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Sample records for oocyte meiotic arrest

  1. Mutations in TUBB8 cause human oocyte meiotic arrest

    PubMed Central

    Feng, Ruizhi; Sang, Qing; Kuang, Yanping; Sun, Xiaoxi; Yan, Zheng; Zhang, Shaozhen; Shi, Juanzi; Tian, Guoling; Luchniak, Anna; Fukuda, Yusuke; Li, Bin; Yu, Min; Chen, Junling; Xu, Yao; Guo, Luo; Qu, Ronggui; Wang, Xueqian; Sun, Zhaogui; Liu, Miao; Shi, Huijuan; Wang, Hongyan; Feng, Yi; Shao, Ruijin; Chai, Renjie; Li, Qiaoli; Xing, Qinghe; Zhang, Rui; Nogales, Eva; Jin, Li; He, Lin; Gupta, Mohan L.; Cowan, Nicholas J.; Wang, Lei

    2016-01-01

    Background Successful human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to human oocyte maturation arrest are unknown. Methods We recruited a rare four-generation family with female infertility as a consequence of oocyte meiosis I arrest. We applied whole-exome and direct Sanger sequencing to an additional 23 patients following identification of mutations in a candidate gene, TUBB8. Expression of TUBB8 and all other β-tubulin isotypes was measured in human oocytes, early embryos, sperm cells and several somatic tissues by qRT-PCR. The effect of the TUBB8 mutations was assessed on α/β tubulin heterodimer assembly in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes via microinjection of the corresponding cRNAs. Results We identified seven mutations in the primate-specific gene TUBB8 that are responsible for human oocyte meiosis I arrest in seven families. TUBB8 expression is unique to oocytes and the early embryo, where this gene accounts for almost all of the expressed β-tubulin. The mutations affect the chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, induce microtubule chaos upon expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle assembly defects and maturation arrest upon expression in mouse and human oocytes. Conclusions TUBB8 mutations function via dominant negative effects that massively disrupt proper microtubule behavior. TUBB8 is a key gene involved in human oocyte meiotic spindle assembly and maturation. PMID:26789871

  2. Mutations in TUBB8 and Human Oocyte Meiotic Arrest.

    PubMed

    Feng, Ruizhi; Sang, Qing; Kuang, Yanping; Sun, Xiaoxi; Yan, Zheng; Zhang, Shaozhen; Shi, Juanzi; Tian, Guoling; Luchniak, Anna; Fukuda, Yusuke; Li, Bin; Yu, Min; Chen, Junling; Xu, Yao; Guo, Luo; Qu, Ronggui; Wang, Xueqian; Sun, Zhaogui; Liu, Miao; Shi, Huijuan; Wang, Hongyan; Feng, Yi; Shao, Ruijin; Chai, Renjie; Li, Qiaoli; Xing, Qinghe; Zhang, Rui; Nogales, Eva; Jin, Li; He, Lin; Gupta, Mohan L; Cowan, Nicholas J; Wang, Lei

    2016-01-21

    Background Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. Methods We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. Results We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. Conclusions TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.). PMID:26789871

  3. Maintenance of meiotic arrest by increasing [cAMP]i may have physiological relevance in bovine oocytes.

    PubMed

    Aktas, H; Wheeler, M B; First, N L; Leibfried-Rutledge, M L

    1995-11-01

    Invasive adenylate cyclase (iAC) reversibly inhibits spontaneous maturation of cumulus-enclosed bovine oocytes by increasing the intracellular concentration of cAMP, [cAMP]i. In this study, physiological aspects of maintaining meiotic arrest in bovine oocytes by iAC were investigated. The maintenance of germinal vesicle arrest by iAC in both cumulus-enclosed and denuded bovine oocytes was concentration dependent (r2 = 0.857). Denuded bovine oocytes were more sensitive to maintenance of meiotic arrest by iAC then were cumulus-enclosed oocytes. At the highest concentration, 70% of the cumulus-enclosed and 90% of the denuded bovine oocytes were maintained in meiotic arrest. The iAC increased [cAMP]i in both intact cumulus-oocyte complexes and enclosed oocytes in a concentration-dependent manner (r2 = 0.795). Cumulus-enclosed oocytes maintained in meiotic arrest by iAC retained developmental competence when subsequently cultured in iAC-free medium and then fertilized. The [cAMP]i in bovine complexes decreased precipitously upon release from follicles and remained low for the next 125 min. However, the [cAMP]i of the enclosed oocytes did not change. Bovine oocytes commit to undergo meiosis in a progressive manner. Approximately 10% of the oocytes were already committed when aspirated. This proportion increased to 40% at 2 h and 70% at 5 h. Use of two inhibitors of cAMP-dependent protein kinase A provided further evidence that cAMP functions in mediating meiotic arrest in bovine oocytes. Bovine oocytes, therefore, are sensitive to different cAMP concentrations, and are developmentally competent after iAC-induced arrest, and complexes containing oocytes exhibit a decrease in [cAMP]i before spontaneous maturation. These results suggest that maintenance of meiotic arrest by iAC is accomplished through modulation of cellular machinery, and regulation of oocyte maturation by [cAMP]i may be physiologically relevant.

  4. Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development.

    PubMed

    Nogueira, D; Ron-El, R; Friedler, S; Schachter, M; Raziel, A; Cortvrindt, R; Smitz, J

    2006-01-01

    Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. We aimed to evaluate the quality of in vitro-matured, germinal vesicle (GV)-stage human oocytes following a prematuration culture (PMC) with a meiotic arrester, phosphodiesterase 3-inhibitor (PDE3-I). Follicles (diameter, 6-12 mm) were retrieved 34-36 h post-hCG administration from informed, consenting patients who had undergone controlled ovarian stimulation. Cumulus-enclosed oocytes (CEOs) presenting moderate expansion or full compaction were placed in PMC with the PDE3-I, Org9935, for 24 or 48 h. Subsequently, oocytes were removed from PMC, denuded of cumulus cells, matured in vitro, and fertilized, and the resulting embryos were cultured. In the presence of PDE3-I, approximately 98% of the oocytes were arrested at the GV stage. Following PDE3-I removal, oocytes acquired a higher maturation rate than oocytes that were immediately denuded of cumulus cells after retrieval and in vitro matured (67% vs. 46%, P = 0.01). In controls, immature CEOs retrieved with moderate expansion reached higher maturation rates compared to fully compacted CEOs, but in PMC groups, high values of maturation were achieved for both morphological classes of CEOs. No effect of PMC on fertilization was observed. A 24-h PMC period proved to be the most effective in preserving embryonic integrity. Similar proportions of nuclear abnormalities were observed in embryos of all in vitro groups. In summary, PMC with the specific PDE3-I had a beneficial effect on human CEOs by enhancing maturation, benefiting mainly the fully compacted CEOs. This resulted in an increased yield of mature oocytes available for insemination without compromising embryonic development. These results suggest that applying an inhibitor to control the rate of nuclear maturity by regulating intraoocyte PDE3 activity may allow the synchronization of nuclear and ooplasmic maturation.

  5. DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint

    PubMed Central

    Collins, Josie K.; Lane, Simon I. R.; Merriman, Julie A.; Jones, Keith T.

    2015-01-01

    Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule–kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis. PMID:26522232

  6. Control of Oocyte Growth and Meiotic Maturation in C. elegans

    PubMed Central

    Kim, Seongseop; Spike, Caroline; Greenstein, David

    2013-01-01

    In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. Chromosome segregation errors in female meiosis I are the leading cause of human birth defects, and age-related changes in the hormonal environment of the ovary are a suggested cause. C. elegans is emerging as a genetic paradigm for studying hormonal control of meiotic maturation. The meiotic maturation processes in C. elegans and mammals share a number of biological and molecular similarities. Major sperm protein (MSP) and luteinizing hormone (LH), though unrelated in sequence, both trigger meiotic resumption using somatic Gαs-adenylate cyclase pathways and soma-germline gap-junctional communication. At a molecular level, the oocyte responses apparently involve the control of conserved protein kinase pathways and post-transcriptional gene regulation in the oocyte. At a cellular level, the responses include cortical cytoskeletal rearrangement, nuclear envelope breakdown, assembly of the acentriolar meiotic spindle, chromosome segregation, and likely changes important for fertilization and the oocyte-to-embryo transition. This chapter focuses on signaling mechanisms required for oocyte growth and meiotic maturation in C. elegans and discusses how these mechanisms coordinate the completion of meiosis and the oocyte-to-embryo transition. PMID:22872481

  7. Cytoplasmic anchoring of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs) is required for meiotic arrest of porcine full-grown and growing oocytes.

    PubMed

    Nishimura, Takanori; Fujii, Wataru; Sugiura, Koji; Naito, Kunihiko

    2014-03-01

    Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. We previously hypothesized that the meiotic incompetence of porcine GOs was attributed to complex spatial-temporal regulation of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs), but found that AKAP1 is not involved in the meiotic incompetence of porcine GOs. In the present study, we cloned porcine cDNAs of AKAP5 and AKAP7alpha, and found that inhibiting the expression of these AKAPs induced PKA translocation into the nucleus and promoted meiotic resumption of porcine GOs without affecting the total PKA activity of GOs, whereas overexpressing these AKAPs had no effect. Because AKAPs regulate PKA localization through binding with regulatory subunits of PKA (PKA-Rs), PKA-R binding with AKAPs was inhibited by AKAP-binding inhibition peptides or PKA-R expression inhibition by antisense RNAs. We found that the expression inhibition and binding inhibition of PRKAR1A, an isoform of mammalian PKA-R, promoted meiotic resumption of porcine GOs, whereas these inhibitions of PRKAR2A, another PKA-R isoform, had no effect. In contrast, the expression inhibition and binding inhibition of PRKAR2A had higher effects than those of PRKAR1A on meiotic resumption of porcine full-grown oocytes. These results suggest that cytoplasmic anchoring of PKA by AKAPs is required for meiotic arrest of oocytes and that the PKA-R isoform working for the maintenance of meiotic arrest changed from PRKAR1A to PRKAR2A during the acquisition of meiotic competence. PMID:24501172

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

    PubMed Central

    Celik, Onder; Celik, Nilufer; Gungor, Sami; Haberal, Esra Tustas; Aydin, Suleyman

    2015-01-01

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

  9. Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro.

    PubMed

    Coy, Pilar; Romar, Raquel; Payton, Rebecca R; McCann, Lisa; Saxton, Arnold M; Edwards, J Lannett

    2005-01-01

    The overall objective was to evaluate the effectiveness of the S-enantiomer of roscovitine (inhibitor of p34cdc2/cyclin B kinase) to maintain bovine cumulus-oocyte complexes at the germinal vesicle (GV) stage for extended times after removal from antral follicles without compromising subsequent maturation, fertilization and embryo development. Oocytes were cultured in 0, 12.5, 25 or 50 micromol/l S-roscovitine for 24 h. Hoechst staining showed that 50 micromol/l S-roscovitine maintained >90% of oocytes at the GV stage and inhibited gonadotropin-induced cumulus expansion. Fewer oocytes underwent nuclear maturation after in vitro maturation (Hoechst staining) when cultured in 50 micromol/l S-roscovitine for 66 versus 21 or 42 h. Zona pellucida (ZP) hardening (pronase resistance), cortical granule types (lens culinaris agglutinin-fluorescein isothiocyanate), nuclear maturation and fertilization with frozen-thawed spermatozoa (Hoechst staining) were assessed after culture of oocytes in 50 micromol/l S-roscovitine for 0, 24 or 48 h. Neither ZP hardening, nor nuclear maturation nor fertilization were altered by roscovitine culture for 48 h. A higher proportion of oocytes had a type III cortical granule pattern (premature translocation to the oolemma) after roscovitine culture for 48 h. However, embryo development was not compromised as cleavage, development to 8-16 cell and blastocyst stages were at least comparable in control and roscovitine-treated oocytes. In conclusion, the studies have shown that S-roscovitine reversibly maintained bovine oocytes at the GV stage for 48 h. However, maintenance of oocytes in static culture for 48 h was not sufficient to improve development above non-treated controls. PMID:15615895

  10. Induction of meiotic maturation in Xenopus oocytes by 12-O-tetradecanoylphorbol 13-acetate

    SciTech Connect

    Stith, B.J.; Maller, J.L.

    1987-04-01

    Fully grown Xenopus oocytes are physiologically arrested at the G2/prophase border of the first meiotic division. Addition in vitro of progesterone or insulin causes release of the G2/prophase block and stimulates meiotic cell division of the oocyte, leading to maturation of the oocyte into an unfertilized egg. The possibility that the products of polyphosphoinositide breakdown, diacylglycerol and inositol-1,4,5-trisphosphate are involved in occyte maturation was investigated. Microinjection of IP/sub 3/ into oocytes just prior to addition of progesterone or insulin accelerated the rate of germinal vesicle breakdown (GVBD) by up to 25%. Half-maximal acceleration occurred at an intracellular IP/sub 3/ concentration of 1 ..mu..M. Treatment of oocytes with the diacylglycerol analog and tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced GVBD in the absence of hormone. Half-maximal induction of GVBD occurred with 150 nM TPA and was blocked by pretreatment of oocytes with 10 nM cholera toxin. Microinjection of highly purified protein kinase C from rat brain oocytes did not induce maturation but markedly accelerated the rate of insulin-induced oocyte maturation. However, injection of the enzyme had no effect on progesterone action. These results indicate that protein kinase C is capable of regulating oocyte maturation of Xenopus.

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

    PubMed Central

    Li, Xuan; Wang, Yan-Kui; Song, Zhi-Qiang; Du, Zhi-Qiang; Yang, Cai-Xia

    2016-01-01

    Meiotic maturation of mammalian oocytes is a precisely orchestrated and complex process. Dimethyl sulfoxide (DMSO), a widely used solvent, drug, and cryoprotectant, is capable of disturbing asymmetric cytokinesis of oocyte meiosis in mice. However, in pigs, DMSO’s effect on oocyte meiosis still remains unknown. We aimed to evaluate if DMSO treatment will affect porcine oocyte meiosis and the underlying molecular changes as well. Interestingly, we did not observe the formation of the large first polar body and symmetric division for porcine oocytes treated with DMSO, contrary to findings reported in mice. 3% DMSO treatment could inhibit cumulus expansion, increase nuclear abnormality, disturb spindle organization, decrease reactive oxygen species level, and elevate mitochondrial membrane potential of porcine oocytes. There was no effect on germinal vesicle breakdown rate regardless of DMSO concentration. 3% DMSO treatment did not affect expression of genes involved in spindle organization (Bub1 and Mad2) and apoptosis (NF-κB, Pten, Bcl2, Caspase3 and Caspase9), however, it significantly decreased expression levels of pluripotency genes (Oct4, Sox2 and Lin28) in mature oocytes. Therefore, we demonstrated that disturbed cumulus expansion, chromosome alignment, spindle organization and pluripotency gene expression could be responsible for DMSO-induced porcine oocyte meiotic arrest and the lower capacity of subsequent embryo development. Our results provide new insights on DMSO’s effect on porcine oocyte meiosis and raise safety concerns over DMSO’s usage on female reproduction in both farm animals and humans. PMID:27348312

  12. Aging predisposes oocytes to meiotic nondisjunction when the cohesin subunit SMC1 is reduced.

    PubMed

    Subramanian, Vijayalakshmi V; Bickel, Sharon E

    2008-11-01

    In humans, meiotic chromosome segregation errors increase dramatically as women age, but the molecular defects responsible are largely unknown. Cohesion along the arms of meiotic sister chromatids provides an evolutionarily conserved mechanism to keep recombinant chromosomes associated until anaphase I. One attractive hypothesis to explain age-dependent nondisjunction (NDJ) is that loss of cohesion over time causes recombinant homologues to dissociate prematurely and segregate randomly during the first meiotic division. Using Drosophila as a model system, we have tested this hypothesis and observe a significant increase in meiosis I NDJ in experimentally aged Drosophila oocytes when the cohesin protein SMC1 is reduced. Our finding that missegregation of recombinant homologues increases with age supports the model that chiasmata are destabilized by gradual loss of cohesion over time. Moreover, the stage at which Drosophila oocytes are most vulnerable to age-related defects is analogous to that at which human oocytes remain arrested for decades. Our data provide the first demonstration in any organism that, when meiotic cohesion begins intact, the aging process can weaken it sufficiently and cause missegregation of recombinant chromosomes. One major advantage of these studies is that we have reduced but not eliminated the SMC1 subunit. Therefore, we have been able to investigate how aging affects normal meiotic cohesion. Our findings that recombinant chromosomes are at highest risk for loss of chiasmata during diplotene argue that human oocytes are most vulnerable to age-induced loss of meiotic cohesion at the stage at which they remain arrested for several years. PMID:19008956

  13. Aging Predisposes Oocytes to Meiotic Nondisjunction When the Cohesin Subunit SMC1 Is Reduced

    PubMed Central

    Subramanian, Vijayalakshmi V.; Bickel, Sharon E.

