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

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

2018-03-20

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

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

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

2013-08-01

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

Maturation, fertilisation and culture of bovine oocytes and embryos in an individually identifiable manner: a tool for studying oocyte developmental competence.

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Matoba, Satoko; Fair, Trudee; Lonergan, Patrick

2010-01-01

The ability to successfully culture oocytes and embryos individually would facilitate the study of the relationship between follicle parameters and oocyte developmental competence, in order to identify markers of competent oocytes, as well as the ability to use small numbers of oocytes from an individual donor such as when ovum pick-up is carried out. Using a total of 3118 oocytes, the aim of the present study was to develop a system capable of supporting the development of immature bovine oocytes to the blastocyst stage in an individually identifiable manner. Initially, post-fertilisation embryo culture in the Well-of-the-Well (WOW) system, on the cell adhesive Cell-Tak or in polyester mesh was tested and shown to result in similar development to embryos cultured in standard group culture. The results demonstrate that it is possible to culture bovine oocytes to the blastocyst stage in an individually identifiable manner in all three culture systems with comparable success rates. This permits the localisation and identification of individual embryos throughout preimplantation development in vitro while retaining the developmental benefits of group culture. In terms of ease of preparation and use, culture in isolation within the strands of a polyester mesh is preferable.

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

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Jolanta Opiela

2014-01-01

Full Text Available The objective of the present study was to evaluate the effect of hyaluronan (HA during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC, and obtained blastocysts. COCs were matured in vitro in control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001 was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01. Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higher Bax mRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

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

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

2014-05-01

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

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

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

2008-12-01

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

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

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

2016-04-01

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

Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP

DEFF Research Database (Denmark)

Hou, Y -p; Liu, Ying; Dai, Y -p

2009-01-01

The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1 h, followed by parthenogenetic activation....... Vitrified-warmed oocytes had a morphologically normal rate similar to that of controls (nonvitrified oocytes cultured in vitro for 24 h; 98.6% vs. 100%, P > 0.05). When vitrified-warmed oocytes were first activated with 7% ethanol for 5 min and then incubated in 6-dimethylaminopurin (6-DMAP) for 4 h...... (for 5 min) or in combination with 6-DMAP (4 h) was used to activate vitrified-warmed oocytes, cleavage rates ranged from 22.3% to 61.1% and blastocyst rates ranged from 1.1% to 30.6%. These rates were optimized when oocytes were treated with 9% ethanol plus 6-DMAP; this was verified in experiments...

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

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

2016-04-01

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

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

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Looft C

2011-01-01

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

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

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V. CARABĂ

2009-05-01

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

Maturation arrest of human oocytes at germinal vesicle stage

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Zhi Qin Chen

2010-01-01

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

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

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Zhengguang Wang

2012-12-01

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

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

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Bilodeau-Goeseels, Sylvie

2011-01-01

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

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

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

2011-06-01

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

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

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

2017-10-01

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

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

Czech Academy of Sciences Publication Activity Database

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

2014-01-01

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

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

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S Modina

2009-08-01

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

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

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

2016-08-02

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

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

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Iris Ferrari

2007-12-01

Full Text Available

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

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

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

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

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Amir Khaki

2014-03-01

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

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

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Z.G. Wang

2007-08-01

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

Maturation of human oocytes in vitro

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Mojca Čižek-Sajko

2007-01-01

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

Induction and inhibition of oocyte maturation by EDCs in zebrafish

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Tokumoto Mika

2005-12-01

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

Replication of somatic micronuclei in bovine enucleated oocytes

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Canel Natalia

2012-11-01

Full Text Available Abstract Background Microcell-mediated chromosome transfer (MMCT was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes. Methods Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+] or not [Micronucleus- injected (−] to a transgene (50 ng/μl pCX-EGFP during 5 min. Enucleated oocytes [Enucleated (+] and parthenogenetic [Parthenogenetic (+] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (−] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (−, Parthenogenetic (− and in vitro fertilized (IVF embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05. Results All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had

Naturally occurring mastitis disrupts developmental competence of bovine oocytes.

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

2013-10-01

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

In vitro maturation of human oocytes for assisted reproduction.

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Jurema, Marcus W; Nogueira, Daniela

2006-11-01

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

Mature Oocyte Cryopreservation for Fertility Preservation.

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Liang, Tina; Motan, Tarek

2016-01-01

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

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

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

1996-08-01

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

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

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Cheng-Jie Zhou

2016-03-01

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

Intraovarian markers of follicular and oocyte maturation.

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

1987-08-01

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

Nicotine supplementation blocks oocyte maturation in Rattus norvegicus

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Meitria Syahadatina Noor

2013-08-01

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

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

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

2005-02-01

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

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

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W.M. Saleh

2017-06-01

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

Vitrification affects nuclear maturation and gene expression of immature human oocytes

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Abbas Shahedi

2017-02-01

Full Text Available Background: Vitrification of oocytes is a fast-freezing technique, which may affect the quality of the human oocyte, and consequently affects the embryo development, pregnancy and birth. The aim of the current study was to investigate the consequence of in-vitro vitrification on maturation status of immature human oocytes, additionally, expression levels of stress, and apoptosis related genes. Materials and Methods: The total of 213 human immature oocytes which routinely discarded from assisted reproduction clinics were collected and divided into two groups including: (I fresh germinal vesicle (GV oocytes (n=106 (matured in-vitro  (fIVM , and  (II GV oocytes (n=107 that initially vitrified, then matured in  in-vitro (vIVM. After 36 hours of incubation, the oocytes were evaluated for nuclear maturation and expression level of DNA methyltransferase (DNMT1, stress related genes (Sod1 and Hsp70, and apoptotic related genes (Bax and Bcl-2 by quantitative Real-Time PCR. Results: Oocyte maturation rates were reduced in vIVM compared to fIVM oocytes (P=0.001. The expression of stress (Sod1 and Hsp70, and apoptotic-related genes (Bax and Bcl-2 in vIVM were significantly higher compared to the fIVM group. Additionally, pro-apoptotic gene up-regulated 4.3 times more than anti-apoptotic gene in vIVM oocyte. However, DNMT1 gene expression was reduced in vIVM oocyte (P = 0.047. Conclusions: The low survival rate of vitrified In-vitro matured GV oocytes could definitely be explained by the alterations of their gene expression profile. 

Maturation of pig oocytes in vitro in a medium with pyruvate

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H. Gonzales-Figueroa

2005-06-01

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

Retinol improves bovine embryonic development in vitro

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Edwards J Lannett

2004-12-01

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

TACC3 Is Important for Correct Progression of Meiosis in Bovine Oocytes

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Mahdipour, Mahdi; Leitoguinho, Ana Rita Canhoto; Zacarias Silva, Ricardo A; van Tol, Helena T A; Stout, Tom A E; Rodrigues, Gabriela; Roelen, Bernard A J

2015-01-01

Transforming acidic coiled-coil (TACC) proteins are key players during mitosis via stabilization of the spindle. The roles of TACCs during meiosis are however less clear. We used bovine oocytes to study the expression and function of TACC3 during meiosis. TACC3 mRNA was detected in bovine oocytes

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

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Ileana Miclea

2016-11-01

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

Intra-follicular interactions affecting mammalian oocyte maturation

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van Tol, H.T.A.|info:eu-repo/dai/nl/313871817

2009-01-01

Nuclear oocyte maturation is defined as reinitiation and progression of the first meiotic division and subsequently formation of the methaphase II (MII) plate. Concomitantly with nuclear maturation, cytoplasmic maturation which is essential for proper fertilization and early embryo development is

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

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F.C. Landim-Alvarenga

2010-02-01

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

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

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

2012-01-01

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

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

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George W. Smith

2010-02-01

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

Closed system for bovine oocyte vitrification

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Helena Ševelová

2012-01-01

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

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

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Borut Kovačič

2002-12-01

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

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

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

2017-11-01

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

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

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Evangelia Kasapi

2017-03-01

Full Text Available Background The use of immature oocytes derived from stimulated cycles could be of great importance, particularly for urgent fertility preservation cases. The current study aimed to determine whether in vitro maturation (IVM was more successful before or after vitrification of these oocytes. Materials and Methods This prospective study was performed in a private in vitro fertilization (IVF center. We collected 318 germinal vesicle (GV oocytes from 104 stimulated oocyte donation cycles. Oocytes were divided into two groups according to whether vitrification was applied at the GV stage (group 1 or in vitro matured to the metaphase II (MII stage and then vitrified (group 2. In the control group (group 3, oocytes were in vitro matured without vitrification. In all three groups, we assessed survival rate after warming, maturation rate, and MII-spindle/chromosome configurations. The chi-square test was used to compare rates between the three groups. Statistical significance was defined at P<0.05 and we used Bonferroni criterion to assess statistical significance regarding the various pairs of groups. The Statistical Package for the Social Sciences version 17.0 was used to perform statistical analysis. Results There was no significant difference in the survival rate after vitrification and warming of GV (93.5% and MII oocytes (90.8%. A significantly higher maturation rate occurred when IVM was performed before vitrification (82.9% compared to after vitrification (51%. There was no significant difference in the incidence of normal spindle/ chromosome configurations among warmed oocytes matured in vitro before (50.0% or after (41.2% vitrification. However, a higher incidence of normal spindle/chromosome configurations existed in the in vitro matured oocytes which were not subjected to vitrification (fresh oocytes, 77.9%. Conclusion In stimulated cycles, vitrification of in vitro matured MII oocytes rather than GV oocytes seems to be more efficient. This

Effects of Crocin Supplementation during In Vitro Maturation of Mouse Oocytes on Glutathione Synthesis and Cytoplasmic Maturation

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Elham Mokhber Maleki

2016-05-01

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

Gene expression and maturation evaluation of sheep oocytes ...

African Journals Online (AJOL)

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

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

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

2017-07-15

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

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

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

2010-03-15

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

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

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Cecilia Dieci

2017-05-01

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

Expression of the CTCF gene in bovine oocytes and preimplantation embryos

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Álvaro F.L. Rios

2007-01-01

Full Text Available The CCCTC - binding factor (CTCF is a protein involved in repression, activation, hormone-inducible gene silencing, functional reading of imprinted genes and X-chromosome inactivation. We analyzed CTCF gene expression in bovine peripheral blood, oocytes and in different cellular stages (2-4 cells, 8-16 cells, 16-32 cells, morulae, and blastocysts of in vitro fertilized embryos. This is the first report of CTCF expression in oocytes and preimplantation bovine embryos and has implications for the production of embryonic stem cells and the development of novel medical technologies for humans.

Investigations of oocyte in vitro maturation within a mouse model.

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Chin, Alexis Heng Boon; Chye, Ng Soon

2004-02-01

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

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

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Melanie L. Walls

2015-08-01

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

Involvement of PLA2, COX and LOX in Rhinella arenarum oocyte maturation.

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Ortiz, Maria Eugenia; Bühler, Marta Inés; Zelarayán, Liliana Isabel

2014-11-01

In Rhinella arenarum, progesterone is the physiological nuclear maturation inducer that interacts with the oocyte surface and starts a cascade of events that leads to germinal vesicle breakdown (GVBD). Polyunsaturated fatty acids and their metabolites produced through cyclooxygenase (COX) and lipoxygenase (LOX) pathways play an important role in reproductive processes. In amphibians, to date, the role of arachidonic acid (AA) metabolites in progesterone (P4)-induced oocyte maturation has not been clarified. In this work we studied the participation of three enzymes involved in AA metabolism - phospholipase A2 (PLA2), COX and LOX in Rhinella arenarum oocyte maturation. PLA2 activation induced maturation in Rhinella arenarum oocytes in a dose-dependent manner. Oocytes when treated with 0.08 μM melittin showed the highest response (78 ± 6% GVBD). In follicles, PLA2 activation did not significantly induce maturation at the assayed doses (12 ± 3% GVBD). PLA2 inhibition with quinacrine prevented melittin-induced GVBD in a dose-dependent manner, however PLA2 inactivation did not affect P4-induced maturation. This finding suggests that PLA2 is not the only phospholipase involved in P4-induced maturation in this species. P4-induced oocyte maturation was inhibited by the COX inhibitors indomethacin and rofecoxib (65 ± 3% and 63 ± 3% GVBD, respectively), although COX activity was never blocked by their addition. Follicles showed a similar response following the addition of these inhibitors. Participation of LOX metabolites in maturation seems to be correlated with seasonal variation in ovarian response to P4. During the February to June period (low P4 response), LOX inhibition by nordihydroguaiaretic acid or lysine clonixinate increased maturation by up to 70%. In contrast, during the July to January period (high P4 response), LOX inhibition had no effect on hormone-induced maturation.

Cumulus cells steroidogenesis is influenced by the degree of oocyte maturation

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Barboni Barbara

2003-05-01

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

The Effects of Progesterone on Oocyte Maturation and Embryo Development

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Saeed Zavareh

2013-01-01

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

Regulation of oocyte maturation in fish.

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Nagahama, Yoshitaka; Yamashita, Masakane

2008-06-01

A period of oocyte growth is followed by a process called oocyte maturation (the resumption of meiosis) which occurs prior to ovulation and is a prerequisite for successful fertilization. Our studies using fish models have revealed that oocyte maturation is a three-step induction process involving gonadotropin (LH), maturation-inducing hormone (MIH), and maturation-promoting factor (MPF). LH acts on the ovarian follicle layer to produce MIH (17alpha, 20beta-dihydroxy-4-pregnen-3-one, 17alpha, 20beta-DP, in most fishes). The interaction of ovarian thecal and granulosa cell layers (two-cell type model), is required for the synthesis of 17alpha,20beta-DP. The dramatic increase in the capacity of postvitellogenic follicles to produce 17alpha,20beta-DP in response to LH is correlated with decreases in P450c17 (P450c17-I) and P450 aromatase (oP450arom) mRNA and increases in the novel form of P450c17 (P450c17-II) and 20beta-hydroxysteroid dehydrogenase (20beta-HSD) mRNA. Transcription factors such as Ad4BP/SF-1, Foxl2, and CREB may be involved in the regulation of expression of these steroidogenic enzymes. A distinct family of G-protein-coupled membrane-bound MIH receptors has been shown to mediate non-genomic actions of 17alpha, 20beta-DP. The MIH signal induces the de novo synthesis of cyclin B from the stored mRNA, which activates a preexisting 35 kDa cdc2 kinase via phosphorylation of its threonine 161 by cyclin-dependent kinase activating kinase, thus producing the 34 kDa active cdc2 (active MPF). Upon egg activation, MPF is inactivated by degradation of cyclin B. This process is initiated by the 26S proteasome through the first cut in its NH(2) terminus at lysine 57.

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

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Ali Abbara

2018-04-01

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

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

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

2017-11-01

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

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

DEFF Research Database (Denmark)

Humblot, P; Holm, Peter; Lonergan, P

2005-01-01

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

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

African Journals Online (AJOL)

STORAGESEVER

2009-10-19

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

Pharmaceutical Options for Triggering of Final Oocyte Maturation in ART

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Juan Carlos Castillo

2014-01-01

Full Text Available Since the pioneering days of in vitro fertilization, hCG has been the gold standard to induce final follicular maturation. We herein reviewed different pharmaceutical options for triggering of final oocyte maturation in ART. The new upcoming agent seems to be GnRHa with its potential advantages over hCG trigger. GnRHa triggering elicits a surge of gonadotropins resembling the natural midcycle surge of gonadotropins, without the prolonged action of hCG, resulting in the retrieval of more mature oocytes and a significant reduction in or elimination of OHSS as compared to hCG triggering. The induction of final follicular maturation using GnRHa represents a paradigm shift in the ovulation triggering concept in ART and, thus, a way to develop a safer IVF procedure. Kisspeptins are key central regulators of the neuroendocrine mechanisms of human reproduction, who have been shown to effectively elicit an LH surge and to induce final oocyte maturation in IVF cycles. This new trigger concept may, therefore, offer a completely new, “natural” pharmacological option for ovulation induction. Whether kisspeptins will be the future agent to trigger ovulation remains to be further explored.

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

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Shuzhen Liu

Full Text Available Acrylamide (ACR is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus-oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus-oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.

Detection of genes associated with developmental competence of bovine oocytes

Czech Academy of Sciences Publication Activity Database

Němcová, Lucie; Jansová, Denisa; Vodičková Kepková, Kateřina; Vodička, Petr; Jeseta, M.; Machatková, M.; Kaňka, Jiří

2016-01-01

Roč. 166, č. 1 (2016), s. 58-71 ISSN 0378-4320 Institutional support: RVO:67985904 Keywords : oocyte * embryo * bovine * developmental competence * transcription Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.605, year: 2016

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

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M Nikseresht

2013-05-01

Full Text Available Abstract Background & aim: Vitrification is a simple and ultra rapid technique for the conservation of fertility. Improving pregnancy rate associate with the use of cryopreserved oocytes would be an important advanced in human assisted reproductive technology (ART. The purpose of this study was to evaluate survival, oocytes maturation and embryo development to the blastocyst stage after vitrification of oocytes germinal vesicle-stage and multi stage Methods: In the present experimental study, germinal vesicle oocytes with or without cumulus cells were transferred to vitrification solution containing 30% (v/v ethylene glycol, 18% (w/v Ficoll-70, and 0.3 M sucrose, either by single step or in a step-wise way. After vitrification and storage in liquid nitrogen, the oocytes were thawed and washed twice in culture medium TCM119, and then subjected to in vitro maturation, fertilization, and culture. Data analysis was performed by using One-way variance and Tukey tests. Results: Oocytes survival, metaphase 2 stage oocyte maturation, fertilization and embryo formed blastocyst in vitrification methods multistage were significantly higher than the single step procedure (P<0/05 Conclusion: The Germinal vesicle stage oocytes vitrified with cumulus cells and stepwise procedure had positive effect on the survival, maturation and developmental rate on blastocyst compared to oocytes without cumulus cell and single step procedure. Key words: Germinal Vesicle Oocyte, Blastocyst, Vitrification, Ethylene glycol

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

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Lin, Zi-Li; Kim, Nam-Hyung

2015-06-01

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

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

African Journals Online (AJOL)

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

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

NARCIS (Netherlands)

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

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

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

DEFF Research Database (Denmark)

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

2013-01-01

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

Effect of Kaempferol on in vitro Maturation of Porcine Oocytes

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Delia Orlovschi

2014-10-01

Full Text Available We investigated the effects of kaempferol on porcine oocytes in vitro maturation. Kaempferol is one the most studied flavonoids and is in research attention on animal cells until 1979. Flavonoids are known as polyphenolic compounds synthesized by the plants. Cumulus-oocyte complexes aspirated from the ovaries were maturated in vitro, fertilized and embryos were cultured in a defined conditioned medium with 5, 15, 25, 35 µg/ml or without kaempferol supplementation. During in vitro maturation with highest kaempferol concentration (35 µg/ml distinct significantly increase the rate of cumulus cell expansion in grad 4 (42.74 vs. 50.96%, p<0.01. The same, addition of 5 µg/ml kaempferol to the in vitro maturation medium increase significantly the rate of expansion compared to 25 µg/ml (42.20 vs. 48.67%, p<0.05 and increase distinct significantly the rate of expansion compared to 35 µg/ml (42.20 vs. 50.96%, p<0.01. Kaempferol supplementation (15 µg/ml vs. 35 µg/ml of the in vitro fertilization medium led to a significant increase in the rate of 4-8 cells formation (0.69 vs. 4.96%, p<0.05. In conclusion, these results demonstrate that supplementation with kaempferol during in vitro maturation improved the developmental competence of porcine oocytes.

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

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

2017-01-01

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

Influence of antral follicle size on oocyte characteristics and embryo development in the bovine

DEFF Research Database (Denmark)

Lequarre, Anne Sophie; Vigneron, Céline; Ribaucour, Fabrice

2005-01-01

The developmental competence of bovine oocytes isolated from antral follicles of different sizes was assessed in three European laboratories (Belgium, UCL; Denmark, DIAS; France, INRA). Using the same protocol for in vitro production of embryos, the oocytes isolated from follicles with a diameter...

GnRHa trigger for final oocyte maturation

DEFF Research Database (Denmark)

Humaidan, Peter; Alsbjerg, Birgit

2014-01-01

Since the introduction of the gonadotrophin-releasing hormone analogues (GnRHa) protocol, it has become possible to trigger final oocyte maturation with a bolus of GnRHa. This leads to a significant reduction or complete elimination of ovarian hyperstimulation syndrome compared with human chorion...

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

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

2011-04-23

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

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

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Silva Liliane FI

2011-04-01

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

Effects of sorbitol on porcine oocyte maturation and embryo development in vitro.

Science.gov (United States)

Lin, Tao; Zhang, Jin Yu; Diao, Yun Fei; Kang, Jung Won; Jin, Dong-Il

2015-04-01

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.

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

African Journals Online (AJOL)

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

Toxicity of beauvericin on porcine oocyte maturation and preimplantation embryo development

NARCIS (Netherlands)

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

2016-01-01

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

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

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M. Matsuo

2017-10-01

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

Quercetin Efficacy on in vitro Maturation of Porcine Oocytes

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Delia Orlovschi

2014-05-01

Full Text Available The present study proposed to examine the effects of a polyphenol (quercetin on in vitro maturated parameters. Quercetin it has been extensively studied by researchers on animals over the 35 years. It is a plant derived flavonoid from fruits and vegetables that has antioxidant action as a free radical scavenger. Immature porcine oocytes were untreated and treated with 5, 15, 25, 35 µg/ml quercetin during in vitro maturation. After then the mature oocytes were fertilized. It was observed that cumulus cell expansion of COCs cultured in maturation media supplemented with 5 µg/ml quercetin in grad 3 could be very significantly increased (p<0.001. In grad 4 could be significantly between different levels of quercetin (5 vs. 25, 5 vs. 35, p<0.001. The rates of embryos cultured in medium supplemented with different levels of quercetin did not presented significantly statistically different. The presence of 25 µg/ml quercetin in the maturation medium increased the percentage of embryos in the morula stage compared with the control. In the morula stage all the concentrations of quercetin resulted percentages increased to control. This results shows that quercetin added during in vitro maturation has a positive effect on future embryos development.

Computer-aided meiotic maturation assay (CAMMA) of zebrafish (danio rerio) oocytes in vitro.

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Lessman, Charles A; Nathani, Ravikanth; Uddin, Rafique; Walker, Jamie; Liu, Jianxiong

2007-01-01

We have developed a new technique called Computer-Aided Meiotic Maturation Assay (CAMMA) for imaging large arrays of zebrafish oocytes and automatically collecting image files at regular intervals during meiotic maturation. This novel method uses a transparency scanner interfaced to a computer with macro programming that automatically scans and archives the image files. Images are stacked and analyzed with ImageJ to quantify changes in optical density characteristic of zebrafish oocyte maturation. Major advantages of CAMMA include (1) ability to image very large arrays of oocytes and follow individual cells over time, (2) simultaneously image many treatment groups, (3) digitized images may be stacked, animated, and analyzed in programs such as ImageJ, NIH-Image, or ScionImage, and (4) CAMMA system is inexpensive, costing less than most microscopes used in traditional assays. We have used CAMMA to determine the dose response and time course of oocyte maturation induced by 17alpha-hydroxyprogesterone (HP). Maximal decrease in optical density occurs around 5 hr after 0.1 micro g/ml HP (28.5 degrees C), approximately 3 hr after germinal vesicle migration (GVM) and dissolution (GVD). In addition to changes in optical density, GVD is accompanied by streaming of ooplasm to the animal pole to form a blastodisc. These dynamic changes are readily visualized by animating image stacks from CAMMA; thus, CAMMA provides a valuable source of time-lapse movies for those studying zebrafish oocyte maturation. The oocyte clearing documented by CAMMA is correlated to changes in size distribution of major yolk proteins upon SDS-PAGE, and, this in turn, is related to increased cyclin B(1) protein.

