report detailed airborne radiation measurements within strong pyroCu taken over boreal forest fires in Saskatchewan, Canada during the Arctic Research of the Composition of the Troposphere from Aircraft and Satellites (ARCTAS) summer field campaign in 2008. We find that the angular distribution of radiance within the pyroCu is closely related to the diffusion domain in water clouds and can be described by very similar simple cosine functions. We demonstrate with Monte Carlo simulations that radiation transport in pyroCu is inherently a 3D phenomenon and must account for particle absorption. However, the simple cosine function promises to offer an easy solution for climate models. The presence of a prominent smoke core, defined by strong extinction in the UV, VIS and NIR, suggests that the core might be an important pathway for emission transport to the upper troposphere and lower stratosphere. We speculate that this plume injection core is generated and sustained by complex processes not yet well understood, but not necessarily related directly to the intense fires that originally initiated the plume rise.
Kumar, Dinesh; Charan Mohanty, Uma; Kumar, Krishan
The cloud processes play an important role in all forms of precipitation. Its proper representation is one of the challenging tasks in mesoscale numerical simulation. Studies have revealed that mesoscale feature require proper initialization which may likely to improve the convective system rainfall forecasts. Understanding the precipitation process, model initial condition accuracy and resolved/sub grid-scale precipitation processes representation, are the important areas which needed to improve in order to represent the mesoscale features properly. Various attempts have been done in order to improve the model performance through grid resolution, physical parameterizations, etc. But it is the physical parameterizations which provide a convective atmosphere for the development and intensification of convective events. Further, physical parameterizations consist of cumulus convection, surface fluxes of heat, moisture, momentum, and vertical mixing in the planetary boundary layer (PBL). How PBL and Cumulus schemes capture the evolution of thunderstorm have been analysed by taking thunderstorm cases occurred over Kolkata, India in the year 2011. PBL and cumulus schemes were customized for WSM-6 microphysics because WSM series has been widely used in operational forecast. Results have shown that KF (PBL scheme) and WSM-6 (Cumulus Scheme) have reproduced the evolution of surface variable such as CAPE, temperature and rainfall very much like observation. Further, KF and WSM-6 scheme also provided the increased moisture availability in the lower atmosphere which was taken to higher level by strong vertical velocities providing a platform to initiate a thunderstorm much better. Overestimation of rain in WSM-6 occurs primarily because of occurrence of melting and freezing process within a deeper layer in WSM-6 scheme. These Schemes have reproduced the spatial pattern and peak rainfall coverage closer to TRMM observation. It is the the combination of WSM-6, and KF schemes
Barlow, Roy W.; Nicholls, S.
On several occasions during the FIRE Marine Stratocumulus IFO off the California coast, small cumulus were observed to form during the morning beneath the main stratocumulus (Sc) deck. This occurs in the type of situation described by Turton and Nicholls (1987) in which there is insufficient generation of turbulent kinetic energy (TKE) from the cloudtop or the surface to sustain mixing throughout the layer, and a separation of the surface and cloud layers occurs. The build up of humidity in the surface layer allows cumuli to form, and the more energetic of these may penetrate back into the Sc deck, reconnecting the layers. The results presented were collected by the UKMO C-130 aircraft flying in a region where these small cumulus had grown to the extent that they had penetrated into the main Sc deck above. The structure of these penetrative cumulus are examined and their implications on the layer flux and radiation budget discussed.
Ma, Jun-Yu; Li, Mo; Luo, Yi-Bo; Song, Shuhui; Tian, Dongmei; Yang, Jin; Zhang, Bing; Hou, Yi; Schatten, Heide; Liu, Zhonghua; Sun, Qing-Yuan
During mouse antral follicle development, the oocyte chromatin gradually transforms from a less condensed state with no Hoechst-positive rim surrounding the nucleolus (NSN) to a fully condensed chromatin state with a Hoechst-positive rim surrounding the nucleolus (SN). Compared with SN oocytes, NSN oocytes display a higher gene transcription activity and a lower rate of meiosis resumption (G2/M transition), and they are mostly arrested at the two-cell stage after in vitro fertilization. To explore the differences between NSN and SN oocytes, and the maternal factors required for oocyte developmental competence, we compared the whole-transcriptome profiles between NSN and SN oocytes. First, we found that the NSN and SN oocytes were different in their metabolic pathways. In the phosphatidylinositol signaling pathway, the SN oocytes tend to produce diacylglycerol, whereas the NSN oocytes tend to produce phosphatidylinositol (3,4,5)-trisphosphate. For energy production, the SN oocytes and NSN oocytes differed in the gluconeogenesis and in the synthesis processes. Second, we also found that the key genes associated with oocyte meiosis and/or preimplantation embryo development were differently expressed in the NSN and SN oocytes. Our results illustrate that during the NSN-SN transition, the oocytes change their metabolic activities and accumulate maternal factors for further oocyte maturation and post-fertilization embryo development.