    2008-01-01

    In humans, meiotic chromosome segregation errors increase dramatically as women age, but the molecular defects responsible are largely unknown. Cohesion along the arms of meiotic sister chromatids provides an evolutionarily conserved mechanism to keep recombinant chromosomes associated until anaphase I. One attractive hypothesis to explain age-dependent nondisjunction (NDJ) is that loss of cohesion over time causes recombinant homologues to dissociate prematurely and segregate randomly during the first meiotic division. Using Drosophila as a model system, we have tested this hypothesis and observe a significant increase in meiosis I NDJ in experimentally aged Drosophila oocytes when the cohesin protein SMC1 is reduced. Our finding that missegregation of recombinant homologues increases with age supports the model that chiasmata are destabilized by gradual loss of cohesion over time. Moreover, the stage at which Drosophila oocytes are most vulnerable to age-related defects is analogous to that at which human oocytes remain arrested for decades. Our data provide the first demonstration in any organism that, when meiotic cohesion begins intact, the aging process can weaken it sufficiently and cause missegregation of recombinant chromosomes. One major advantage of these studies is that we have reduced but not eliminated the SMC1 subunit. Therefore, we have been able to investigate how aging affects normal meiotic cohesion. Our findings that recombinant chromosomes are at highest risk for loss of chiasmata during diplotene argue that human oocytes are most vulnerable to age-induced loss of meiotic cohesion at the stage at which they remain arrested for several years. PMID:19008956

  14. LSD1 is essential for oocyte meiotic progression by regulating CDC25B expression in mice

    PubMed Central

    Kim, Jeesun; Singh, Anup Kumar; Takata, Yoko; Lin, Kevin; Shen, Jianjun; Lu, Yue; Kerenyi, Marc A.; Orkin, Stuart H.; Chen, Taiping

    2015-01-01

    Mammalian oocytes are arrested at prophase I until puberty when hormonal signals induce the resumption of meiosis I and progression to meiosis II. Meiotic progression is controlled by CDK1 activity and is accompanied by dynamic epigenetic changes. Although the signalling pathways regulating CDK1 activity are well defined, the functional significance of epigenetic changes remains largely unknown. Here we show that LSD1, a lysine demethylase, regulates histone H3 lysine 4 di-methylation (H3K4me2) in mouse oocytes and is essential for meiotic progression. Conditional deletion of Lsd1 in growing oocytes results in precocious resumption of meiosis and spindle and chromosomal abnormalities. Consequently, most Lsd1-null oocytes fail to complete meiosis I and undergo apoptosis. Mechanistically, upregulation of CDC25B, a phosphatase that activates CDK1, is responsible for precocious meiotic resumption and also contributes to subsequent spindle and chromosomal defects. Our findings uncover a functional link between LSD1 and the major signalling pathway governing meiotic progression. PMID:26626423

  15. XGef Mediates Early CPEB Phosphorylation during Xenopus Oocyte Meiotic Maturation

    PubMed Central

    Martínez, Susana E.; Yuan, Lei; Lacza, Charlemagne; Ransom, Heather; Mahon, Gwendolyn M.; Whitehead, Ian P.; Hake, Laura E.

    2005-01-01

    Polyadenylation-induced translation is an important regulatory mechanism during metazoan development. During Xenopus oocyte meiotic progression, polyadenylation-induced translation is regulated by CPEB, which is activated by phosphorylation. XGef, a guanine exchange factor, is a CPEB-interacting protein involved in the early steps of progesterone-stimulated oocyte maturation. We find that XGef influences early oocyte maturation by directly influencing CPEB function. XGef and CPEB interact during oogenesis and oocyte maturation and are present in a c-mos messenger ribonucleoprotein (mRNP). Both proteins also interact directly in vitro. XGef overexpression increases the level of CPEB phosphorylated early during oocyte maturation, and this directly correlates with increased Mos protein accumulation and acceleration of meiotic resumption. To exert this effect, XGef must retain guanine exchange activity and the interaction with CPEB. Overexpression of a guanine exchange deficient version of XGef, which interacts with CPEB, does not enhance early CPEB phosphorylation. Overexpression of a version of XGef that has significantly reduced interaction with CPEB, but retains guanine exchange activity, decreases early CPEB phosphorylation and delays oocyte maturation. Injection of XGef antibodies into oocytes blocks progesterone-induced oocyte maturation and early CPEB phosphorylation. These findings indicate that XGef is involved in early CPEB activation and implicate GTPase signaling in this process. PMID:15635100

  16. Depletion of the LINC complex disrupts cytoskeleton dynamics and meiotic resumption in mouse oocytes

    PubMed Central

    Luo, Yibo; Lee, In-Won; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

    2016-01-01

    The SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) proteins constitute the linker of nucleoskeleton and cytoskeleton (LINC) complex on the nuclear envelope. To date, the SUN1/KASH5 complex is known to function as meiotic-specific factors. In this study, gene-silencing methods were used to explore the roles of SUN1 and KASH5 in mouse oocytes after prophase. SUN1 was detected throughout the nucleus; however, KASH5 was dispersed through the cell. After germinal vesicle breakdown (GVBD), SUN1 and KASH5 migrated during spindle formation and localized to the spindle poles at the MII stage. Most oocytes were arrested at the germinal vesicle (GV) stage after depletion of either SUN1 or KASH5. The DNA damage response was triggered in SUN1-depleted oocytes and thus gave rise to the G2/M checkpoint protein, p-CHK1. Oocytes that underwent GVBD had relatively small and abnormal spindles and lower levels of cytoplasm F-actin mesh. Immunofluorescence results also indicated the dislocation of pericentrin and P150Glued after SUN1 or KASH5 depletion. Furthermore, KASH5 localized exclusively near the oocyte cortex after SUN1 depletion, but SUN1 localization was unaffected in KASH5-depleted oocytes. Taken together, the results suggest that SUN1 and KASH5 are essential factors in the regulation of meiotic resumption and spindle formation. PMID:26842404

  17. Cep55 regulates spindle organization and cell cycle progression in meiotic oocyte

    PubMed Central

    Xu, Zhao-Yang; Ma, Xue-Shan; Qi, Shu-Tao; Wang, Zhen-Bo; Guo, Lei; Schatten, Heide; Sun, Qing-Yuan; Sun, Ying-Pu

    2015-01-01

    Cep55 is a relatively novel member of the centrosomal protein family. Here, we show that Cep55 is expressed in mouse oocytes from the germinal vesicle (GV) to metaphase II (MII) stages. Immuostaining and confocal microscopy as well as time lapse live imaging after injection of mRNA encoding fusion protein of Cep55 and GFP identified that Cep55 was localized to the meiotic spindle, especially to the spindle poles at metaphase, while it was concentrated at the midbody in telophase in meiotic oocytes. Knockdown of Cep55 by specific siRNA injection caused the dissociation of γ-tubulin from the spindle poles, resulting in severely defective spindles and misaligned chromosomes, leading to metaphase I arrest and failure of first polar body (PB1) extrusion. Correspondingly, cyclin B accumulation and spindle assembly checkpoint (SAC) activation were observed in Cep55 knockdown oocytes. Our results suggest that Cep55 may act as an MTOC-associated protein regulating spindle organization, and thus cell cycle progression during mouse oocyte meiotic maturation. PMID:26582107

  18. Analyses of the involvement of PKA regulation mechanism in meiotic incompetence of porcine growing oocytes.

    PubMed

    Nishimura, Takanori; Fujii, Wataru; Kano, Kiyoshi; Sugiura, Koji; Naito, Kunihiko

    2012-09-01

    Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. In the present study, we cloned cDNAs of cAMP-dependent protein-kinase (PKA) subunits from porcine oocytes and analyzed the involvement of the PKA regulation mechanism in the meiotic incompetence of GOs at the molecular level. We found a cAMP-independent high PKA activity in GOs throughout the in vitro culture using a porcine PKA assay system we established, and inhibition of the activity by injection of the antisense RNA of the PKA catalytic subunit (PKA-C) induced meiotic resumption in GOs. Then we examined the possibility that the amount of the PKA regulatory subunit (PKA-R), which can bind and inhibit PKA-C, was insufficient to suppress PKA activity in GOs because of the overexpression of two PKA-Rs, PRKAR1A and PRKAR2A. We found that neither of them affected PKA activity and induced meiotic resumption in GO although PRKAR2A could inhibit PKA activity and induce meiosis in cAMP-treated full-grown oocytes (FGOs). Finally, we analyzed the subcellular localization of PKA subunits and found that all the subunits were localized in the cytoplasm during meiotic arrest and that PKA-C and PRKAR2A, but not PRKAR1A, entered into the nucleus just before meiotic resumption in FGOs, whereas all of them remained in the cytoplasm in GOs throughout the culture period. Our findings suggest that the continuous high PKA activity is a primary cause of the meiotic incompetence of porcine GOs and that this PKA activity is not simply caused by an insufficient expression level of PKA-R, but can be attributed to more complex spatial-temporal regulation mechanisms. PMID:22674394

  19. Mitofusin-2 is required for mouse oocyte meiotic maturation

    PubMed Central

    Zhang, Jing-Hua; Zhang, Teng; Gao, Si-Hua; Wang, Ke; Yang, Xiu-Yan; Mo, Fang-Fang; Na Yu; An, Tian; Li, Yu-Feng; Hu, Ji-Wei; Jiang, Guang-Jian

    2016-01-01

    Mitofusin-2 (Mfn2) is essential for embryonic development, anti-apoptotic events, protection against free radical-induced lesions, and mitochondrial fusion in many cells. However, little is known about its mechanism and function during oocyte maturation. In this study, we found that Mfn2 was expressed in the cytoplasm during different stages of mouse oocyte maturation. Mfn2 was mainly associated with α-tubulin during oocyte maturation. Knockdown of Mfn2 by specific siRNA injection into oocytes caused the mitochondrial morphology and quantity to change, resulting in severely defective spindles and misaligned chromosomes. This led to metaphase I arrest and the failure of first polar body extrusion. Furthermore, Mfn2 depletion from GV stage oocytes caused the redistribution of p38 MAPK in oocyte cytoplasm. These findings provide insights into potential mechanisms of Mfn2-mediated cellular alterations, which may have significant implications for oocyte maturation. PMID:27485634

  20. H3 Thr3 phosphorylation is crucial for meiotic resumption and anaphase onset in oocyte meiosis.

    PubMed

    Wang, Qian; Wei, Haojie; Du, Juan; Cao, Yan; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Chen, Dandan; Ma, Wei

    2016-01-01

    Haspin-catalyzed histone H3 threonine 3 (Thr3) phosphorylation facilitates chromosomal passenger complex (CPC) docking at centromeres, regulating indirectly chromosome behavior during somatic mitosis. It is not fully known about the expression and function of H3 with phosphorylated Thr3 (H3T3-P) during meiosis in oocytes. In this study, we investigated the expression and sub-cellular distribution of H3T3-P, as well as its function in mouse oocytes during meiotic division. Western blot analysis revealed that H3T3-P expression was only detected after germinal vesicle breakdown (GVBD), and gradually increased to peak level at metaphase I (MI), but sharply decreased at metaphase II (MII). Immunofluorescence showed H3T3-P was only brightly labeled on chromosomes after GVBD, with relatively high concentration across the whole chromosome axis from pro-metaphase I (pro-MI) to MI. Specially, H3T3-P distribution was exclusively limited to the local space between sister centromeres at MII stage. Haspin inhibitor, 5-iodotubercidin (5-ITu), dose- and time-dependently blocked H3T3-P expression in mouse oocytes. H3T3-P inhibition delayed the resumption of meiosis (GVBD) and chromatin condensation. Moreover, the loss of H3T3-P speeded up the meiotic transition to MII of pro-MI oocytes in spite of the presence of non-aligned chromosomes, even reversed MI-arrest induced with Nocodazole. The inhibition of H3T3-P expression distinguishably damaged MAD1 recruitment on centromeres, which indicates the spindle assembly checkpoint was impaired in function, logically explaining the premature onset of anaphase I. Therefore, Haspin-catalyzed histone H3 phosphorylation is essential for chromatin condensation and the following timely transition from meiosis I to meiosis II in mouse oocytes during meiotic division. PMID:26636626

  1. New elements in the C-type natriuretic peptide signaling pathway inhibiting swine in vitro oocyte meiotic resumption.

    PubMed

    Santiquet, Nicolas; Papillon-Dion, Emilie; Djender, Nadjib; Guillemette, Christine; Richard, François J

    2014-07-01

    C-type natriuretic peptide (CNP) and its cognate receptor, natriuretic peptide receptor (NPR) B, have been shown to promote cGMP production in granulosa/cumulus cells. Once transferred to the oocyte through the gap junctions, the cGMP inhibits oocyte meiotic resumption. CNP has been shown to bind another natriuretic receptor, NPR-C. NPR-C is known to interact with and degrade bound CNP, and has been reported to possess signaling functions. Therefore, NPR-C could participate in the control of oocyte maturation during swine in vitro maturation (IVM). Here, we examine the effect of CNP signaling on meiotic resumption, the amount of cGMP and gap junctional communication (GJC) regulation during swine IVM. The results show an inhibitory effect of CNP in inhibiting oocyte meiotic resumption in follicle-stimulating hormone (FSH)-stimulated IVM. We also found that an NPR-C-specific agonist (cANP([4-23])) is likely to play a role in maintaining meiotic arrest during porcine IVM when in the presence of a suboptimal dose of CNP. Moreover, we show that, even if CNP can increase intracellular concentration of cGMP in cumulus-oocyte complexes, cANP((4-23)) had no impact on cGMP concentration, suggesting a potential cGMP-independent signaling pathway related to NPR-C activation. These data support a potential involvement of cANP((4-23)) through NPR-C in inhibiting oocyte meiotic resumption while in the presence of a suboptimal dose of CNP. The regulation of GJC was not altered by CNP, cANP((4-23)), or the combination of CNP and cANP((4-23)), supporting their potential contribution in sending signals to the oocytes. These findings offer promising insights in to new elements of the signaling pathways that may be involved in inhibiting resumption of meiosis during FSH-stimulated swine IVM.