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

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Christopher B Ball

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

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

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

2017-10-01

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

Use of pregnant mare’s sera gonadotropin (PMSG in media in vitro maturation of cow oocytes

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Zaituni Udin

2007-03-01

Full Text Available It is known that hormone addition in media helps in vitro maturation of oocyte. This research was aimmed to determine the effect of PMSG in media to maturation rate and nucleous developvement of cow oocyte. Ovaries were obtainned from local slaughterhouse. The media used for in vitro maturation of oocyte was TCM- 199 and the treatment was 3 levels of PMSG: 0, 10 and 20 mg/ml. Result of this research showed that the dose of PMSG in maturation media was significantly affected (P<0.05 nucleolus development of oocytes and maturation rate. The average of germinal vesicle (GV stage in 3 levels of PMSG 0, 10 and 20 mg/ml were 38.33; 12.64 and 9.64%, respectivelly. There was no germinal vesicle breakdown (GVBD found in 3 levels of PMSG addition. The nucleous development of metaphase–I (M-I were 7.64; 20.2 and 22.00%, but the average of maturation rate (M-II was 16.32; 48.10 and 35.34% for 3 levels of PMSG: 0, 10 and 20 mg/ml, respectivelly. It is concluded that 10 mg/ml PMSG in media of in vitro maturation resuls in the highest maturation rate of cow oocyte.

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

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Kwon, Sojung; Lim, Hyunjung J

2011-03-01

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

Adnexal Torsion during Pregnancy after Oocyte In Vitro Maturation and Intracytoplasmic Sperm Injection Cycle

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Simone Giulini

2010-01-01

Full Text Available We report a case of right adnexal torsion during pregnancy after an oocyte in vitro maturation and intracitoplasmic sperm injection cycle in patient with polycystic ovary syndrome. A 31-year-old woman with a typical clinical disorder of polycystic ovarian syndrome was included in an oocyte in vitro maturation program. Right adnexal torsion occurred two days after embryo transfer, and laparoscopy detorsion was successfully performed with preservation of adnexa. The patient had a full-term pregnancy and delivered a healthy infant at 40 weeks of gestation. To our knowledge this is the first report of adnexal torsion after an oocyte in vitro maturation and intracitoplasmic sperm injection program.

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

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Fábio Gallas Leivas

2004-02-01

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

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

International Nuclear Information System (INIS)

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

2016-01-01

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

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

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

2016-06-01

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

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

Science.gov (United States)

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

2006-02-25

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

Pharmaceutical Options for Triggering of Final Oocyte Maturation in ART

DEFF Research Database (Denmark)

Castillo, Juan Carlos; Humaidan, Peter; Bernabéu, Rafael

2014-01-01

Since the pioneering days of in vitro fertilization, hCG has been the gold standard to induce final follicular maturation. We herein reviewed different pharmaceutical options for triggering of final oocyte maturation in ART. The new upcoming agent seems to be GnRHa with its potential advantages o...

DNA damage response during mouse oocyte maturation

Czech Academy of Sciences Publication Activity Database

Mayer, Alexandra; Baran, Vladimír; Sakakibara, Y.; Brzáková, Adéla; Ferencová, Ivana; Motlík, Jan; Kitajima, T.; Schultz, R. M.; Šolc, Petr

2016-01-01

Roč. 15, č. 4 (2016), s. 546-558 ISSN 1538-4101 R&D Projects: GA MŠk LH12057; GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : double strand DNA breaks * DNA damage * MRE11 * meiotic maturation * mouse oocytes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.530, year: 2016

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

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A. M. Luciano

2009-12-01

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

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

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Ghaffari Novin M.

2015-06-01

Full Text Available In some cases of infertility in women, human oocytes fail to mature when they reach the metaphase II (MII stage. Mitochondria plays an important role in oocyte maturation. A large number of mitochondrial DNA (mtDNA, copied in oocytes, is essential for providing adenosine triphosphate (ATP during oocyte maturation. The purpose of this study was to identify the relationship between transcript expression levels of the mitochondrial encoded gene (MT-CO1 and two nuclear encoded genes, nuclear respiratory factor 1 (NRF1 and mitochondrial transcription factor A (TFAM in various stages of human oocyte maturation. Nine consenting patients, age 21-35 years old, with male factors were selected for ovarian stimulation and intracytoplasmic sperm injection (ICSI procedures. mRNA levels of mitochondrial- related genes were performed by singlecell TaqMan® quantitative real-time polymerase chain reaction (qRT-PCR. There was no significant relationship between the relative expression levels in germinal vesicle (GV stage oocytes (p = 0.62. On the contrary, a significant relationship was seen between the relative expression levels of TFAM and NRF1 and the MT-CO1 genes at the stages of metaphase I (MI and MII (p = 0.03 and p = 0.002. A relationship exists between the transcript expression levels of TFAM and NRF1, and MT-CO1 genes in various stages of human oocyte maturation.

Genome-wide, Single-Cell DNA Methylomics Reveals Increased Non-CpG Methylation during Human Oocyte Maturation

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Bo Yu

2017-07-01

Full Text Available The establishment of DNA methylation patterns in oocytes is a highly dynamic process marking gene-regulatory events during fertilization, embryonic development, and adulthood. However, after epigenetic reprogramming in primordial germ cells, how and when DNA methylation is re-established in developing human oocytes remains to be characterized. Here, using single-cell whole-genome bisulfite sequencing, we describe DNA methylation patterns in three different maturation stages of human oocytes. We found that while broad-scale patterns of CpG methylation have been largely established by the immature germinal vesicle stage, localized changes continue into later development. Non-CpG methylation, on the other hand, undergoes a large-scale, generalized remodeling through the final stage of maturation, with the net overall result being the accumulation of methylation as oocytes mature. The role of the genome-wide, non-CpG methylation remodeling in the final stage of oocyte maturation deserves further investigation.

Comparative Proteomic Analysis of Mature and Immature Oocytes of the Swamp Buffalo (Bubalus bubalis

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Qiang Fu

2016-01-01

Full Text Available Maternal protein components change markedly during mammalian oogenesis. Many of these proteins have yet to be characterized and verified. In this study, a proteomics approach was used to evaluate changes in proteins during oogenesis in the Swamp Buffalo (Bubalus bubalis. Proteins from 500 immature oocytes and 500 in vitro matured oocytes were subjected to two-dimensional electrophoresis, and more than 400 spots were detected. Image analysis indicated that 17 proteins were differentially expressed between the two groups. Eight proteins were identified by mass spectrometry. In mature oocytes, three proteins were down-regulated: major vault protein (MVP, N-acetyllactosaminide β-1,6-N-acetylglucosaminyl-transferase (GCNT-2, and gem-associated protein (GEMIN8, whereas five other proteins, heat shock protein (HSP60, Ras-responsive element-binding protein 1 (RREB-1, heat shock cognate 71 kDa protein (HSC71, hemoglobin subunit α (HBA, and BMP-2-inducible protein kinase (BMP-2K, were up-regulated. The expression profiles of HSP60 and GEMIN8 were further verified by Western blotting. The changes in HSP60 protein expression demonstrate the increasing need for mitochondrial protein importation to facilitate macromolecular assembly during oocyte maturation. The down-regulation of GEMIN8 production implies that RNA splicing is impaired in mature oocytes.

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

Science.gov (United States)

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

2014-04-01

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

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

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Kátia R L Schwarz

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

Optimal doses of EGF and GDNF act as biological response modifiers to improve porcine oocyte maturation and quality

DEFF Research Database (Denmark)

Valleh, Mehdi Vafaye; Zandi, Nahid Karimi; Rasmussen, Mikkel Aabech

2017-01-01

It is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action of the opti......It is well documented that both epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) are critical for porcine oocyte maturation, however, little information is known about their mechanism of action in vitro. To gain insight into the mechanisms of action...... of the optimum doses of EGF and GDNF on porcine oocyte maturation, porcine cumulus-oocyte complexes (COCs) were matured in defined porcine oocyte medium supplemented with EGF, GDNF or a combination of both at varying concentrations (0-100 ng/ml) for 44 h. Nuclear and cytoplasmic maturation were determined...

Effect of melatonin on maturation capacity and fertilization of Nili-Ravi buffalo (Bubalus bubalis oocytes

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G. Nagina

2016-08-01

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.

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

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Jung-Taek Kang

2016-03-01

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

In vitro production of bovine embryos

DEFF Research Database (Denmark)

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

2015-01-01

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

Toxic effects and possible mechanisms of hydrogen sulfide and/or ammonia on porcine oocyte maturation in vitro.

Science.gov (United States)

Yang, Lei-Lei; Zhao, Yong; Luo, Shi-Ming; Ma, Jun-Yu; Ge, Zhao-Jia; Shen, Wei; Yin, Shen

2018-03-15

Previous studies suggest that hydrogen sulfide (H 2 S) and ammonia (NH 3 ) are two major air pollutants which can cause damage to porcine health. However, the mechanisms underlying toxic effects of these compounds on porcine oocyte maturation are not clear. To clarify the mechanism, we evaluated the oocyte quality by detecting some events during oocytes maturation. In our study, porcine oocytes were cultured with different concentrations of Na 2 S and/or NH 4 Cl in vitro and the rate of the first polar body extrusion decreased significantly. Also, actin filament was seriously disrupted to damage the cytoskeleton which resulted in reduced rate of oocyte maturation. We explored the reactive oxygen species (ROS) generation and found that the ROS level was increased significantly after Na 2 S treatment but not after NH 4 Cl treatment. Moreover, early stage apoptosis rate was significantly increased and autophagy protein LC3 B expression level was higher in oocytes treated with Na 2 S and/or NH 4 Cl, which might be caused by ROS elevation. Additionally, exposure to Na 2 S and/or NH 4 Cl also caused ROS generation and early apoptosis in cumulus cells, which might further affect oocyte maturation in vitro. In summary, our data suggested that exposure to H 2 S and/or NH 3 decreased porcine oocyte maturation in vitro, which might be caused by actin disruption, ROS generation, early apoptosis and autophagy. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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R mahmoudi

2014-09-01

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

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

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Jung Kyu Choi

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

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

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ShuZhen Liu

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

Injurious Effects of Curcumin on Maturation of Mouse Oocytes, Fertilization and Fetal Development via Apoptosis

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Wen-Hsiung Chan

2012-04-01

Full Text Available Curcumin, a common dietary pigment and spice, is a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa. Previously, we reported a cytotoxic effect of curcumin on mouse embryonic stem cells and blastocysts and its association with defects in subsequent development. In the present study, we further investigated the effects of curcumin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, curcumin induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with curcumin during in vitro maturation (IVM led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments with an in vivo mouse model disclosed that consumption of drinking water containing 40 μM curcumin led to decreased oocyte maturation and in vitro fertilization as well as early embryonic developmental injury. Finally, pretreatment with a caspase-3-specific inhibitor effectively prevented curcumin-triggered injury effects, suggesting that embryo impairment by curcumin occurs mainly via a caspase-dependent apoptotic process.

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

Science.gov (United States)

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

2010-10-01

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

Dog cloning with in vivo matured oocytes obtained using electric chemiluminescence immunoassay-predicted ovulation method.

Science.gov (United States)

Lee, Seunghoon; Zhao, Minghui; No, Jingu; Nam, Yoonseok; Im, Gi-Sun; Hur, Tai-Young

2017-01-01

Radioactive immunoassay (RIA) is a traditional serum hormone assay method, but the application of the method in reproductive studies is limited by the associated radioactivity. The aim of present study was to evaluate the reliability of RIA and to compare its canine serum progesterone concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). In vivo matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum progesterone levels were assessed to accurately determine ovulation and oocyte maturation. Canine serum progesterone concentrations during both proestrus and estrus were analyzed by RIA and ECLI to determine the ovulation day. Although both methods detected similar progesterone levels before ovulation, the mean progesterone concentration determined using ECLI was significantly higher than of RIA three days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of progesterone were used for determination of ovulation. A high percentage of mature oocytes was observed using ECLI when 6-15 ng/mL of progesterone was used for ovulation determination. To determine whether ECLI could be used for canine cloning, six canines were selected as oocyte donors, and two puppies were obtained after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning.

AKT (protein kinase B) is implicated in meiotic maturation of porcine oocytes

Czech Academy of Sciences Publication Activity Database

Kalous, Jaroslav; Kubelka, Michal; Šolc, Petr; Šušor, Andrej; Motlík, Jan

2009-01-01

Roč. 138, - (2009), s. 645-654 ISSN 1470-1626 R&D Projects: GA ČR GA204/06/1297; GA ČR GA523/03/0857; GA ČR GA524/07/1087 Institutional research plan: CEZ:AV0Z50450515 Keywords : protein kinase * porcine oocyte * oocyte maturation Subject RIV: CE - Biochemistry Impact factor: 2.579, year: 2009

Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells.

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Leon, P M M; Campos, V F; Kaefer, C; Begnini, K R; McBride, A J A; Dellagostin, O A; Seixas, F K; Deschamps, J C; Collares, T

2013-08-01

The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

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

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Ai Tsuji

2017-03-01

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

Stability of the cytoskeleton of matured buffalo oocytes pretreated with cytochalasin B prior to vitrification.

Science.gov (United States)

Wang, C L; Xu, H Y; Xie, L; Lu, Y Q; Yang, X G; Lu, S S; Lu, K H

2016-06-01

Stabilizing the cytoskeleton system during vitrification can improve the post-thaw survival and development of vitrified oocytes. The cytoskeleton stabilizer cytochalasin B (CB) has been used in cryopreservation to improve the developmental competence of vitrified oocytes. To assess the effect of pretreating matured buffalo oocytes with CB before vitrification, we applied 0, 4, 8, or 12 μg/mL CB for 30 min. The optimum concentration of CB treatment (8 μg/mL for 30 min) was then used to evaluate the distribution of microtubules and microfilaments, the expression of the cytoskeleton proteins actin and tubulin, and the developmental potential of matured oocytes that were vitrified-warmed by the Cryotop method. Western blotting demonstrated that vitrification significantly decreased tubulin expression, but that the decrease was attenuated for oocytes pretreated with 8 μg/mL CB before vitrification. After warming and intracytoplasmic sperm injection, oocytes that were pretreated with 8 μg/mL CB before vitrification yielded significantly higher 8-cell and blastocyst rates than those that were vitrified without CB pretreatment. The values for the vitrified groups in all experiments were significantly lower (P < 0.01) than those of the control groups. In conclusion, pretreatment with 8 μg/mL CB for 30 min significantly improves the cytoskeletal structure, expression of tubulin, and development capacity of vitrified matured buffalo oocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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Trounson Alan

2008-12-01

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

Biochanin a and Daidzein Influence Meiotic Maturation of Pig Oocytes in a Different Manner

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Hošková K.

2014-09-01

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.

In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model.

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Barbara Ambruosi

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

In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model.

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Ambruosi, Barbara; Uranio, Manuel Filioli; Sardanelli, Anna Maria; Pocar, Paola; Martino, Nicola Antonio; Paternoster, Maria Stefania; Amati, Francesca; Dell'Aquila, Maria Elena

2011-01-01

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 embryos

In vitro and in vivo Development of Cloned Ovine Embryos using in vitro and in vivo Matured Oocytes

DEFF Research Database (Denmark)

Holm, P; Nagashima, H; Sun, F-J

1995-01-01

Cloning of sheep embryos by nucleus transplantation can be achieved by using in vivo matured (oviductal) oocytes and in vivo culture. However, these steps involve cumbersome procedures. Therefore, the effects of in vivo vs. the equivalent in vitro procedures on the pre-implantation development...... matured oocytes were enucleated and fused with inserted blastomeres from donor embryos. In vitro matured oocytes were enucleated and allowed to age prior to blastomere insertion and electrofusion. Fused embryos were cultured for approximately 132 h either in vivo in ligated sheep oviducts or in vitro...

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

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Pivko Juraj

2003-12-01

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

Hydrostatic pressure affects in vitro maturation of oocytes and follicles and increases granulosa cell death.

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Rashidi, Zahra; Azadbakht, Mehri; Amini, Ali; Karimi, Isac

2014-01-01

This study examines the effects of hydrostatic pressure on in vitro maturation (IVM) of oocytes derived from in vitro grown follicles. 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. The percentage of metaphase II oocytes (MII) increased in hydrostatic pressuretreated follicles compared to controls (phydrostatic pressure-treated follicles compared to controls (pHydrostatic pressure, by inducing apoptosis in cumulus cells, participates in the cumulus oocyte coupled relationship with oocyte maturation.

The effect of hepatocyte growth factor on mouse oocyte in vitro maturation and subsequent fertilization and embryo development

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Mohammad H. Bahadori

2011-05-01

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

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

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

2011-02-01

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

Increased stress tolerance of matured pig oocytes after high hydrostatic presure

DEFF Research Database (Denmark)

Pribenszky, Cs; Du, Y; Molnár, M

2008-01-01

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

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

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Jorgelina Buschiazzo

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

X-ray induced dominant lethal mutations in mature and immature oocytes of guinea-pigs and golden hamsters

International Nuclear Information System (INIS)

Cox, B.D.; Lyon, M.F.

1975-01-01

The induction of dominant lethal mutations by doses of 100-400 rad X-rays in oocytes of the guinea-pig and golden hamster was studied using criteria of embryonic mortality. For both species higher yields were obtained from mature than from immature oocytes. Data on fertility indicated that in the golden hamster immature oocytes were more sensitive to killing by X-rays than mature oocytes but that the converse was true in the guinea-pig. The dose-response relationship for mutation to dominant lethals in pre-ovulatory oocytes of guinea-pigs and golden hamsters was linear, both when based on pre- and post-implantation loss only. The rate per unit dose was higher for the golden hamster, and the old golden hamsters were possibly slightly more sensitive than young ones

Comparison of Gene Expression Profiles in Human Germinal Vesicle Before and After Cytoplasmic Transfer From Mature Oocytes in Iranian Infertile Couples

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Fatemeh Sadat Hoseini

2016-08-01

Full Text Available Objective: To evaluate the effect of cytoplasm transfer from mature oocytes to germinal vesicle(GVs on promoting the maturation of cytoplasm of GV at the mRNA level.Materials and methods: Sixty six in vitro fertilization (IVF operations between June 2012 and November 2013 were included in this study. Totally 120 GVs were obtained. Normal GVs were categorized into 3 groups (n = 40 randomly: the first comprised oocytes that did not receive the cytoplasm of mature oocytes; the second group comprised oocytes that did not receive the cytoplasm of mature oocytes but were incubated for 24 h; and the third group comprised oocytes that received 10-15% the cytoplasm of mature oocytes and were then incubated for 24 h. Each group was separately analyzed by quantitative polymerase chain reaction (qPCR and the expression levels of selected genes were assessed.Results: The expression levels of genes involved in the cytoplasmic maturity, and energy-producing mitochondria were significantly higher in the pooled oocytes of 2nd control group than those of the 1st control and intervention groups (p < 0.001. The genes involved in the meiosis, spindle check point, DNA repairing and cell cycle checkpoint did not have any expression in the 1st and intervention groups; however, these genes were expressed in the 2nd group, significantly. In the 2nd group, the highest expression level was observed for genes involved in the DNA repairing and cell cycle checkpoint. In the intervention group, none of the genes were expressed except for energy-producing mitochondria gene; even in this case, the expression level of this gene in this group of oocytes was significantly lower than that in other groups (p < 0.001. After 24 h meiosis assumption was significantly higher in the third group than in the second group (95% vs. 68%, p < 0.001.Conclusion: The cytoplasm transfer technique is not effective in cytoplasmic maturity of the recipient GV oocytes. In contrast, 24-hr in

In vitro maturation, fertilization, embryo development & clinical outcome of human metaphase-I oocytes retrieved from stimulated intracytoplasmic sperm injection cycles

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Cristina Álvarez

2013-01-01

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

A Role of Lipid Metabolism during Cumulus-Oocyte Complex Maturation: Impact of Lipid Modulators to Improve Embryo Production

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E. G. Prates

2014-01-01

Full Text Available Oocyte intracellular lipids are mainly stored in lipid droplets (LD providing energy for proper growth and development. Lipids are also important signalling molecules involved in the regulatory mechanisms of maturation and hence in oocyte competence acquisition. Recent studies show that LD are highly dynamic organelles. They change their shape, volume, and location within the ooplasm as well as their interaction with other organelles during the maturation process. The droplets high lipid content has been correlated with impaired oocyte developmental competence and low cryosurvival. Yet the underlying mechanisms are not fully understood. In particular, the lipid-rich pig oocyte might be an excellent model to understand the role of lipids and fatty acid metabolism during the mammalian oocyte maturation and their implications on subsequent monospermic fertilization and preimplantation embryo development. The possibility of using chemical molecules to modulate the lipid content of oocytes and embryos to improve cryopreservation as well as its biological effects during development is here described. Furthermore, these principles of lipid content modulation may be applied not only to germ cells and embryo cryopreservation in livestock production but also to biomedical fundamental research.

Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey.

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Chen, N; Liow, S-L; Abdullah, R Bin; Embong, W Khadijah Wan; Yip, W-Y; Tan, L-G; Tong, G-Q; Ng, S-C

2007-02-01

Somatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 degrees C) without CO(2) supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo

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

Science.gov (United States)

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

2016-07-15

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

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

NARCIS (Netherlands)

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

2004-01-01

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

Vitrification of in vitro matured oocytes collected from surplus ovarian medulla tissue resulting from fertility preservation of ovarian cortex tissue

DEFF Research Database (Denmark)

Yin, Huiqun; Jiang, Hong; Kristensen, Stine Gry

2016-01-01

PURPOSE: The aim of the study was to investigate the maturation rate of immature oocytes collected from ovarian medulla tissue normally discarded during preparation of ovarian cortical tissue for fertility preservation. Further we evaluated survival of derived MII oocytes following vitrification...... and warming. METHODS: 36 patients aged from 8 to 41 years who had one ovary excised for fertility preservation were included. Oocytes were collected from the medulla tissue and matured in vitro 44-48 h followed by vitrification. Number of oocytes collected, the rates of maturation and post-warming survival...... of cortical tissue may pose a risk of relapse, but the IVM approach is currently too inefficient to be the only method used for fertility preservation....

Comparing the effects of different in vitro maturation media on IVM outcomes of MI oocytes

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Farzaneh Fesahat

2017-09-01

Conclusion: While the immature oocytes rescued from stimulated cycles based on specific conditions of patients can be useful for an alternative IVM intervention, it seems that different commercial culture media and longer incubation time has no beneficial effects on maturation, fertilization and embryo development on oocytes at MI stage.

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

Science.gov (United States)

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

2016-02-01

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

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

Science.gov (United States)

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

2015-01-01

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

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

Science.gov (United States)

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

2008-10-01

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

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

OpenAIRE

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

2016-01-01

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

The efficiency of in vitro ovine embryo production using an undefined or a defined maturation medium is determined by the source of the oocyte.