Lord, Tessa; Aitken, R John
With extended periods of time following ovulation, the metaphase II stage oocyte experiences deterioration in quality referred to as post-ovulatory oocyte ageing. Post-ovulatory ageing occurs both in vivo and in vitro and has been associated with reduced fertilization rates, poor embryo quality, post-implantation errors and abnormalities in the offspring. Although the physiological consequences of post-ovulatory oocyte ageing have largely been established, the molecular mechanisms controlling this process are not well defined. This review analyses the relationships between biochemical changes exhibited by the ageing oocyte and the symptoms associated with the ageing phenotype. We also discuss molecular events that are potentially involved in orchestrating post-ovulatory ageing with a particular focus on the role of oxidative stress. We propose that oxidative stress may act as the initiator for a cascade of events that create the aged oocyte phenotype. Specifically, oxidative stress has the capacity to cause a decline in levels of critical cell cycle factors such as maturation-promoting factor, impair calcium homoeostasis, induce mitochondrial dysfunction and directly damage multiple intracellular components of the oocyte such as lipids, proteins and DNA. Finally, this review addresses current strategies for delaying post-ovulatory oocyte ageing with a particular focus on the potential use of compounds such as caffeine or selected antioxidants in the development of more refined media for the preservation of oocyte integrity during IVF procedures.
Malchau, Sara S; Loft, Anne; Larsen, Elisabeth C;
To describe perinatal outcomes in children born after oocyte donation (OD) compared with in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and spontaneous conception (SC).......To describe perinatal outcomes in children born after oocyte donation (OD) compared with in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and spontaneous conception (SC)....
Demeestere, Isabelle; Streiff, Agathe K; Suzuki, João; Al-Khabouri, Shaima; Mahrous, Enas; Tan, Seang Lin; Clarke, Hugh J
During folliculogenesis, oocytes grow and acquire developmental competence in a mutually dependent relationship with their adjacent somatic cells. Follicle-stimulating hormone (FSH) plays an essential and well-established role in the differentiation of somatic follicular cells, but its function in the development of the oocyte has still not been elucidated. We report here that oocytes of Fshb(-/-) mice, which cannot produce FSH, grow at the same rate and reach the same size as those of wild-type mice. Consistent with this observation, the granulosa cells of Fshb(-/-) mice express the normal quantity of mRNA encoding Kit ligand, which has been implicated in oocyte growth. Oocytes of Fshb(-/-) mice also accumulate normal quantities of cyclin B1 and CDK1 proteins and mitochondrial DNA. Moreover, they acquire the ability to complete meiotic maturation in vitro and undergo transition from non-surrounded nucleolus to surrounded nucleolus. However, these events of late oocyte development are significantly delayed. Following in vitro maturation and fertilization, only a small number of embryos derived from oocytes of Fshb(-/-) mice reach the blastocyst stage. Administration of equine chorionic gonadotropin, which provides FSH activity, 48 h before in vitro maturation increases the number of blastocysts obtained subsequently. These results indicate that FSH is not absolutely required for oocyte development in vivo but that this process occurs more rapidly in its presence. We suggest that FSH may coordinate the development of the germline and somatic compartments of the follicle, ensuring that ovulation releases a developmentally competent egg.
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.
Greenstein, David; Lee, Laura A
The transition from oocyte to embryo is among the most enthralling events in developmental biology. Recent studies of this transition in the nematode Caenorhabditis elegans have revealed how conserved kinases administer the destruction of key oocyte meiotic regulators to create an embryo.
Monti, Manuela; Calligaro, Alberto; Behr, Barry; Pera, Renee Rejo; Redi, Carlo Alberto; Wossidlo, Mark
In vivo maturation (IVM) of human oocytes is a technique used to increase the number of usable oocytes for in vitro fertilization (IVF) and represents a necessity for women with different ovarian pathologies. During IVM the oocytes progress from the germinal vesicle stage (GV) through the metaphase II and during this journey both nuclear and cytoplasmic rearrangements must be obtained to increase the probability to get viable and healthy zygotes/embryos after IVF. As the successful clinical outcomes of this technique are a reality, we wanted to investigate the causes behind oocytes maturation arrest. For obvious ethical reasons, we were able to analyze only few human immature oocytes discarded and donated to research by transmission electron microscopy showing that, as in the mouse, they have different chromatin and cytoplasmic organizations both essential for further embryo development.
Full Text Available The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing.
Devroey, P; Wisanto, A; Camus, M; Van Waesberghe, L; Bourgain, C; Liebaers, I; Van Steirteghem, A C
The clinical, hormonal and cytogenetic findings in 36 women with primary ovarian failure, referred for oocyte or embryo donations are reported. Fifteen women were suffering from ovarian dysgenesis and 11 from premature menopause. Six of these 26 patients showed X-chromosome abnormalities. One patient had a Noonan syndrome. The remaining 10 had surgical menopause. The mean duration of their infertility was 6.5 +/- 3.2 years (+/- SD). All patients had elevated serum gonadotrophins within the menopausal range. Hypothalamic, pituitary and thyroid function were found to be intact. In one of the 15 ovarian biopsies on the patients with chromosomal competent ovarian failure, primordial follicles were found. Hysterosalpingograms revealed a normal uterine cavity in all patients. In view of oocyte donation, careful evaluation of the obstetric risk was mandatory in the six patients with X-chromosome aberrations and in the patient with the Noonan syndrome, because of their short stature and possible concomitant cardiovascular and renal disease. After substitution therapy with oestradiol valerate and natural progesterone, 13 pregnancies were established, seven patients delivered (one set of twins), eight healthy children were born, three pregnancies aborted and three pregnancies are progressing normally.