  2. Non-meiotic chromosome instability in human immature oocytes

    PubMed Central

    Daina, Gemma; Ramos, Laia; Rius, Mariona; Obradors, Albert; del Rey, Javier; Giralt, Magda; Campillo, Mercedes; Velilla, Esther; Pujol, Aïda; Martinez-Pasarell, Olga; Benet, Jordi; Navarro, Joaquima

    2014-01-01

    Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25–45 years of age) and 24 IVF oocyte donors (18–33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes. PMID:23695274

  3. The release from metaphase arrest in blue mussel oocytes.

    PubMed

    Néant, I; Dufresne, L; Morasse, J; Gicquaud, C; Guerrier, P; Dubé, F

    1994-09-01

    In Mytilus edulis, shed oocytes are arrested at metaphase I of meiosis until fertilization. We previously suggested (Dubé and Dufresne, J. Exp. Zool. 256:323-332, 1990) that such a metaphase arrest depends upon a continuous synthesis of short-lived proteins, the destruction of which is sufficient to induce meiosis resumption. We further investigated the mechanism of metaphase release in blue mussel oocytes as triggered either by fertilization or by inhibition of protein synthesis (emetine) or phosphorylation (6-dimethylaminopurine, 6-DMAP). Treatment of unfertilized oocytes (UF) with emetine induces completion of the first meiotic cycle including extrusion of the polar body, followed by chromosome decondensation and by the formation of large membrane-bound nuclei, as visualized by Hoechst staining and transmission electron microscopy (TEM). Inhibition of protein phosphorylation with 6-DMAP induces directly chromosome decondensation and the formation of multiple nuclei surrounded by nuclear membrane. These interphasic nuclei exhibit continuous 3H-thymidine incorporation. p13 precipitation of p34 and associated proteins reveals "putative" cyclins in UF, no longer detected after metaphase/anaphase transition due to fertilization or emetine treatment. In the presence of 6-DMAP, new migrating forms are observed. The phosphorylated p34cdc2 homolog becomes dephosphorylated after fertilization or emetine treatment, whereas 6-DMAP induces its phosphorylation on tyrosine. Histone H1 kinase activity is reduced after these treatments, compared to the UF sample. Our results suggest that the metaphase/anaphase transition triggered by fertilization in blue mussel oocytes is induced by the rapid destruction of a set of continuously synthesized proteins accompanied by decreased histone H1 kinase activity. These events can be mimicked by inhibiting protein synthesis. Inhibition of protein phosphorylation would drive the cell to interphase without commitment to meiosis I. PMID

  4. Intranuclear membranes and the formation of the first meiotic spindle in Xenos peckii (Acroschismus wheeleri) oocytes

    PubMed Central

    1983-01-01

    The ultrastructure of spindle formation during the first meiotic division in oocytes of the Strepsipteran insect Xenos peckii Kirby (Acroschismus wheeleri Pierce) was examined in serial thick (0.25- micron) and thin sections. During late prophase the nuclear envelope became extremely convoluted and fenestrated. At this time vesicular and tubular membrane elements permeated the nucleoplasm and formed a thin fusiform sheath, 5-7 micron in length, around each of the randomly oriented and condensing tetrads. These membrane elements appeared to arise from the nuclear envelope and/or in association with annulate lamellae in the nuclear region. All of the individual tetrads and their associated fusiform sheaths became aligned within the nucleus subsequent to the breakdown of the nuclear envelope. Microtubules (MTs) were found associated with membranes of the meiotic apparatus only after the nuclear envelope had broken down. Kinetochores, with associated MTs, were first recognizable as electron-opaque patches on the chromosomes at this time. The fully formed metaphase arrested Xenos oocyte meiotic apparatus contained an abundance of membranes and had diffuse poles that lacked distinct polar MT organizing centers. From these observations we conclude that the apparent individual chromosomal spindles--seen in the light microscope to form around each Xenos tetrad during "intranuclear prometaphase" (Hughes-Schrader, S., 1924, J. Morphol. 39:157-197)--actually form during late prophase, lack MTs, and are therefore not complete miniature bipolar spindles, as had been commonly assumed. Thus, the unique mode of spindle formation in Xenos oocytes cannot be used to support the hypothesis that chromosomes (kinetochores) induce the polymerization of their associated MTs. Our observation that MTs appeared in association with and parallel to tubular membrane components of the Xenos meiotic apparatus after these membranes became oriented with respect to the tetrads, is consistent with the

  5. Axin-1 Regulates Meiotic Spindle Organization in Mouse Oocytes

    PubMed Central

    Liu, Rui; Liu, Yu; Zhang, Fei; Zhang, Zhen; Shen, Yu-Ting; Xu, Lin; Chen, Ming-Huang; Wang, Ya-Long; Xu, Bai-Hui; Yang, Xiang-Jun; Wang, Hai-Long

    2016-01-01

    Axin-1, a negative regulator of Wnt signaling, is a versatile scaffold protein involved in centrosome separation and spindle assembly in mitosis, but its function in mammalian oogenesis remains unknown. Here we examined the localization and function of Axin-1 during meiotic maturation in mouse oocytes. Immunofluorescence analysis showed that Axin-1 was localized around the spindle. Knockdown of the Axin1 gene by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in severely defective spindles, misaligned chromosomes, failure of first polar body (PB1) extrusion, and impaired pronuclear formation. However, supplementing the culture medium with the Wnt pathway activator LiCl improved spindle morphology and pronuclear formation. Downregulation of Axin1 gene expression also impaired the spindle pole localization of γ-tubulin/Nek9 and resulted in retention of the spindle assembly checkpoint protein BubR1 at kinetochores after 8.5 h of culture. Our results suggest that Axin-1 is critical for spindle organization and cell cycle progression during meiotic maturation in mouse oocytes. PMID:27284927

  6. BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes.

    PubMed

    Yu, Chao; Ji, Shu-Yan; Sha, Qian-Qian; Dang, Yujiao; Zhou, Jian-Jie; Zhang, Yin-Li; Liu, Yang; Wang, Zhong-Wei; Hu, Boqiang; Sun, Qing-Yuan; Sun, Shao-Chen; Tang, Fuchou; Fan, Heng-Yu

    2016-05-01

    The mRNAs stored in oocytes undergo general decay during the maternal-zygotic transition (MZT), and their stability is tightly interconnected with meiotic cell-cycle progression. However, the factors that trigger decay of maternal mRNA and couple this event to oocyte meiotic maturation remain elusive. Here, we identified B-cell translocation gene-4 (BTG4) as an MZT licensing factor in mice. BTG4 bridged CNOT7, a catalytic subunit of the CCR4-NOT deadenylase, to eIF4E, a key translation initiation factor, and facilitated decay of maternal mRNA. Btg4-null females produced morphologically normal oocytes but were infertile, owing to early developmental arrest. The intrinsic MAP kinase cascade in oocytes triggered translation of Btg4 mRNA stored in fully grown oocytes by targeting the 3' untranslated region, thereby coupling CCR4-NOT deadenylase-mediated decay of maternal mRNA with oocyte maturation and fertilization. This is a key step in oocyte cytoplasmic maturation that determines the developmental potential of mammalian embryos.

  7. Chromosome Cohesion Established by Rec8-Cohesin in Fetal Oocytes Is Maintained without Detectable Turnover in Oocytes Arrested for Months in Mice

    PubMed Central

    Burkhardt, Sabrina; Borsos, Máté; Szydlowska, Anna; Godwin, Jonathan; Williams, Suzannah A.; Cohen, Paula E.; Hirota, Takayuki; Saitou, Mitinori; Tachibana-Konwalski, Kikuë

    2016-01-01

    Summary Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation in mitosis and meiosis [1]. Rec8-containing cohesin, bound to Smc3/Smc1α or Smc3/Smc1β, maintains bivalent cohesion in mammalian meiosis [2, 3, 4, 5, 6]. In females, meiotic DNA replication and recombination occur in fetal oocytes. After birth, oocytes arrest at the prolonged dictyate stage until recruited to grow into mature oocytes that divide at ovulation. How cohesion is maintained in arrested oocytes remains a pivotal question relevant to maternal age-related aneuploidy. Hypothetically, cohesin turnover regenerates cohesion in oocytes. Evidence for post-replicative cohesion establishment mechanism exists, in yeast and invertebrates [7, 8]. In mouse fetal oocytes, cohesin loading factor Nipbl/Scc2 localizes to chromosome axes during recombination [9, 10]. Alternatively, cohesion is maintained without turnover. Consistent with this, cohesion maintenance does not require Smc1β transcription, but unlike Rec8, Smc1β is not required for establishing bivalent cohesion [11, 12]. Rec8 maintains cohesion without turnover during weeks of oocyte growth [3]. Whether the same applies to months or decades of arrest is unknown. Here, we test whether Rec8 activated in arrested mouse oocytes builds cohesion revealed by TEV cleavage and live-cell imaging. Rec8 establishes cohesion when activated during DNA replication in fetal oocytes using tamoxifen-inducible Cre. In contrast, no new cohesion is detected when Rec8 is activated in arrested oocytes by tamoxifen despite cohesin synthesis. We conclude that cohesion established in fetal oocytes is maintained for months without detectable turnover in dictyate-arrested oocytes. This implies that women’s fertility depends on the longevity of cohesin proteins that established cohesion in utero. PMID:26898469

  8. The Microtubule-Associated Protein ASPM Regulates Spindle Assembly and Meiotic Progression in Mouse Oocytes

    PubMed Central

    Xu, Xiao-Ling; Ma, Wei; Zhu, Yu-Bo; Wang, Chao; Wang, Bing-Yuan; An, Na; An, Lei; Liu, Yan; Wu, Zhong-Hong; Tian, Jian-Hui

    2012-01-01

    The microtubule-associated protein ASPM (abnormal spindle-like microcephaly-associated) plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes. PMID:23152892

  9. Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing--a review article.

    PubMed

    Chen, S U; Lien, Y R; Chao, K H; Ho, H N; Yang, Y S; Lee, T Y

    2003-04-28

    Embryo freezing has been a successful practice, but oocyte cryopreservation formerly achieved poorer results. This was mainly due to low rates of survival, fertilization, and development. The major dissimilarities for oocytes to embryos are the character of the plasma membrane, the presence of cortical granules, at the metaphase of meiosis II with the spindle system. In addition, the oocytes must be fertilized by sperm at the appropriate time. To improve the survival rate, a refined slow freezing method with increased sucrose concentration would dehydrate oocytes more sufficiently. Vitrification is another approach to prevent ice crystal formation. Intracytoplasmic sperm injection is used to overcome possible zona hardening from the release of cortical granules. The microtubules of meiotic spindles are vulnerable to the thermal changes and would depolymerize. Cryopreserved oocytes exhibited serious disturbances of the microtubules immediately after thawing. Fertilization of oocytes with disorganized spindles could lead to chromosomal aneuploidy, digyny, and arrest of cleavage. After incubation, the microtubules would repolymerize in a time-dependent way. Normal fertilization and development of cryopreserved oocytes improved after appropriate incubation and timing of insemination, compatible with recovery of the spindles. With the improvement of survival, fertilization, and cleavage, oocyte cryopreservation would gain an imperative role.

  10. EZH2 is required for mouse oocyte meiotic maturation by interacting with and stabilizing spindle assembly checkpoint protein BubRI

    PubMed Central

    Qu, Yi; Lu, Danyu; Jiang, Hao; Chi, Xiaochun; Zhang, Hongquan

    2016-01-01

    Enhancer of zeste homolog 2 (EZH2) trimethylates histone H3 Lys 27 and plays key roles in a variety of biological processes. Stability of spindle assembly checkpoint protein BubR1 is essential for mitosis in somatic cells and for meiosis in oocytes. However, the role of EZH2 in oocyte meiotic maturation was unknown. Here, we presented a mechanism underlying EZH2 control of BubR1 stability in the meiosis of mouse oocytes. We identified a methyltransferase activity-independent function of EZH2 by demonstrating that EZH2 regulates spindle assembly and the polar body I extrusion. EZH2 was increased with the oocyte progression from GVBD to MII, while EZH2 was concentrated on the chromosomes. Interestingly, inhibition of EZH2 methyltranferase activity by DZNep or GSK343 did not affect oocyte meiotic maturation. However, depletion of EZH2 by morpholino led to chromosome misalignment and abnormal spindle assembly. Furthermore, ectopic expression of EZH2 led to oocyte meiotic maturation arrested at the MI stage followed by chromosome misalignment and aneuploidy. Mechanistically, EZH2 directly interacted with and stabilized BubR1, an effect driving EZH2 into the concert of meiosis regulation. Collectively, we provided a paradigm that EZH2 is required for mouse oocyte meiotic maturation. PMID:27226494

  11. Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors.

    PubMed

    Nagaoka, So Iha; Hodges, Craig A; Albertini, David F; Hunt, Patricia Ann

    2011-04-26

    Segregation of homologs at the first meiotic division (MI) is facilitated by crossovers and by a physical constraint imposed on sister kinetochores that facilitates monopolar attachment to the MI spindle. Recombination failure or premature separation of homologs results in univalent chromosomes at MI, and univalents constrained to form monopolar attachments should be inherently unstable and trigger the spindle assembly checkpoint (SAC). Although univalents trigger cell-cycle arrest in the male, this is not the case in mammalian oocytes. Because the spindle assembly portion of the SAC appears to function normally, two hypotheses have been proposed to explain the lack of response to univalents: (1) reduced stringency of the oocyte SAC to aberrant chromosome behavior, and (2) the ability of univalents to satisfy the SAC by forming bipolar attachments. The present study of Mlh1 mutant mice demonstrates that metaphase alignment is not a prerequisite for anaphase onset and provides strong evidence that MI spindle stabilization and anaphase onset require stable bipolar attachment of a critical mass--but not all--of chromosomes. We postulate that subtle differences in SAC-mediated control make the human oocyte inherently error prone and contribute to the age-related increase in aneuploidy.

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

    PubMed

    Kalous, Jaroslav; Kubelka, Michal; Solc, Petr; Susor, Andrej; Motlík, Jan

    2009-10-01

    The aim of this study was to investigate the involvement of the serine/threonine protein kinase AKT (also called protein kinase B) in the control of meiosis of porcine denuded oocytes (DOs) matured in vitro. Western blot analysis revealed that the two principal AKT phosphorylation sites, Ser473 and Thr308, are phosphorylated at different stages of meiosis. In freshly isolated germinal vesicle (GV)-stage DOs, Ser473 was already phosphorylated. After the onset of oocyte maturation, the intensity of the Ser473 phosphorylation increased, however, which declined sharply when DOs underwent GV breakdown (GVBD) and remained at low levels in metaphase I- and II-stage (MI- and MII-stage). In contrast, phosphorylation of Thr308 was increased by the time of GVBD and reached maximum at MI-stage. A peak of AKT activity was noticed around GVBD and activity of AKT declined at MI-stage. To assess the role of AKT during meiosis, porcine DOs were cultured in 50 microM SH-6, a specific inhibitor of AKT. In SH-6-treated DOs, GVBD was not inhibited; on the contrary, a significant acceleration of meiosis resumption was observed. The dynamics of the Ser473 phosphorylation was not affected; however, phosphorylation of Thr308 was reduced, AKT activity was diminished at the time of GVBD, and meiotic progression was arrested in early MI-stage. Moreover, the activity of the cyclin-dependent kinase 1 (CDK1) and MAP kinase declined when SH-6-treated DOs underwent GVBD, indicating that AKT activity is involved in the regulation of CDK1 and MAP kinase. These results suggest that activity of AKT is not essential for induction of GVBD in porcine oocytes but plays a substantial role during progression of meiosis to MI/MII-stage.