Science.gov (United States)

Cocero, M J; Alabart, J L; Hammami, S; Martí, J I; Lahoz, B; Sánchez, P; Echegoyen, E; Beckers, J F; Folch, J

2011-06-01

In vitro oocyte maturation can be influenced by oocyte source and maturation media composition. The aim of the present study was to compare the efficiency of a defined in vitro maturation medium (TCM199 supplemented with cysteamine and epidermal growth factor; Cys + EGF) with an undefined medium (TCM199 supplemented with follicle-stimulating hormone and follicular fluid; FSH + FF) for in vitro production (IVP) of ovine embryos, using oocytes obtained by laparoscopic ovum pick-up from FSH-stimulated [n=11; 158 cumulus-oocyte complexes (COCs)] and non-stimulated (n=16; 120 COCs) live ewes, as well as abattoir-derived oocytes (170 COCs). The produced blastocysts were vitrified and some of them were transferred to synchronized recipients. The best and the worst final yields of embryo IVP observed in this study were obtained using oocytes from FSH-stimulated ewes matured in FSH + FF (41.3%; 33/80) and in Cys + EGF (19.2%; 15/78) medium, respectively (p<0.01). No significant differences between both media were attained in the blastocyst development rate or in the final yield of embryo IVP using oocytes from non-stimulated ewes or abattoir-derived oocytes. The overall in vivo survival rate of the transferred vitrified blastocysts was 13.1% (8/61), without significant differences between oocyte sources or maturation media. In conclusion, under the experimental conditions of the present study, TCM199 supplemented with cysteamine and EGF is a convenient defined maturation medium for IVP of embryos from oocytes of live non-stimulated ewes or from oocytes of abattoir-derived ovaries. However, the best final yield of embryo IVP observed in this study was attained when oocytes came from FSH-stimulated donors and TCM199 was supplemented with FSH and follicular fluid. © 2010 Blackwell Verlag GmbH.

Endogenously produced hydrogen sulfide is involved in porcine oocyte maturation in vitro

Czech Academy of Sciences Publication Activity Database

Nevoral, J.; Žalmanová, T.; Zámostná, K.; Kott, T.; Kučerová-Chrpová, K.; Bodart, J. F.; Gelaude, A.; Procházka, Radek; Orsák, M.; Šulc, M.; Klein, P.; Dvořáková, M.; Weingartová, I.; Víghová, A.; Hošková, K.; Krejčová, T.; Jílek, F.; Petr, J.

2015-01-01

Roč. 51, č. 1 (2015), s. 24-35 ISSN 1089-8603 R&D Projects: GA MZe(CZ) QJ1510138 Institutional support: RVO:67985904 Keywords : oocyte * GVBD * meiotic maturation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.760, year: 2015

Effect of Rat Medicated Serum Containing You Gui Wan on Mouse Oocyte In Vitro Maturation and Subsequent Fertilization Competence

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Xiao-Hui Jiang

2014-01-01

Full Text Available You Gui Wan (YGW is a classic herbal formula in traditional Chinese medicine (TCM used for the clinical treatment of infertility. This study was to explore whether YGW has an impact on mouse oocyte maturation in vitro and subsequent fertilization competence. Rat medicated serum containing YGW was prepared by orally administrating YGW. Mouse immature oocytes were cultured with YGW medicated serum and compared to those cultured with or without normal rat serum or follicle-stimulating hormone (FSH. YGW medicated serum significantly increased the percentages of matured oocytes when compared to the groups with or without normal rat serum (P < 0.01. Furthermore, YGW medicated serum increased the rate of in vitro fertilization (IVF when compared to the groups treated with FSH and with or without normal rat serum (P < 0.001. YGW medicated serum also had significant effects on the mRNA expressions of PKA, CREB, MAPK, PKC, PKG, and MPF and the concentrations of cAMP, cGMP, and NO in matured oocytes. These results indicate that YGW can promote mouse oocyte maturation and IVF in vitro. Signaling pathways, such as the cAMP/PKA/MAPK, the PKC-MAPK, and the NO-cGMP-PKG pathway, which are similar to those induced by FSH, may be responsible for this action.

The effects analysis of two neonicotinoid insecticides on in vitro maturation of porcine oocytes using hanging drop monoculture method.

Science.gov (United States)

Ishikawa, Sadamasa; Hiraga, Kou; Hiradate, Yuuki; Tanemura, Kentaro

2015-06-01

Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure.

Feed restriction and insulin-like growth factor-I (IGF-I) affect the oocyte maturation in matrinxã Brycon amazonicus.

Science.gov (United States)

Montrezor, Luís Henrique; Urbinati, Elisabeth Criscuolo

2017-02-01

The feeding and nutrition of breeders are crucial aspects in the reproductive process. During the maturation period, metabolic changes occur aiming at mobilizing energy for growth and follicular development. The involvement of IGF-1 in metabolic and reproductive events is important. The aim of this work was to evaluate if alternate feed restriction and re-feeding have permissive effects on in vitro actions of IGF-1 on oocytes development of matrinxã. In vivo experiments were performed during vitellogenesis period. Females (n = 60) were fed with a commercial feed (2% of biomass) and they were divided into two treatments: fish receiving food daily (control - fed), and fish submitted to cycles of 3 days of feed restriction and 2 days of re-feeding (no-fed group). For the in vitro experiments, oocytes (n = 20) were obtained from the ovaries removed at the end of the in vivo experiment and were divided into four groups: fed -IGF-1; fed +IGF-1; no-fed -IGF-1 and no-fed +IGF-1. Fish under restriction had lower body weights, decreased plasma glucose, increased triglycerides levels, and their final maturation and mature oocyte were reduced and the atresic ones were in higher number. Moreover, IGF-1, in vitro, increased the percentage of mature oocytes in fed females and decreased the atresic ones. In no-fed females, IGF-1 increased the final maturation and mature oocytes and reduced the atresic ones. This study demonstrates the importance of the feeding management of female breeders of matrinxã during the vitellogenesis period.

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

Science.gov (United States)

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

2006-10-01

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

The effect of 24 hours delay in oocyte maturation triggering in IVF/ICSI cycles with antagonist protocol and not-elevated progesterone: A randomized control trial

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Robab Davar

2017-08-01

Full Text Available Background: The best time of final oocyte maturation triggering in assisted reproduction technology protocols is unknown. This time always estimated by combined follicular size and blood progesterone level. Objective: The aim of this study was evaluation of the effect of delaying oocyte maturation triggering by 24 hr on the number of mature oocytes (MII and other in vitro fertilization cycle characteristics in antagonist protocols with not-elevated progesterone (p ≤1 ng/ml. Materials and Methods: All patients' candidate for assisted reproduction technology underwent controlled ovarian hyperstimulation by antagonist protocol. When at least 3 follicles with ≥18 mm diameters were seen by vaginal ultrasonography; blood progesterone level was measured. The patients who had progesterone level ≤1 ng/dl entered the study. The participants' randomizations were done and patients were divided into two groups. In the first group, final oocyte maturation was done by human chorionic gonadotropin at the same day, but in the second group, this was performed 24 hr later. Oocytes retrieval was done 36 hr after human chorionic gonadotropin trigger by transvaginal ultrasound guide. Results: Number of retrieved oocytes, mature oocytes (MII, fertilized oocytes (2PN, embryos formation, number of transferred embryos and embryos quality has not significant differences between two groups. Also, fertilization and implantation rate, chemical and clinical pregnancy did not differ between groups. Conclusion: Delaying of triggering oocyte maturation by 24 hr in antagonist protocol with not-elevated progesterone (progesterone ≤1 ng/ml have not beneficial nor harmful effect on the number of mature oocytes (MII and other in vitro fertilization cycle characteristics

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

Science.gov (United States)

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

1996-04-15

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

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

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Sérgio Galbinski

2003-09-01

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

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

Science.gov (United States)

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

2016-04-01

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

Melatonin has dose-dependent effects on folliculogenesis, oocyte maturation capacity and steroidogenesis

International Nuclear Information System (INIS)

Adriaens, I.; Jacquet, P.; Cortvrindt, R.; Janssen, K.; Smitz, J.

2006-01-01

Chemo and/or radiotherapy applied to young cancer patients most often have severe effects upon female fertility. Today, few options are available to protect ovarian function in females. However, these options are either ineffective, belong to the field of experimental research or/and are not applicable to all patients. Drugs that could protect the oocyte and its surrounding feeder cells from damage can be of great importance. Melatonin, being an important indirect antioxidant and a powerful direct free radical scavenger could be such a reagent. This paper reports the direct effects of different melatonin concentrations (range: 1 nM to 2 mM) on folliculogenesis and oogenesis of in vitro cultured mouse ovarian follicles. Early secondary mouse follicles were cultured in vitro for 12 days under different melatonin regimes. Every fourth day, survival rates were scored, follicles were morphologically evaluated and medium was collected for steroid analyses. On day 12, in vitro ovulation was induced by hCG/EGF. Eighteen hours later, oocytes were measured, oocyte maturation was evaluated and normality of spindle and chromosomes ascertained. Results obtained in this study indicated that 2 mM melatonin is toxic. One mM negatively influenced oocyte maturation capacity. In the presence of 100 μM melatonin, androstenedione and progesterone were increased whereas estradiol was not influenced. Lower melatonin concentrations had no effect on the evaluated parameters. These data indicate an effect of melatonin on theca cell steroidogenesis. For prophylactic use, a dose of 10 μM could be suitable to reduce oxidative stress in cultured follicles

On-chip enucleation of an oocyte by untethered microrobots

International Nuclear Information System (INIS)

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

2014-01-01

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

The role of cilostazol, a phosphodiesterase 3 inhibitor, on oocyte maturation and subsequent pregnancy in mice.

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Min Li

Full Text Available It is important to identify effective contraceptive drugs that cause minimal disruption to physiological processes. Phosphodiesterase 3 (PDE3 inhibitors suppress meiosis in oocytes by decreasing the level of cAMP and blocking the extrusion of the first polar body. In this study, we tested the PDE3 inhibitor, cilostazol, as a potential contraceptive agent. The effects of cilostazol treatment in vitro and in vivo on the suppression of oocyte maturation in a mouse model were investigated. The results indicated that treatment with increasing concentrations of cilostazol led to a dose-dependent arrest in meiosis progression. The effective in vitro concentration was 1 µM and was 300 mg/kg in vivo. The effect of cilostazol was reversible. After removal of the drug, meiosis resumed and mouse oocytes matured in vitro, and showed normal chromosome alignment and spindle organization. After fertilization using an ICSI method, the oocytes showed normal morphology, fertilization rate, embryo cleavage, blastocyst formation, and number of viable pups when compared with controls. The offspring showed similar body weight and fertility. In vivo, the mice became infertile if the drug was injected sequentially, and became pregnant following discontinuation of cilostazol. More importantly, no side effects of cilostazol were observed in treated female mice as demonstrated by blood pressure and heart rate monitoring. It is concluded that cilostazol, a drug routinely used for intermittent claudication, can effectively inhibit oocyte maturation in vitro and in vivo, does not affect the developmental potential of oocytes following drug removal and has few side effects in female mice treated with this drug. These findings suggest that cilostazol may be a potential new contraceptive agent that may facilitate an efficacy and safety study of this drug.

Effect of cysteine supplementation on in vitro maturation of bovine ...

African Journals Online (AJOL)

B Rahim, S Jalal, N Yosef ... Cumulus-oocyte complexes (COCs) from abattoir ovaries were matured in vitro in Hepes-TCM 199 supplemented with 0.2 mM sodium pyruvate, 1 μg/ml 17-β-estradiol, 10% fetal calf serum (FCS), 0.5 μg/ml bFSH and 0 (control) and 100 or 500 μM/ml of cysteine for 24 h. When COCs matured in ...

Repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes

International Nuclear Information System (INIS)

Matsuda, Yoichi; Tobari, Izuo

1989-01-01

To study the repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes, the potentiating effects of 3 well-known repair inhibitors, arabinofuranosyl cytosine (ara-C), 3-aminobenzamide (3AB) and caffeine, on the frequency of induced chromosome aberrations were examined in eggs fertilized with X-irradiated sperm or in eggs irradiated with X-rays at the mature oocyte stage immediately before fertilization. Gametic treatment, fertilization and embryo culture wer carried out in vitro. Ara-C treatment was done only in the pre-DNA replication period, while treatment with 3AB and caffeine was continuous from fertilization to the first-cleavage metaphase. The induction of chromosome aberrations by exposing sperm or oocytes to X-rays was remarkably potentiated by post-treatment incubation in the presence of each of the 3 inhibitors. This result indicates the possibility that X-ray damage induced in sperm or oocytes is reparable in the fertilized eggs and that various types of repair processes are involved. (author). 39 refs.; 3 figs.; 5 tabs

Mesenchymal Stem Cell-Conditioned Medium Modulates Apoptotic and Stress-Related Gene Expression, Ameliorates Maturation and Allows for the Development of Immature Human Oocytes after Artificial Activation

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Hakimeh Akbari

2017-12-01

Full Text Available The aim of the present study was to determine whether mesenchymal stem cell-conditioned medium (MSC-CM modulates apoptotic and stress-related gene expression, and ameliorates maturation and developmental potential of immature human oocytes after artificial activation. A total of 247 surplus immature germinal vesicle (GV oocytes obtained from infertile women were allocated into two in vitro maturation (IVM groups: 1: GV oocytes (n = 116 matured in vitro (fIVM, and 2: GV oocytes (n = 131 that were vitrified, then in vitro matured (vIVM. Also, two maturation media were used: Alpha-minimum essential medium (α-MEM and human umbilical cord-derived MSCs (hUCM. After 36 h of incubation, the IVM oocytes were examined for nuclear maturation. In IVM-matured oocytes, cytoplasmic maturation was evaluated after artificial activation through Ionomycin. Moreover, the quantitative expressions of B-cell CLL/lymphoma 2 (BCL2, BCL2-associated X protein (BAX, superoxide dismutase (SOD, and Heat shock proteins (HSP70 in matured oocytes were assessed by quantitative Real-time polymerase chain reaction (qRT-PCR and compared with fresh and vitrified in vivo matured oocytes, which were used as fIVM and vIVM controls, respectively. The highest maturation rate was found in hUCM in fIVM, and the lowest maturation rate was found using α-MEM in vIVM (85.18% and 71.42%, respectively. The cleavage rate in fIVM was higher than that in vIVM (83.4% vs. 72.0%. In addition, the cleavage rate in α-MEM was lower than that in the hUCM (66.0% vs. 89.4%. Furthermore, the difference between parthenote embryo arrested in 4–8 cells (p < 0.04 and the quality of embryo arrested in 8-cell (p < 0.007 were significant. The developmental stages of parthenote embryos in hUCM versus α-MEM were as follows: 2–4 cell (89.45% vs. 66.00%, respectively, 4–8 cell (44.31% vs. 29.11%, respectively, morula (12.27% vs. 2.63%, respectively, and blastocysts (2.5% vs. 0%, respectively. The messenger

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

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Shujuan Wang

2017-07-01

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

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

Science.gov (United States)

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

1998-10-01

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

Evaluation of the Effect of Low-Frequency Electromagnetic Fields on in Vitro Growth and Maturation of Mouse Oocytes

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F Barzegari Firouzabadi

2012-05-01

Full Text Available Introduction: Access to modern methods for increasing the percentage of in vitro human and animal mature oocytes can be useful in the treatment of some forms of human infertility as well as proliferation of many domestic and wild animals which generation is endangered. Effect of low- frequency electromagnetic fields on in vitro growth and maturation of mouse oocytes is recently considered as a new approach. In this study we evaluated the effect of low- frequency electromagnetic field on in vitro growth and maturation of mouse oocyte. Methods: In this study electromagnetic fields with frequencies of 5, 50 and 100 Hz and 2mT intensity were used. For observation of the effect of electromagnetic field four groups were selected: Group 1 as control group, which included 35 prenatal follicles (immature oocytes. Groups 2, 3 and 4were exposed to 5, 50 and 100 Hz electromagnetic fields, respectively. Results: Prenatal follicles exposed to 5 and 50 Hz frequencies showed no significant changes in diameter and survival rates. In contrast at a frequency of 100 Hz in 72-hour culture period a significant increase in diameter(155μm, follicles livability power(59%, oocyte maturation(52% and GVBD(39% was shown in comparison to other experimental groups and control group(P <0.05. Conclusion: Low-frequency magnetic field effects gene expression and thus protein synthesis, cell division, proliferation and behavior. Although this effect can be temporary, it can increase the percentage of ovulation for in vitro environment along with other environmental factors.

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

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M.R. Souza

2003-10-01

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

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

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Gabriella Mamede Andrade

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

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

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Clelland Eric

2005-09-01

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

Casein kinase 1 alpha regulates chromosome congression and separation during mouse oocyte meiotic maturation and early embryo development.

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Lu Wang

Full Text Available Casein kinase I alpha (CK1α is a member of serine/threonine protein kinase, generally present in all eukaryotes. In mammals, CK1α regulates the transition from interphase to metaphase in mitosis. However, little is known about its role in meiosis. Here we examined Ck1α mRNA and protein expression, as well as its subcellular localization in mouse oocytes from germinal vesicle to the late 1-cell stage. Our results showed that the expression level of CK1α was increased in metaphase. Immunostaining results showed that CK1α colocalized with condensed chromosomes during oocyte meiotic maturation and early embryo development. We used the loss-of-function approach by employing CK1α specific morpholino injection to block the function of CK1α. This functional blocking leads to failure of polar body 1 (PB1 extrusion, chromosome misalignment and MII plate incrassation. We further found that D4476, a specific and efficient CK1 inhibitor, decreased the rate of PB1 extrusion. Moreover, D4476 resulted in giant polar body extrusion, oocyte pro-MI arrest, chromosome congression failure and impairment of embryo developmental potential. In addition, we employed pyrvinium pamoate (PP, an allosteric activator of CK1α, to enhance CK1α activity in oocytes. Supplementation of PP induced oocyte meiotic maturation failure, severe congression abnormalities and misalignment of chromosomes. Taken together, our study for the first time demonstrates that CK1α is required for chromosome alignment and segregation during oocyte meiotic maturation and early embryo development.

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

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Schmalzing, G.; Eckard, P.; Kroener, S.P.; Passow, H.

1990-01-01

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

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

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

2011-09-15

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

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

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

2011-04-01

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

In Vitro Maturation and Fertilization of Cryopreserved Germinal Vesicle Stage Oocytes in NMRI Mice, Using Ethylene Glycol and DMSO

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O Mayahi

2011-10-01

Full Text Available Background & Aim: Cryopreservation of oocytes is an essential part of reproductive biotechnology. The objective of the present study was to investigate the effects of exposure to combination of cryoprotectants and vitrification on immature mouse oocytes with or without cumulus cells. Methods: This was an experimental study conducted at Yasouj University of Medical Sciences in 2010. Immature oocytes with and without cumulus cells were isolated from ovaries of mice 4-6 weeks of age. They were vitrified in conventional straw using ethylene glycol (EG, dimethyl sulfoxide (DMSO and sucrose as vitrification solution or exposed to vitrification solution without subjected to liquid nitrogen. After warming, oocytes were assessed for nuclear maturation and fertilization. The collected data were analyzed with one-way ANOVA and Tukey test. Results: Survival and fertilization rates in vitrified oocytes with cumulus cells were significantly lower than the control group (p<0.05. Maturation rates in exposure groups were significantly lower than the vitrified and control groups (p<0.05. The fertilization rate increased significantly in all experiment and control groups with cumulus cells in comparison with denuded oocytes (p<0.05. Conclusion: Germinal vesicle stage oocytes in the presence or absence of cumulus cells can be vitrified successfully. Exposure to cryoprotectants can decrease the developmental competence of GV oocytes. Presence of cumulus cells can increase the fertilization rate in IVF procedure.

The Effect of Insulin-like Growth Factor-1, Cysteamine and β-Mercaptoethanol on the In Vitro Maturation of Immature Mice Oocytes

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A Dehghan Manshadi

2015-11-01

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.

IVF versus ICSI for the fertilization of in-vitro matured human oocytes.

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Walls, M; Junk, S; Ryan, J P; Hart, R

2012-12-01

Traditional dogma suggests that intracytoplasmic sperm injection (ICSI) should be performed to ensure successful oocyte fertilization in an in-vitro maturation (IVM) cycle. This study postulated that there would be no difference in the fertilization rate when ICSI was compared with IVF. This hypothesis was tested in a randomized trial of IVF versus ICSI in IVM. A total of 150 immature oocytes were collected in eight cycles of IVM for patients diagnosed with polycystic ovarian syndrome (PCOS). Patients were primed with minimal FSH before transvaginal oocyte aspiration. Sibling oocytes were inseminated by 50% IVF and 50% ICSI. There was no significant difference in fertilization, useable or total blastocyst development between the two insemination technique groups. Clinical pregnancy results for combined fresh and cryopreserved transfers were identical between the two insemination techniques with a total of two fresh and five cryopreserved IVF-inseminated embryos resulting in three clinical pregnancies (42.9%) and five fresh and two cryopreserved ICSI-derived embryos resulting in three clinical pregnancies (42.9%). This research has shown IVF to be a legitimate fertilization technique for IVM oocytes in PCOS patients and provides a greater awareness of the use of a fertilization method previously not utilized with IVM. In-vitro maturation (IVM) is an alternative treatment method to traditional IVF. Due to the minimal use of stimulating hormones in this treatment, IVM has a lower risk of ovarian hyperstimulation syndrome, it can be used for fertility preservation in cancer patients and it is more cost conservative. Early research into the effects of IVM showed a hardening effect on the membrane surrounding the egg (the zona pellucida). It was initially believed that, to overcome this hardening in order to allow the egg to be fertilized, spermatozoa would need to be injected into the egg using intracytoplasmic sperm injection. Due to recent advances in hormonal

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

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

2011-10-01

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

The effects of EGF and IGF-1 on FSH-mediated in vitro maturation of domestic cat oocytes derived from follicular and luteal stages.

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Yıldırım, Koray; Vural, M Rıfat; Küplülü, Sükrü; Ozcan, Ziya; Polat, I Mert

2014-04-01

The objective of this study was to evaluate the influence of epidermal growth factor (EGF) and insulin like growth factor-I (IGF-1) on the in vitro maturation of cat oocytes recovered from follicular and luteal stage ovaries. Oocytes from follicular (n=580) and luteal (n=209) stages were harvested and divided into four groups, which were cultured in FSH-mediated maturation medium supplemented with: (1) EGF alone (25ng/mL); (2) IGF-1 alone (100ng/mL); (3) EGF+IGF-1 (25ng/mL EGF+100ng/mL IGF-I); or (4) no growth factor (control). The proportion of follicular stage oocytes reaching the metaphase II stage was significantly higher than that of oocytes obtained at the luteal stage in both control and study groups (pIGF-1, and 78.1% in EGF+IGF-1 groups, whereas the respective values for gametes collected from luteal stage ovaries were 12.5%, 17.5%, 12.5%, and 16.9%. Additionally, the differences between the study and control groups were significant in the case of follicular stage oocytes. Finally, supplementing the maturation medium with EGF and/or IGF-1 significantly enhanced the meiotic maturation of oocytes recovered from follicular stage ovaries. The present study also demonstrated that the combination of EGF and IGF-I provides an additional or synergic effect on meiotic maturation of oocytes recovered from the follicular stage. Copyright © 2014 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.