Chang, P; Pérez-Mongiovi, D; Houliston, E
The division of the Xenopus oocyte cortex into structurally and functionally distinct "animal" and "vegetal" regions during oogenesis provides the basis of the organisation of the early embryo. The vegetal region of the cortex accumulates specific maternal mRNAs that specify the development of the endoderm and mesoderm, as well as functionally-defined "determinants" of dorso-anterior development, and recognisable "germ plasm" determinants that segregate into primary germ cells. These localised elements on the vegetal cortex underlie both the primary animal-vegetal polarity of the egg and the organisation of the developing embryo. The animal cortex meanwhile becomes specialised for the events associated with fertilisation: sperm entry, calcium release into the cytoplasm, cortical granule exocytosis, and polarised cortical contraction. Cortical and subcortical reorganisations associated with meiotic maturation, fertilisation, cortical rotation, and the first mitotic cleavage divisions redistribute the vegetal cortical determinants, contributing to the specification of dorso-anterior axis and segregation of the germ line. In this article we consider what is known about the changing organisation of the oocyte and egg cortex in relation to the mechanisms of determinant localisation, anchorage, and redistribution, and show novel ultrastructural views of cortices isolated at different stages and processed by the rapid-freeze deep-etch method. Cortical organisation involves interactions between the different cytoskeletal filament systems and internal membranes. Associated proteins and cytoplasmic signals probably modulate these interactions in stage-specific ways, leaving much to be understood.
Berkowitz, Carl M.; Berg, Larry K.; Ogren, J. A.; Hostetler, Chris A.; Ferrare, Richard
This white paper presents the scientific motivation and preliminary logistical plans for a proposed ASP field campaign to be carried out in the summer of 2007. The primary objective of this campaign is to use the DOE Gulfstream-1 aircraft to make measurements characterizing the chemical, physical and optical properties of aerosols below, within and above large fields of fair weather cumulus and to use the NASA Langley Research Center’s High Spectral Resolution Lidar (HSRL) to make independent measurements of aerosol backscatter and extinction profiles in the vicinity of these fields. Separate from the science questions to be addressed by these observations will be information to add in the development of a parameterized cumulus scheme capable of including multiple cloud fields within a regional or global scale model. We will also be able to compare and contrast the cloud and aerosol properties within and outside the Oklahoma City plume to study aerosol processes within individual clouds. Preliminary discussions with the Cloud and Land Surface Interaction Campaign (CLASIC) science team have identified overlap between the science questions posed for the CLASIC Intensive Operation Period (IOP) and the proposed ASP campaign, suggesting collaboration would benefit both teams.
Shuyan LIU; Wei GAO; Min XU; Xueyuan WANG; Xin-Zhong LIANG
RegCM3 (REGional Climate Model) simulations of precipitation in China in 1991 and 1998 are very sensitive to the cumulus parameterization. Among the four schemes available, none has superior skills over the whole of China, but each captures certain observed signals in distinct regions. The Grell scheme with the FritschChappell closure produces the smallest biases over the North; the Grell scheme with the Arakawa-Schubert closure performs the best over the southeast of 100°E;the Anthes-Kuo scheme is superior over the northeast; and the Emanuel scheme is more realistic over the southwest of 100~E and along the Yangtze River Basin. These differences indicate a strong degree of independence and complementarity between the parameterizations. As such,an ensemble is developed from the four schemes, whose relative contributions or weights are optimized locally to yield overall minimum root-mean-square errors from observed daily precipitation. The skill gain is evaluated by applying the identical distribution of the weights in a different period. It is shown that the ensemble always produces gross biases that are smaller than the individual schemes in both 1991 and 1998. The ensemble, however,cannot eliminate the large rainfall deficits over the southwest of 100°E and along the Yangtze River Basin that are systematic across all schemes. Further improvements can be made by a super-ensemble based on more cumulus schemes and/or multiple models.
Narasimha, Roddam; Diwan, Sourabh Suhas; Duvvuri, Subrahmanyam; Sreenivas, K R; Bhat, G S
Clouds are the largest source of uncertainty in climate science, and remain a weak link in modeling tropical circulation. A major challenge is to establish connections between particulate microphysics and macroscale turbulent dynamics in cumulus clouds. Here we address the issue from the latter standpoint. First we show how to create bench-scale flows that reproduce a variety of cumulus-cloud forms (including two genera and three species), and track complete cloud life cycles--e.g., from a "cauliflower" congestus to a dissipating fractus. The flow model used is a transient plume with volumetric diabatic heating scaled dynamically to simulate latent-heat release from phase changes in clouds. Laser-based diagnostics of steady plumes reveal Riehl-Malkus type protected cores. They also show that, unlike the constancy implied by early self-similar plume models, the diabatic heating raises the Taylor entrainment coefficient just above cloud base, depressing it at higher levels. This behavior is consistent with cloud-dilution rates found in recent numerical simulations of steady deep convection, and with aircraft-based observations of homogeneous mixing in clouds. In-cloud diabatic heating thus emerges as the key driver in cloud development, and could well provide a major link between microphysics and cloud-scale dynamics.