  13. The role of Rad51 in safeguarding mitochondrial activity during the meiotic cell cycle in mammalian oocytes

    PubMed Central

    Kim, Kyeoung-Hwa; Park, Ji-Hoon; Kim, Eun-Young; Ko, Jung-Jae; Park, Kyung-Soon; Lee, Kyung-Ah

    2016-01-01

    Rad51 is a conserved eukaryotic protein that mediates the homologous recombination repair of DNA double-strand breaks that occur during mitosis and meiosis. In addition, Rad51 promotes mitochondrial DNA synthesis when replication stress is increased. Rad51 also regulates cell cycle progression by preserving the G2/M transition in embryonic stem cells. In this study, we report a novel function of Rad51 in regulating mitochondrial activity during in vitro maturation of mouse oocytes. Suppression of Rad51 by injection of Rad51 dsRNA into germinal vesicle-stage oocytes resulted in arrest of meiosis in metaphase I. Rad51-depleted oocytes showed chromosome misalignment and failures in spindle aggregation, affecting the completion of cytokinesis. We found that Rad51 depletion was accompanied by decreased ATP production and mitochondrial membrane potential and increased DNA degradation. We further demonstrated that the mitochondrial defect activated autophagy in Rad51-depleted oocytes. Taken together, we concluded that Rad51 functions to safeguard mitochondrial integrity during the meiotic maturation of oocytes. PMID:27677401

  14. Structural damage to meiotic chromosomes impairs DNA recombination and checkpoint control in mammalian oocytes.

    PubMed

    Wang, Hong; Höög, Christer

    2006-05-22

    Meiosis in human oocytes is a highly error-prone process with profound effects on germ cell and embryo development. The synaptonemal complex protein 3 (SYCP3) transiently supports the structural organization of the meiotic chromosome axis. Offspring derived from murine Sycp3(-)(/)(-) females die in utero as a result of aneuploidy. We studied the nature of the proximal chromosomal defects that give rise to aneuploidy in Sycp3(-)(/)(-) oocytes and how these errors evade meiotic quality control mechanisms. We show that DNA double-stranded breaks are inefficiently repaired in Sycp3(-)(/)(-) oocytes, thereby generating a temporal spectrum of recombination errors. This is indicated by a strong residual gammaH2AX labeling retained at late meiotic stages in mutant oocytes and an increased persistence of recombination-related proteins associated with meiotic chromosomes. Although a majority of the mutant oocytes are rapidly eliminated at early postnatal development, a subset with a small number of unfinished crossovers evades the DNA damage checkpoint, resulting in the formation of aneuploid gametes. PMID:16717125

  15. Mouse oocytes depend on BubR1 for proper chromosome segregation but not for prophase I arrest

    PubMed Central

    Touati, Sandra A.; Buffin, Eulalie; Cladière, Damien; Hached, Khaled; Rachez, Christophe; van Deursen, Jan M.; Wassmann, Katja

    2015-01-01

    Mammalian female meiosis is error prone, with rates of meiotic chromosome missegregations strongly increasing towards the end of the reproductive lifespan. A strong reduction of BubR1 has been observed in oocytes of women approaching menopause and in ovaries of aged mice, which led to the hypothesis that a gradual decline of BubR1 contributes to age-related aneuploidization. Here we employ a conditional knockout approach in mouse oocytes to dissect the meiotic roles of BubR1. We show that BubR1 is required for diverse meiotic functions, including persistent spindle assembly checkpoint activity, timing of meiosis I and the establishment of robust kinetochore-microtubule attachments in a meiosis-specific manner, but not prophase I arrest. These data reveal that BubR1 plays a multifaceted role in chromosome segregation during the first meiotic division and suggest that age-related decline of BubR1 is a key determinant of the formation of aneuploid oocytes as women approach menopause. PMID:25897860

  16. FSH Modulates PKAI and GPR3 Activities in Mouse Oocyte of COC in a Gap Junctional Communication (GJC)-Dependent Manner to Initiate Meiotic Resumption

    PubMed Central

    Xia, Guoliang

    2012-01-01

    Many studies have shown that cyclic adenosine-5′-monophosphate (cAMP)-dependent protein kinase A (PKA) and G-protein-coupled receptor 3 (GPR3) are crucial for controlling meiotic arrest in oocytes. However, it is unclear how gonadotropins modulate these factors to regulate oocyte maturation, especially by gap junctional communication (GJC). Using an in vitro meiosis-arrested mouse cumulus-oocyte complex (COC) culture model, we showed that there is a close relationship between follicle-stimulating hormone (FSH) and the PKA type I (PKAI) and GPR3. The effect of FSH on oocyte maturation was biphasic, initially inhibitory and then stimulatory. During FSH-induced maturation, rapid cAMP surges were observed in both cumulus cells and oocyte. Most GJC between cumulus cells and oocyte ceased immediately after FSH stimulation and recommenced after the cAMP surge. FSH-induced maturation was blocked by PKAI activator 8-AHA-cAMP. Levels of PKAI regulatory subunits and GPR3 decreased and increased, respectively, after FSH stimulation. In the presence of the GJC inhibitor carbenoxolone (CBX), FSH failed to induce the meiotic resumption and the changes in PKAI, GPR3 and cAMP surge in oocyte were no longer detected. Furthermore, GPR3 was upregulated by high cAMP levels, but not by PKAI activation. When applied after FSH stimulation, the specific phosphodiesterase 3A (PDE3A) inhibitor cilostamide immediately blocked meiotic induction, regardless of when it was administered. PKAI activation inhibited mitogen-activated protein kinase (MAPK) phosphorylation in the oocytes of COCs, which participated in the initiation of FSH-induced meiotic maturation in vitro. Just before FSH-induced meiotic maturation, cAMP, PKAI, and GPR3 returned to basal levels, and PDE3A activity and MAPK phosphorylation increased markedly. These experiments show that FSH induces a transient increase in cAMP levels and regulates GJC to control PKAI and GPR3 activities, thereby creating an inhibitory phase. After

  17. Assessment of meiotic spindle configuration and post-warming bovine oocyte viability using polarized light microscopy.

    PubMed

    Caamaño, J N; Díez, C; Trigal, B; Muñoz, M; Morató, R; Martín, D; Carrocera, S; Mogas, T; Gómez, E

    2013-06-01

    The objectives of this study were to assess the efficiency of polarized light microscopy (PLM) in detecting microtubule-polymerized protein in in vitro-matured bovine oocytes; to examine its effects on oocyte developmental competence; and to assess the meiotic spindle of in vitro-matured oocytes after vitrification/warming and further assessment of oocyte developmental competence. In the first experiment, the presence of microtubule-polymerized protein (MPP) was confirmed as a positive PLM signal detected in 99.1% of analysed oocytes (n = 115), which strongly correlated (r = 1; p < 0.0001) with the presence of MPP as confirmed by immunostaining. In the second experiment, oocytes (n = 651) were exposed or not (controls) to PLM for 10 min and then fertilized and cultured in vitro. Oocytes exposed to PLM did not significantly differ from controls with regard to cleavage, total blastocyst and expanded blastocyst rates and cell numbers. In the third experiment, meiotic spindles were detected in 145 of 182 oocytes (79.6%) following vitrification and warming. Interestingly, after parthenogenetic activation and in vitro culture, oocytes that displayed a positive PLM signal PLM(+) differed significantly from PLM(-) in cleavage and Day 8 blastocyst rates. These results suggest that polarized light microscopy is an efficient system to detect microtubule-polymerized protein in in vitro-matured bovine oocytes and does not exert detrimental effects on bovine oocyte developmental competence. Moreover, PLM could be used as a tool to assess post-warming viability in vitrified bovine oocytes.

  18. Lipopolysaccharide initiates inflammation in bovine granulosa cells via the TLR4 pathway and perturbs oocyte meiotic progression in vitro

    PubMed Central

    Bromfield, John J.; Sheldon, I. Martin

    2012-01-01

    Infections of the reproductive tract or mammary gland with Gram-negative bacteria perturb ovarian function, follicular growth and fecundity in cattle. We hypothesised that lipopolysaccharide (LPS) from Gram-negative bacteria stimulates an inflammatory response by ovarian granulosa cells that is mediated by TLR4. The present study tested the capacity of bovine ovarian granulosa cells to initiate an inflammatory response to pathogen associated molecular patterns (PAMPs), and determined subsequent effects on the in vitro maturation of oocytes. Granulosa cells elicited an inflammatory response to PAMPs (LPS, lipoteichoic acid, peptidoglycan or Pam3CSK4) with accumulation of the cytokine IL-6, and the chemokine IL-8, in a time- and dose-dependent manner. Granulosa cells responded acutely to LPS with rapid phosphorylation of TLR signaling components, p38 and ERK, and increased expression of IL6 and IL8 mRNA, although nuclear translocation of p65 was not evident. Targeting TLR4 with siRNA, attenuated granulosa cell accumulation of IL-6 in response to LPS. Endocrine function of granulosa cells is regulated by FSH, but here FSH also enhanced responsiveness to LPS, increasing IL-6 and IL-8 accumulation. Furthermore, LPS stimulated IL-6 secretion and expansion by cumulus-oocyte complexes (COCs), and increased rates of meiotic arrest and germinal vesicle breakdown failure. In conclusion, bovine granulosa cells initiate an innate immune response to LPS via the TLR4 pathway leading to inflammation and to perturbation of meiotic competence. PMID:21990308

  19. Effect of temporary nuclear arrest by phosphodiesterase 3-inhibitor on morphological and functional aspects of in vitro matured mouse oocytes.

    PubMed

    Vanhoutte, Leen; Nogueira, Daniela; Gerris, Jan; Dhont, Marc; De Sutter, Petra

    2008-06-01

    The present study aimed to analyze detailed morphological and functional characteristics of mouse in vitro matured oocytes after a pre-maturation culture (PMC) by temporary nuclear arrest with the specific phosphodiesterase 3-inhibitor (PDE3-I) Cilostamide. In a first experiment the lowest effective dose of Cilostamide was determined. Cumulus-oocyte complexes (COCs), isolated from small antral follicles, were exposed to different concentrations of Cilostamide (ranging from 0 (control) to 10 microM) for 24 hr. Afterwards, oocytes were removed from PDE3-I-containing medium and underwent in vitro maturation (IVM) for 16-18 hr. A concentration of 1 microM Cilostamide was the lowest effective dose for maximum level of inhibition and reversibility of meiosis inhibition. This concentration was used in further experiments to evaluate oocyte quality following IVM in relation to different parameters: kinetics of meiotic progression, metaphase II (MII) spindle morphology, aneuploidy rate, fertilization, and embryonic developmental rates. The results were compared to nonarrested (in vitro control) and in vivo matured oocytes (in vivo control). Following withdrawal of the inhibitor, the progression of meiosis was more synchronous and accelerated in arrested when compared to nonarrested oocytes. A PMC resulted in a significant increase in the number of oocytes constituting a MII spindle of normal morphology. None of the oocytes exposed to PDE3-I showed numerical chromosome alterations. In addition, fertilization and embryonic developmental rates were higher in the PMC group compared to in vitro controls, but lower than in vivo controls. These results provide evidence that induced nuclear arrest by PDE3-I is a safe and reliable method to improve oocyte quality after IVM.

  20. Timing of meiotic progression in bovine oocytes and its effect on early embryo development.

    PubMed

    Dominko, T; First, N L

    1997-08-01

    This study was designed to investigate the effect of the kinetics of nuclear maturation in bovine oocytes on early embryo development and to examine whether the time of insemination of mature oocytes affects the oocytes' ability to support events of early embryo development. The time required for completion of nuclear maturation was influenced by gonadotropins used to supplement the maturation medium. Luteinizing hormone (LH) enhanced the speed of nuclear maturation when compared to follicle-stimulating hormone (FSH). Oocytes completing their nuclear maturation early (by 16 hours after the initiation of culture) were more likely to complete the first embryonic cell cycle (78% in LH vs. 43% in FSH) and develop to the blastocyst stage (47% in LH vs. 34% in FSH). As the age of the oocytes at the time of MII arrest increased (extrusion of the polar body by 20 or 24 hours), a decrease in their ability to cleave and develop to the blastocyst stage was observed. Differences in the oocyte's ability to decondense chromatin and form pronuclei were also observed. Early maturing oocytes started forming pronuclei earlier than their later maturing counterparts. The time of insemination of mature oocytes played an equally important role. Generally, when insemination of mature oocytes was delayed for 8 hours, higher proportions of fertilized oocytes developed to advanced preimplantation stages than did the oocytes inseminated immediately after metaphase II arrest. PMID:9211431

  1. Timing of meiotic progression in bovine oocytes and its effect on early embryo development.

    PubMed

    Dominko, T; First, N L

    1997-08-01

    This study was designed to investigate the effect of the kinetics of nuclear maturation in bovine oocytes on early embryo development and to examine whether the time of insemination of mature oocytes affects the oocytes' ability to support events of early embryo development. The time required for completion of nuclear maturation was influenced by gonadotropins used to supplement the maturation medium. Luteinizing hormone (LH) enhanced the speed of nuclear maturation when compared to follicle-stimulating hormone (FSH). Oocytes completing their nuclear maturation early (by 16 hours after the initiation of culture) were more likely to complete the first embryonic cell cycle (78% in LH vs. 43% in FSH) and develop to the blastocyst stage (47% in LH vs. 34% in FSH). As the age of the oocytes at the time of MII arrest increased (extrusion of the polar body by 20 or 24 hours), a decrease in their ability to cleave and develop to the blastocyst stage was observed. Differences in the oocyte's ability to decondense chromatin and form pronuclei were also observed. Early maturing oocytes started forming pronuclei earlier than their later maturing counterparts. The time of insemination of mature oocytes played an equally important role. Generally, when insemination of mature oocytes was delayed for 8 hours, higher proportions of fertilized oocytes developed to advanced preimplantation stages than did the oocytes inseminated immediately after metaphase II arrest.