High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification

DEFF Research Database (Denmark)

Du, Y; Pribenszky, C S; Molnár, M

2008-01-01

The purpose of the present study was to improve cryotolerance using high hydrostatic pressure (HHP) pretreatment of porcine in vitro matured (IVM) oocytes, to facilitate their further developmental competence after parthenogenetic activation. A total of 1668 porcine IVM oocytes were used in our...... present study. The pressure tolerance and optimal duration of recovery after HHP treatment were determined. Oocytes were treated with either 20 or 40 MPa (200 and 400 times greater than atmospheric pressure) for 60 min, with an interval of 10, 70, and 130 min between pressure treatment and subsequent...... vitrification under each pressure parameter. Oocytes from all vitrification groups had much lower developmental competence than fresh oocytes (Ppressure, with either 70...

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

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

1982-01-01

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

The crucial role of the proto-oncogene c-mos in regulation of oocyte maturation

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Irena Jałocha

2010-12-01

Full Text Available Meiosis arrest before fertilization is a common and unique feature of oogenesis in many animal species. On account of the unclear biological significance of meiosis arrest at various stages and for different durations in different animal species, this process and its regulation are the subject of many scientific studies. Studies on the development of ovarian teratomas proved to be helpful in defining the role of particular genes and biochemical cycles in control of the cell cycle in animals. These benign tumors are a valuable source of information on oocyte maturation. The [i]c-mos[/i] proto-oncogene, which is specifically expressed in female and male germ cells, plays a crucial role in control of meiotic cell division in mammals. Its product – Mos protein kinase – acting through mitogen-activated protein kinases (MAPKs regulates critical cellular functions required for homeostasis and decides about cell survival or apoptosis. The MAPK kinase kinase – MAPK kinase – MAPK (MKKK-MKK-MAPK phosphorelay system, in view of its role in cells, seems to be the ideal target for therapeutic intervention in cancer and other diseases. The recent research on human oocytes suggests that the basic mechanisms regulating various stages of oocyte maturation are similar to those described in animals.

Exposure to α-Tocopherol, Lutein or Ascorbic Acid improve Cumulus Expansion, Viability and Maturation of Swine Oocytes

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Ileana Miclea

2010-05-01

Full Text Available Protection of the fatty acid and lipid components of oocytes that render them susceptible to free radical or other oxidative injury may prevent the damage currently associated with culture. The goal of this study was to establish the influence of several α-tocopherol, lutein and ascorbic acid concentrations on swine oocyte maturation, viability and the function of cumulus cells in order to improve culture media. Pig oocytes were cultured for 45 hours at 37°C in 5% CO2 atmosphere; in M199 containing several α-tocopherol (5, 10, 20, 40, 80 μM, lutein (2.5, 4, 5, 8, 10 M or ascorbic acid (50, 150, 250, 500, 750 μM concentrations and cumulus expansion was assessed. Afterwards oocytes were coloured using FDA, PI and Hoechst 33258. The differences between treatments were analyzed by the analysis of variance and interpreted using the Newman-Keuls method. When cultured in α-tocopherol supplemented medium the number of expanded COCs to be scored as 3 was significantly greater (p<0.05 for the 5 and 40 μM concentrations. The addition of 8 M lutein to the maturation medium lead to a significant (p<0.05 increase in the number of COCs that were scored at 4. For both α-tocopherol and lutein additions the numbers of oocytes stained by FDA, as well as those stained by Hoechst were greater than the control without being statistically significant. When cultured in 150 and 500 μM ascorbic acid the percentages of COCs scored at 4 were significantly lower (p<0.05 than the control. Also, significantly (p<0.05 fewer oocytes were stained with FDA when matured in 500 μM. Differences between the control and the several concentrations were significant (p<0.05 for 150 and 750 μM and distinctly significant (p<0.01 for 250 μM.

Effect of cysteine supplementation on in vitro maturation of bovine ...

African Journals Online (AJOL)

Parham

2011-11-09

Nov 9, 2011 ... or more layer of cumulus cells and homogeneous granular ooplasm were selected for IVM procedures (Badr, 2009). In vitro maturation of oocytes. The basic medium for IVM was HEPES-buffered tissue culture medium 199 supplemented with 0.2 mM sodium pyruvate, 1 µg/ml. 17-β-estradiol, 10% fetal calf ...

In vivo effect of interleukin-1beta and interleukin-1RA on oocyte cytoplasmic maturation, ovulation, and early embryonic development in the mare

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Gérard Nadine

2005-06-01

Full Text Available Abstract A growing body of evidence suggests that the interleukin-1 system is involved in periovulatory events. Previous work from our lab demonstrated that in the mare, interleukin-1beta (IL-1beta increases the ovulatory rate of metaphase II oocytes. The present study was conducted to analyze in vivo the effect of IL-1 on oocyte cytoplasmic maturation, ovulation and pregnancy rate. In the present work, IL-1beta (experiment 1, n = 13; experiment 2, n = 25 and interleukin-1RA (IL-1RA; experiment 1, n = 25 were injected intrafollicularly by using the transvaginal ultrasound-guided injection method. Injections were performed on cyclic mares when the diameter of the growing dominant follicle reached 30–34 mm. In experiment 1, mares were inseminated the day of the treatment and all the other day until ovulation. The time of ovulation was determined and a pregnancy diagnosis was performed 14 days after ovulation of the injected follicle. In experiment 2, the cumulus-oocyte complex from each injected follicle was collected by transvaginal ultrasound-guided aspiration 38 h after the intrafollicular injection. Oocyte nuclear stage and oocyte cytoplasmic maturation were assessed by analyzing chromatin configuration, cortical granules migration and mitochondria distribution under a confocal microscope. The results from experiment 1 confirm that an intrafollicular injection of 1 microgram IL-1beta induces ovulation in the mare whereas IL-1RA has no effect at the dose used in the present study. Furthemore, we demonstrated, that in our experimental conditions, IL-1beta and IL-1RA induced a decrease in embryo development. Experiment 2 leads us to observe that IL-1beta is unable to induce cortical granules migration and remodelling of mitochondria, that commonly occurs during oocyte maturation, whereas it acts on nuclear maturation. This result may explain the decrease in embryo development we observed after IL-1beta intrafollicular injection. In conclusion

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes.

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Kyozuka, Keiichiro; Chun, Jong T; Puppo, Agostina; Gragnaniello, Gianni; Garante, Ezio; Santella, Luigia

2008-08-15

Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca2+ release in the cell cortex. Here, we show that this Ca2+ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca2+ release during meiotic maturation. The Ca2+ wave was inhibited by the classical antagonists of the InsP(3)-linked Ca2+ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca2+ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP(3), despite the massive release of Ca2+, implying that exocytosis of the cortical granules requires not only a Ca2+ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca2+ signals and in regulating cortical granule exocytosis.

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

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E.T Mergawati

2000-12-01

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

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

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Swamy K Tripurani

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

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

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Trudee Fair

2016-06-01

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

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

Czech Academy of Sciences Publication Activity Database

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

2012-01-01

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

Nicotine-induced Disturbances of Meiotic Maturation in Cultured Mouse Oocytes: Alterations of Spindle Integrity and Chromosome Alignment

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Zenzes Maria

2004-09-01

Full Text Available Abstract We investigated whether nicotine exposure in vitro of mouse oocytes affects spindle and chromosome function during meiotic maturation (M-I and M-II. Oocytes in germinal vesicle (GV stage were cultured in nicotine for 8 h or for 16 h, to assess effects in M-I and in metaphase II (M-II. The latter culture setting used the three protocols: 8 h nicotine then 8 h medium (8N + 8M; 16 h nicotine (16N; 8 h medium then 8 h nicotine (8M + 8N. Non-toxic concentrations of nicotine at 1.0, 2.5, 5.0 and 10.0 mmol/L were used. Spindle-chromosome configurations were analyzed with wide-field optical sectioning microscopy. In 8 h cultures, nicotine exposure resulted in dose-related increased proportions of M-I oocytes with defective spindle-chromosome configurations. A dose-related delayed entry into anaphase I was also detected. In 16 h cultures, nicotine exposure for the first 8 h (8N + 8M, or for 16 h (16N, resulted in dose- and time-related increased proportions of oocytes arrested in M-I (10 mmol/L; 8 h: 53.2%, controls 9.6%; 16 h: 87.6%, controls 8.5%. Defects in M-I spindles and chromosomes caused M-I arrest leading to dose-related decreased proportions of oocytes that reached metaphase-II (10 mmol/L 8 h: 46.8%, controls 90.4%;16 h: 12.4%, controls 91.5%. A delayed anaphase-I affected the normal timing of M-II, leading to abnormal oocytes with dispersed chromosomes, or with double spindles and no polar body. Nicotine exposure during the second 8 h (8M + 8N resulted in dose-related, increased proportions of M-II oocytes with defective spindles and chromosomes (10 mmol/L: 42.9%, controls 2.0%. Nicotine has no adverse effects on GV break down, but induces spindle and chromosome defects compromising oocyte meiotic maturation and development.

Developmental capacity of in vitro-matured human oocytes retrieved from polycystic ovary syndrome ovaries containing no follicles larger than 6 mm.

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Guzman, Luis; Ortega-Hrepich, Carolina; Albuz, Firas K; Verheyen, Greta; Devroey, Paul; Smitz, Johan; De Vos, Michel

2012-08-01

To test the developmental competence of oocytes in a nonhCG-triggered in vitro maturation (IVM) system when oocyte-cumulus complexes (OCC) are retrieved from antral follicles with a diameter of polycystic ovaries/polycystic ovary syndrome underwent 239 IVM cycles in total. In 58 of these cycles (44 patients), all antral follicles had a diameter of <6 mm on the day of oocyte retrieval. NonhCG-triggered IVM of oocytes, fresh or vitrified/warmed embryo transfer (ET). Oocyte diameter, maturation rate, fertilization rate, embryo development and morphology, implantation rate, clinical pregnancy rate, ongoing pregnancy rate. Oocyte retrieval yielded 16.7 OCC/cycle, and 50.8% of oocytes completed IVM. The mean oocyte diameter increased from 108.8 ± 4.3 μm to 111.9 ± 4.1 μm after IVM. Mean fertilization rate was 63.7%, and 45.4% of 2-pronuclei oocytes developed into a morphologically good-quality embryo on day 3 after intracytoplasmic sperm injection. Fresh ET resulted in two ongoing pregnancies (2/37; 5.4%). Deferred vitrified-warmed ET led to an ongoing pregnancy rate of 34.6% (9/24). Three healthy babies were born and eight pregnancies were still ongoing. Oocytes retrieved from follicles with a diameter of <6 mm grow during a 40-hour IVM culture can acquire full competence in vitro, as illustrated by their development into healthy offspring. Endometrial quality appears to be a crucial determinant of pregnancy after nonhCG-triggered IVM. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

In Vitro Maturation (IVM of Human Oocytes: Promising Potential, Challenges and Chances for Improvement

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Roza silvia

2015-05-01

Full Text Available AbstractIn Vitro Maturation (IVM of human oocytes is an innovation in Assisted Reproductive Technology (ART. It is believed more patient-friendly than conventional In Vitro Fertilization (IVF method. It is a simple protocol that needs only less injection of ovarian stimulation for the patients and fewer blood sample and ultrasound scans, so this technique may become more favorable. Patients are also prevented from higher cost treatments and quite long control in the hospital. However, there are some problems to be addressed, such as how to improve the success rate, how to assure the safety and to avoid the health risk for the offsprings. Modification in IVM medium and optimizing the IVM protocols have increased the results in some studies. However, further investigation related to all aspects influencing the human oocyte maturation in vitro is still needed to make it enable to be a routine practice in ART centers for a defined group.Kata kunci: in vitro maturation, human oocyte, in vitro fertilization, assisted reproductive technology AbstrakMaturasi oosit in vitro atau In Vitro Maturation (IVM terhadap oosit manusia merupakan suatu inovasi dalam Teknologi Reproduksi Berbantu (TRB. Teknik ini dianggap lebih nyaman bagi pasien dibandingkan dengan metode Fertilisasi In Vitro (FIV konvensional. Metode IVM ini sederhana dan hanya membutuhkan lebih sedikit penyuntikan obat stimulasi ovarium ke pasien serta lebih sedikit pemeriksaan darah dan ultrasonografi, sehingga memungkinkan untuk menjadi suatu pilihan yang disukai oleh pasien. Pasien juga bisa terhindar dari biaya terapi yang lebih mahal serta waktu kontrol yang lama di rumah sakit. Namun demikian, terdapat beberapa masalah yang perlu ditangani terkait metode ini, seperti bagaimana meningkatkan angka keberhasilan serta memastikan keamanan dan mencegah resiko kesehatan pada anak yang akan dilahirkan. Modifikasi pada medium IVM serta pengoptimalan protokol IVM telah meningkatkan hasil pada

Advancing maternal age predisposes to mitochondrial damage and loss during maturation of equine oocytes invitro

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Rambags, B. P B; van Boxtel, D. C J; Tharasanit, T.; Lenstra, J. A.; Colenbrander, B.; Stout, T. A E

2014-01-01

In many mammalian species, reproductive success decreases with maternal age. One proposed contributor to this age-related decrease in fertility is a reduction in the quantity or functionality of mitochondria in oocytes. This study examined whether maternal age or (in vitro maturation). IVM affect

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

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Md Mahmodul Hasan Sohel

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

Effect of Somatic Cell Types and Culture Medium on in vitro Maturation, Fertilization and Early Development Capability of Buffalo Oocytes

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H. Jamil*, H. A. Samad, N. Rehman, Z. I. Qureshi and L. A. Lodhi

2011-04-01

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.

The human cumulus--oocyte complex gene-expression profile

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

2006-01-01

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

Eccentric localization of catalase to protect chromosomes from oxidative damages during meiotic maturation in mouse oocytes.

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Park, Yong Seok; You, Seung Yeop; Cho, Sungrae; Jeon, Hyuk-Joon; Lee, Sukchan; Cho, Dong-Hyung; Kim, Jae-Sung; Oh, Jeong Su

2016-09-01

The maintenance of genomic integrity and stability is essential for the survival of every organism. Unfortunately, DNA is vulnerable to attack by a variety of damaging agents. Oxidative stress is a major cause of DNA damage because reactive oxygen species (ROS) are produced as by-products of normal cellular metabolism. Cells have developed eloquent antioxidant defense systems to protect themselves from oxidative damage along with aerobic metabolism. Here, we show that catalase (CAT) is present in mouse oocytes to protect the genome from oxidative damage during meiotic maturation. CAT was expressed in the nucleus to form unique vesicular structures. However, after nuclear envelope breakdown, CAT was redistributed in the cytoplasm with particular focus at the chromosomes. Inhibition of CAT activity increased endogenous ROS levels, but did not perturb meiotic maturation. In addition, CAT inhibition produced chromosomal defects, including chromosome misalignment and DNA damage. Therefore, our data suggest that CAT is required not only to scavenge ROS, but also to protect DNA from oxidative damage during meiotic maturation in mouse oocytes.

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

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

2016-04-01

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

Role of gap junctions and protein kinase A during the development of oocyte maturational competence in Ayu (Plecoglossus altivelis)

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Yamamoto, Y.; Yoshizaki, G.; Takeuchi, T.; Soyano, K.; Patino, R.

2008-01-01

Meiotic resumption in teleost oocytes is induced by a maturation-inducing hormone (MIH). The sensitivity of oocytes to MIH, also known as oocyte maturational competence (OMC), is induced by LH via mechanisms that are not fully understood. A previous study of Ayu (Plecoglossus altivelis) showed the presence of functional heterologous gap junctions (GJs) between oocytes and their surrounding granulosa cells. The objectives of this study were to determine the role of ovarian GJs and of protein kinase A (PKA) during the acquisition of OMC. We examined the effects of the specific GJ inhibitor carbenoxolone (CBX) and 18??-glycyrrhetinic acid (??-GA) on the LH-(hCG)-dependent acquisition of OMC and on MIH-(17,20??-dihydroxy-4-pregnen-3-one)-dependent meiotic resumption; measured the cAMP content of ovarian follicles during the hCG-dependent acquisition of OMC; and determined the effects of PK activators and inhibitors on hCG-dependent OMC. Production of follicular cAMP increased during the hCG-dependent acquisition of OMC. Both GJ inhibitors and the PKA inhibitor H8-dihydrochloride, but not the PKC inhibitor GF109203X, suppressed the hCG-dependent acquisition of OMC in a dose-dependent manner. The PKA activator forskolin induced OMC with a similar potency to hCG. Unlike previous observations with teleosts where disruption of heterologous GJ either blocks or stimulates meiotic resumption, treatment with GJ inhibitors did not affect MIH-dependent meiotic resumption in maturationally competent follicles of Ayu. These observations suggest that ovarian GJs are essential for LH-dependent acquisition of OMC but not for MIH-dependent meiotic resumption, and that the stimulation of OMC by LH is mediated by cAMP-dependent PKA. They are also consistent with the view that a precise balance between GJ-mediated signals (positive or negative) and oocyte maturational readiness is required for hormonally regulated meiotic resumption. ?? 2007 Elsevier Inc. All rights reserved.

Paxillin and embryonic PolyAdenylation Binding Protein (ePABP) engage to regulate androgen-dependent Xenopus laevis oocyte maturation - A model of kinase-dependent regulation of protein expression.

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Miedlich, Susanne U; Taya, Manisha; Young, Melissa Rasar; Hammes, Stephen R

2017-06-15

Steroid-triggered Xenopus laevis oocyte maturation is an elegant physiologic model of nongenomic steroid signaling, as it proceeds completely independent of transcription. We previously demonstrated that androgens are the main physiologic stimulator of oocyte maturation in Xenopus oocytes, and that the adaptor protein paxillin plays a crucial role in mediating this process through a positive feedback loop in which paxillin first enhances Mos protein translation, ensued by Erk2 activation and Erk-dependent phosphorylation of paxillin on serine residues. Phosphoserine-paxillin then further augments Mos protein translation and downstream Erk2 activation, resulting in meiotic progression. We hypothesized that paxillin enhances Mos translation by interacting with embryonic PolyAdenylation Binding Protein (ePABP) on polyadenylated Mos mRNA. Knockdown of ePABP phenocopied paxillin knockdown, with reduced Mos protein expression, Erk2 and Cdk1 activation, as well as oocyte maturation. In both Xenopus oocytes and mammalian cells (HEK-293), paxillin and ePABP constitutively interacted. Testosterone (Xenopus) or EGF (HEK-293) augmented ePABP-paxillin binding, as well as ePABP binding to Mos mRNA (Xenopus), in an Erk-dependent fashion. Thus, ePABP and paxillin work together in an Erk-dependent fashion to enhance Mos protein translation and promote oocyte maturation. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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

1999-01-01

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

Luteal-phase ovarian stimulation increases the number of mature oocytes in older women with severe diminished ovarian reserve.

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Rashtian, Justin; Zhang, John

2018-03-22

In older women with severe diminished ovarian response (DOR), in vitro fertilization (IVF) treatment is much less successful due to the low number of mature oocytes collected. The objective of this study was to assess whether follicular-phase stimulation (FPS) and luteal-phase stimulation (LPS) in the same menstrual cycle (double ovarian stimulation) in older women with severe DOR will produce a higher number of oocytes compared to FPS alone. Women with DOR (n = 69; mean age = 42.4) who underwent double ovarian stimulation for IVF were included. Women underwent ovarian stimulation in FPS using clomiphene citrate, letrozole, and gonadotropins followed by oocyte retrieval. The next day following oocyte retrieval, women underwent a second ovarian stimulation (LPS) using the same medications followed by a second oocyte retrieval. T-test was performed in order to compare the clinical characteristics and outcome in the same participant between FPS and LPS. Although antral follicle count at the start of FPS tended to be higher than at the start of the LPS cycle, there was no statistically significant difference between the duration of ovarian stimulation, peak estradiol levels, number of small (FPS alone. The addition of LPS to the conventional FPS increases the number of mature oocytes retrieved in the same IVF cycle, thus potentially increasing the chances of pregnancy in older women with severe DOR. AFC: antral follicle count; BMI: body mass index; DOR: diminished ovarian reserve; E2: estradiol; FPS: follicular-phase stimulation; FSH: follicle stimulating hormone; GnRH: gonadotropin-releasing hormone; HCG: human chorionic gonadotropin; IRB: institutional review board; IVF: in vitro fertilization; LH: luteinizing hormone; LPS: luteal-phase stimulation; MII: metaphase II.

Comparison of the Developmental Potential and Clinical Results of In Vivo Matured Oocytes Cryopreserved with Different Vitrification Media

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Mei Li

2015-01-01

Full Text Available Background: Oocyte vitrification is widely used throughout the world, but its clinical efficacy is inconsistent and depends on the vitrification media. This study compared the developmental potential and clinical results of in vivo matured oocytes cryopreserved with different vitrification media. Methods: This retrospective study involved vitrified-warmed oocytes at one in vitro fertilization laboratory. Vitrification media kits comprised the MC kit (ethylene glycol [EG] plus 1,2-propanediol [PROH], the KT kit (EG plus dimethyl sulphoxide [DMSO], and the Modified kit (EG plus DMSO and PROH kit. Rates of oocyte survival and subsequent developmental potential were recorded and analyzed. The t-test and the Chi-square test were used to evaluate each method′s efficacy. Results: Oocyte survival rate was significantly higher for the Modified kit (92.0% than for the MC kit (88.2% (P 0.05. The high-quality embryo rate per warmed oocyte was significantly higher (23.4% in the Modified kit group than in the other groups (P 0.05. Conclusions: Modified vitrification media are efficient for oocyte vitrification and, with further verification, may be able to replace commercially available media in future clinical applications.

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

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

2016-12-01

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

Follicular 17β-estradiol and progesterone concentrations and degree of cumulus cell expansion as predictors of in vivo-matured oocyte developmental competence in superstimulated heifers

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Aardema, Hilde; Roelen, Bernard A J; van Tol, Helena T A; Oei, Christine H Y; Gadella, Bart M; Vos, Peter L A M

2013-01-01

The quality of an oocyte is crucial for successful generation of offspring, but few selection parameters have been identified that reliably predict oocyte developmental competence. The objective of the present study was to determine whether the developmental competence of in vivo-matured oocytes

Adverse Effects of High Concentrations of Fluoride on Characteristics of the Ovary and Mature Oocyte of Mouse

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Yin, Songna; Song, Chao; Wu, Haibo; Chen, Xin; Zhang, Yong

2015-01-01

Reproductive toxicity has been an exciting topic of research in reproductive biology in recent years. Soluble fluoride salts are toxic at high concentrations; their reproductive toxicity was assessed in this study by administering different fluoride salt concentrations to mice. Continuous feeding for five weeks resulted in damage to the histological architecture of ovaries. The expression of genes, including Dazl, Stra8, Nobox, Sohlh1, and ZP3 gene, associated with oocyte formation were much lower in the experimental group as compared with the control group. The number of in vitro fertilization of mature oocytes were also much lower in the experimental group as compared with control. Moreover, the fertility of female mice, as assessed by mating with normal male mice, was also lower in experimental compared with control groups. The expression of the oocyte-specific genes: Bmp15, Gdf9, H1oo, and ZP2, which are involved in oocyte growth and the induction of the acrosome reaction, decreased with the fluoride administration. DNA methylation and histone acetylation (H3K18ac and H3K9ac) are indispensable for germline development and genomic imprinting in mammals, and fluoride administration resulted in reduced levels of H3K9ac and H3K18ac in the experimental group as compared with the control group, as detected by immunostaining. Our results indicate that the administration of high concentrations of fluoride to female mice significantly reduced the number of mature oocytes and hampered their development and fertilization. Thus, this study lays a foundation for future studies on fluoride-induced reproductive disorders in women. PMID:26053026

Adverse Effects of High Concentrations of Fluoride on Characteristics of the Ovary and Mature Oocyte of Mouse.