Peter, J. R.; Siems, S. T.; Jensen, J. B.; Gras, J. L.; Ishizaka, Y.; Hacker, J. M.
Airborne measurements, obtained during the Asian Aerosol Characterisation Experiment (ACE-Asia), of SO2 and condensation nuclei (CN) concentrations were made in the local environment of a cumulus cloud band. Conserved quantities, wet equivalent potential temperature θq, and total water content Q, were used to identify the sources of air detrained on the downwind side of the cumulus band. It was found that ~65% of the detrained air originated from below cloud base and the remainder was air that had been entrained from the free troposphere upwind of the cloud and subsequently been detrained. Calculation of the sources of the detrained air parcels enabled a prediction of the concentration of SO2 and CN, assuming that SO2 and CN experienced no processing within cloud. A comparison of the predicted concentration of SO2 and CN was made with those observed. The concentration of SO2 observed was less than predicted and the amount of SO2 scavenged within cloud was calculated. The CN concentration observed was also less than predicted and, moreover, inclusion of the loss of CN to cloud droplets due to Brownian scavenging resulted in an enhanced decrease of the number concentration of CN predicted. Clear air regions around the cloud exhibited no indication of being a major source of new particles. It was concluded that new particles were formed within cloud.
The term complexity derives etymologically from the Latin plexus, which means interwoven. Intuitively, this implies that something complex is composed by elements that are difficult to separate. This difficulty arises from the relevant interactions that take place between components. This lack of separability is at odds with the classical scientific method - which has been used since the times of Galileo, Newton, Descartes, and Laplace - and has also influenced philosophy and engineering. In recent decades, the scientific study of complexity and complex systems has proposed a paradigm shift in science and philosophy, proposing novel methods that take into account relevant interactions.
Voltage-clamp techniques were used to study the membrane currents elicited by follicle stimulating hormone (FSH) and acetylcholine (ACh) in follicle-enclosed oocytes of Xenopus laevis (follicles). Both agonists caused complex responses that were more evident when the follicles were in hypotonic Ringer solution (HR; 190.4 mosM). In this medium, currents activated by FSH regularly showed three phases whereas currents activated by ACh displayed three to six phases. At a holding potential of -60 mV, FSH, and ACh responses involved combinations of inward and outward currents. Both FSH and ACh responses included a slow smooth inward component that was associated with an increase in membrane conductance, mainly to Cl- (S(in)). This current was strongly dependent on the osmolarity of the external solution: an increase in osmolarity of the HR solution of 18-20 mosM caused a 50% decrease in S(in). In contrast, a fast and transient Cl- current (F(in)) specifically elicited by ACh was not dependent on osmolarity. Both, F(in) and S(in) currents required the presence of follicular cells, since defolliculation using three different methods abolished all the response to FSH and at least four components of the ACh responses. The membrane channels carrying F(in) and oscillatory Cl- currents elicited by stimulation of ACh or serum receptors, were much more permeable to I- and Br- than Cl-, whereas S(in) channels were equally permeable to these anions. Unlike the oscillatory Cl- currents generated in the oocyte itself, S(in) and F(in) currents in follicle-enclosed oocytes were not abolished by chelation of intracellular Ca2+, either with EGTA or BAPTA, which suggests that intracellular Ca2+ does not play a critical role in the activation of these currents. Our experiments show that S(in) and F(in) currents are quite distinct from the previously characterized oscillatory Cl- responses of oocytes. Moreover, the results strongly suggest that the FSH and ACh receptors, the Cl- channels
Riepsamen, Angelique; Wu, Lindsay; Lau, Laurin; Listijono, Dave; Ledger, William; Sinclair, David; Homer, Hayden
Following exit from meiosis I, mammalian oocytes immediately enter meiosis II without an intervening interphase, accompanied by rapid reassembly of a bipolar spindle that maintains condensed chromosomes in a metaphase configuration (metaphase II arrest). Here we study the effect of nicotinamide (NAM), a non-competitive pan-sirtuin inhibitor, during meiotic maturation in mouse oocytes. Sirtuins are a family of seven NAD+-dependent deacetylases (Sirt1-7), which are involved in multiple cellular processes and are emerging as important regulators in oocytes and embryos. We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1. GVBD was also inhibited by the Sirt2-specific inhibitor, AGK2, and in a very similar pattern to NAM, supporting the notion that as in somatic cells, NAM inhibits sirtuins in oocytes. NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE). Unexpectedly, however, in the majority of oocytes with a polar body, chromatin was decondensed and a nuclear structure was present. An identical phenotype was observed when flavopiridol was used to induce Cdk1 inactivation during late meiosis I prior to PBE, but not if Cdk1 was inactivated after PBE when metaphase II arrest was already established, altogether indicating that NAM impaired establishment rather than maintenance of metaphase II arrest. During meiosis I exit in NAM-treated medium, we found that cyclin B1 levels were lower and inhibitory Cdk1 phosphorylation was increased compared with controls. Although activation of the anaphase-promoting complex-Cdc20 (APC-Cdc20) occurred on-time in NAM-treated oocytes, Cdc20 levels were higher in very late meiosis I, pointing to exaggerated APC-Cdc20-mediated proteolysis as a reason for lower cyclin B1 levels. Collectively, therefore, our data indicate that by disrupting Cdk1 regulation, NAM impairs entry into meiosis I and