  2. SLX2 interacting with BLOS2 is differentially expressed during mouse oocyte meiotic maturation.

    PubMed

    Zhuang, Xin-Jie; Shi, Yu-Qiang; Xu, Bo; Chen, Lei; Tang, Wen-Hao; Huang, Jin; Lian, Ying; Liu, Ping; Qiao, Jie

    2014-01-01

    Gametogenesis is a complex biological process of producing cells for sexual reproduction. Xlr super family members containing a conserved COR1 domain play essential roles in gametogenesis. In the present study, we identified that Slx2, a novel member of Xlr super family, is specifically expressed in the meiotic oocytes, which is demonstrated by western blotting and immunohistochemistry studies. In the first meiotic prophase, SLX2 is unevenly distributed in the nuclei of oocytes, during which phase SLX2 is partly co-localized with SYCP3 in synaptonemal complex and γH2AX in the nucleus of oocytes. Interestingly, the localization of SLX2 was found to be switched into the cytoplasm of oocytes after prometaphase I during oocyte maturation. Furthermore, yeast two-hybrid and coimmunoprecipitation studies demonstrated that SLX2 interacts with BLOS2, which is a novel centrosome-associated protein, and co-localized with γ-Tubulin, which is a protein marker of chromosome segregation in meiosis. These results indicated that SLX2 might get involved in chromosomes segregation during meiosis by interaction with BLOS2. In conclusion, SLX2 might be a novel gametogenesis-related protein that could play multiple roles in regulation of meiotic processes including synaptonemal complex assembly and chromosome segregation. PMID:24870619

  3. Reduction of nitric oxide level leads to spontaneous resumption of meiosis in diplotene-arrested rat oocytes cultured in vitro

    PubMed Central

    Pandey, Ashutosh N

    2015-01-01

    The present study was aimed to investigate whether a decrease of nitric oxide (NO) level is beneficial for sponateous resumptiom of meiosis in diplotene-arrested oocytes cultured in vitro. For this purpose, diplotene-arrested oocytes were collected from ovary of immature female rats after a single subcutaneous injection of 20 IU pregnant mare’s serum gonadotropins (PMSG) for 48 h. In vitro effects of S-nitroso-l-acetyl penicillamine (SNAP; an NO donor) and aminoguanidine (AG; an inducible NOS [iNOS] inhibitor), intracellular NO, cyclic guanosine monophosphate (cGMP), Cdc25B, Thr-14/Tyr-15 and Thr-161 phosphorylated cyclin-dependent kinase-1 (CDK1), and cyclin B1 levels were analyzed. The SNAP inhibited spontaneous meiotic resumption form diplotene arrest in a concentration-dependent manner, while AG-induced meiotic resumption form diplotene in 0.1 mmol/L 3-isobutyl-1-methylxanthine (IBMX)-treated oocytes in a concentration-dependent manner. The intracellular NO as well as cGMP levels were decreased significantly during spontaneous meiotic resumption from diplotene arrest. The reduction of Cdc25B expression level was associated with the accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. However, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels were reduced significantly during meiotic resumption from diplotene arrest. Taken together, these data suggest that the inhibition of iNOS expression leads to a decrease of NO and cGMP levels thereby decreasing Cdc25B level. The reduced CDC25 B level leads to accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. As a result, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels are decreased leading to maturation-promoting factor (MPF) inactivation. The inactive MPF finally induced meiotic resumption from diplotene stage in rat oocytes cultured in vitro. PMID:25092827

  4. Histone H2AFX Links Meiotic Chromosome Asynapsis to Prophase I Oocyte Loss in Mammals

    PubMed Central

    Cloutier, Jeffrey M.; Mahadevaiah, Shantha K.; ElInati, Elias; Nussenzweig, André; Tóth, Attila; Turner, James M. A.

    2015-01-01

    Chromosome abnormalities are common in the human population, causing germ cell loss at meiotic prophase I and infertility. The mechanisms driving this loss are unknown, but persistent meiotic DNA damage and asynapsis may be triggers. Here we investigate the contribution of these lesions to oocyte elimination in mice with chromosome abnormalities, e.g. Turner syndrome (XO) and translocations. We show that asynapsed chromosomes trigger oocyte elimination at diplonema, which is linked to the presence of phosphorylated H2AFX (γH2AFX). We find that DNA double-strand break (DSB) foci disappear on asynapsed chromosomes during pachynema, excluding persistent DNA damage as a likely cause, and demonstrating the existence in mammalian oocytes of a repair pathway for asynapsis-associated DNA DSBs. Importantly, deletion or point mutation of H2afx restores oocyte numbers in XO females to wild type (XX) levels. Unexpectedly, we find that asynapsed supernumerary chromosomes do not elicit prophase I loss, despite being enriched for γH2AFX and other checkpoint proteins. These results suggest that oocyte loss cannot be explained simply by asynapsis checkpoint models, but is related to the gene content of asynapsed chromosomes. A similar mechanistic basis for oocyte loss may operate in humans with chromosome abnormalities. PMID:26509888

  5. MBTD1 is associated with Pr-Set7 to stabilize H4K20me1 in mouse oocyte meiotic maturation.

    PubMed

    Luo, Yi-Bo; Ma, Jun-Yu; Zhang, Qing-Hua; Lin, Fei; Wang, Zhong-Wei; Huang, Lin; Schatten, Heide; Sun, Qing-Yuan

    2013-04-01

    H4K20me1 is a critical histone lysine methyl modification in eukaryotes. It is recognized and "read" by various histone lysine methyl modification binding proteins. In this study, the function of MBTD1, a member of the Polycomb protein family containing four MBT domains, was comprehensively studied in mouse oocyte meiotic maturation. The results showed that depletion of MBTD1 caused reduced expression of histone lysine methyl transferase Pr-Set7 and H4K20me1 as well as increased oocyte arrest at the GV stage. Increased γH2AX foci were formed, and DNA damage repair checkpoint protein 53BP1 was downregulated. Furthermore, depletion of MBTD1 activated the cell cycle checkpoint protein Chk1 and downregulated the expression of cyclin B1 and cdc2. MBTD1 knockdown also affected chromosome configuration in GV stage oocytes and chromosome alignment at the MII stage. All these phenotypes were reproduced when the H4K20 methyl transferase Pr-Set7 was depleted. Co-IP demonstrated that MBTD1 was correlated with Pr-Set7 in mouse oocytes. Our results demonstrate that MBTD1 is associated with Pr-Set7 to stabilize H4K20me1 in mouse oocyte meiotic maturation. PMID:23475131

  6. Live Imaging of Intracellular Dynamics During Meiotic Maturation in Mouse Oocytes.

    PubMed

    Yoshida, Shuhei; Sakakibara, Yogo; Kitajima, Tomoya S

    2016-01-01

    Fluorescence live imaging is a powerful approach to study intracellular dynamics during cellular events such as cell division. By applying automated confocal live imaging to mouse oocytes, in which meiotic maturation can be induced in vitro after the introduction of fluorescent proteins through microinjection, the meiotic dynamics of intracellular structures, such as chromosomes, can be monitored at high resolution. A combination of this method with approaches for the perturbation of specific proteins opens up opportunities for understanding the molecular and intracellular basis of mammalian meiosis. PMID:27557586

  7. Effect of gap junction uncoupling in full-grown Bufo arenarum ovarian follicles: participation of cAMP in meiotic arrest.

    PubMed

    Villecco, E I; Aybar, M J; Genta, S B; Sánchez, S S; Sánchez Riera, A N

    2000-05-01

    The aim of the present study was to determine the presence of the connexins Cx43, Cx32 and Cx26 in Bufo arenarum ovarian follicles during the breeding season as well as to analyse the possible alterations in the meiotic process when connexins are blocked by specific antibodies. Western blot analysis revealed that the Cx43 and Cx32 proteins were present but not Cx26. We demonstrated that the anti-Cx43 and anti-Cx32 antibodies produced the uncoupling of the gap junctions. When these junctions are blocked the maturation process is triggered in the oocytes. We determined that dbcAMP exerts an inhibitory effect on the maturation induced by the uncoupling of the gap junctions when the oocytes are injected or pretreated with this metabolite. We propose the idea that cAMP is the regulatory molecule in meiotic arrest in this amphibian species. PMID:10857588

  8. Oocyte heterogeneity with respect to the meiotic silencing of unsynapsed X chromosomes in the XY female mouse.

    PubMed

    Taketo, Teruko; Naumova, Anna K

    2013-10-01

    In the XY pachytene spermatocyte, the sex chromosomes do not synapse except for the pseudoautosomal region and become transcriptionally silenced. It has been suggested that the meiotic silencing of unsynapsed chromatin (MSUC) also occurs in oocytes. In the XY sex-reversed female mouse, the sex chromosomes fail to pair in the majority of oocytes and a greater number of oocytes are eliminated during the meiotic prophase compared to the XX female. Yet, many XY oocytes survive to reach the second meiotic metaphase. The goal of our current study was to determine whether the single X chromosome shows the characteristics of asynapsis and meiotic silencing in a proportion of XY oocytes, which can explain the survival of the remaining oocytes. We first examined the accumulation of markers associated with asynapsis or transcriptional silencing, i.e., BRCA1, γH2AX, H3K9me3, and H3K27me3, at the single X chromosome in the XY oocyte. We found that γH2AX and BRCA1 were enriched on the single X chromosome whereas H3K9me3 was not, and H3K27me3 was enriched at all chromosomes in the majority of XY oocytes. We next examined the meiotic silencing of the single X chromosome using enrichment of the X-encoded ATRX protein. On average, ATRX enrichment was lower in XY oocytes than in XX oocytes as expected from its half gene dosage. However, the intensity of ATRX staining in XY oocytes harboring γH2AX domains showed a remarkable heterogeneity. We conclude that MSUC occurs with varying consequences, resulting in a heterogeneous population of oocytes with respect to protein enrichment in the XY female mouse. PMID:23760560

  9. Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes

    SciTech Connect

    Peaucellier, G.; Andersen, A.C.; Kinsey, W.H. )

    1990-04-01

    We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.

  10. PDK1 is required for the hormonal signaling pathway leading to meiotic resumption in starfish oocytes.

    PubMed

    Hiraoka, Daisaku; Hori-Oshima, Sawako; Fukuhara, Takeshi; Tachibana, Kazunori; Okumura, Eiichi; Kishimoto, Takeo

    2004-12-15

    Meiotic resumption is generally under the control of an extracellular maturation-inducing hormone. It is equivalent to the G2-M phase transition in somatic cell mitosis and is regulated by cyclin B-Cdc2 kinase. However, the complete signaling pathway from the hormone to cyclin B-Cdc2 is yet unclear in any organism. A model system to analyze meiotic resumption is the starfish oocyte, in which Akt/protein kinase B (PKB) plays a key mediator in hormonal signaling that leads to cyclin B-Cdc2 activation. Here we show in starfish oocytes that when PDK1 activity is inhibited by a neutralizing antibody, maturation-inducing hormone fails to induce cyclin B-Cdc2 activation at the meiotic G2-M phase transition, even though PDK2 activity becomes detectable. These observations assign a novel role to PDK1 for a hormonal signaling intermediate toward meiotic resumption. They further support that PDK2 is a molecule distinct from PDK1 and Akt, and that PDK2 activity is not sufficient for the full activation of Akt in the absence of PDK1 activity. PMID:15581868

  11. Rejuvenation of Meiotic Cohesion in Oocytes during Prophase I Is Required for Chiasma Maintenance and Accurate Chromosome Segregation

    PubMed Central

    Weng, Katherine A.; Jeffreys, Charlotte A.; Bickel, Sharon E.

    2014-01-01

    Chromosome segregation errors in human oocytes are the leading cause of birth defects, and the risk of aneuploid pregnancy increases dramatically as women age. Accurate segregation demands that sister chromatid cohesion remain intact for decades in human oocytes, and gradual loss of the original cohesive linkages established in fetal oocytes is proposed to be a major cause of age-dependent segregation errors. Here we demonstrate that maintenance of meiotic cohesion in Drosophila oocytes during prophase I requires an active rejuvenation program, and provide mechanistic insight into the molecular events that underlie rejuvenation. Gal4/UAS inducible knockdown of the cohesion establishment factor Eco after meiotic S phase, but before oocyte maturation, causes premature loss of meiotic cohesion, resulting in destabilization of chiasmata and subsequent missegregation of recombinant homologs. Reduction of individual cohesin subunits or the cohesin loader Nipped B during prophase I leads to similar defects. These data indicate that loading of newly synthesized replacement cohesin rings by Nipped B and establishment of new cohesive linkages by the acetyltransferase Eco must occur during prophase I to maintain cohesion in oocytes. Moreover, we show that rejuvenation of meiotic cohesion does not depend on the programmed induction of meiotic double strand breaks that occurs during early prophase I, and is therefore mechanistically distinct from the DNA damage cohesion re-establishment pathway identified in G2 vegetative yeast cells. Our work provides the first evidence that new cohesive linkages are established in Drosophila oocytes after meiotic S phase, and that these are required for accurate chromosome segregation. If such a pathway also operates in human oocytes, meiotic cohesion defects may become pronounced in a woman's thirties, not because the original cohesive linkages finally give out, but because the rejuvenation program can no longer supply new cohesive linkages

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

    PubMed

    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. PMID:24671882

  13. Factors affecting meiotic and developmental competence of primary spermatocyte nuclei injected into mouse oocytes.

    PubMed

    Kimura, Y; Tateno, H; Handel, M A; Yanagimachi, R

    1998-10-01

    Mature mouse oocytes that have received the nuclei of pachytene primary spermatocytes (or metaphase I chromosomes of primary spermatocytes) can develop into fertile offspring. However, success rate in this study was low. No more than 3.8% of transferred 2-cell embryos arising from spermatocyte-injected oocytes developed to full term. Nevertheless, the birth of normal offspring seems to suggest that at least in some primary spermatocytes the functional genomic imprinting is complete before transfer and/or consolidated after the transfer. Although injected spermatocyte nuclei could undergo two successive meiotic divisions within oocytes, abnormalities of both divisions were commonly observed, and sister chromatids often separated prematurely during the second meiotic division. Chromosome breakage/rearrangements were also frequently seen before the first cleavage. Such abnormalities of chromosome behavior are probably the major causes of the poor preimplantation development of zygotes arising from primary spermatocyte-injected oocytes. Thus, clinical use of primary spermatocytes as substitutes for spermatozoa in assisted fertilization is not advisable until the causes of chromosomal abnormalities are better understood through extensive animal studies. PMID:9746737

  14. The effect of temporary meiotic attenuation on the in vitro maturation outcome of bovine oocytes.

    PubMed

    Farghaly, T; Khalifa, E; Mostafa, S; Hussein, M; Bedaiwy, M; Ahmady, A

    2015-08-01

    The aim of this study was to investigate the effect of delaying maturation by extended culture of immature bovine oocytes in prematuration medium (PMC) containing single maturation inhibitor on their meiotic resumption and embryonic development. Bovine immature oocytes were cultured in M199 containing 10 μM of either inhibitor (roscovitine, cilostamide, or forskolin) for either 72 or 120 h followed by up to 48 h in maturation media supplemented with 7.5 IU follicle-stimulating hormone (FSH)/luteinizing hormone (LH). Two control groups were used. In untreated control, immature oocytes were cultured in the same medium as the experimental group without any inhibitors. In the FSH/LH control group, oocytes were cultured directly in the maturation medium supplemented with FSH/LH up to 48 h. In vitro matured oocytes were then inseminated with frozen-thawed bull sperm. Fertilization, defined as two-cell division 48 h post-insemination, and blastocyst formation were recorded. Total maturation rate for the 72-h group was 73%, 70%, 66%, and 69% for roscovitine, forskolin, cilostamide, and FSH/LH control, respectively, with no significant difference indicating that inhibitors have no negative effect on the oocyte maturation rate. The total fertilization rate for the 72-h group revealed that cilostamide (47%) and roscovitine (35%) were significantly higher than FSH/LH control (20%). The total blastocyst formation rates per inseminated oocytes revealed that among treatment groups, roscovitine (20%) had significantly higher rate than forskolin (9%). Overall, 72-h exposure period had better outcomes than 120 h in all the treated groups. In conclusion, prematuration culture of the bovine oocytes in the presence of maturation inhibitor for 72-h period at 10 μM concentration is sufficient in improving the bovine oocyte developmental competence.