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Songna Yin

Full Text Available Reproductive toxicity has been an exciting topic of research in reproductive biology in recent years. Soluble fluoride salts are toxic at high concentrations; their reproductive toxicity was assessed in this study by administering different fluoride salt concentrations to mice. Continuous feeding for five weeks resulted in damage to the histological architecture of ovaries. The expression of genes, including Dazl, Stra8, Nobox, Sohlh1, and ZP3 gene, associated with oocyte formation were much lower in the experimental group as compared with the control group. The number of in vitro fertilization of mature oocytes were also much lower in the experimental group as compared with control. Moreover, the fertility of female mice, as assessed by mating with normal male mice, was also lower in experimental compared with control groups. The expression of the oocyte-specific genes: Bmp15, Gdf9, H1oo, and ZP2, which are involved in oocyte growth and the induction of the acrosome reaction, decreased with the fluoride administration. DNA methylation and histone acetylation (H3K18ac and H3K9ac are indispensable for germline development and genomic imprinting in mammals, and fluoride administration resulted in reduced levels of H3K9ac and H3K18ac in the experimental group as compared with the control group, as detected by immunostaining. Our results indicate that the administration of high concentrations of fluoride to female mice significantly reduced the number of mature oocytes and hampered their development and fertilization. Thus, this study lays a foundation for future studies on fluoride-induced reproductive disorders in women.

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

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Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

2010-02-01

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

Molecular and structural aspects of oocyte maturation

NARCIS (Netherlands)

Hölzenspies, J.J.

2009-01-01

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

Caffeine and oocyte vitrification: Sheep as an animal model

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Adel R. Moawad

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

Diving into the oocyte pool

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

2017-01-01

of the signaling pathways activating dormant follicles and breakthroughs in techniques for autologous transfer of mitochondria have opened new doors to unexploited sources of oocytes and attractive ways of revitalizing oocytes. Extended numbers of mature oocytes may be obtained by in-vitro activation of dormant...... for revitalizing deficient oocytes may transform ART, and potentially enhance both quantity and quality of fertilizable oocytes; hereby augmenting the pregnancy potential of women with poor reproductive performance....

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

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

Human oocyte cryopreservation and the fate of cortical granules.

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

2006-07-01

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

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

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

2018-01-01

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

Cysteamine supplementation during in vitro maturation of slaughterhouse- and opu-derived bovine oocytes improves embryonic development without affecting cryotolerance, pregnancy rate, and calf characteristics.

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Merton, J S; Knijn, H M; Flapper, H; Dotinga, F; Roelen, B A J; Vos, P L A M; Mullaart, E

2013-09-01

Optimization of ovum pick up (OPU) followed by in vitro embryo production (IVP) is strongly driven by the needs of both beef and dairy cattle breeders to enhance genetic improvement. The rapidly growing use of genomic selection in cattle has increased the interest in using OPU-IVP technology to increase the number of embryos and offspring per donor, thus allowing enhanced selection intensity for the next generation. The aim of this study was to optimize embryo production through supplementation of cysteamine during in vitro maturation (IVM) and in vitro culture (IVC) of both slaughterhouse- and OPU-derived oocytes. The effects on embryo production and on embryo cryotolerance, post-transfer embryo survival, and calf characteristics, including gestation length, birth weight, perinatal mortality, and sex ratio were studied. In study 1, immature slaughterhouse-derived cumulus-oocyte complexes (COCs) were matured in IVM medium supplemented with or without 0.1 mM cysteamine, fertilized and cultured for 7 days in 0.5 ml SOFaaBSA. In study 2, cysteamine was present during both IVM (0.1 mM) and IVC (0.01, 0.05, 0.1 mM) from Days 1 to 4. In study 3, OPU-derived COCs were matured in medium supplemented with or without 0.1 mM cysteamine in a 2 × 2 factorial design (OPU week and cysteamine treatment). Embryos were evaluated for stage and grade on Day 7 and, depending on the number of transferable embryos and recipients available, the embryos were transferred either fresh or frozen-thawed at a later date. The presence of cysteamine during IVM significantly increased the embryo production rate with slaughterhouse-derived COCs (24.0% vs. 19.4%). The higher number of embryos at Day 7 was due to an increased number of blastocysts, whereas the distribution of embryos among different quality grades and cryotolerance was not affected. Embryo production rate was negatively affected when cysteamine was present during both the processes of IVM and IVC during Days 1 to 4 of culture (13

The fate and role of macromolecules synthesized during mammalian oocyte meiotic maturation. I. Autoradiographic topography of newly synthesized RNA and protein in the germinal vesicle of the pig and rabbit

International Nuclear Information System (INIS)

Motlik, J.; Kopecny, V.; Pivko, J.

1978-01-01

Pig and rabbit oocytes were cytoautoradiographically checked for their synthetic activities during meiotic maturation. Tritiated uridine and lysine or 35 S-methionine were introduced into the culture medium in which the oocytes were maintained either immediately at the beginning of the germinal vesicle breakdown in vitro or after reaching a more advanced stage of this process in vitro or in vivo. Some oocytes were maintained thereafter in a cold medium to trace the metabolism of the labelled protein. In addition to uridine- 3 H incorporation into the nucleolus and nucleoplasm, during pig oocyte maturation it was found that an intensive RNA synthesis site appeared in association with condensing chromocentres of the GV II. A considerable proportion of oocytes from slaughterhouse material did not show intensive GV activity in RNA synthesis during maturation in vitro. In the pig and rabbit oocyte it was shown that the newly synthesized 3 H-lysine-labelled protein accumulated to a high degree in the GV and in the nucleolus. The labelled protein accumulated in the GV up to the stage of GV IV (pig) and persisted during the chase period in the ooplasm; it was found to be associated with chromosomes of metaphase I (pig) or metaphase II (rabbit) of the meiotic division. The process of protein accumulation in the GV was not influenced by meiotic arrest during oocyte culture in autologous follicular fluid. A similar accumulation of the label in the GV was detected in oocytes which were cultured in a medium enriched by 35 S-methionine. In some oocytes the labelled protein failed to accumulate in the nucleolar area during maturation in vitro

Toxicity effect of Auxemma oncocalyx fraction and its active principle oncocalyxone A on in vitro culture of caprine secondary follicles and in vitro oocyte maturation

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Johanna Leiva-Revilla

2017-06-01

Full Text Available Crude extract of the heartwood of Auxemma oncocalyx (A. oncocalyx and its main component i.e., Oncocalyxone A (onco A, have elevated antioxidant and anti-tumoral activity, but studies on the action of these drugs regarding folliculogenesis are lacking. The aim of this study was to evaluate the effect of A. oncocalyx and onco A on the in vitro culture of isolated secondary follicles and on the in vitro maturation of oocytes from caprine antral follicles grown in vivo. Isolated secondary follicles were randomly distributed in six groups; the non-cultured control was immediately fixed upon isolation. The remaining follicles were cultured for 7 days in ?-MEM+ alone (control or supplemented with DMSO, doxorrubicin, A. oncocalyx or onco A. After culture, follicles were evaluated for antrum formation, growth rate, apoptosis (TUNEL and cellular proliferation (PCNA, as well as gene expression of Bcl2 and Bax. Additionally, cumulus oocyte complexes (COCs were aspirated and allocated into five treatments for in vitro maturation: control, cultured only in maturation base medium (TCM 199+; or supplemented with DMSO; DXR; A. oncocalyx or onco A. After in vitro maturation, oocyte chromatin configuration and viability were assessed. After 7 days of culture, there was a reduction (P < 0.05 in the percentage of morphologically intact follicles, antrum formation, growth rate and number of PCNA positive granulosa cells in DXR treatment compared to the other treatments. In the DXR treatment a higher percentage (P < 0.05 of TUNEL positive follicles and higher (P < 0.05 relative BAX:BCL2 mRNA ratio’s were observed. After in vitro maturation of the COCs DXR, A. oncocalyx and onco A treatments had a greater (P < 0.05 percentage of abnormal oocytes and a lower (P < 0.05 percentage of viable oocytes as compared with the control group. However, only DXR and onco A treatments increased (P < 0.05 the percentage of alive oocytes with

Differential expression of gonadotropin and estrogen receptors and oocyte cytology during follicular maturation associated with egg viability in European eel (Anguilla anguilla)

DEFF Research Database (Denmark)

da Silva, Filipa F. G.; Tveiten, Helge; Maugars, Gersende

2018-01-01

In captivity, oogenesis and ovarian follicle maturation in European eel can be induced experimentally using hormonal therapy. The follicle's ability to respond effectively to the induction of maturation and ovulation, resulting in viable eggs, depends on the oocyte stage at the time of induction....

Effects of RU486 and indomethacin on meiotic maturation, formation of extracellular matrix, and progesterone production by porcine oocyte-cumulus complexes.

Science.gov (United States)

Nagyova, E; Scsukova, S; Kalous, J; Mlynarcikova, A

2014-07-01

This study was designed to determine whether inhibition of either cyclooxygenase-2 (COX-2) by indomethacin or progesterone receptor (PR) by PR antagonist, RU486, affects oocyte maturation, progesterone production, and covalent binding between hyaluronan (HA) and heavy chains of inter-α trypsin inhibitor, as well as expression of cumulus expansion-associated proteins (HA-binding protein, tumor necrosis factor α-induced protein 6, pentraxin 3) in oocyte-cumulus complexes (OCCs). The experiments were based on freshly isolated porcine OCC cultures in which the consequences of PR and COX-2 inhibition on the final processes of oocyte maturation were determined. Granulosa cells (GCs) and OCCs were cultured in medium supplemented with FSH/LH (both 100 ng/mL) in the presence/absence of RU486 or indomethacin. Western blot analysis, (3)H-glucosamine hydrochloride assay, immunofluorescence, and radioimmunoassay were performed. Only treatment with RU486 (25 μM) caused a decrease in the number of oocytes that reached germinal vesicle breakdown and metaphase II stage compared with indomethacin (100 μM) or FSH/LH treatment alone after 44 h. All treated OCCs synthesized an almost equal amount of HA. Heavy chains (of inter-α trypsin inhibitor)-HA covalent complexes were formed during in vitro FSH/LH-stimulated expansion in RU486- or indomethacin-treated OCCs. Follicle-stimulating hormone/LH-induced progesterone production by OCCs was increased in the presence of RU486 after 44 h. In contrast, a decrease of FSH/LH-stimulated progesterone production by GCs was detected in the presence of either RU486 or indomethacin after 72 h. We suggest that the PR-dependent pathway may be involved in the regulation of oocyte maturation. Both PR and COX-2 regulate FSH/LH-stimulated progesterone production by OCCs and GCs. Copyright © 2014 Elsevier Inc. All rights reserved.

Kit ligand promotes first polar body extrusion of mouse preovulatory oocytes

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Ye Yinghui

2009-04-01

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

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

Czech Academy of Sciences Publication Activity Database

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

2012-01-01

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

Molecular Basis of Meiotic Maturation and Apoptosis of Oocytes, Sperm-Oocyte Interactions and Early Cleavage of Embryos in Mice, Role of Phosphatidylinositol 3-Kinase, Mos, Fas-Fas Ligand, Integrinα6 and MAP Kinase

OpenAIRE

Yumi Hoshino; Ken-ichi Yamanaka; Ikuo Tomioka; Noritaka Fukunaga; Mehdi Abbasi; Eimei Sato

2005-01-01

The interaction between molecular biology and embryology made an extensive progress in the research on gametogenesis, fertilization and early embryogenesis in mice. In this article, molecules involving in meiotic maturation and apoptosis of oocytes, sperm-oocyte interactions and early cleavage of fertilized embryos in mice are described including our recent following experiments. 1) Phosphatidylinositol 3-kinase and Akt participate in the follicle stimulating hormone-induced meiotic maturatio...

Supplementation with nanomolar concentrations of verbascoside during in vitro maturation improves embryo development by protecting the oocyte against oxidative stress: a large animal model study.

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Martino, Nicola Antonio; Ariu, Federica; Bebbere, Daniela; Uranio, Manuel Filioli; Chirico, Adriana; Marzano, Giuseppina; Sardanelli, Anna Maria; Cardinali, Angela; Minervini, Fiorenza; Bogliolo, Luisa; Dell'Aquila, Maria Elena

2016-10-01

The effects of verbascoside (VB), added at nanomolar concentrations during in vitro maturation (IVM) of juvenile sheep oocytes, on in vitro embryo development and its mechanisms of action at the oocyte level were analyzed. Developmental rates, after IVM in the presence/absence of VB (1nM for 24h; 1nM for 2h; 10nM for 2h), were evaluated. The bioenergetic/oxidative status of oocytes matured after IVM in the presence/absence of 1nM VB for 24h was assessed by confocal analysis of mitochondria and reactive oxygen species (ROS), lipid peroxidation (LPO) assay, and quantitative PCR of bioenergy/redox-related genes. The addition of 1nM VB during 24h IVM significantly increased blastocyst formation and quality. Verbascoside reduced oocyte ROS and LPO and increased mitochondria/ROS colocalization while keeping mitochondria activity and gene expression unchanged. In conclusion, supplementation with nanomolar concentrations of VB during IVM, in the juvenile sheep model, promotes embryo development by protecting the oocyte against oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

In vitro and in vivo Development of Cloned Ovine Embryos using in vitro and in vivo Matured Oocytes

DEFF Research Database (Denmark)

Holm, P; Nagashima, H; Sun, F-J

1995-01-01

Cloning of sheep embryos by nucleus transplantation can be achieved by using in vivo matured (oviductal) oocytes and in vivo culture. However, these steps involve cumbersome procedures. Therefore, the effects of in vivo vs. the equivalent in vitro procedures on the pre-implantation development of...

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

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Wahyuningsih, S.; Ihsan, M. N.

2018-02-01

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

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

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Joanna Budna

2017-12-01

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

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

Science.gov (United States)

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

2003-01-01

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

“Positive Regulation of RNA Metabolic Process” Ontology Group Highly Regulated in Porcine Oocytes Matured In Vitro: A Microarray Approach

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Piotr Celichowski

2018-01-01

Full Text Available The cumulus-oocyte complexes (COCs growth and development during folliculogenesis and oogenesis are accompanied by changes involving synthesis and accumulation of large amount of RNA and proteins. In this study, the transcriptomic profile of genes involved in “oocytes RNA synthesis” in relation to in vitro maturation in pigs was investigated for the first time. The RNA was isolated from oocytes before and after in vitro maturation (IVM. Interactions between differentially expressed genes/proteins belonging to “positive regulation of RNA metabolic process” ontology group were investigated by STRING10 software. Using microarray assays, we found expression of 12258 porcine transcripts. Genes with fold change higher than 2 and with corrected p value lower than 0.05 were considered as differentially expressed. The ontology group “positive regulation of RNA metabolic process” involved differential expression of AR, INHBA, WWTR1, FOS, MEF2C, VEGFA, IKZF2, IHH, RORA, MAP3K1, NFAT5, SMARCA1, EGR1, EGR2, MITF, SMAD4, APP, and NR5A1 transcripts. Since all of the presented genes were downregulated after IVM, we suggested that they might be significantly involved in regulation of RNA synthesis before reaching oocyte MII stage. Higher expression of “RNA metabolic process” related genes before IVM indicated that they might be recognized as important markers and specific “transcriptomic fingerprint” of RNA template accumulation and storage for further porcine embryos growth and development.

A second dose of kisspeptin-54 improves oocyte maturation in women at high risk of ovarian hyperstimulation syndrome: a Phase 2 randomized controlled trial.

Science.gov (United States)

Abbara, Ali; Clarke, Sophie; Islam, Rumana; Prague, Julia K; Comninos, Alexander N; Narayanaswamy, Shakunthala; Papadopoulou, Deborah; Roberts, Rachel; Izzi-Engbeaya, Chioma; Ratnasabapathy, Risheka; Nesbitt, Alexander; Vimalesvaran, Sunitha; Salim, Rehan; Lavery, Stuart A; Bloom, Stephen R; Huson, Les; Trew, Geoffrey H; Dhillo, Waljit S

2017-09-01

Can increasing the duration of LH-exposure with a second dose of kisspeptin-54 improve oocyte maturation in women at high risk of ovarian hyperstimulation syndrome (OHSS)? A second dose of kisspeptin-54 at 10 h following the first improves oocyte yield in women at high risk of OHSS. Kisspeptin acts at the hypothalamus to stimulate the release of an endogenous pool of GnRH from the hypothalamus. We have previously reported that a single dose of kisspeptin-54 results in an LH-surge of ~12-14 h duration, which safely triggers oocyte maturation in women at high risk of OHSS. Phase-2 randomized placebo-controlled trial of 62 women at high risk of OHSS recruited between August 2015 and May 2016. Following controlled ovarian stimulation, all patients (n = 62) received a subcutaneous injection of kisspeptin-54 (9.6 nmol/kg) 36 h prior to oocyte retrieval. Patients were randomized 1:1 to receive either a second dose of kisspeptin-54 (D; Double, n = 31), or saline (S; Single, n = 31) 10 h thereafter. Patients, embryologists, and IVF clinicians remained blinded to the dosing allocation. Study participants: Sixty-two women aged 18-34 years at high risk of OHSS (antral follicle count ≥23 or anti-Mullerian hormone level ≥40 pmol/L). Setting: Single centre study carried out at Hammersmith Hospital IVF unit, London, UK. Primary outcome: Proportion of patients achieving an oocyte yield (percentage of mature oocytes retrieved from follicles ≥14 mm on morning of first kisspeptin-54 trigger administration) of at least 60%. Secondary outcomes: Reproductive hormone levels, implantation rate and OHSS occurrence. A second dose of kisspeptin-54 at 10 h following the first induced further LH-secretion at 4 h after administration. A higher proportion of patients achieved an oocyte yield ≥60% following a second dose of kisspeptin-54 (Single: 14/31, 45%, Double: 21/31, 71%; absolute difference +26%, CI 2-50%, P = 0.042). Patients receiving two doses of kisspeptin-54 had a variable LH

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

Science.gov (United States)

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

2018-01-15

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

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

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

2012-05-01

Full Text Available This study was conducted to evaluate the effect of different cryopreservation methods (slow-freezing and vitrification on structural changes of bovine immature oocytes. Bovine ovaries were collected from local abattoirs. Cumulus-oocyte-complexes (COCs were retrieved using aspiration method from 2-6 mm follicles. In Experiment 1, selected oocytes were randomly divided into 4 treatment groups namely freezing solution-exposed, frozen-thawed, vitrification solution-exposed and vitrified-thawed and then oocytes abnormalities were examined under a stereomicroscope. In Experiment 2, oocytes were randomly allocated to the same grouping as experiment 1 plus control group. Following freezing or vitrification, all oocytes were fixed in glutaraldehyde and processed for transmission electron microscopy. In experiment 1, there was a higher incidence of abnormalities in the frozen-thawed and vitrified-warmed oocytes compared to those in freezing solution and vitrification solution-exposed groups (P<0.05. In experiment 2, there were marked alterations in the perivitelline space, microvilli and vesicles of frozen-thawed and vitrified-warmed oocytes characterized by loss of elasticity and integrity of cytoplasmic processes and microvilli following cooling and warming. In conclusion, ethylene glycol-based freezing and vitrification solutions are suitable choices for cryopreservation of immature oocytes and most organelles are able to retain their normal morphology following cryopreservation and thawing processes.

Bovine cumulus-oocyte disconnection in vitro

DEFF Research Database (Denmark)

Maddox-Hyttel, Poul

1987-01-01

Cumulus-oocyte complexes were obtained from cows by aspiration of small (1-6 mm in diameter) antral follicles after slaughter. Complexes with a compact multilayered cumulus investment were cultured and processed for transmission electron microscopy after different periods of culture including a 0...

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-12-01

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

Cumulus cells gene expression profiling in terms of oocyte maturity in controlled ovarian hyperstimulation using GnRH agonist or GnRH antagonist.

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Devjak, Rok; Fon Tacer, Klementina; Juvan, Peter; Virant Klun, Irma; Rozman, Damjana; Vrtačnik Bokal, Eda

2012-01-01

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.

Successful ongoing pregnancies after vitrification of oocytes.

Science.gov (United States)

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

2006-01-01

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

Transmembrane Signal Transduction in Oocyte Maturation and Fertilization: Focusing on Xenopus laevis as a Model Animal

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Ken-ichi Sato

2014-12-01

Full Text Available Fertilization is a cell biological phenomenon of crucial importance for the birth of new life in a variety of multicellular and sexual reproduction species such as algae, animal and plants. Fertilization involves a sequence of events, in which the female gamete “egg” and the male gamete “spermatozoon (sperm” develop, acquire their functions, meet and fuse with each other, to initiate embryonic and zygotic development. Here, it will be briefly reviewed how oocyte cytoplasmic components are orchestrated to undergo hormone-induced oocyte maturation and sperm-induced activation of development. I then review how sperm-egg membrane interaction/fusion and activation of development in the fertilized egg are accomplished and regulated through egg coat- or egg plasma membrane-associated components, highlighting recent findings and future directions in the studies using Xenopus laevis as a model experimental animal.

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

International Nuclear Information System (INIS)

Pedersen, R.A.; Brandriff, B.

1979-01-01

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

Release of sICAM-1 in oocytes and in vitro fertilized human embryos.

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Monica Borgatti

Full Text Available During the last years, several studies have reported the significant relationship between the production of soluble HLA-G molecules (sHLA-G by 48-72 hours early embryos and an increased implantation rate in IVF protocols. As consequence, the detection of HLA-G modulation was suggested as a marker to identify the best embryos to be transferred. On the opposite, no suitable markers are available for the oocyte selection.The major finding of the present paper is that the release of ICAM-1 might be predictive of oocyte maturation. The results obtained are confirmed using three independent methodologies, such as ELISA, Bio-Plex assay and Western blotting. The sICAM-1 release is very high in immature oocytes, decrease in mature oocytes and become even lower in in vitro fertilized embryos. No significant differences were observed in the levels of sICAM-1 release between immature oocytes with different morphological characteristics. On the contrary, when the mature oocytes were subdivided accordingly to morphological criteria, the mean sICAM-I levels in grade 1 oocytes were significantly decreased when compared to grade 2 and 3 oocytes.The reduction of the number of fertilized oocytes and transferred embryos represents the main target of assisted reproductive medicine. We propose sICAM-1 as a biochemical marker for oocyte maturation and grading, with a possible interesting rebound in assisted reproduction techniques.

Cryopreservation of oocytes

International Nuclear Information System (INIS)

Jadoon, S.

2015-01-01

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

Apoptosis maintains oocyte quality in aging Caenorhabditis elegans females.

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Sara Andux

2008-12-01

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

In vitro growth and maturation of isolated caprine preantral follicles: Influence of insulin and FSH concentration, culture dish, coculture, and oocyte size on meiotic resumption.

Science.gov (United States)

Silva, G M; Brito, I R; Sales, A D; Aguiar, F L N; Duarte, A B G; Araújo, V R; Vieira, L A; Magalhães-Padilha, D M; Lima, L F; Alves, B G; Silveira, L B R; Lo Turco, E G; Rodrigues, A P; Campello, C C; Wheeler, M B; Figueiredo, J R

2017-03-01

The aims of this study were: (1) to evaluate the effect of different insulin concentrations, alone or in combination with either a fixed FSH concentration or increasing FSH concentrations on the in vitro culture of isolated caprine preantral follicles and (2) to analyze the efficiency of two IVM media and maturation culture systems (with or without coculture with in vivo grown oocytes) on the meiosis resumption. Secondary follicles were cultured for 18 days in a basic medium supplemented with low- or high-insulin concentration alone or with a fixed FSH concentration or with increasing FSH concentrations. Oocytes grown in vivo or in vitro were matured alone or cocultured. The high-insulin concentration associated with fixed FSH treatment had higher meiotic resumption rate (P media. In conclusion, a basic medium supplemented with 10-μg/mL insulin and 100-μg/mL FSH throughout the culture period improved meiotic resumption rate and produced MII oocytes from caprine preantral follicles cultured in vitro. The MII rate was similar between in vivo and in vitro grown oocytes ≥110 μm. Copyright © 2016 Elsevier Inc. All rights reserved.