Qi, Shu-Tao; Wang, Zhen-Bo; Ouyang, Ying-Chun; Zhang, Qing-Hua; Hu, Meng-Wen; Huang, Xin; Ge, Zhaojia; Guo, Lei; Wang, Ya-Peng; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan
Chromosome segregation in mammalian oocyte meiosis is an error-prone process, and any mistake in this process may result in aneuploidy, which is the main cause of infertility, abortion and many genetic diseases. It is now well known that shugoshin and protein phosphatase 2A (PP2A) play important roles in the protection of centromeric cohesion during the first meiosis. PP2A can antagonize the phosphorylation of rec8, a member of the cohesin complex, at the centromeres and thus prevent cleavage of rec8 and so maintain the cohesion of chromatids. SETβ is a protein that physically interacts with shugoshin and inhibits PP2A activity. We thus hypothesized that SETβ might regulate cohesion protection and chromosome segregation during oocyte meiotic maturation. Here we report for the first time the expression, subcellular localization and functions of SETβ during mouse oocyte meiosis. Immunoblotting analysis showed that the expression level of SETβ was stable from the germinal vesicle stage to the MII stage of oocyte meiosis. Immunofluorescence analysis showed SETβ accumulation in the nucleus at the germinal vesicle stage, whereas it was targeted mainly to the inner centromere area and faintly localized to the interchromatid axes from germinal vesicle breakdown to MI stages. At the MII stage, SETβ still localized to the inner centromere area, but could relocalize to kinetochores in a process perhaps dependent on the tension on the centromeres. SETβ partly colocalized with PP2A at the inner centromere area. Overexpression of SETβ in mouse oocytes caused precocious separation of sister chromatids, but depletion of SETβ by RNAi showed little effects on the meiotic maturation process. Taken together, our results suggest that SETβ, even though it localizes to centromeres, might not be essential for chromosome separation during mouse oocyte meiotic maturation, although its forced overexpression causes premature chromatid separation.
Nusser, K D; Mitalipov, S; Widmann, A; Gerami-Naini, B; Yeoman, R R; Wolf, D P
Oocyte quantity and quality are critical to assisted reproductive technology (ART), yet few assessments beyond counting metaphase II (MII) oocytes exist. In this study, 30 +/- 2 oocytes per cycle were recovered from rhesus monkeys subjected to follicular stimulation with human gonadotrophins, of which 15 +/- 1 were MII. Oocyte quality was investigated by monitoring the developmental potential of oocytes subjected to intracytoplasmic sperm injection (ICSI). Despite uniform fertilization rates (71 +/- 4%), progression of embryos to blastocysts varied when expressed as a monthly average, from 20 to 85%, with lows from February to April and again in October, which could be attributed to developmental failure of a significant number of oocyte cohorts (14 of 55). Blastocyst rates, after elimination of failed cohorts, were uniform over time (59 +/- 4%). Neither culture conditions, the number of follicular stimulations, nor the individual sperm or oocyte donor were associated specifically with developmental failure, suggesting that intrinsic differences between stimulation cycles account for the observed variation in developmental potential. The in-vivo developmental competence of ICSI-produced embryos grown to blastocysts in vitro was also assessed. Two ongoing pregnancies and the birth of a normal female, 'Blastulina', represent landmarks in efforts to expand the use of ART in the rhesus monkey.
Telfer, Evelyn E; McLaughlin, Marie
Many young cancer patients are now being given the option to store ovarian cortical biopsies before undergoing potentially damaging chemo- or radiotherapy. This tissue mainly contains large numbers of immature primordial follicles. Currently the only option to restore fertility using this tissue is by transplantation which may not be a viable option for all patients. Greater options to realise the potential of this tissue to restore fertility could be achieved by the development of in vitro systems that support oocyte development. The ability to develop human oocytes from the most immature stages of follicles (primordial) through to maturation and fertilisation in vitro would revolutionise fertility preservation practice. This has been achieved in mouse where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. However, developing IVG systems to support complete development of human oocytes has been more difficult because of differences in scale of timing and size. Our lab has been working on a multi-step culture system to support growth and development of bo-vine and human oocytes from primordial through to fully grown, using fresh and cryopreserved ovarian cortical tissue. This review outlines the approaches being taken to obtain complete in vitro development of human oocytes and strategies for assessing the health and viability of IVG oocytes.