  15. The effect of temporary meiotic attenuation on the in vitro maturation outcome of bovine oocytes.

    PubMed

    Farghaly, T; Khalifa, E; Mostafa, S; Hussein, M; Bedaiwy, M; Ahmady, A

    2015-08-01

    The aim of this study was to investigate the effect of delaying maturation by extended culture of immature bovine oocytes in prematuration medium (PMC) containing single maturation inhibitor on their meiotic resumption and embryonic development. Bovine immature oocytes were cultured in M199 containing 10 μM of either inhibitor (roscovitine, cilostamide, or forskolin) for either 72 or 120 h followed by up to 48 h in maturation media supplemented with 7.5 IU follicle-stimulating hormone (FSH)/luteinizing hormone (LH). Two control groups were used. In untreated control, immature oocytes were cultured in the same medium as the experimental group without any inhibitors. In the FSH/LH control group, oocytes were cultured directly in the maturation medium supplemented with FSH/LH up to 48 h. In vitro matured oocytes were then inseminated with frozen-thawed bull sperm. Fertilization, defined as two-cell division 48 h post-insemination, and blastocyst formation were recorded. Total maturation rate for the 72-h group was 73%, 70%, 66%, and 69% for roscovitine, forskolin, cilostamide, and FSH/LH control, respectively, with no significant difference indicating that inhibitors have no negative effect on the oocyte maturation rate. The total fertilization rate for the 72-h group revealed that cilostamide (47%) and roscovitine (35%) were significantly higher than FSH/LH control (20%). The total blastocyst formation rates per inseminated oocytes revealed that among treatment groups, roscovitine (20%) had significantly higher rate than forskolin (9%). Overall, 72-h exposure period had better outcomes than 120 h in all the treated groups. In conclusion, prematuration culture of the bovine oocytes in the presence of maturation inhibitor for 72-h period at 10 μM concentration is sufficient in improving the bovine oocyte developmental competence. PMID:25784604

  16. Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes.

    PubMed

    Fan, Heng-Yu; Huo, Li-Jun; Meng, Xiao-Qian; Zhong, Zhi-Sheng; Hou, Yi; Chen, Da-Yuan; Sun, Qing-Yuan

    2003-11-01

    Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins

  17. Kinetochore Fibers Are Not Involved in the Formation of the First Meiotic Spindle in Mouse Oocytes, but Control the Exit from the First Meiotic M Phase

    PubMed Central

    Brunet, Stéphane; Maria, Angélica Santa; Guillaud, Philippe; Dujardin, Denis; Kubiak, Jacek Z.; Maro, Bernard

    1999-01-01

    During meiosis, two successive divisions occur without any intermediate S phase to produce haploid gametes. The first meiotic division is unique in that homologous chromosomes are segregated while the cohesion between sister chromatids is maintained, resulting in a reductional division. Moreover, the duration of the first meiotic M phase is usually prolonged when compared with mitotic M phases lasting 8 h in mouse oocytes. We investigated the spindle assembly pathway and its role in the progression of the first meiotic M phase in mouse oocytes. During the first 4 h, a bipolar spindle forms and the chromosomes congress near the equatorial plane of the spindle without stable kinetochore– microtubule end interactions. This late prometaphase spindle is then maintained for 4 h with chromosomes oscillating in the central region of the spindle. The kinetochore–microtubule end interactions are set up at the end of the first meiotic M phase (8 h after entry into M phase). This event allows the final alignment of the chromosomes and exit from metaphase. The continuous presence of the prometaphase spindle is not required for progression of the first meiotic M phase. Finally, the ability of kinetochores to interact with microtubules is acquired at the end of the first meiotic M phase and determines the timing of polar body extrusion. PMID:10402455

  18. The Role of Microfilaments in Early Meiotic Maturation of Mouse Oocytes

    NASA Astrophysics Data System (ADS)

    Calarco, Patricia G.

    2005-04-01

    Mouse oocyte microfilaments (MF) were perturbed by depolymerization (cytochalasin B) or stabilization (jasplakinolide) and correlated meiotic defects examined by confocal microscopy. MF, microtubules, and mitochondria were vitally stained; centrosomes ([gamma]-tubulin), after fixation. MF depolymerization by cytochalasin in culture medium did not affect central migration of centrosomes, mitochondria, or nuclear breakdown (GVBD); some MF signal was localized around the germinal vesicle (GV). In maturation-blocking medium (containing IBMX), central movement was curtailed and cortical MF aggregations made the plasma membrane wavy. Occasional long MF suggested that not all MF were depolymerized. MF stabilization by jasplakinolide led to MF aggregations throughout the cytoplasm. GVBD occurred (unless IBMX was present) but no spindle formed. Over time, most oocytes constricted creating a dumbbell shape with MF concentrated under one-half of the oocyte cortex and on either side of the constriction. In IBMX medium, the MF-containing half of the dumbbell over time sequestered the GV, MF, mitochondria, and one to two large cortical centrosomes; the non-MF half appeared empty. Cumulus processes contacted the oocyte surface (detected by microtubule content) and mirrored MF distribution. Results demonstrated that MF play an essential role in meiosis, primarily through cortically mediated events, including centrosome localization, spindle (or GV) movement to the periphery, activation of (polar body) constriction, and establishment of oocyte polarity. The presence of a cortical “organizing pole” is hypothesized.

  19. Kinesin-1 Prevents Capture of the Oocyte Meiotic Spindle by the Sperm Aster

    PubMed Central

    McNally, Karen L.P.; Fabritius, Amy S.; Ellefson, Marina L.; Flynn, Jonathan R.; Milan, Jennifer A.; McNally, Francis J.

    2012-01-01

    Centrioles are lost during oogenesis and inherited from the sperm at fertilization. In the zygote, the centrioles recruit pericentriolar proteins from the egg to form a mature centrosome that nucleates a sperm aster. The sperm aster then captures the female pronucleus to join the maternal and paternal genomes. Because fertilization occurs before completion of female meiosis, some mechanism must prevent capture of the meiotic spindle by the sperm aster. Here we show that in wild-type Caenorhabditis elegans zygotes, maternal pericentriolar proteins are not recruited to the sperm centrioles until after completion of meiosis. Depletion of kinesin-1 heavy chain or its binding partner resulted in premature centrosome maturation during meiosis and growth of a sperm aster that could capture the oocyte meiotic spindle. Kinesin prevents recruitment of pericentriolar proteins by coating the sperm DNA and centrioles and thus prevents triploidy by a non-motor mechanism. PMID:22465668

  20. Activation of ADF/cofilin by phosphorylation-regulated Slingshot phosphatase is required for the meiotic spindle assembly in Xenopus laevis oocytes

    PubMed Central

    Iwase, Shohei; Sato, Ryuhei; De Bock, Pieter-Jan; Gevaert, Kris; Fujiki, Saburo; Tawada, Toshinobu; Kuchitsu, Miyako; Yamagishi, Yuka; Ono, Shoichiro; Abe, Hiroshi

    2013-01-01

    We identify Xenopus ADF/cofilin (XAC) and its activator, Slingshot phosphatase (XSSH), as key regulators of actin dynamics essential for spindle microtubule assembly during Xenopus oocyte maturation. Phosphorylation of XSSH at multiple sites within the tail domain occurs just after germinal vesicle breakdown (GVBD) and is accompanied by dephosphorylation of XAC, which was mostly phosphorylated in immature oocytes. This XAC dephosphorylation after GVBD is completely suppressed by latrunculin B, an actin monomer–sequestering drug. On the other hand, jasplakinolide, an F-actin–stabilizing drug, induces dephosphorylation of XAC. Effects of latrunculin B and jasplakinolide are reconstituted in cytostatic factor–arrested extracts (CSF extracts), and XAC dephosphorylation is abolished by depletion of XSSH from CSF extracts, suggesting that XSSH functions as an actin filament sensor to facilitate actin filament dynamics via XAC activation. Injection of anti-XSSH antibody, which blocks full phosphorylation of XSSH after GVBD, inhibits both meiotic spindle formation and XAC dephosphorylation. Coinjection of constitutively active XAC with the antibody suppresses this phenotype. Treatment of oocytes with jasplakinolide also impairs spindle formation. These results strongly suggest that elevation of actin dynamics by XAC activation through XSSH phosphorylation is required for meiotic spindle assembly in Xenopus laevis. PMID:23615437

  1. Dynamic secretion during meiotic reentry integrates the function of the oocyte and cumulus cells.

    PubMed

    Cakmak, Hakan; Franciosi, Federica; Zamah, A Musa; Cedars, Marcelle I; Conti, Marco

    2016-03-01

    The differentiation of the female gamete into a developmentally competent oocyte relies on the protected environment of the ovarian follicle. The oocyte plays a key role in establishing this microenvironment by releasing paracrine factors that control the functions of surrounding somatic cells. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are secreted during follicle growth and play pivotal roles in this local regulation. The current view is that the function of these secreted factors declines in the periovulatory period when the oocyte reenters the meiotic cell cycle. Here, we provide evidence that oocyte reentry into meiosis is instead associated with a shift in the pattern of secretion with a new set of bioactive molecules synthesized before ovulation. Using interleukin 7 (IL7) as a prototypic secreted factor, we show that its secretion is dependent on activation of mRNA translation in synchrony with the cell cycle and that its translation is under the control of somatic cells. IL7 is part of a local feedback loop with the soma because it regulates cumulus cell replication. Similar conclusions are reached when IL7 secretion is measured in human follicular fluid during in vitro fertilization cycles. IL7 concentration in the follicular fluid correlates with the oocyte ability to reach the MII stage of maturation. These findings are consistent with the hypothesis that a new set of local factors is secreted by the oocyte during ovulation. These dynamic secretions are likely critical for promoting the final stages of maturation and oocyte developmental competence. PMID:26864200

  2. Dynamic secretion during meiotic reentry integrates the function of the oocyte and cumulus cells

    PubMed Central

    Cakmak, Hakan; Franciosi, Federica; Zamah, A. Musa; Cedars, Marcelle I.; Conti, Marco

    2016-01-01

    The differentiation of the female gamete into a developmentally competent oocyte relies on the protected environment of the ovarian follicle. The oocyte plays a key role in establishing this microenvironment by releasing paracrine factors that control the functions of surrounding somatic cells. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are secreted during follicle growth and play pivotal roles in this local regulation. The current view is that the function of these secreted factors declines in the periovulatory period when the oocyte reenters the meiotic cell cycle. Here, we provide evidence that oocyte reentry into meiosis is instead associated with a shift in the pattern of secretion with a new set of bioactive molecules synthesized before ovulation. Using interleukin 7 (IL7) as a prototypic secreted factor, we show that its secretion is dependent on activation of mRNA translation in synchrony with the cell cycle and that its translation is under the control of somatic cells. IL7 is part of a local feedback loop with the soma because it regulates cumulus cell replication. Similar conclusions are reached when IL7 secretion is measured in human follicular fluid during in vitro fertilization cycles. IL7 concentration in the follicular fluid correlates with the oocyte ability to reach the MII stage of maturation. These findings are consistent with the hypothesis that a new set of local factors is secreted by the oocyte during ovulation. These dynamic secretions are likely critical for promoting the final stages of maturation and oocyte developmental competence. PMID:26864200

  3. Identification of microtubule-associated proteins in the meiotic spindle of surf clam oocytes

    PubMed Central

    1980-01-01

    Meiotic spindles isolated from surf clam oocytes to morphological purity are biochemically complex, consisting of many polypeptides. These proteins fall into two classes: (a) polypeptides that are apparently cytoplasmic proteins and are not specifically associated with the spindle; and (b) polypeptides that are specifically associated with the spindle. A subset of the spindle-associated proteins, including a 250,000 mol wt component, remain with spindle tubulin through cycles of cold depolymerization and warm polymerization, showing that they are microtubule-associated proteins. PMID:7189754

  4. Persistence of histone H2AX phosphorylation after meiotic chromosome synapsis and abnormal centromere cohesion in Poly (ADP-ribose) polymerase (Parp-1) null oocytes

    PubMed Central

    Yang, Feikun; Baumann, Claudia; De La Fuente, Rabindranath

    2009-01-01

    In spite of the impact of aneuploidy on human health little is known concerning the molecular mechanisms involved in the formation of structural or numerical chromosome abnormalities during meiosis. Here, we provide novel evidence indicating that lack of PARP-1 function during oogenesis predisposes the female gamete to genome instability. During prophase I of meiosis, a high proportion of Parp-1 (−/−) mouse oocytes exhibit a spectrum of meiotic defects including incomplete homologous chromosome synapsis or persistent histone H2AX phosphorylation in fully synapsed chromosomes at the late pachytene stage. Moreover, the X chromosome bivalent is also prone to exhibit persistent double strand DNA breaks (DSBs). In striking contrast, such defects were not detected in mutant pachytene spermatocytes. In fully-grown wild type oocytes at the germinal vesicle stage, PARP-1 protein associates with nuclear speckles and upon meiotic resumption, undergoes a striking re-localization towards spindle poles as well as pericentric heterochromatin domains at the metaphase II stage. Notably, a high proportion of in vivo matured Parp-1 (−/−) oocytes show lack of recruitment of the kinetochore-associated protein BUB3 to centromeric domains and fail to maintain metaphase II arrest. Defects in chromatin modifications in the form of persistent histone H2AX phosphorylation during prophase I of meiosis and deficient sister chromatid cohesion during metaphase II predispose mutant oocytes to premature anaphase II onset upon removal from the oviductal environment. Our results indicate that PARP-1 plays a critical role in the maintenance of chromosome stability at key stages of meiosis in the female germ line. Moreover, in the metaphase II stage oocyte PARP-1 is required for the regulation of centromere structure and function through a mechanism that involves the recruitment of BUB3 protein to centromeric domains. PMID:19463809

  5. APCFZR1 prevents nondisjunction in mouse oocytes by controlling meiotic spindle assembly timing

    PubMed Central

    Holt, Janet E.; Lane, Simon I. R.; Jennings, Phoebe; García-Higuera, Irene; Moreno, Sergio; Jones, Keith T.

    2012-01-01

    FZR1 is an anaphase-promoting complex (APC) activator best known for its role in the mitotic cell cycle at M-phase exit, in G1, and in maintaining genome integrity. Previous studies also established that it prevents meiotic resumption, equivalent to the G2/M transition. Here we report that mouse oocytes lacking FZR1 undergo passage through meiosis I that is accelerated by ∼1 h, and this is due to an earlier onset of spindle assembly checkpoint (SAC) satisfaction and APCCDC20 activity. However, loss of FZR1 did not compromise SAC functionality; instead, earlier SAC satisfaction was achieved because the bipolar meiotic spindle was assembled more quickly in the absence of FZR1. This novel regulation of spindle assembly by FZR1 led to premature bivalent attachment to microtubules and loss of kinetochore-bound MAD2. Bivalents, however, were observed to congress poorly, leading to nondisjunction rates of 25%. We conclude that in mouse oocytes FZR1 controls the timing of assembly of the bipolar spindle and in so doing the timing of SAC satisfaction and APCCDC20 activity. This study implicates FZR1 as a major regulator of prometaphase whose activity helps to prevent chromosome nondisjunction. PMID:22918942

  6. A Maternal Screen for Genes Regulating Drosophila Oocyte Polarity Uncovers New Steps in Meiotic Progression

    PubMed Central

    Barbosa, Vitor; Kimm, Naomi; Lehmann, Ruth

    2007-01-01

    Meiotic checkpoints monitor chromosome status to ensure correct homologous recombination, genomic integrity, and chromosome segregation. In Drosophila, the persistent presence of double-strand DNA breaks (DSB) activates the ATR/Mei-41 checkpoint, delays progression through meiosis, and causes defects in DNA condensation of the oocyte nucleus, the karyosome. Checkpoint activation has also been linked to decreased levels of the TGFα-like molecule Gurken, which controls normal eggshell patterning. We used this easy-to-score eggshell phenotype in a germ-line mosaic screen in Drosophila to identify new genes affecting meiotic progression, DNA condensation, and Gurken signaling. One hundred eighteen new ventralizing mutants on the second chromosome fell into 17 complementation groups. Here we describe the analysis of 8 complementation groups, including Kinesin heavy chain, the SR protein kinase cuaba, the cohesin-related gene dPds5/cohiba, and the Tudor-domain gene montecristo. Our findings challenge the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR/Mei-41 kinase and instead reveal a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation, and Gurken protein synthesis. PMID:17507684

  7. Cellular and molecular events during oocyte maturation in mammals: molecules of cumulus-oocyte complex matrix and signalling pathways regulating meiotic progression.