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

International Nuclear Information System (INIS)

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

1976-01-01

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

Rapamycin Rescues the Poor Developmental Capacity of Aged Porcine Oocytes

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Seung Eun Lee

2014-05-01

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

Comparison of The Effects of Vitrification on Gene Expression of Mature Mouse Oocytes Using Cryotop and Open Pulled Straw

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Fardin Amidi

2018-01-01

Full Text Available Background Oocyte cryopreservation is an essential part of the assisted reproductive technology (ART, which was recently introduced into clinical practice. This study aimed to evaluate the effects of two vitrification systems-Cryotop and Open Pulled Straw (OPS-on mature oocytes gene expressions. Materials and Methods In this experimental study, the survival rate of metaphase II (MII mouse oocytes were assessed after cryopreservation by vitrification via i. OPS or ii. Cryotop. Then we compared the fertilization rate of oocytes produced via these two methods. In the second experiment, we determined the effects of the two vitrification methods on the expression of Hspa1a, mn-Sod, and ß-actin genes in vitrified-warmed oocytes. Denuded MII oocytes were vitrified in two concentrations of vitrification solution (VS1 and VS2 by Cryotop and straw. We then compared the results using the two vitrification methods with fresh control oocytes. Results mn-Sod expression increased in the vitrified-warmed group both in OPS and Cryotop compared with the con- trols. We only detected Hspa1a in VS1 and control groups using Cryotop. The survival rate of the oocytes was 91.2% (VS1 and 89.2% (VS2 in the Cryotop groups (P=0.902 and 85.5% (VS1 and 83.6% (VS2 in the OPS groups (P=0.905. There were no significant differences between the Cryotop and the OPS groups (P=0.927. The survival rate in the Cryotop or the OPS groups was, nevertheless, significantly lower than the control group (P<0.001. The fertilization rates of the oocytes were 39% (VS1 and 34% (VS2 in the Cryotop groups (P=0.902 and 29 %( VS1 and 19.7% (VS2 in the OPS groups (P=0.413. The fertilization rates were achieved without significant differences among the Cryotop and OPS groups (P=0.755. Conclusion Our results indicated that Cryotop vitrification increases both cooling and warming rates, but both Cryo- top and OPS techniques have the same effect on the mouse oocytes after vitrification.

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

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Valeriu Carabă

2011-05-01

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

Metformin therapy in a hyperandrogenic anovulatory mutant murine model with polycystic ovarian syndrome characteristics improves oocyte maturity during superovulation

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Sabatini Mary E

2011-05-01

Full Text Available Abstract Background Metformin, an oral biguanide traditionally used for the treatment of type 2 diabetes, is widely used for the management of polycystic ovary syndrome (PCOS-related anovulation. Because of the significant prevalence of insulin resistance and glucose intolerance in PCOS patients, and their putative role in ovulatory dysfunction, the use of metformin was touted as a means to improve ovulatory function and reproductive outcomes in PCOS patients. To date, there has been inconsistent evidence to demonstrate a favorable effect of metformin on oocyte quality and competence in women with PCOS. Given the heterogeneous nature of this disorder, we hypothesized that metformin may be beneficial in mice with aberrant metabolic characteristics similar to a significant number of PCOS patients. The aim of this study was to gain insight into the in vitro and in vivo effects of metformin on oocyte development and ovulatory function. Methods We utilized metformin treatment in the transgenic ob/ob and db/db mutant murine models which demonstrate metabolic and reproductive characteristics similar to women with PCOS. Results: Metformin did not improve in vitro oocyte maturation nor did it have an appreciable effect on in vitro granulosa cell luteinization (progesterone production in any genotype studied. Although both mutant strains have evidence of hyperandrogenemia, anovulation, and hyperinsulinemia, only db/db mice treated with metformin had a greater number of mature oocytes and total overall oocytes compared to control. There was no observed impact on body mass, or serum glucose and androgens in any genotype. Conclusions Our data provide evidence to suggest that metformin may optimize ovulatory performance in mice with a specific reproductive and metabolic phenotype shared by women with PCOS. The only obvious difference between the mutant murine models is that the db/db mice have elevated leptin levels raising the questions of whether their

The dynamics of connexin expression, degradation and localisation are regulated by gonadotropins during the early stages of in vitro maturation of swine oocytes.

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Nicolas Santiquet

Full Text Available Gap junctional communication (GJC plays a primordial role in oocyte maturation and meiotic resumption in mammals by directing the transfer of numerous molecules between cumulus cells and the oocyte. Gap junctions are made of connexins (Cx, proteins that regulate GJC in numerous ways. Understanding the dynamic regulation of connexin arrangements during in vitro maturation (IVM could provide a powerful tool for controlling meiotic resumption and consequently in vitro development of fully competent oocytes. However, physiological events happening during the early hours of IVM may still be elucidated. The present study reports the dynamic regulation of connexin expression, degradation and localization during this stage. Cx43, Cx45 and Cx60 were identified as the main connexins expressed in swine COC. Cx43 and Cx45 transcripts were judged too static to be a regulator of GJC, while Cx43 protein expression was highly responsive to gonadotropins, suggesting that it might be the principal regulator of GJC. In addition, the degradation of Cx43 expressed after 4.5 h of IVM in response to equine chorionic gonadotropin appeared to involve the proteasomal complex. Cx43 localisation appeared to be associated with GJC. Taken together, these results show for the first time that gonadotropins regulate Cx43 protein expression, degradation and localisation in porcine COC during the first several hours of IVM. Regulation of Cx43 may in turn, via GJC, participate in the development of fully competent oocytes.

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

DEFF Research Database (Denmark)

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

2013-01-01

Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human...... oocyte in the quiescent state and at the pinnacle of maturity at ovulation. In silico bioinformatic comparisons were made between the transcriptome of human oocytes from dormant primordial follicles and that of human metaphase II (MII) oocytes and granulosa cells and unique gene expression profiles were...... identified as well as functional and pathway enrichments associated with the oocytes from the two developmental hallmarks. A total of 729 genes were highly enriched in oocytes from primodial follicles and 1456 genes were highly enriched in MII oocytes (>10-fold, P...

Effects of selected endocrine disruptors on meiotic maturation, cumulus expansion, syntesis of hyaluronan and progesterone by porcine oocyte-cumulus complexes

Czech Academy of Sciences Publication Activity Database

Mlynarčíková, A.; Nagyová, Eva; Ficková, M.; Scsuková, S.

2009-01-01

Roč. 23, - (2009), s. 371-377 ISSN 0887-2333 R&D Projects: GA ČR GA305/05/0960 Grant - others:VEGA(SK) 2/6171/26; EU(XE) QLK4-CT-2002-02637 Institutional research plan: CEZ:AV0Z50450515 Keywords : oocyte-cumulus complex * meiotic maturation * cumulus expansion Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.060, year: 2009

Proteomic Analysis of Porcine Oocytes During in vitro Maturation Reveals Essential Role for the Ubiquitin C- terminal hydrolase-L1

Czech Academy of Sciences Publication Activity Database

Šušor, Andrej; Ellederová, Zdeňka; Jelínková, Lucie; Halada, Petr; Kavan, Daniel; Kubelka, Michal; Kovářová, Hana

2007-01-01

Roč. 134, č. 4 (2007), s. 559-568 ISSN 1470-1626 R&D Projects: GA ČR GA204/04/0571; GA AV ČR 1QS500450568 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50200510 Keywords : porcine oocyte * in vitro maturation * proteome Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.962, year: 2007

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

Science.gov (United States)

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

2016-09-01

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

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

African Journals Online (AJOL)

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

Analysis of mRNA Associated factors During Bovine Oocyte Maturation and Early Embryonic Development

Czech Academy of Sciences Publication Activity Database

Siemer, C.; Smiljakovic, T.; Bhojawni, M.; Leiding, C.; Kanitz, W.; Kubelka, Michal; Tomek, W.

2009-01-01

Roč. 76, č. 12 (2009), s. 1208-1219 ISSN 1040-452X R&D Projects: GA ČR GA524/07/1087 Institutional research plan: CEZ:AV0Z50450515 Keywords : In Vitro maturation * Poly(A) binding protein * Initiation-factor 4E Subject RIV: ED - Physiology Impact factor: 2.041, year: 2009

Lipid characterization of individual porcine oocytes by dual mode DESI-MS and data fusion

International Nuclear Information System (INIS)

Pirro, Valentina; Oliveri, Paolo; Ferreira, Christina Ramires; González-Serrano, Andrés Felipe; Machaty, Zoltan; Cooks, Robert Graham

2014-01-01

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

Glycosylated chicken ZP2 accumulates in the egg coat of immature oocytes and remains localized to the germinal disc region of mature eggs.

Science.gov (United States)

Nishio, Shunsuke; Kohno, Yoshinori; Iwata, Yuki; Arai, Mayumi; Okumura, Hiroki; Oshima, Kenzi; Nadano, Daita; Matsuda, Tsukasa

2014-11-01

Vertebrate eggs are surrounded by an egg coat, which is a specific extracellular egg matrix consisting of several glycoproteins with a conserved zona pellucida (ZP) domain. Two mammalian egg coat subunits, ZP2 and ZP3, have been suggested to act as sperm receptors. In bird eggs, however, ZP2 has never been identified in the egg coat of mature oocytes and ovulated eggs. Here we report that chicken ZP2 is expressed in immature small follicles and remains as an egg-coat component locally in the germinal disc region of mature eggs. RT-PCR analysis indicated marked expression of the ZP2 and ZP4 genes in the granulosa cells of immature white follicles, whereas the ZP3 and ZPD genes showed marked expression in the cells of maturing yellow follicles. ZP2 was identified in the egg coat isolated from immature follicles as a heavily N-glycosylated glycoprotein of ∼200 kDa, which was enzymatically converted to a 70-kDa deglycosylated form. Immunoblotting and immunohistological analyses showed that ZP2 was localized around the germinal disc region of mature follicles. ZP2 was accumulated in the egg coat of immature white follicles at the earlier stages of oocyte development and became a minor component in the egg coat of maturing yellow follicles, except for the germinal disc region. Localization of ZP2 in the germinal disc region of mature eggs, where sperm bind to the egg coat at high density, suggests some role for ZP2 in the preferential binding and penetration of sperm in the germinal disc region of bird eggs. © 2014 by the Society for the Study of Reproduction, Inc.

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

KAUST Repository

Maddirevula, Sateesh

2017-09-29

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

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

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A.C. Bertagnolli

2004-08-01

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

Two Dimensional Finite Element Model to Study Calcium Distribution in Oocytes

Science.gov (United States)

Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

2015-06-01

Cytosolic free calcium concentration is a key regulatory factor and perhaps the most widely used means of controlling cellular function. Calcium can enter cells through different pathways which are activated by specific stimuli including membrane depolarization, chemical signals and calcium depletion of intracellular stores. One of the important components of oocyte maturation is differentiation of the Ca2+ signaling machinery which is essential for egg activation after fertilization. Eggs acquire the ability to produce the fertilization-specific calcium signal during oocyte maturation. The calcium concentration patterns required during different stages of oocyte maturation are still not completely known. Also the mechanisms involved in calcium dynamics in oocyte cell are still not well understood. In view of above a two dimensional FEM model has been proposed to study calcium distribution in an oocyte cell. The parameters such as buffers, ryanodine receptor, SERCA pump and voltage gated calcium channel are incorporated in the model. Based on the biophysical conditions the initial and boundary conditions have been framed. The model is transformed into variational form and Ritz finite element method has been employed to obtain the solution. A program has been developed in MATLAB 7.10 for the entire problem and executed to obtain numerical results. The numerical results have been used to study the effect of buffers, RyR, SERCA pump and VGCC on calcium distribution in an oocyte cell.

Effect of melatonin on maturation capacity and fertilization of Nili ...

African Journals Online (AJOL)

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 ...

Multiple requirements of PLK1 during mouse oocyte maturation.

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Petr Solc

Full Text Available Polo-like kinase 1 (PLK1 orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1's functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC. Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.

Ultrastructure and mitochondrial numbers in pre- and postpubertal pig oocytes

DEFF Research Database (Denmark)

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

2016-01-01

Prepubertal pig oocytes are associated with lower developmental competence. The aim of this experiment was to conduct an exhaustive survey of oocyte ultrastructure and to use a design-unbiased stereological approach to quantify the numerical density and total number of mitochondria in oocytes...... with different diameters from pre- and postpubertal pigs. The ultrastructure of smaller prepubertal immature oocytes indicated active cells in close contact with cumulus cells. The postpubertal oocytes were more quiescent cell types. The small prepubertal oocytes had a lower total mitochondrial number......, but no differences were observed in mitochondrial densities between groups. Mature postpubertal oocytes adhered to the following characteristics: presence of metaphase II, lack of contact between cumulus cells and oocyte, absence of rough endoplasmic reticulum and Golgi complexes, peripheral location of cortical...

Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.

Science.gov (United States)

Yang, Mei; Cao, Xueyan; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing; Wu, Junrui

2017-09-01

Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.

Mammalian oocyte growth and development in vitro.

Science.gov (United States)

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

1996-06-01

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

Methyl parathion inhibits the nuclear maturation, decreases the cytoplasmic quality in oocytes and alters the developmental potential of embryos of Swiss albino mice

Energy Technology Data Exchange (ETDEWEB)

Nair, Ramya; Singh, Vikram Jeet; Salian, Sujith Raj [Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal 576 104 (India); Kalthur, Sneha Guruprasad; D' Souza, Antony Sylvan [Department of Anatomy, Kasturba Medical College, Manipal University, Manipal 576 104 (India); Shetty, Pallavi K.; Mutalik, Srinivas [Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576 104 (India); Kalthur, Guruprasad, E-mail: guru.kalthur@manipal.edu [Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal 576 104 (India); Adiga, Satish Kumar [Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal 576 104 (India)

2014-09-15

Methyl parathion (MP) is one of the most commonly used and extremely toxic organophosphorous group of pesticide. A large number of studies in the literature suggest that it has adverse effects on the male reproductive system. However, there is limited information about its toxicity to the female reproductive system. In the present study we report the toxic effects of methyl parathion on the female reproductive system using Swiss albino mice as the experimental model. The female mice were administered orally with 5, 10 and 20 mg/kg of MP. One week later, the mice were superovulated with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to study the quality of the oocytes, spindle organization, developmental potential of early embryos and the DNA integrity in blastocysts. MP exposure resulted in a non-significant decrease in the number of primordial follicles and increased DNA damage in granulosa cells. Though MP did not have any effect on the ovulation it had a significant inhibitory effect on the nuclear maturity of oocytes which was associated with spindle deformity. In addition, the oocytes had higher cytoplasmic abnormalities with depleted glutathione level. Even though it did not have any effect on the fertilization and blastocyst rate at lower doses, at 20 mg/kg MP it resulted in a significant decrease in blastocyst hatching, decrease in cell number and high DNA damage. While low body weight gain was observed in F1 generation from 5 mg/kg group, at higher dose, the body weight in F1 generation was marginally higher than control. Post-natal death in F1 generation was observed only in mice treated with 20 mg/kg MP. In conclusion, we report that MP has adverse effects on the oocyte quality, developmental potential of the embryo and reproductive outcome. - Highlights: • Methyl parathion induces severe cytoplasmic abnormalities in oocytes. • Inhibits nuclear maturation and spindle damage • Poor blastocyst quality and high DNA

Methyl parathion inhibits the nuclear maturation, decreases the cytoplasmic quality in oocytes and alters the developmental potential of embryos of Swiss albino mice

International Nuclear Information System (INIS)

Nair, Ramya; Singh, Vikram Jeet; Salian, Sujith Raj; Kalthur, Sneha Guruprasad; D'Souza, Antony Sylvan; Shetty, Pallavi K.; Mutalik, Srinivas; Kalthur, Guruprasad; Adiga, Satish Kumar

2014-01-01

Methyl parathion (MP) is one of the most commonly used and extremely toxic organophosphorous group of pesticide. A large number of studies in the literature suggest that it has adverse effects on the male reproductive system. However, there is limited information about its toxicity to the female reproductive system. In the present study we report the toxic effects of methyl parathion on the female reproductive system using Swiss albino mice as the experimental model. The female mice were administered orally with 5, 10 and 20 mg/kg of MP. One week later, the mice were superovulated with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to study the quality of the oocytes, spindle organization, developmental potential of early embryos and the DNA integrity in blastocysts. MP exposure resulted in a non-significant decrease in the number of primordial follicles and increased DNA damage in granulosa cells. Though MP did not have any effect on the ovulation it had a significant inhibitory effect on the nuclear maturity of oocytes which was associated with spindle deformity. In addition, the oocytes had higher cytoplasmic abnormalities with depleted glutathione level. Even though it did not have any effect on the fertilization and blastocyst rate at lower doses, at 20 mg/kg MP it resulted in a significant decrease in blastocyst hatching, decrease in cell number and high DNA damage. While low body weight gain was observed in F1 generation from 5 mg/kg group, at higher dose, the body weight in F1 generation was marginally higher than control. Post-natal death in F1 generation was observed only in mice treated with 20 mg/kg MP. In conclusion, we report that MP has adverse effects on the oocyte quality, developmental potential of the embryo and reproductive outcome. - Highlights: • Methyl parathion induces severe cytoplasmic abnormalities in oocytes. • Inhibits nuclear maturation and spindle damage • Poor blastocyst quality and high DNA

Superoxide dismutase and taurine supplementation improves in vitro blastocyst yield from poor-quality feline oocytes.

Science.gov (United States)

Ochota, Małgorzata; Pasieka, Anna; Niżański, Wojciech

2016-03-15

Blastocyst production in vitro seems to be crucial part of assisted reproduction techniques in feline species. However, the results of cats' oocyte maturation and embryo development are still lower than those in other species. The aim of this study was to evaluate whether the supplementation with superoxide dismutase (SOD) and taurine during maturation or culture would improve the blastocyst yield obtained from lower grades of oocytes, that are usually discarded, as not suitable for further in vitro purposes. To investigate the effect of antioxidants' addition, the good- and poor-quality oocytes, were cultured with the addition of 10-mmol taurine and 600 UI/mL SOD. The nuclear maturity, embryo development, and blastocyst quality were subsequently assessed. In control group, without antioxidant supplementation, significantly less poor-quality oocytes matured (42% vs. 62%) and more degenerated (35% vs. 20%), comparing to the experimental group supplemented with SOD and taurine. The amount of obtained blastocyst was much higher, when poor quality oocytes were supplemented with SOD and taurine (supplementation to IVM-4%; supplementation to IVC-5.5%; supplementation to IVM and IVC-5.9% of blastocyst), comparing to not supplemented control group (1.3%). The best blastocysts were obtained when poor oocytes had antioxidants added only during embryo culture (185 ± 13.4 blastomeres vs. 100 ± 1.5 in control). In the present study, we reported that the lower grades of oocytes can better mature and form significantly more blastocysts with better quality, when cultured with addition of SOD and taurine. Copyright © 2016 Elsevier Inc. All rights reserved.

Birth after human chorionic gonadotropin-primed oocyte in vitro maturation and fertilization with testicular sperm in a normo-ovulatory patient

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Claudia González-Ortega

2016-01-01

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.

The relationships between the arrangement of teeth, root resorption, and dental maturity in bovine mandibular incisors

Science.gov (United States)

An, Jin-kyu; Ono, Takashi

2017-01-01

Objective The objective of this study is to investigate the eruption pattern and root resorption of the bovine anterior dentition in relation to growth-related parameters based on dental maturity. Methods A cross-sectional study was conducted on 110 bovine anterior mandibles by using standard radiography, cone-beam computed tomography (CBCT), and actual measurements. We determined the relationships between the stages of dental maturity by using a modification of Demirjian's method and various growth-related parameters, such as the activity of the root-resorbing tissue and mobility of the deciduous teeth. The correlation of growth-related parameters with interdental spacing and distal unusual root resorption (DRR) of the deciduous fourth incisor was assessed. The cause of mesial unusual root resorption (MRR) of the deciduous fourth incisor was determined on the basis of the arrangement of the permanent third incisor. Results An independent t-test and chi-square test indicated significant differences in growth-related parameters associated with dental arch length discrepancy and factors related to the shedding of deciduous teeth between the low and high dental maturity groups. The samples with interdental spacing and DRR showed a larger sum of mesiodistal permanent crown widths and higher dental maturity than did the respective controls. Samples with MRR tended to show a lingually rotated distal tip of the adjacent tooth crown. Conclusions Dental maturity has relevance to the interdental spaces and unusual root resorption of mixed dentition. The position of the adjacent tooth crown on CBCT may be correlated with the occurrence of unusual root resorption of the incisor. PMID:29090124

Expression stability of two housekeeping genes (18S rRNA and G3PDH) during in vitro maturation of follicular oocytes in buffalo (Bubalus bubalis).

Science.gov (United States)

Aswal, Ajay Pal Singh; Raghav, Sarvesh; De, Sachinandan; Thakur, Manish; Goswami, Surender Lal; Datta, Tirtha Kumar

2008-01-15

The present study was undertaken to evaluate the expression stability of two housekeeping genes (HKGs), 18S rRNA and G3PDH during in vitro maturation (IVM) of oocytes in buffalo, which qualifies their use as internal controls for valid qRT-PCR estimation of other oocyte transcripts. A semi quantitative RT-PCR system was used with optimised qRT-PCR parameters at exponential PCR cycle for evaluation of temporal expression pattern of these genes over 24 h of IVM. 18S rRNA was found more stable in its expression pattern than G3PDH.

Different gonadotropin releasing hormone agonist doses for the final oocyte maturation in high-responder patients undergoing in vitro fertilization/intra-cytoplasmic sperm injection

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Emre Goksan Pabuccu

2015-01-01

Full Text Available Context: Efficacy of gonadotropin releasing hormone agonists (GnRH-a for ovulation in high-responders. Aims: The aim of the current study is to compare the impact of different GnRH-a doses for the final oocyte maturation on cycle outcomes and ovarian hyperstimulation syndrome (OHSS rates in high-responder patients undergoing ovarian stimulation. Settings And Designs: Electronic medical records of a private in vitro fertilization center, a retrospective analysis. Subjects and Methods: A total of 77 high-responder cases were detected receiving GnRH-a. Group I consisted of 38 patients who received 1 mg of agonist and Group II consisted of 39 patients who received 2 mg of agonist. Statistical Analysis: In order to compare groups, Student′s t-test, Mann-Whitney U-test, Pearson′s Chi-square test or Fisher′s exact test were used where appropriate. A P < 0.05 was considered as statistically significant. Result: Number of retrieved oocytes (17.5 vs. 15.0, P = 0.510, implantation rates (46% vs. 55.1%, P = 0.419 and clinical pregnancy rates (42.1% vs. 38.5%, P = 0.744 were similar among groups. There were no mild or severe OHSS cases detected in Group I. Only 1 mild OHSS case was detected in Group II. Conclusion: A volume of 1 or 2 mg leuprolide acetate yields similar outcomes when used for the final oocyte maturation in high-responder patients.