Yamochi, T; Hashimoto, S; Amo, A; Goto, H; Yamanaka, M; Inoue, M; Nakaoka, Y; Morimoto, Y
Meiotic maturation of oocytes requires a variety of ATP-dependent reactions, such as germinal vesicle breakdown, spindle formation, and rearrangement of plasma membrane structure, which is required for fertilization. Mitochondria are accordingly expected be localized to subcellular sites of energy utilization. Although microtubule-dependent cellular traffic for mitochondria has been studied extensively in cultured neuronal (and some other somatic) cells, the molecular mechanism of their dynamics in mammalian oocytes at different stages of maturation remains obscure. The present work describes dynamic aspects of mitochondria in porcine oocytes at the germinal vesicle stage. After incubation of oocytes with MitoTracker Orange followed by centrifugation, mitochondria-enriched ooplasm was obtained using a glass needle and transferred into a recipient oocyte. The intracellular distribution of the fluorescent mitochondria was then observed over time using a laser scanning confocal microscopy equipped with an incubator. Kinetic analysis revealed that fluorescent mitochondria moved from central to subcortical areas of oocytes and were dispersed along plasma membranes. Such movement of mitochondria was inhibited by either cytochalasin B or cytochalasin D but not by colcemid, suggesting the involvement of microfilaments. This method of visualizing mitochondrial dynamics in live cells permits study of the pathophysiology of cytoskeleton-dependent intracellular traffic of mitochondria and associated energy metabolism during meiotic maturation of oocytes.
Grøndahl, M L; Andersen, Claus Yding; Bogstad, J
The development competence of human oocytes declines with increasing age. The objective of this study was to investigate the effect of age on gene expression profile in mature human oocytes.......The development competence of human oocytes declines with increasing age. The objective of this study was to investigate the effect of age on gene expression profile in mature human oocytes....
Desrosiers, R.R.; Romanik, E.A.; O' Connor, C.M. (Worcester Foundation for Experimental Biology, Shrewsbury, MA (USA))
The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-(methyl-3H)methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-(methyl-3H)methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly.
Grøndahl, M L; Andersen, C Yding; Bogstad, J;
During folliculogenesis the granulosa cells differentiate into two cell types: Cumulus cells (CC) and mural granulosa cells (MGC). The objective of the study was to generate and compare the transcriptomes of MGC and CC from the pre-ovulatory follicle to characterize the detailed profile of the two...
... decline as a result of the transaction. Cumulus must divest: WRSR, WCAT, and the Federal Communications... is required to determine not whether a particular decree is the one that will best serve society, but.... Gillette Co., 406 F. Supp. 713, 716 (D. Mass. 1975) (noting that, in this way, the court is constrained...
Full Text Available Enucleation is a crucial step in cloning. In order to achieve automatic blind enucleation, we should detect the polar body of the oocyte automatically. The conventional polar body detection approaches have low success rate or low efficiency. We propose a polar body detection method based on machine learning in this paper. On one hand, the improved Histogram of Oriented Gradient (HOG algorithm is employed to extract features of polar body images, which will increase success rate. On the other hand, a position prediction method is put forward to narrow the search range of polar body, which will improve efficiency. Experiment results show that the success rate is 96% for various types of polar bodies. Furthermore, the method is applied to an enucleation experiment and improves the degree of automatic enucleation.
Ozernyuk, N D; Zotin, A I
It is shown that the constant k in the equation QO2 equals apk and the constant b in the equation qo2 equals aP-b change during the oogenesis of the loach. Hence, the growth of oocytes differs considerably from the growth of animals, where the constants k and b do not change with increase in weight. It is suggested that the relationship between the respiration rate and weight of the oocytes is due to the change in the amount of mitochondria in the oocytes.
Seung Eun Lee
Full Text Available Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 μM rapamycin/24 h, 47.52±5.68 or control oocytes (44 h IVM; 42.14±4.40 significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68 (p<0.05. Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK, and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1 compared with untreated, 24 h-aged IVM oocytes (p<0.05. Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS activity and DNA fragmentation (p<0.05, and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05 and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1, anti-apoptosis (BCL2L1 and BIRC5; p<0.05, and development (NANOG and SOX2; p<0.05 genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3 compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.
Full Text Available The assessment of oocytes showing only one pronucleus during assisted reproduction is associated with uncertainty. A compilation of data on the genetic constitution of different developmental stages shows that affected oocytes are able to develop into haploid, diploid, and mosaic embryos with more or less complex chromosomal compositions. In the majority of cases (~80%, haploidy appears to be caused by gynogenesis, whereas parthenogenesis or androgenesis is less common. Most of the diploid embryos result from a fertilization event involving asynchronous formation of the two pronuclei or pronuclear fusion at a very early stage. Uniparental diploidy may sometimes occur if one pronucleus fails to develop and the other pronucleus already contains a diploid genome or alternatively a haploid genome undergoes endoreduplication. In general, the chance of obtaining a biparental diploid embryo appears higher after conventional in vitro fertilization than after intracytoplasmic sperm injection. If a transfer of embryos obtained from monopronuclear oocytes is envisaged, it should be tried to culture them up to the blastocyst since most haploid embryos are not able to reach this stage. Comprehensive counselling of patients on potential risks is advisable before transfer and a preimplantation genetic diagnosis could be offered if available.