    PubMed

    Kimura, N; Hoshino, Y; Totsukawa, K; Sato, E

    2007-01-01

    Mammalian oocytes acquire their intrinsic ability in a stepwise manner through ovarian folliculogenesis, ultimately reaching the competence to undergo complete oocyte maturation at the final stage of Graafian follicle development. The fully-grown oocyte is tightly surrounded by compact layers of specialized granulosa cells (cumulus cells) to form a cumulus-oocyte complex (COC). After a preovulatory gonadotrophin surge, the COCs rapidly organize a special muco-elastic extracellular matrix (ECM) consisting of large amounts of hyaluronan (HA) and HA binding matrix glycoproteins. Simultaneously, the oocytes undergo meiotic resumption and cytoplasmic modification and attain the fertilizable metaphase II (MII) stage. These cellular events that immediately occur in COCs in the ovulatory phase are strictly regulated by pituitary hormones, steroids, growth factors and so on. Knowledge of the efficient mechanisms and the downstream cascades of the key molecules controlling oocyte maturation may gradually lead to improvement of the present oocyte/ embryo culture systems and gamete biotechnology. Recent studies by our group imply that i) the interaction of HA-CD44 identified in the porcine COC matrix is likely to participate in gap junctional communication and meiotic progression, and that ii) phosphatidylinositol 3-kinase (P13-K) and Akt contribute to the progress of follicle stimulating hormone (FSH)-induced meiosis in mice. Furthermore, this review focuses on the current understanding of biosynthetic regulation, the presumptive role of COC matrix molecules and the signalling pathways for meiotic modulators, such as the protein kinase A (PKA) pathway, the P13-K/Akt pathway and the mitogen activated protein kinase (MAPK) pathway.

  8. KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly

    PubMed Central

    Connolly, Amy A.; Sugioka, Kenji; Chuang, Chien-Hui; Lowry, Joshua B.

    2015-01-01

    During oocyte meiotic cell division in many animals, bipolar spindles assemble in the absence of centrosomes, but the mechanisms that restrict pole assembly to a bipolar state are unknown. We show that KLP-7, the single mitotic centromere–associated kinesin (MCAK)/kinesin-13 in Caenorhabditis elegans, is required for bipolar oocyte meiotic spindle assembly. In klp-7(−) mutants, extra microtubules accumulated, extra functional spindle poles assembled, and chromosomes frequently segregated as three distinct masses during meiosis I anaphase. Moreover, reducing KLP-7 function in monopolar klp-18(−) mutants often restored spindle bipolarity and chromosome segregation. MCAKs act at kinetochores to correct improper kinetochore–microtubule (k–MT) attachments, and depletion of the Ndc-80 kinetochore complex, which binds microtubules to mediate kinetochore attachment, restored bipolarity in klp-7(−) mutant oocytes. We propose a model in which KLP-7/MCAK regulates k–MT attachment and spindle tension to promote the coalescence of early spindle pole foci that produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly. PMID:26370499

  9. Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1(Ser137) involvement in spindle formation and REC8 cleavage.

    PubMed

    Du, Juan; Cao, Yan; Wang, Qian; Zhang, Nana; Liu, Xiaoyu; Chen, Dandan; Liu, Xiaoyun; Xu, Qunyuan; Ma, Wei

    2015-01-01

    Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1(Ser137) and pPlk1(Thr210)) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1(Ser137) with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1(Thr210) was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1(Ser137) was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1(Thr210) was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1(Ser137) accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1(Ser137) and pPlk1(Thr210), as well as the subcellular distribution of pPlk1(Thr210), were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1(Ser137) is the action executor, in mouse oocytes during meiotic division. PMID:26654596

  10. Perturbing microtubule integrity blocks AMP-activated protein kinase-induced meiotic resumption in cultured mouse oocytes

    PubMed Central

    Ya, Ru; Downs, Stephen M.

    2014-01-01

    AMP-activated protein kinase (AMPK) is an important cellular energy sensor that is activated by a low AMP-to-ATP ratio. AMPK is involved in meiotic induction of mouse oocytes and also promotes the completion of maturation, but suppress premature activation. This study was conducted to further examine the activity and localization of AMPK in relation to microtubule (MT) integrity. Immunostaining with tubulin revealed that active AMPK localized with gamma tubulin during metaphase I and II, while it localized at the spindle midzone during anaphase. This discrete localization pattern was dependent on MT integrity. Treatment with nocodazole, which depolymerized spindle MTs, led to disruption of proper spindle pole localization of active AMPK. In contrast, active AMPK was localized at each pole and with small asters when treated with paclitaxel, which induced excessive polymerization of spindle MTs. Disturbing spindle integrity not only influenced active AMPK localization, but also blocked AMPK activity, as both hormone- and AMPK activator- induced oocyte maturation were blocked by MT-disrupting agents. In concert with these data, the treatments inhibited active AMPK germinal vesicle staining as well, which appears before germinal vesicle breakdown (GVB) in meiotically induced oocytes. Targeting actin filament polymerization had only a marginal effect on meiotic induction. Although oocytes stimulated by AMPK activator increased the rate of GVB, spindle formation and PB extrusion, blocking AMPK activity did not influence peripheral movement of the spindle. These data suggest that the meiosis-inducing action and localization of AMPK are regulated by MT spindle integrity during mouse oocyte maturation. PMID:23199370

  11. Effects of granulosa coculture on in-vitro oocyte meiotic maturation within a putatively less competent murine model.

    PubMed

    Heng, Boon Chin; Tong, Guo Qing; Ng, Soon Chye

    2004-09-15

    A less competent murine in vitro maturation (IVM) model was achieved by shortening the standard duration of in vivo PMSG stimulation from 48 to 24 h and selecting only naked/partially naked GV oocytes from a mixture of large and small follicles. Porcine granulosa coculture enhanced meiotic maturation within such a less competent model (37.3% versus 23.1%, P<0.05), while no significant enhancement was observed with macaque and murine granulosa coculture. Culture of porcine granulosa on extracellular matrix (ECM) gel resulted in a more differentiated morphology, but did not significantly further enhance the beneficial effects it already had on meiotic maturation. Increased concentrations of serum as well as the supplementation of gonadotrophins and follicular fluid within the culture milieu did not enhance IVM under both cell-free and coculture conditions. Porcine granulosa-conditioned medium also enhanced meiotic maturation (36.5% versus 26.7%, P<0.05), which was not diminished upon freeze-thawing (35.8% versus 22.6%, P<0.05). Enhancement of meiotic maturation by porcine granulosa coculture did not however translate to significant improvements in developmental competence, as assessed by in vitro fertilization (IVF) and embryo culture to the blastocyst stage, followed by total cell counts. ECM gel had a detrimental effect on fertilization and developmental competence, even though it had no detrimental effect on meiotic maturation itself. PMID:15289048

  12. Proteolytic activity of the 26S proteasome is required for the meiotic resumption, germinal vesicle breakdown, and cumulus expansion of porcine cumulus-oocyte complexes matured in vitro.

    PubMed

    Yi, Young-Joo; Nagyova, Eva; Manandhar, Gaurishankar; Procházka, Radek; Sutovsky, Miriam; Park, Chang-Sik; Sutovsky, Peter

    2008-01-01

    The resumption of oocyte meiosis in mammals encompasses the landmark event of oocyte germinal vesicle (GV) breakdown (GVBD), accompanied by the modification of cell-to-cell communication and adhesion between the oocyte and surrounding cumulus cells. The concomitant cumulus expansion relies on microfilament-cytoskeletal remodeling and extracellular matrix (ECM) deposition. We hypothesized that this multifaceted remodeling event requires substrate-specific proteolysis by the ubiquitin-proteasome pathway (UPP). We evaluated meiotic progression, cytoskeletal dynamics, and the production of cumulus ECM in porcine cumulus-oocyte complexes (COCs) cultured with or without 10-200 microM MG132, a specific proteasomal inhibitor, for the first 22 h of in vitro maturation, followed by 22 h of culture with or without MG132. Treatment with 10 microM MG132 arrested 28.4% of oocytes in GV stage (vs. 1.3% in control), 43.1% in prometaphase I, and 16.2% in metaphase I, whereas 83.7% of control ova reached metaphase II (0% of MG132 reached metaphase II). The proportion of GV-stage ova increased progressively to >90% with increased concentration of MG132 (20-200 microM). Furthermore, MG132 blocked the extrusion of the first polar body and degradation of F-actin-rich transzonal projections (TZP) interconnecting cumulus cells with the oocyte. The microfilament disruptor cytochalasin E (CE) prevented cumulus expansion but accelerated the breakdown of TZPs. Ova treated with a combination of 10 microM MG132 and 10 microM CE underwent GVBD, despite the inhibition of proteasomal activity. However, 90.0% of cumulus-free ova treated with 10 microM MG132 remained in GV stage, compared with 16.7% GV ova in control. Cumulus expansion, retention of hyaluronic acid, and the deposition of cumulus ECM relying on the covalent transfer of heavy chains of inter-alpha trypsin inhibitor (IalphaI) were also inhibited by MG132. Cumulus expansion in control COCs was accompanied by the degradation of ubiquitin

  13. Effects of Simulated Weightlessness on Mammalian Development. Part 2: Meiotic Maturation of Mouse Oocytes During Clinostat Rotation

    NASA Technical Reports Server (NTRS)

    Wolgemuth, D. J.; Grills, G. S.

    1985-01-01

    In order to understand the role of gravity in basic cellular processes that are important during development, the effects of a simulated microgravity environment on mammalian gametes and early embryos cultured in vitro are examined. A microgravity environment is simulated by use of a clinostat, which essentially reorients cells relative to the gravity vector. Initial studies have focused on assessing the effects of clinostat rotation on the meiotic progression of mouse oocytes. Modifications centered on providing the unique in vitro culture of the clinostat requirements of mammalian oocytes and embryos: 37 C temperature, constant humidity, and a 5% CO2 in air environment. The oocytes are observed under the dissecting microscope for polar body formation and gross morphological appearance. They are then processed for cytogenetic analysis.

  14. Nitric oxide donor s-nitroso-n-acetyl penicillamine (SNAP) alters meiotic spindle morphogenesis in Xenopus oocytes.

    PubMed

    Gelaude, Armance; Marin, Matthieu; Cailliau, Katia; Jeseta, Michal; Lescuyer-Rousseau, Arlette; Vandame, Pauline; Nevoral, Jan; Sedmikova, Marketa; Martoriati, Alain; Bodart, Jean-François

    2015-11-01

    Nitric Oxide (NO) has been involved in both intra- and extra-cellular signaling pathways in a wide range of organisms, and can be detected in some reproductive tissues. Based upon previous results reporting that NO-donor SNAP (s-nitroso-n-acetyl penicillamine) promoted the release from the metaphase II-anaphase II block in amphibian eggs, the aim of the present study was to assess the influence of SNAP on the activation of the molecular mechanisms triggering meiotic resumption of Xenopus oocytes, analogous to G2/M transition of the cell cycle. A high concentration of SNAP (2.5 mM) was found to inhibit the appearance of the white spot (meiotic resumption) and promoted alteration of spindle morphogenesis leading to atypical structures lacking bipolarity and correct chromosomes equatorial alignment. The medium acidification (pH = 4) promoted by SNAP specifically impacted the white spot occurrence. However, even when pH was restored to 7.4 in SNAP medium, observed spindles remained atypical (microtubule disorganization), suggesting SNAP impacted spindle assembly regardless of the pH. n-Acetyl-d,l-penicillamine disulfide, a degradation product of SNAP with the same molecular characteristics, albeit without release of NO, yielded spindle assemblies typical of metaphase II suggesting the specificity of NO action on meiotic spindle morphogenesis in Xenopus oocytes.

  15. Neonatal bisphenol A exposure induces meiotic arrest and apoptosis of spermatogenic cells

    PubMed Central

    Xie, Meina; Bu, Pengli; Li, Fengjie; Lan, Shijian; Wu, Hongjuan; Yuan, Lu; Wang, Ying

    2016-01-01

    Bisphenol A (BPA) is a widely used industrial plasticizer, which is ubiquitously present in the environment and organisms. As an endocrine disruptor, BPA has caused significant concerns regarding its interference with reproductive function. However, little is known about the impact of BPA exposure on early testicular development. The aim of the present study was to investigate the influence of neonatal BPA exposure on the first wave of spermatogenesis. Newborn male mice were subcutaneously injected with BPA (0.01, 0.1 and 5 mg/kg body weight) daily from postnatal day (PND) 1 to 21. Histological analysis of testes at PND 22 revealed that BPA-treated testes contained mostly spermatogonia and spermatocytes with markedly less round spermatids, indicating signs of meiotic arrest. Terminal dUTP nick-end labeling (TUNEL) assay showed that BPA treatment significantly increased the number of apoptotic germ cells per tubule, which corroborated the observation of meiotic arrest. In addition, BPA caused abnormal proliferation of germ cells as revealed by Proliferating Cell Nuclear Antigen (PCNA) immunohistochemical staining. Mechanistically, BPA-treated testes displayed a complete lack of BOULE expression, which is a conserved key regulator for spermatogenesis. Moreover, BPA significantly increased the expression of estrogen receptor (ER) α and β in the developing testis. The present study demonstrated that neonatal BPA exposure disrupted meiosis progression during the first wave of spermatogenesis, which may be, at least in part, due to inhibition of BOULE expression and/or up-regulation of ERα/β expression in BPA-exposed developing testis. PMID:26863571

  16. Neonatal bisphenol A exposure induces meiotic arrest and apoptosis of spermatogenic cells.

    PubMed

    Xie, Meina; Bu, Pengli; Li, Fengjie; Lan, Shijian; Wu, Hongjuan; Yuan, Lu; Wang, Ying

    2016-03-01

    Bisphenol A (BPA) is a widely used industrial plasticizer, which is ubiquitously present in the environment and organisms. As an endocrine disruptor, BPA has caused significant concerns regarding its interference with reproductive function. However, little is known about the impact of BPA exposure on early testicular development. The aim of the present study was to investigate the influence of neonatal BPA exposure on the first wave of spermatogenesis. Newborn male mice were subcutaneously injected with BPA (0.01, 0.1 and 5 mg/kg body weight) daily from postnatal day (PND) 1 to 21. Histological analysis of testes at PND 22 revealed that BPA-treated testes contained mostly spermatogonia and spermatocytes with markedly less round spermatids, indicating signs of meiotic arrest. Terminal dUTP nick-end labeling (TUNEL) assay showed that BPA treatment significantly increased the number of apoptotic germ cells per tubule, which corroborated the observation of meiotic arrest. In addition, BPA caused abnormal proliferation of germ cells as revealed by Proliferating Cell Nuclear Antigen (PCNA) immunohistochemical staining. Mechanistically, BPA-treated testes displayed a complete lack of BOULE expression, which is a conserved key regulator for spermatogenesis. Moreover, BPA significantly increased the expression of estrogen receptor (ER) α and β in the developing testis. The present study demonstrated that neonatal BPA exposure disrupted meiosis progression during the first wave of spermatogenesis, which may be, at least in part, due to inhibition of BOULE expression and/or up-regulation of ERα/β expression in BPA-exposed developing testis. PMID:26863571

  17. Regulation of the G2/M Transition in Rodent Oocytes

    PubMed Central

    Downs, Stephen M.