Ultrastructure of the human preovulatory oocyte.

Science.gov (United States)

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

1986-08-01

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

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

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NURBARIAH

2011-12-01

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

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

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Liyuan Fu

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

Endocrine profiles after triggering of final oocyte maturation with GnRH agonist after cotreatment with the GnRH antagonist ganirelix during ovarian hyperstimulation for in vitro fertilization

NARCIS (Netherlands)

B.C.J.M. Fauser (Bart); D. de Jong (Danielle); F. Olivennes; H. Wramsby; C. Tay; J. Itskovitz-Eldor; H.G. van Hooren

2002-01-01

textabstractIn a randomized multicenter study, the efficacies of two different GnRH agonists were compared with that of hCG for triggering final stages of oocyte maturation after ovarian hyperstimulation for in vitro fertilization. Ovarian stimulation was conducted by recombinant

Highly efficient vitrification method for cryopreservation of human oocytes.

Science.gov (United States)

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

2005-09-01

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

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

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Cai-Rong Yang

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

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

Science.gov (United States)

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

2010-12-07

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

Ovarian follicle maturation and ovulation: An integrated perspective

Science.gov (United States)

Patino, R.; Thomas, P.; Yoshizaki, G.

2003-01-01

Numerous studies with teleosts have addressed the regulation and mechanisms of oocyte maturation, but largely at the exclusion of ovulation. A smaller but still considerable number of studies have focused on ovulation, and ignored maturation. Consequently, little is known about the mechanistic linkages between these two events. New information is presented here indicating that luteinizing hormone regulates the acquisition not only of oocyte maturational competence, but also ovulatory competence. The thesis is presented that maturation and ovulation are closely integrated and overlapping events that are best viewed conceptually and experimentally as parts of a functional whole. ?? 2004 Kluwer Academic Publishers.

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

Science.gov (United States)

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

2017-07-01

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

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

African Journals Online (AJOL)

Jane

2011-08-01

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

bromopropane on maturation of mouse oocytes, fertilization and ...

African Journals Online (AJOL)

DR. NJ TONUKARI

2012-05-31

May 31, 2012 ... to prevent the hazardous effects of 2-BP on embryos derived from pretreated oocytes. Key words: 2-Bromopropane, ... E-mail: whchan@cycu.edu.tw. Tel: ..... Huang F, Ning H, Xin QQ, Huang Y, Wang H, Zhang ZH, Xu DX,. Ichihara G .... Surh YJ, Hurh YJ, Kang JY, Lee E, Kong G, Lee SJ (1999). Resveratrol,.

Effect of hCG priming on embryonic development of immature oocytes collected from unstimulated women with polycystic ovarian syndrome

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Zheng Xiaoying

2012-05-01

Full Text Available Abstract Backgroud The effect of hCG priming on oocyte maturation and subsequently outcome in IVM cycles has remained a debated issue. A randomized controlled study was performed to investigate whether or not hCG priming prior to oocyte aspiration can improve the developmental competence of immature oocytes from unstimulated ovaries in women with polycystic ovarian syndrome (PCOS. Methods Eighty two patients with PCOS underwent IVM cycles. Each patient was randomly assigned to the hCG-primed (10,000 IU or non-primed groups 36–38 hours before oocyte retrieval depending on the computerized random table. After the oocytes had in vitro matured, fertilization, culture and embryo transfer were performed. Results The average number of cumulus-oocyte complexes (COCs recovered was 13.80 and 14.35 in the hCG-primed and non-primed groups, respectively (p > 0.05. The maturation rate of COCs was significantly improved in the hCG-primed group (55.43% vs. 42.29%; p  Conclusions While a significant improvement in the nuclear maturation rate of immature oocytes was observed in hCG-primed IVM cycles with PCOS patients, the use of hCG prior to oocyte retrieval did not improve the subsequent embryo developmental competence. The high rate of pregnancy loss in IVM cycles should receive more attention.

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

Science.gov (United States)

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

2009-03-01

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

Apoptosis in mammalian oocytes: a review.

Science.gov (United States)

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

2015-08-01

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

Age-dependent radiosensitivity of mouse oocytes

International Nuclear Information System (INIS)

Koehler, C.

1976-01-01

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

Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes.

Science.gov (United States)

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

2017-12-01

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

Triggering of final oocyte maturation with gonadotropin-releasing hormone agonist or human chorionic gonadotropin. Live birth after frozen-thawed embryo replacement cycles

DEFF Research Database (Denmark)

Griesinger, Georg; Kolibianakis, E M; Papanikolaou, E G

2007-01-01

OBJECTIVE: To report the outcome of frozen-thawed embryo replacement cycles after GnRH-agonist triggering of final oocyte maturation in the collecting cycle with GnRH-antagonist. DESIGN: Prospective, observational, multicentric clinical study. SETTING: Tertiary university-affiliated IVF centers...... a total of 228 participants. Surplus embryos or oocytes at the pronuclear stage were cryopreserved in 53 patients after hCG administration and 32 patients after GnRH-agonist administration on the basis of patient choice, pronuclear/embryo availability, and local laws. INTERVENTION(S): Transfer of frozen......-thawed embryos. MAIN OUTCOME MEASURE(S): Live birth rate. RESULT(S): Thirty-one and 23 patients after administration of hCG and GnRH-agonist, respectively, started a frozen-embryo replacement cycle by September 2005, with 25 and 16 patients eventually undergoing at least one frozen-thawed ET. Live birth rate per...

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

Directory of Open Access Journals (Sweden)

Rinaudo Paolo F

2009-04-01

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

Gas6 downregulation impaired cytoplasmic maturation and pronuclear formation independent to the MPF activity.

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Kyeoung-Hwa Kim

Full Text Available Previously, we found that the growth arrest-specific gene 6 (Gas6 is more highly expressed in germinal vesicle (GV oocytes than in metaphase II (MII oocytes using annealing control primer (ACP-PCR technology. The current study was undertaken to investigate the role of Gas6 in oocyte maturation and fertilization using RNA interference (RNAi. Interestingly, despite the specific and marked decrease in Gas6 mRNA and protein expression in GVs after Gas6 RNAi, nuclear maturation including spindle structures and chromosome segregation was not affected. The only discernible effect induced by Gas6 RNAi was a change in maturation promoting factor (MPF activity. After parthenogenetic activation, Gas6 RNAi-treated oocytes at the MII stage had not developed further and arrested at MII (90.0%. After stimulation with Sr(2+, Gas6-silenced MII oocytes had markedly reduced Ca(2+ oscillation and exhibited no exocytosis of cortical granules. In these oocytes, sperm penetration occurred during fertilization but not pronucleus (PN formation. By roscovitine and colcemid treatment, we found that the Gas6 knockdown affected cytoplasmic maturation directly, independent to the changed MPF activity. These results strongly suggest that 1 the Gas6 signaling itself is important to the cytoplasmic maturation, but not nuclear maturation, and 2 the decreased Gas6 expression and decreased MPF activity separately or mutually influence sperm head decondensation and PN formation.

Enucleolation of porcine oocytes

Czech Academy of Sciences Publication Activity Database

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

2003-01-01

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

Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring.

Science.gov (United States)

Watkins, Adam J; Wilkins, Adrian; Cunningham, Colm; Perry, V Hugh; Seet, Meei J; Osmond, Clive; Eckert, Judith J; Torrens, Christopher; Cagampang, Felino R A; Cleal, Jane; Gray, William P; Hanson, Mark A; Fleming, Tom P

2008-04-15

Early embryonic development is known to be susceptible to maternal undernutrition, leading to a disease-related postnatal phenotype. To determine whether this sensitivity extended into oocyte development, we examined the effect of maternal normal protein diet (18% casein; NPD) or isocaloric low protein diet (9% casein; LPD) restricted to one ovulatory cycle (3.5 days) prior to natural mating in female MF-1 mice. After mating, all females received NPD for the remainder of gestation and all offspring were litter size adjusted and fed standard chow. No difference in gestation length, litter size, sex ratio or postnatal growth was observed between treatments. Maternal LPD did, however, induce abnormal anxiety-related behaviour in open field activities in male and female offspring (P size or nephron number was altered by diet treatment (P < 0.05). These data demonstrate the sensitivity of mouse maturing oocytes in vivo to maternal protein undernutrition and identify both behavioural and cardiovascular postnatal outcomes, indicative of adult disease. These outcomes probably derive from a direct effect of protein restriction, although indirect stress mechanisms may also be contributory. Similar and distinct postnatal outcomes were observed here compared with maternal LPD treatment during post-fertilization preimplantation development which may reflect the relative contribution of the paternal genome.

Wolbachia infect ovaries in the course of their maturation: last minute passengers and priority travellers?

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Lise-Marie Genty

Full Text Available Wolbachia are widespread endosymbiotic bacteria of arthropods and nematodes. Studies on such models suggest that Wolbachia's remarkable aptitude to infect offspring may rely on a re-infection of ovaries from somatic tissues instead of direct cellular segregation between oogonia and oocytes. In the terrestrial isopod Armadillidium vulgare, Wolbachia are vertically transmitted to the host offspring, even though ovary cells are cyclically renewed. Using Fluorescence in situ hybridization (FISH, we showed that the proportion of infected oocytes increased in the course of ovary and oocyte maturation, starting with 31.5% of infected oocytes only. At the end of ovary maturation, this proportion reached 87.6% for the most mature oocytes, which is close to the known transmission rate to offspring. This enrichment can be explained by a secondary acquisition of the bacteria by oocytes (Wolbachia can be seen as last minute passengers and/or by a preferential selection of oocytes infected with Wolbachia (as priority travellers.

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

Science.gov (United States)

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

1996-10-15

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

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos Melatonina no meio de maturação não melhorou as taxas de maturação dos ovócitos, de desenvolvimento embrionário e a fragmentação do DNA dos embriões bovinos

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Luciana Takada

2010-08-01

Full Text Available Melatonin (MEL acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM on bovine embryos. This study tested the addition of MEL to maturation medium (MM with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum - FCS at 39ºC and 5% CO2 in air. After 24 hours of culture in MM with 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH; 10-9 M MEL or 10-9 M MEL, 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10-9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM.Melatonin (MEL atua como um potente redutor de radicais livres. Efeito direto da MEL na função gonadal também foi observado. Existem poucos estudos relacionados ao efeito da MEL durante a maturação no desenvolvimento embrionário in vitro. Avaliou-se a adição de MEL no meio de maturação (sem gonadotrofinas nas taxas de maturação nuclear e de desenvolvimento embrionário e na incidência de fragmentação do DNA nos embriões produzidos in vitro

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

Science.gov (United States)

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

2008-08-01

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

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

OpenAIRE

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

2015-01-01

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

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

Science.gov (United States)

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

2011-11-01

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

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

Directory of Open Access Journals (Sweden)

F. Perecin

2007-04-01

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

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

Science.gov (United States)

Kitaev, E M; Pimenova, M N

1980-12-01

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

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

Czech Academy of Sciences Publication Activity Database

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

2016-01-01

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

Psychological stress on female mice diminishes the developmental potential of oocytes: a study using the predatory stress model.

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Yu-Xiang Liu

Full Text Available Although the predatory stress experimental protocol is considered more psychological than the restraint protocol, it has rarely been used to study the effect of psychological stress on reproduction. Few studies exist on the direct effect of psychological stress to a female on developmental competence of her oocytes, and the direct effect of predatory maternal stress on oocytes has not been reported. In this study, a predatory stress system was first established for mice with cats as predators. Beginning 24 h after injection of equine chorionic gonadotropin, female mice were subjected to predatory stress for 24 h. Evaluation of mouse responses showed that the predatory stress system that we established increased anxiety-like behaviors and plasma cortisol concentrations significantly and continuously while not affecting food and water intake of the mice. In vitro experiments showed that whereas oocyte maturation and Sr(2+ activation or fertilization were unaffected by maternal predatory stress, rate of blastocyst formation and number of cells per blastocyst decreased significantly in stressed mice compared to non-stressed controls. In vivo embryo development indicated that both the number of blastocysts recovered per donor mouse and the average number of young per recipient after embryo transfer of blastocysts with similar cell counts were significantly lower in stressed than in unstressed donor mice. It is concluded that the predatory stress system we established was both effective and durative to induce mouse stress responses. Furthermore, predatory stress applied during the oocyte pre-maturation stage significantly impaired oocyte developmental potential while exerting no measurable impact on nuclear maturation, suggesting that cytoplasmic maturation of mouse oocytes was more vulnerable to maternal stress than nuclear maturation.

Function of JARID2 in bovines during early embryonic development

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Yao Fu

2017-12-01

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

The Sea Cucumber Body Wall Extract Promoted in Vitro Maturation of NMRI Mice Follicles at Germinal Vesicle Stage

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Mitra Khalilzadeh

2016-07-01

Full Text Available Background IVM (in vitro maturation appear to be certified high pregnancy outcome. Therefore, attempt to find an appropriate culture system using natural products for increasing developmental competencies fascinating. Objectives This experiment is aimed to evaluate the effect of sea cucumber methanol extract on in vitro maturation of immature mouse oocyte. Methods In this experimental study, Preantral follicles at germinal vesicle stage were collected from 24 - 26 days NMRI female mouse ovaries and transferred to IVM medium supplemented with different concentration of sea cucumber methanol extract (5, 10, 20, 30 µg/mL. Antioxidant capacity of sea cucumber extract was evaluated using DPPH (2, 2-diphenyl-1-picrylhydrazyl assay. Further, oocyte maturation and TNF-α expression were recorded till day 10th. Data were analyzed by one-way analysis of variance (ANOVA through SPSS 16. Results The percentage of arrested oocyte at germinal vesicle stage in the control group was significantly (P < 0.05 higher than treated group (20 µg/mL. There were significant (P < 0.01 differences in percentage of maturated oocyte to metaphase II (MII stage between treated and control group. DPPH radical scavenging capacity and reduction of TNF-α expression in treated oocyte demonstrated that sea cucumber increased rate of oocyte maturation. In addition, the treated oocyte (20 µg/mL achieved the highest percentage of MII stage. Conclusions It was concluded that the sea cucumber extract with optimum concentration can improve oocyte maturation. Sea cucumber extract treatment can be suggested as a novel therapeutic strategy to be used in infertility modality in the future.

An improved vitrification protocol for equine immature oocytes, resulting in a first live foal

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Ortiz-Escribano, N.; Bogado Pascottini, O.; Woelders, H.; Vandenberghe, L.; Schauwer, De C.; Govaere, J.; Abbeel, Van den E.; Vullers, T.; Ververs, C.; Roels, K.; De Velde, Van M.; Soom, van A.; Smits, K.

2018-01-01

Background: The success rate for vitrification of immature equine oocytes is low. Although vitrified-warmed oocytes are able to mature, further embryonic development appears to be compromised. Objectives: The aim of this study was to compare two vitrification protocols, and to examine the effect of

Oocyte size, egg index, and body lipid content in relation to body size in the solitary bee Megachile rotundata

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Kevin M. O’Neill

2014-03-01

Full Text Available Females of solitary, nest-provisioning bees have relatively low fecundity, but produce large eggs as part of their overall strategy of investing substantially in each offspring. In intraspecific comparisons of several species of solitary, nest-provisioning bees and wasps, the size of the mature eggs produced increases with female body size. We further examined oocyte size–body size correlations in the solitary bee Megachile rotundata (F., an important crop pollinator. We hypothesized that larger females carry larger basal oocytes (i.e., those next in line to be oviposited but that body size–oocyte size correlations would be absent soon after emergence, before their first eggs fully matured. Because egg production is likely affected by the quantity of stored lipids carried over from the bees’ immature stages, we also tested the hypothesis that female body size is correlated with the body lipid content at adult emergence, the time during which oocyte growth accelerates. We found significant correlations of body size with oocyte size variables chosen to reflect: (1 the magnitude of the investment in the next egg to be laid (i.e., the length and volume of the basal oocyte and (2 the longer term potential to produce mature oocytes (i.e., the summed lengths and volumes of the three largest oocytes in each female. Positive correlations existed throughout the nesting season, even during the first week following adult emergence. The ability to produce and carry larger oocytes may be linked to larger females starting the nesting season with greater lipid stores (which we document here or to greater space within the abdomen of larger females. Compared to other species of solitary bees, M. rotundata appears to have (1 smaller oocytes than solitary nest-provisioning bees in general, (2 comparable oocyte sizes relative to congeners, and (3 larger oocytes than related brood parasitic megachilids.

Oocyte size, egg index, and body lipid content in relation to body size in the solitary bee Megachile rotundata.

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O'Neill, Kevin M; Delphia, Casey M; O'Neill, Ruth P

2014-01-01

Females of solitary, nest-provisioning bees have relatively low fecundity, but produce large eggs as part of their overall strategy of investing substantially in each offspring. In intraspecific comparisons of several species of solitary, nest-provisioning bees and wasps, the size of the mature eggs produced increases with female body size. We further examined oocyte size-body size correlations in the solitary bee Megachile rotundata (F.), an important crop pollinator. We hypothesized that larger females carry larger basal oocytes (i.e., those next in line to be oviposited) but that body size-oocyte size correlations would be absent soon after emergence, before their first eggs fully matured. Because egg production is likely affected by the quantity of stored lipids carried over from the bees' immature stages, we also tested the hypothesis that female body size is correlated with the body lipid content at adult emergence, the time during which oocyte growth accelerates. We found significant correlations of body size with oocyte size variables chosen to reflect: (1) the magnitude of the investment in the next egg to be laid (i.e., the length and volume of the basal oocyte) and (2) the longer term potential to produce mature oocytes (i.e., the summed lengths and volumes of the three largest oocytes in each female). Positive correlations existed throughout the nesting season, even during the first week following adult emergence. The ability to produce and carry larger oocytes may be linked to larger females starting the nesting season with greater lipid stores (which we document here) or to greater space within the abdomen of larger females. Compared to other species of solitary bees, M. rotundata appears to have (1) smaller oocytes than solitary nest-provisioning bees in general, (2) comparable oocyte sizes relative to congeners, and (3) larger oocytes than related brood parasitic megachilids.

The use of CR1aa for ovine in vitro embryo production

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Yulnawati

2006-06-01

Full Text Available The aim of this study was to investigate the capacity of CR1aa as a simple medium for maturation, fertilization and culture of ovine embryo in vitro. Oocytes were collected by slicing method in Phosphate Buffer Saline (PBS supplemented with 5% Fetal Bovine Serum (FBS and 100 IU/ml penicillin streptomycin. Oocytes were matured in Tissue Culture Medium (TCM-199 as control or CR1aa as treatment medium. Both maturation medium were supplemented with 10% Fetal Bovine Serum (FBS, 10 IU/ml Follicle Stimulating Hormone (FSH, 10 IU/ml Luteinizing Hormone (LH, 1 μg/ml Estradiol and 100 IU/ml penicillin-streptomycin. Oocytes were incubated in 5% CO2 incubator, 38˚C for 24 h. Matured oocytes were fertilized in BO or CR1aa medium, supplemented with 2.5 mM caffeine benzoate and 20 mg /ml heparin. After 18 h in vitro fertilization, oocytes were cultured in TCM-199 or CR1aa medium, both supplemented with 5% FBS, 5 mg/ml insulin and 100 IU/ml penicillin streptomycin. Results showed that the highest maturation rate was found in TCM-199 medium (73.27% and significantly different (P0.05 between cleavage rate of ovine embryos in TCM-199 and CR1aa medium (39.45% vs 50.94%. In conclusion, optimum result on ovine in vitro embryo production can be achieved from a combination of TCM-199 as maturation medium and CR1aa as fertilization and culture medium.

Activation of dormant ovarian follicles to generate mature eggs.

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Li, Jing; Kawamura, Kazuhiro; Cheng, Yuan; Liu, Shuang; Klein, Cynthia; Liu, Shu; Duan, En-Kui; Hsueh, Aaron J W

2010-06-01

Although multiple follicles are present in mammalian ovaries, most of them remain dormant for years or decades. During reproductive life, some follicles are activated for development. Genetically modified mouse models with oocyte-specific deletion of genes in the PTEN-PI3K-Akt-Foxo3 pathway exhibited premature activation of all dormant follicles. Using an inhibitor of the Phosphatase with TENsin homology deleted in chromosome 10 (PTEN) phosphatase and a PI3K activating peptide, we found that short-term treatment of neonatal mouse ovaries increased nuclear exclusion of Foxo3 in primordial oocytes. After transplantation under kidney capsules of ovariectomized hosts, treated follicles developed to the preovulatory stage with mature eggs displaying normal epigenetic changes of imprinted genes. After in vitro fertilization and embryo transfer, healthy progeny with proven fertility were delivered. Human ovarian cortical fragments from cancer patients were also treated with the PTEN inhibitor. After xeno-transplantation to immune-deficient mice for 6 months, primordial follicles developed to the preovulatory stage with oocytes capable of undergoing nuclear maturation. Major differences between male and female mammals are unlimited number of sperm and paucity of mature oocytes. Thus, short-term in vitro activation of dormant ovarian follicles after stimulation of the PI3K-Akt pathway allows the generation of a large supply of mature female germ cells for future treatment of infertile women with a diminishing ovarian reserve and for cancer patients with cryo-preserved ovaries. Generation of a large number of human oocytes also facilitates future derivation of embryonic stem cells for regenerative medicine.

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

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

2014-09-01

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

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

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Akihiko Ichikawa

2013-06-01

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

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

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

2016-01-01

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

On gonadic maturation and reproductive strategy in deep-sea benthic octopus Graneledone macrotyla

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Guerra, Ángel; Sieiro, María Pilar; Roura, Álvaro; Portela, Julio M.; del Río, José Luís

2013-09-01

The new information reported in this paper is based on five maturing and mature females of the large-tuberculate octopus Graneledone macrotyla. These specimens were caught in bottom trawl surveys ATLANTIS 2009 (February 24 to April 1, 2009) and ATLANTIS 2010 (March 9 to April 5, 2010) carried out off the Argentinean Economic Exclusive Zone. Capture depth ranged from 475 to 921 m and sea bottom temperature between 2.8 and 3.1 °C. Development of the complex ovary, oviducts, and oviducal glands during gonadic maturation is described. The absence of spermathecae in the oviducal glands and the presence of fertilized eggs inside the ovary suggested that fertilization took place within the ovary. Histological techniques showed the presence of four types of oocytes. Maturing oocyte size-frequency distribution was polymodal. Fluorescence reaction showed that atresia occurred in both early and later oocyte maturation stages. Atresia affected 48-55 % of the initial number of oocytes. The maximum observed potential fecundity was estimated at 250-300 eggs. G. macrotyla showed a group-synchronous ovulation pattern, regulative atresia, and a batching spawning pattern with a few egg batches spawned intermittently over an extended period of spawning.

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

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

2015-01-01

Transvaginal ultrasound-guided follicle aspiration is one method of obtaining recipient oocytes for equine somatic cell nuclear transfer (SCNT). This study was conducted: (1) to evaluate the possibility of oocyte aspiration from pre-ovulatory follicles using a short disposable needle system (14-G) by comparing the oocyte recovery rate with that of a long double lumen needle (12-G); (2) to investigate the developmental competence of recovered oocytes after SCNT and embryo transfer. The recovery rates with the short disposable needle vs. the long needle were not significantly different (47.5% and 35.0%, respectively). Twenty-six SCNT embryos were transferred to 13 mares, and one mare delivered a live offspring at Day 342. There was a perfect identity match between the cloned foal and the cell donor after analysis of microsatellite DNA, and the mitochondrial DNA of the cloned foal was identical with that of the oocyte donor. These results demonstrated that the short disposable needle system can be used to recover oocytes to use as cytoplasts for SCNT, in the production of cloned foals and for other applications in equine embryology.