CLEMENT O BEWAJI
Schiff bases and their complex compounds have been studied for their .... establishing coordination of the N–(2 – hydroxybenzyl) - L - α - valine Schiff base ..... (1967); “Spectrophotometric Identification of Organic Compounds”, Willey, New.
Vogt, E; Kirsch-Volders, M; Parry, J; Eichenlaub-Ritter, U
The spindle assembly checkpoint (SAC) monitors attachment to microtubules and tension on chromosomes in mitosis and meiosis. It represents a surveillance mechanism that halts cells in M-phase in the presence of unattached chromosomes, associated with accumulation of checkpoint components, in particular, Mad2, at the kinetochores. A complex between the anaphase promoting factor/cylosome (APC/C), its accessory protein Cdc20 and proteins of the SAC renders APC/C inactive, usually until all chromosomes are properly assembled at the spindle equator (chromosome congression) and under tension from spindle fibres. Upon release from the SAC the APC/C can target proteins like cyclin B and securin for degradation by the proteasome. Securin degradation causes activation of separase proteolytic enzyme, and in mitosis cleavage of cohesin proteins at the centromeres and arms of sister chromatids. In meiosis I only the cohesin proteins at the sister chromatid arms are cleaved. This requires meiosis specific components and tight regulation by kinase and phosphatase activities. There is no S-phase between meiotic divisions. Second meiosis resembles mitosis. Mammalian oocytes arrest constitutively at metaphase II in presence of aligned chromosomes, which is due to the activity of the cytostatic factor (CSF). The SAC has been identified in spermatogenesis and oogenesis, but gender-differences may contribute to sex-specific differential responses to aneugens. The age-related reduction in expression of components of the SAC in mammalian oocytes may act synergistically with spindle and other cell organelles' dysfunction, and a partial loss of cohesion between sister chromatids to predispose oocytes to errors in chromosome segregation. This might affect dose-response to aneugens. In view of the tendency to have children at advanced maternal ages it appears relevant to pursue studies on consequences of ageing on the susceptibility of human oocytes to the induction of meiotic error by
Bang, Soyoung; Lee, Geun-Kyung; Shin, Hyejin; Suh, Chang Suk; Lim, Hyunjung Jade
Autophagy contributes to the clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified mouse oocytes show acute increases in autophagy during warming. Herein, we investigate the potential role of Atg7 in oocyte vitrification by using an oocyte-specific deletion model of the Atg7 gene, a crucial upstream gene in the autophagic pathway. Oocyte-specific Atg7 deficient mice were generated by crossing Atg7 floxed mice and Zp3-Cre transgenic mice. The oocytes were vitrified-warmed and then subjected to in vitro fertilization and development. The rates of survival, fertilization, and development were assessed in the Atg7 deficient oocytes in comparison with the wildtype oocytes. Light chain 3 (LC3) immunofluorescence staining was performed to determine whether this method effectively evaluates the autophagy status of oocytes. The survival rate of vitrified-warmed Atg7(f/f) ;Zp3-Cre (Atg7(d/d) ) metaphase II (MII) oocytes was not significantly different from that of the wildtype (Atg7(f/f) ) oocytes. Fertilization and development in the Atg7(d/d) oocytes were significantly lower than the Atg7(f/f) oocytes, comparable to the Atg5(d/d) oocytes previously described. Notably, the developmental rate improved slightly in vitrified-warmed Atg7(d/d) MII oocytes when compared to fresh Atg7(d/d) oocytes. LC3 immunofluorescence staining showed that this method can be reliably used to assess autophagic activation in oocytes. We confirmed that the LC3-positive signal is nearly absent in Atg7(d/d) oocytes. While autophagy is induced during the warming process after vitrification of MII oocytes, the Atg7 gene is not essential for survival of vitrified-warmed oocytes. Thus, induction of autophagy during warming of vitrified MII oocytes seems to be a natural response to manage cold or other cellular stresses.
Full Text Available Trade wind cumulus clouds often organize in along-wind cloud streets and across-wind mesoscale arcs. We present a benchmark large-eddy simulation which resolves the individual clouds as well as the mesoscale organization on scales of O(10 km. Different methods to quantify organization of cloud fields are applied and discussed. Using perturbed physics large-eddy simulations experiments the processes leading to the formation of cloud clusters and the mesoscale arcs are revealed. We find that both cold pools as well as the sub-cloud layer moisture field are crucial to understand the organization of precipitating shallow convection. Further sensitivity studies show that microphysical assumptions can have a pronounced impact on the onset of cloud organization.