Interspecies somatic cell nuclear transfer in Asiatic cheetah using nuclei derived from post-mortem frozen tissue in absence of cryo-protectant and in vitro matured domestic cat oocytes.

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Moulavi, F; Hosseini, S M; Tanhaie-Vash, N; Ostadhosseini, S; Hosseini, S H; Hajinasrollah, M; Asghari, M H; Gourabi, H; Shahverdi, A; Vosough, A D; Nasr-Esfahani, M H

2017-03-01

Recent accomplishments in the field of somatic cell nuclear transfer (SCNT) hold tremendous promise to prevent rapid loss of animal genetic resources using ex situ conservation technology. Most of SCNT studies use viable cells for nuclear transfer into recipient oocytes. However, preparation of live cells in extreme circumstances, in which post-mortem material of endangered/rare animals is improperly retained frozen, is difficult, if not impossible. This study investigated the possibility of interspecies-SCNT (iSCNT) in Asiatic cheetah (Acinonyx jubatus venaticus), a critically endangered subspecies, using nuclei derived from frozen tissue in absence of cryo-protectant at -20 °C and in vitro matured domestic cat oocytes. No cells growth was detected in primary culture of skin and tendon pieces or following culture of singled cells prepared by enzymatic digestion. Furthermore, no live cells were detected following differential viable staining and almost all cells had ruptured membrane. Therefore, direct injection of donor nuclei into enucleated cat oocytes matured in vitro was carried out for SCNT experiments. Early signs of nuclear remodeling were observed as early as 2 h post-iSCNT and significantly increased at 4 h post-iSCNT. The percentages of iSCNT reconstructs that cleaved and developed to 4-16 cell and morula stages were 32.3 ± 7.3, 18.2 ± 9.8 and 5.9 ± 4.3%, respectively. However, none of the iSCNT reconstructs developed to the blastocyst stage. When domestic cat somatic and oocytes were used for control SCNT and parthenogenetic activation, the respective percentages of oocytes that cleaved (51.3 ± 13.9 and 77.3 ± 4.0%) and further developed to the blastocyst stage (11.3 ± 3.3 and 16.8 ± 3.8%) were comparable. In summary, this study demonstrated that enucleated cat oocytes can partially remodel and reactivate non-viable nuclei of Asiatic cheetah and support its reprogramming back to the embryonic stage. To our knowledge, this is

Gonadotropin-releasing hormone agonist triggering of oocyte maturation in assisted reproductive technology cycles

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Engin Türkgeldi

2015-06-01

Full Text Available Gonadotropin-releasing hormone agonists (GnRHa have gained increasing attention in the last decade as an alternative trigger for oocyte maturation in patients at high risk for ovarian hyperstimulation syndrome (OHSS. They provide a short luteinizing hormone (LH peak that limits the production of vascular endothelial growth factor, which is the key mediator leading to increased vascular permeability, the hallmark of OHSS. Initial studies showed similar oocyte yield and embryo quality compared with conventional human chorionic gonadotropin (hCG triggering; however, lower pregnancy rates and higher miscarriage rates were alarming in GnRHa triggered groups. Therefore, two approaches have been implemented to rescue the luteal phase in fresh transfers. Intensive luteal phase support (iLPS involves administiration of high doses of progesterone and estrogen and active patient monitoring. iLPS has been shown to provide satisfactory fertilization and clinical pregnancy rates, and to be especially useful in patients with high endogenous LH levels, such as in polycystic ovary syndrome. The other method for luteal phase rescue is low-dose hCG administiration 35 hours after GnRHa trigger. Likewise, this method results in statistically similar ongoing pregnancy rates (although slightly lower than to those of hCG triggered cycles. GnRHa triggering decreased OHSS rates dramatically, however, none of the rescue methods prevent OHSS totally. Cases were reported even in patients who underwent cryopreservation and did not receive hCG. GnRH triggering induces a follicle stimulating hormone (FSH surge, similar to natural cycles. Its possible benefits have been investigated and dual triggering, GnRHa trigger accompanied by a simultaneous low-dose hCG injection, has produced promising results that urge further exploration. Last of all, GnRHa triggering is useful in fertility preservation cycles in patients with hormone sensitive tumors. In conclusion, GnRHa triggering

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

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Eduardo Paulino da Costa

2002-06-01

. Treatment 0 (control: frozen-thawed undesnude oocytes; treatment 1, immature vitrificated undesnude oocytes dehydrated for 5 minutes in each of the following solutions of 20, 20 and 40% of ethylene glycol, respectively, associated to 0.3 Mol l-1 of trehalose and 20% of PVP, in media Talp Hepes, and, treatment 2, the same as treatment 1, but desnudes oocytes. After frozen-thawed of the oocytes (imersion in water bath at 30ºC for 20 seconds, the oocytes were gradually rehydrated, in the following sequence of solutions: media Talp Hepes with 20% of ethylene glycol + 0.3 Mol l-1 of trehalose + 10% of PVP and media Talp Hepes without ethylene glycol, trehalose and PVP, were washed three times. Ultimately, the oocytes were cultured at 38.5ºC, with 95% umidity and atmosphere of 5% of CO2 for 24 hours. After culture, the oocytes were fertilized and the embryos cultured in vitro for seven days. The nuclear maturation were 81 (68/84, 19 (7/36 and 0% (0/31, for treatments 0, 1 and 2, respectively. The cleavage and development rates were: 56.4(102/181 and 54,9% (56/102, 1,7. (1/60 and 0,0% (1/60, 0,0 (0/71 and 0,0% (0/71, for the treatments 1, 2 e 3, respectively. These results show that the vitrification procedures, by the used protocols, are not indicated for bovine oocytes cryopreservation.

Bovine somatic cell nuclear transfer.

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Ross, Pablo J; Cibelli, Jose B

2010-01-01

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

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

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Abd-Allah, Saber Mohammed

2009-01-01

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

In vitro production of buffalo embryos from stepwise vitrified immature oocytes

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Saber Mohammed Abd-Allah

2009-09-01

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

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

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

2010-05-01

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

Oocyte-like cells induced from mouse spermatogonial stem cells.

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

2012-08-06

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

Oocyte-like cells induced from mouse spermatogonial stem cells

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Wang Lu

2012-08-01

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

Calcium and actin in the saga of awakening oocytes

Energy Technology Data Exchange (ETDEWEB)

Santella, Luigia, E-mail: santella@szn.it; Limatola, Nunzia; Chun, Jong T.

2015-04-24

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

Calcium and actin in the saga of awakening oocytes

International Nuclear Information System (INIS)

Santella, Luigia; Limatola, Nunzia; Chun, Jong T.

2015-01-01

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

Effect of Heat Stress on Reproduction in Dairy Cows: Insights into the Cellular and Molecular Responses of the Oocyte.

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Roth, Zvi

2017-02-08

Among the components of the female reproductive tract, the ovarian pool of follicles and their enclosed oocytes are highly sensitive to hyperthermia. Heat-induced alterations in small antral follicles can be expressed later as compromised maturation and developmental capacity of the ovulating oocyte. This review summarizes the most up-to-date information on the effects of heat stress on the oocyte with an emphasis on unclear points and open questions, some of which might involve new research directions, for instance, whether preantral follicles are heat resistant. The review focuses on the follicle-enclosed oocytes, provides new insights into the cellular and molecular responses of the oocyte to elevated temperature, points out the role of the follicle microenvironment, and discusses some mechanisms that might underlie oocyte impairment. Mechanisms include nuclear and cytoplasmic maturation, mitochondrial function, apoptotic pathways, and oxidative stress. Understanding the mechanism by which heat stress compromises fertility might enable development of new strategies to mitigate its effects.

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

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

2004-11-01

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

GnRH agonist trigger for the induction of oocyte maturation in GnRH antagonist IVF cycles: a SWOT analysis.

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Engmann, Lawrence; Benadiva, Claudio; Humaidan, Peter

2016-03-01

Gonadotrophin releasing hormone agonist (GnRHa) trigger is effective in the induction of oocyte maturation and prevention of ovarian hyperstimulation syndrome during IVF treatment. This trigger concept, however, results in early corpora lutea demise and consequently luteal phase dysfunction and impaired endometrial receptivity. The aim of this strenghths, weaknesses, opportunities and threats analysis was to summarize the progress made over the past 15 years to optimize ongoing pregnancy rates after GnRHa trigger. The advantages and potential drawbacks of this type of triggering are reviewed. The current approach to the management of GnRHa trigger in autologous cycles is based on the peak serum oestradiol level or follicle number and aims at a fresh embryo transfer or a segmentation approach with elective cryopreservation policy. We recommend intensive luteal support with transdermal oestradiol and intramuscular progesterone alone if peak serum oestradiol is 4000 or more pg/ml after GnRHa trigger or dual trigger with GnRHa and HCG 1000 IU if peak serum oestradiol is less than 4000 pg/mL. On the contrary, we recommend HCG 1500 IU 35 h after GnRHa trigger if there are less than 25 follicles, or freeze all oocytes or embryos if there are over 25 follicles. Copyright © 2015. Published by Elsevier Ltd.

Macroenvironment effects on oocytes and embryos in swine.

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Foxcroft, G R; Vinsky, M D; Paradis, F; Tse, W-Y; Town, S C; Putman, C T; Dyck, M K; Dixon, W T

2007-09-01

As in other domestic mammals, the interaction between genotype and environment in swine has profound effects on the ultimate phenotype of the individual born. Interactions within the litter in utero add an additional level of complexity in a litter-bearing species like the pig. Nutritional manipulations during the preovulatory period affect the maturity of the follicle and enclosed oocyte, and the metabolic and endocrine mechanisms potentially mediating these effects have been described. Extensive research on lactational catabolism in the first parity sow has established an association between the development of immature follicles and oocytes, and the reduced fertility of these sows when bred at the first postweaning estrus. This negative impact of lactational catabolism appears to be exaggerated in contemporary dam-lines by a minimal delay between weaning and first estrus, further limiting the maturity of the follicle and oocyte at the time of ovulation. Metabolic programming may induce gender-specific loss of embryos by Day 30 and affects embryonic development directly, without significant effects on placental size. In contrast, inadvertent crowding of embryos in utero, particularly evident in a sub-population of mature sows with high ovulation rates and moderate to high embryonic survival to Day 30, significantly limits placental development of crowded litters. However, even at Day 30, moderate crowding in utero also appears to affect myogenesis in the embryo in a gender-specific manner. In the absence of compensatory placental growth after Day 30, classic measures of IUGR are evident in surviving fetuses at Day 90 and at term.

Multiple Requirements of PLK1 during Mouse Oocyte Maturation

Czech Academy of Sciences Publication Activity Database

Šolc, Petr; Kitajima, T.; Yoshida, S.; Brzáková, Adéla; Kaido, M.; Baran, V.; Mayer, Alexandra; Šámalová, P.; Motlík, Jan; Ellenberg, J.

2015-01-01

Roč. 10, č. 2 (2015) E-ISSN 1932-6203 R&D Projects: GA MŠk LH12057; GA ČR(CZ) GPP301/11/P081; GA ČR(CZ) GC301/09/J036; GA ČR GAP502/11/0593; GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : PLK1 * meiosis * mouse oocytes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.057, year: 2015

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

DEFF Research Database (Denmark)

Skovsgaard, Hanne; Li, Rong; Liu, Ying

2013-01-01

Most of the porcine oocytes used for in vitro studies are collected from gilts. Our aims were to study development capacity of gilt v. sow oocytes (pre- and postpubertal respectively) using 2 techniques illustrating development competence [parthenogenetic activation (PA) and somatic cell nuclear...... transfer (SCNT)], and to describe a simple method to select the most competent oocytes. Inside-ZP diameter of in vitro-matured gilt oocytes was measured (µm; small ≤110; medium >110; large ≥120). Gilt and sow oocytes were morphologically grouped as good (even cytoplasm, smooth cell membrane, visible...

Fertilization and Embryo Development of Fresh and Cryopreserved Sibling Oocytes

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Robert F. Casper

2010-01-01

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

Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence

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Yokoi Hayato

2011-04-01

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

Clathrin heavy chain 1 is required for spindle assembly and chromosome congression in mouse oocytes.

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Zhao, Jie; Wang, Lu; Zhou, Hong-Xia; Liu, Li; Lu, Angeleem; Li, Guang-Peng; Schatten, Heide; Liang, Cheng-Guang

2013-10-01

Clathrin heavy chain 1 (CLTC) has been considered a “moonlighting protein” which acts in membrane trafficking during interphase and in stabilizing spindle fibers during mitosis. However, its roles in meiosis, especially in mammalian oocyte maturation, remain unclear. This study investigated CLTC expression and function in spindle formation and chromosome congression during mouse oocyte meiotic maturation. Our results showed that the expression level of CLTC increased after germinal vesicle breakdown (GVBD) and peaked in the M phase. Immunostaining results showed CLTC distribution throughout the cytoplasm in a cell cycle-dependent manner. Appearance and disappearance of CLTC along with β-tubulin (TUBB) could be observed during spindle dynamic changes. To explore the relationship between CLTC and microtubule dynamics, oocytes at metaphase were treated with taxol or nocodazole. CLTC colocalized with TUBB at the enlarged spindle and with cytoplasmic asters after taxol treatment; it disassembled and distributed into the cytoplasm along with TUBB after nocodazole treatment. Disruption of CLTC function using stealth siRNA caused a decreased first polar body extrusion rate and extensive spindle formation and chromosome congression defects. Taken together, these results show that CLTC plays an important role in spindle assembly and chromosome congression through a microtubule correlation mechanism during mouse oocyte maturation.

Gonadotropin-dependent oocyte maturational competence requires activation of the protein kinase A pathway and synthesis of RNA and protein in ovarian follicles of Nibe, Nibea mitsukurii (Teleostei, Sciaenidae)

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Yoshizaki, G.; Shusa, M.; Takeuchi, T.; Patino, R.

2002-01-01

Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage; whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH [17,20??-dihydroxy-4-pregnen-3-one or 17,20??,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.

Differential expression and localization of glycosidic residues in in vitro- and in vivo-matured cumulus-oocyte complexes in equine and porcine species.

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Accogli, Gianluca; Douet, Cécile; Ambruosi, Barbara; Martino, Nicola Antonio; Uranio, Manuel Filioli; Deleuze, Stefan; Dell'Aquila, Maria Elena; Desantis, Salvatore; Goudet, Ghylène

2014-12-01

Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and βN-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models. © 2014 Wiley Periodicals, Inc.

Comparison of Different Vitrification Procedures on Developmental Competence of Mouse Germinal Vesicle Oocytes in the Presence or Absence of Cumulus Cells

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Mojdeh Salehnia

2009-01-01

Full Text Available Background: An evaluation of the developmental competence of vitrified mouse germinal vesicle(GV oocytes with various equilibration and vitrification times; in the presence or absence ofcumulus cells and by comparison between the cryotop method and straws was performed.Materials and Methods: Mouse GV oocytes were considered in cumulus-denuded oocytes(CDOs and cumulus-oocyte complexes (COCs groups. Their survival and developmental rateswere studied in the following experiments: (I exposure to different equilibration times (0, 3 and5 minutes and vitrification (1, 3 and 5 minutes without plunging in LN2 as toxicity tests, (IIoocytes were vitrified using straws followed by exposure to equilibration solution for 0, 3 and 5minutes and vitrification solution for 1 and 3 minutes, and (III oocytes were vitrified by cryotopfollowing exposure to equilibration for 5 minutes and vitrification for 1 minute, respectively.Results: Maturation and developmental rates of the COCs were higher than CDOs in the nonvitrifiedgroup (p<0.05. The survival and maturation rates were low in all oocytes exposed tovitrification solution for 5 minutes (p <0.05. In vitrified CDOs and COCs using straws, the survivalrates ranged from 56.9% to 85.4% and 44.0% to 84.5%, and the maturation rates from 35.3% to56.8% and 25.8% to 56.2%, respectively; which were lower than non-vitrified samples (p <0.05.Cryotop vitrified oocytes showed higher survival, maturation and fertilization rates when comparedto straw in both CDOs and COCs (p <0.05.Conclusion: The presence of cumulus cells improves developmental competence of GV oocytesin control groups but it did not affect the vitrified group. Vitrification of mouse GV oocytes usingcryotop was more effective than straws, however both vitrification techniques did not improve thecleavage rate.

Oocyte cryopreservation for fertility preservation in postpubertal female children at risk for premature ovarian failure due to accelerated follicle loss in Turner syndrome or cancer treatments.

Science.gov (United States)

Oktay, K; Bedoschi, G

2014-12-01

To preliminarily study the feasibility of oocyte cryopreservation in postpubertal girls aged between 13 and 15 years who were at risk for premature ovarian failure due to the accelerated follicle loss associated with Turner syndrome or cancer treatments. Retrospective cohort and review of literature. Academic fertility preservation unit. Three girls diagnosed with Turner syndrome, 1 girl diagnosed with germ-cell tumor. and 1 girl diagnosed with lymphoblastic leukemia. Assessment of ovarian reserve, ovarian stimulation, oocyte retrieval, in vitro maturation, and mature oocyte cryopreservation. Response to ovarian stimulation, number of mature oocytes cryopreserved and complications, if any. Mean anti-müllerian hormone, baseline follical stimulating hormone, estradiol, and antral follicle counts were 1.30 ± 0.39, 6.08 ± 2.63, 41.39 ± 24.68, 8.0 ± 3.2; respectively. In Turner girls the ovarian reserve assessment indicated already diminished ovarian reserve. Ovarian stimulation and oocyte cryopreservation was successfully performed in all female children referred for fertility preservation. A range of 4-11 mature oocytes (mean 8.1 ± 3.4) was cryopreserved without any complications. All girls tolerated the procedure well. Oocyte cryopreservation is a feasible technique in selected female children at risk for premature ovarian failure. Further studies would be beneficial to test the success of oocyte cryopreservation in young girls. Copyright © 2014 North American Society for Pediatric and Adolescent Gynecology. Published by Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2015-06-01

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

Are there optimal numbers of oocytes, spermatozoa and embryos in assisted reproduction?

Science.gov (United States)

Milachich, Tanya; Shterev, Atanas

2016-08-01

The aim of this overview is to discuss the current information about the search for the optimum yield of gametes in assisted reproduction, as one of the major pillars of IVF success. The first topic is focused on the number of male gametes and the possible impact of some genetic traits on these parameters. The number of spermatozoa did not seem to be crucial when there is no severe male factor of infertility. Genetic testing prior to using those sperm cells is very important. Different methods were applied in order to elect the "best" spermatozoa according to specific indications. The next problem discussed is the importance of the number of oocytes collected. Several studies have agreed that "15 oocytes is the perfect number," as the number of mature oocytes is more important. However, if elective single embryo transfer is performed, the optimal number of oocytes will enable a proper embryo selection. The third problem discussed concerns fertility preservation. Many educational programs promote and encourage procreation at maternal ages between 20-35 years, since assisted reproduction is unable to fully overcome the effects of female aging and fertility loss after that age. It is also strongly recommended to ensure a reasonable number of cryopreserved mature oocytes, preferably in younger ages (average of two stimulation cycles are likely required. For embryo cryopreservation, the "freeze all" strategy suggests the vitrification of good embryos, therefore quality is prior to number and patient recruitment for this strategy should be performed cautiously.

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

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

2016-02-01

In spite of great efforts, the mechanisms for postovulatory oocyte aging are not fully understood. Although our previous work showed that the FasL/Fas signaling facilitated oocyte aging, the intra-oocyte signaling pathways are unknown. Furthermore, the mechanisms by which oxidative stress facilitates oocyte aging and the causal relationship between Ca2+ rises and caspase-3 activation and between the cell cycle and apoptosis during oocyte aging need detailed investigations. Our aim was to address these issues by studying the intra-oocyte signaling pathways for Fas/FasL to accelerate oocyte aging. The results indicated that sFasL released by cumulus cells activated Fas on the oocyte by increasing reactive oxygen species via activating NADPH oxidase. The activated Fas triggered Ca2+ release from the endoplasmic reticulum by activating phospholipase C-γ pathway and cytochrome c pathway. The cytoplasmic Ca2+ rises activated calcium/calmodulin-dependent protein kinase II (CaMKII) and caspase-3. While activated CaMKII increased oocyte susceptibility to activation by inactivating maturation-promoting factor (MPF) through cyclin B degradation, the activated caspase-3 facilitated further Ca2+releasing that activates more caspase-3 leading to oocyte fragmentation. Furthermore, caspase-3 activation and fragmentation were prevented in oocytes with a high MPF activity, suggesting that an oocyte must be in interphase to undergo apoptosis.

CFP1 Regulates Histone H3K4 Trimethylation and Developmental Potential in Mouse Oocytes

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Chao Yu

2017-08-01

Full Text Available Trimethylation of histone H3 at lysine-4 (H3K4me3 is associated with eukaryotic gene promoters and poises their transcriptional activation during development. To examine the in vivo function of H3K4me3 in the absence of DNA replication, we deleted CXXC finger protein 1 (CFP1, the DNA-binding subunit of the SETD1 histone H3K4 methyltransferase, in developing oocytes. We find that CFP1 is required for H3K4me3 accumulation and the deposition of histone variants onto chromatin during oocyte maturation. Decreased H3K4me3 in oocytes caused global downregulation of transcription activity. Oocytes lacking CFP1 failed to complete maturation and were unable to gain developmental competence after fertilization, due to defects in cytoplasmic lattice formation, meiotic division, and maternal-zygotic transition. Our study highlights the importance of H3K4me3 in continuous histone replacement for transcriptional regulation, chromatin remodeling, and normal developmental progression in a non-replicative system.

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

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

2016-06-02

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

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

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Hayashi Ken-go

2011-03-01

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

The phosphodiesterase 3 inhibitor ORG 9935 inhibits oocyte maturation in the naturally selected dominant follicle in rhesus macaques.

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Jensen, Jeffrey T; Zelinski, Mary B; Stanley, Jessica E; Fanton, John W; Stouffer, Richard L

2008-04-01

The study was conducted to determine whether the phosphodiesterase (PDE) 3 inhibitor ORG 9935 prevents the resumption of meiosis in primate oocytes during natural menstrual cycles. Regularly cycling adult female macaques (n=8) were followed during the follicular phase and then started on a 2-day treatment regimen of human recombinant gonadotropins to control the timing of ovulation. Monkeys received no further treatment (controls) or ORG 9935. Oocytes were recovered by laparoscopic follicle aspiration 27 h after an ovulatory stimulus, cultured in vitro in the absence of inhibitor and inseminated. The primary outcome was the meiotic stage of the oocyte. In six ORG 9935 cycles, five of the recovered oocytes were germinal vesicle (GV)-intact, and one exhibited GV breakdown (GVBD). In contrast, all three oocytes that recovered during control cycles were GVBD (pORG 9935-treated oocytes underwent fertilization compared with 2/3 (67%) from controls. These results demonstrate that ORG 9935 blocks resumption of meiosis in the naturally selected dominant follicle in primates and suggest that PDE3 inhibitors have potential clinical use as contraceptives in women.

From fresh heterologous oocyte donation to autologous oocyte banking.

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Stoop, D

2012-01-01

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

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

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

2001-01-01

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