Hann-Ming Henry Juang
Full Text Available Massively parallel computing, using a message passing interface (MPI, has been implemented into a three-dimensional version of the Goddard Cumulus Ensemble (GCE model. The implementation uses the domainresemble concept to design a code structure for both the whole domain and sub-domains after decomposition. Instead of inserting a group of MPI related statements into the model routine, these statements are packed into a single routine. In other words, only a single call statement to the model code is utilized once in a place, thus there is minimal impact on the original code. Therefore, the model is easily modified and/or managed by the model developers and/or users, who have little knowledge of massively parallel computing.
Alexandrov, Mikhail D.; Cairns, Brian; Emde, Claudia; Ackerman, Andrew S.; Ottaviani, Matteo; Wasilewski, Andrzej P.
The Research Scanning Polarimeter (RSP) is an airborne instrument, whose measurements have been extensively used for retrievals of microphysical properties of clouds. In this study we show that for cumulus clouds the information content of the RSP data can be extended by adding the macroscopic parameters of the cloud, such as its geometric shape, dimensions, and height above the ground. This extension is possible by virtue of the high angular resolution and high frequency of the RSP measurements, which allow for geometric constraint of the cloud's 2D cross section between a number of tangent lines of view. The retrieval method is tested on realistic 3D radiative transfer simulations and applied to actual RSP data.
Weger, R. C.; Lee, J.; Zhu, Tianri; Welch, R. M.
The current controversy existing in reference to the regularity vs. clustering in cloud fields is examined by means of analysis and simulation studies based upon nearest-neighbor cumulative distribution statistics. It is shown that the Poisson representation of random point processes is superior to pseudorandom-number-generated models and that pseudorandom-number-generated models bias the observed nearest-neighbor statistics towards regularity. Interpretation of this nearest-neighbor statistics is discussed for many cases of superpositions of clustering, randomness, and regularity. A detailed analysis is carried out of cumulus cloud field spatial distributions based upon Landsat, AVHRR, and Skylab data, showing that, when both large and small clouds are included in the cloud field distributions, the cloud field always has a strong clustering signal.
Morse, S. A.
As Maaløe noted in 1976 the basal Skaergaard cumulates commonly contain plagioclase crystals with zoning and resorption that preceded their accumulation on the floor. This conclusion follows from the observation that neighboring grains are unassociated with each other in their zoning. Such crystals, he reasoned, have been tossed about from cooler to hotter regimes in a way that we might call a hailstone history. By contrast, associated grains zoned from trapped liquid have a common junction, easily signaled in troctolites by a scrap of augite or opaque oxide. In a compilation of the An-range (max - min) in Kiglapait grain mounts, samples sorted by increasing values when examined reveal a dominance of unassociated pre-cumulus grains up to values of 5.5 to 7.5 (median 6.65) mole % in the An range, beyond which increasing evidence for intercumulus growth sets in. The pre-cumulus samples occur among other types in stratigraphic levels from 10 to 83 PCS and include rare samples with cumulus augite. A conservative threshold for intercumulus growth is taken as 6 mole %, above which the An-range minus 6 defines residual porosity p(r) >0 (Morse, AGU JA Abstr. 2009). In the presence of pre-cumulus zoning, the threshold criterion for adcumulus growth rises even higher, for example to An range 8 or even 9 mole %, allowing for the fact that even imperfect adcumulates have had a major amount of isothermal adcumulus growth before or after trapping a small amount of residual liquid. At the conservative end of this range, 75 % of the Kiglapait cumulates are practical adcumulates, 17 % are mesocumulates, and 8% are orthocumulates, the latter dominating both lowermost and highest parts of the stratigraphy. From this intrusion-wide distribution, the overall probability that a given rock sample is an adcumulate is 0.75. But the scarcity of mesocumulates in the LZ above 30 PCS and in the UZ from 95 to 99.9 PCS means that the probability of finding an adcumulate is much greater
Parker, Lindsay; Welch, R. M.; Musil, D. J.
Aircraft observations and high resolution Landsat MSS digital data are used to determine the sizes of spatial inhomogeneities ('holes') in cumulus clouds. The majority of holes are found near cloud edges, but the larger holes tend to be found in cloud interiors. Aircraft measurements show these cloud spatial inhomogeneities in the range of 100 to 500 m, while Landsat data show them in the range of 100 m to 3 km. The number of holes per cloud decreases exponentially with increasing hole diameter. Small clouds not only have smaller holes, but also fewer holes than large clouds. Large clouds have large holes in them, as well as large numbers of the smaller holes. The total cloud area occupied by holes increases with increasing cloud size.
Alexandrov, Mikhail D.; Cairns, Brian; Emde, Claudia; Ackerman, Andrew S.; Ottaviani, Matteo; Wasilewski, Andrzej P.
The Research Scanning Polarimeter (RSP) is an airborne instrument, whose measurements have been extensively used for retrievals of microphysical properties of clouds. In this study we show that for cumulus clouds the information content of the RSP data can be extended by adding the macroscopic parameters of the cloud, such as its geometric shape, dimensions, and height above the ground. This extension is possible by virtue of the high angular resolution and high frequency of the RSP measurements, which allow for geometric constraint of the cloud's 2D cross section between a number of tangent lines of view.