    2014-01-01

    Regulation of maturation in meiotically competent mammalian oocytes is a complex process involving the carefully coordinated exchange of signals between the somatic and germ cell compartments of the ovarian follicle via paracrine and cell-cell coupling pathways. This review highlights recent advances in our understanding of how such signaling controls both meiotic arrest and gonadotropin-triggered meiotic resumption in competent oocytes and relates them to the historical context. Emphasis will be on rodent systems, where many of these new findings have taken place. A regulatory scheme is then proposed that integrates this information into an overall framework for meiotic regulation that demonstrates the complex interplay between different follicular compartments. PMID:20578061

  18. High Fat Diet Induced Developmental Defects in the Mouse: Oocyte Meiotic Aneuploidy and Fetal Growth Retardation/Brain Defects

    PubMed Central

    Purcell, Scott H.; Chi, Maggie; Jimenez, Patricia T.; Grindler, Natalia; Schedl, Tim; Moley, Kelle H.

    2012-01-01

    Background Maternal obesity is associated with poor outcomes across the reproductive spectrum including infertility, increased time to pregnancy, early pregnancy loss, fetal loss, congenital abnormalities and neonatal conditions. Furthermore, the proportion of reproductive-aged woman that are obese in the population is increasing sharply. From current studies it is not clear if the origin of the reproductive complications is attributable to problems that arise in the oocyte or the uterine environment. Methodology/Principal Findings We examined the developmental basis of the reproductive phenotypes in obese animals by employing a high fat diet mouse model of obesity. We analyzed very early embryonic and fetal phenotypes, which can be parsed into three abnormal developmental processes that occur in obese mothers. The first is oocyte meiotic aneuploidy that then leads to early embryonic loss. The second is an abnormal process distinct from meiotic aneuploidy that also leads to early embryonic loss. The third is fetal growth retardation and brain developmental abnormalities, which based on embryo transfer experiments are not due to the obese uterine environment but instead must be from a defect that arises prior to the blastocyst stage. Conclusions/Significance Our results suggest that reproductive complications in obese females are, at least in part, from oocyte maternal effects. This conclusion is consistent with IVF studies where the increased pregnancy failure rate in obese women returns to the normal rate if donor oocytes are used instead of autologous oocytes. We postulate that preconceptional weight gain adversely affects pregnancy outcomes and fetal development. In light of our findings, preconceptional counseling may be indicated as the preferable, earlier target for intervention in obese women desiring pregnancy and healthy outcomes. PMID:23152876

  19. The kinesinlike protein Subito contributes to central spindle assembly and organization of the meiotic spindle in Drosophila oocytes.

    PubMed

    Jang, J K; Rahman, T; McKim, K S

    2005-10-01

    In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila melanogaster oocyte chromosomes have a major role in nucleating microtubules, which precedes the bundling and assembly of these microtubules into a bipolar spindle. Here we present evidence that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. Subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. Subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but our results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. We propose that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle. PMID:16055508

  20. Oocyte development, meiosis and aneuploidy.

    PubMed

    MacLennan, Marie; Crichton, James H; Playfoot, Christopher J; Adams, Ian R

    2015-09-01

    Meiosis is one of the defining events in gametogenesis. Male and female germ cells both undergo one round of meiotic cell division during their development in order to reduce the ploidy of the gametes, and thereby maintain the ploidy of the species after fertilisation. However, there are some aspects of meiosis in the female germline, such as the prolonged arrest in dictyate, that appear to predispose oocytes to missegregate their chromosomes and transmit aneuploidies to the next generation. These maternally-derived aneuploidies are particularly problematic in humans where they are major contributors to miscarriage, age-related infertility, and the high incidence of Down's syndrome in human conceptions. This review will discuss how events that occur in foetal oocyte development and during the oocytes' prolonged dictyate arrest can influence meiotic chromosome segregation and the incidence of aneuploidy in adult oocytes.

  1. [The effect of in-situ nerve growth factor from different biological sources on the reinitiation of mouse oocyte meiotic maturation in culture and on parthenogenetic activation].

    PubMed

    Fedorushchenko, A N; Koval', T Iu; Khamidov, D Kh

    1999-01-01

    We studied the capacity of mouse oocytes to complete meiotic maturation in vitro and form the female pronucleus upon parthenogenetic activation by cycloheximide, in response to a single injection into the mouse ovaries in situ of a purified fraction of 2.5 S NGF from mouse submaxillary glands and beta-NGF from bovine sperm. Injection of NGF from both sources at 10 ng/ml with subsequent incubation of the ovaries for 1 h increased the capacity of matured oocytes for parthenogenetic formation of the pronucleus. The frequency of pronucleus formation in both "naked oocyte" and oocytes surrounded by the cumulus cells was four times that in the control. PMID:10624718

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

    PubMed Central

    Redi, Carlo Alberto; Zuccotti, Maurizio

    2014-01-01

    In the mammalian oocyte, distinct patterns of centromeres and pericentromeric heterochromatin localisation correlate with the gamete's developmental competence. Mouse antral oocytes display two main types of chromatin organisation: SN oocytes, with a ring of Hoechst-positive chromatin surrounding the nucleolus, and NSN oocytes lacking this ring. When matured to MII and fertilised, only SN oocytes develop beyond the 2-cell, and reach full term. To give detailed information on the dynamics of the SN or NSN chromatin during meiosis resumption, we performed a 9 hr time-lapse observation. The main significant differences recorded are: (1) reduction of the nuclear area only in SN oocytes; (2) ~17 min delay of GVBD in NSN oocytes; (3) chromatin condensation, after GVBD, in SN oocytes; (4) formation of 4-5 CHCs in SN oocytes; (5) increase of the perivitelline space, ~57 min later in NSN oocytes; (6) formation of a rosette-like disposition of CHCs, ~84 min later in SN oocytes; (7) appearance of the MI plate ~40 min later in NSN oocytes. Overall, we described a pathway of transition from the GV to the MII stage that is punctuated of discrete recordable events showing their specificity and occurring with different time kinetics in the two types of oocytes. PMID:24864231

  3. Effects of simulated weightlessness on mammalian development. Part 1: Development of clinostat for mammalian tissue culture and use in studies on meiotic maturation of mouse oocytes

    NASA Technical Reports Server (NTRS)

    Wolegemuth, D. J.; Grills, G. S.

    1984-01-01

    The effects of weightlessness on three aspects of mammalian reproduction: oocyte development, fertilization, and early embryogenesis was studied. Zero-gravity conditions within the laboratory by construction of a clinostat designed to support in vitro tissue culture were simulated and the effects of simulated weightlessness on meiotic maturation in mammalian oocytes using mouse as the model system were studied. The timing and frequency of germinal vesicule breakdown and polar body extrusion, and the structural and numerical properties of meiotic chromosomes at Metaphase and Metaphase of meiosis are assessed.

  4. Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse.

    PubMed

    Kim, Jeesun; Zhao, Hongbo; Dan, Jiameng; Kim, Soojin; Hardikar, Swanand; Hollowell, Debra; Lin, Kevin; Lu, Yue; Takata, Yoko; Shen, Jianjun; Chen, Taiping

    2016-04-01

    Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression. PMID:27070551

  5. Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse

    PubMed Central

    Dan, Jiameng; Kim, Soojin; Hardikar, Swanand; Hollowell, Debra; Lin, Kevin; Lu, Yue; Takata, Yoko; Shen, Jianjun; Chen, Taiping

    2016-01-01

    Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression. PMID:27070551

  6. CRL4–DCAF1 ubiquitin E3 ligase directs protein phosphatase 2A degradation to control oocyte meiotic maturation

    PubMed Central

    Yu, Chao; Ji, Shu-Yan; Sha, Qian-Qian; Sun, Qing-Yuan; Fan, Heng-Yu

    2015-01-01

    Oocyte meiosis is a specialized cell cycle that gives rise to fertilizable haploid gametes and is precisely controlled in various dimensions. We recently found that E3 ubiquitin ligase CRL4 is required for female fertility by regulating DNA hydroxymethylation to maintain oocyte survival and to promote zygotic genome reprogramming. However, not all phenotypes of CRL4-deleted oocytes could be explained by this mechanism. Here we show that CRL4 controls oocyte meiotic maturation by proteasomal degradation of protein phosphatase 2A scaffold subunit, PP2A-A. Oocyte-specific deletion of DDB1 or DCAF1 (also called VPRBP) results in delayed meiotic resumption and failure to complete meiosis I along with PP2A-A accumulation. DCAF1 directly binds to and results in the poly-ubiquitination of PP2A-A. Moreover, combined deletion of Ppp2r1a rescues the meiotic defects caused by DDB1/DCAF1 deficiency. These results provide in vivo evidence that CRL4-directed PP2A-A degradation is physiologically essential for regulating oocyte meiosis and female fertility. PMID:26281983

  7. Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling

    PubMed Central

    Navarro-Costa, Paulo; McCarthy, Alicia; Prudêncio, Pedro; Greer, Christina; Guilgur, Leonardo G.; Becker, Jörg D.; Secombe, Julie; Rangan, Prashanth; Martinho, Rui G.

    2016-01-01

    Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I. PMID:27507044

  8. Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling.

    PubMed

    Navarro-Costa, Paulo; McCarthy, Alicia; Prudêncio, Pedro; Greer, Christina; Guilgur, Leonardo G; Becker, Jörg D; Secombe, Julie; Rangan, Prashanth; Martinho, Rui G

    2016-08-10

    Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I.

  9. Presence of an extra chromosome alters meiotic double-stranded break repair dynamics and MLH1 foci distribution in human oocytes.

    PubMed

    Robles, P; Roig, I; Garcia, R; Brieño-Enríquez, M; Martin, M; Cabero, Ll; Toran, N; Garcia Caldés, M

    2013-03-01

    Studies performed on human trisomic 21 oocytes have revealed that during meiosis, the three homologues 21 synapse and, in some cases, achieve what looks like a trivalent. This implies that meiotic recombination takes place among the three homologous chromosomes 21, and to some extent, crossovers form between them. To see how meiotic recombination is in the presence of an extra chromosome 21, we analyzed the distribution of three recombination markers (γH2AX, RPA, and MLH1) on trisomic 21 oocytes at pachynema and, in particular, on chromosomes 21. Results clearly show how the presence of an extra chromosome 21 alters meiotic recombination progression, leading to the presence of a higher number of early recombination markers at pachynema. Moreover, the distribution on these chromosomes 21 of some of these markers is different in aneuploid oocytes. Finally, there is a substantial increase in the number of MLH1 foci, a marker of most crossovers in mammals, which is related to the number of synapsed chromosomes in pachynema. Thus, bivalents 21 had fewer MLH1 foci than partial or total trivalents, suggesting a close relationship between synapsis and crossover designation. All of the data presented suggest that the presence of an extra chromosome alters meiotic recombination globally in aneuploid human oocytes. PMID:23283390

  10. Oocyte development, meiosis and aneuploidy

    PubMed Central

    MacLennan, Marie; Crichton, James H.; Playfoot, Christopher J.; Adams, Ian R.

    2015-01-01

    Meiosis is one of the defining events in gametogenesis. Male and female germ cells both undergo one round of meiotic cell division during their development in order to reduce the ploidy of the gametes, and thereby maintain the ploidy of the species after fertilisation. However, there are some aspects of meiosis in the female germline, such as the prolonged arrest in dictyate, that appear to predispose oocytes to missegregate their chromosomes and transmit aneuploidies to the next generation. These maternally-derived aneuploidies are particularly problematic in humans where they are major contributors to miscarriage, age-related infertility, and the high incidence of Down's syndrome in human conceptions. This review will discuss how events that occur in foetal oocyte development and during the oocytes’ prolonged dictyate arrest can influence meiotic chromosome segregation and the incidence of aneuploidy in adult oocytes. PMID:26454098

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

    PubMed

    Mlynarcíková, Alzbeta; Nagyová, Eva; Ficková, Mária; Scsuková, Sona

    2009-04-01

    In most mammals, before ovulation, cumulus cells synthesize a large amount of hyaluronan (HA) that is organized into an extracellular matrix (ECM), which provides an essential microenvironment for in vivo oocyte fertilization. This process is called cumulus expansion. The present study assessed effects of selected endocrine disruptors (bisphenol A, BPA; 4-chloro-3-methyl phenol, CMP; di(2-ethylhexyl) phthalate, DEHP; and benzyl butyl phthalate, BBP) in a range of 100pM-100microM, on follicle-stimulating hormone (FSH)-induced meiotic maturation and cumulus expansion of porcine oocyte-cumulus complexes (OCC) cultured in vitro. Moreover, FSH-stimulated production of hyaluronic acid (HA) and progesterone by cumulus cells was measured. Both phenols, BPA and CMP (100microM), significantly affected meiotic maturation of oocytes. The number of oocytes that underwent germinal vesicle breakdown (GVBD) (78.7% and 72.4%, respectively) as well as the rate of oocytes that reached metaphase II stage (MII) (50% and 53.6%, respectively) after 44h culture were decreased compared to control (89.6% for GVBD and 81.5% for MII). FSH-stimulated expansion of cumulus was altered by the highest concentration of BPA and CMP (70% and 64%, respectively vs. 80.3% in control). Although BPA did not alter FSH-stimulated HA synthesis by cumulus cells, its incorporation within the complex was reduced to a half of control value. Progesterone production by OCC was significantly changed in the presence of BPA or DEHP. Finally, our results provide valuable information that oocyte meiotic progression was adversely affected during in vitro culture with endocrine disruptors. PMID:19162163

  12. Progesterone regulation of AVEN protects bovine oocytes from apoptosis during meiotic maturation.

    PubMed

    O'Shea, Lynne C; Hensey, Carmel; Fair, Trudee

    2013-12-01

    Inhibition of progesterone (P4) synthesis by cumulus cells during bovine in vitro oocyte maturation (IVM) causes a decrease in subsequent embryo development, indicating that P4 intracellular signaling within the cumulus oocyte complex (COC) is important for oocyte developmental competence. The aim of the present study was to further elucidate, on a protein level, the downstream signaling pathway involved in P4 regulation of oocyte developmental competence. COCs were subjected to IVM for 24 h in the presence or absence of trilostane, aglepristone, or promegestone (R5020). These altered IVM conditions resulted in dynamic changes in protein expression of the progesterone receptors and the cell death-regulated proteins AVEN, BCL-xL, and active caspase 3. In addition, AVEN protein localization, caspase 3 activation, and mitochondrial distribution were studied by immunofluorescence. Inhibition of progesterone synthesis (trilostane treatment) resulted in changes in AVEN localization within the COC, corresponding to caspase 3 activation and altered mitochondrial distribution. AVEN was also found to bind BCL-xL in COCs, but this interaction was lost following treatment with trilostane.

  13. The behavior of the X- and Y-chromosomes in the oocyte during meiotic prophase in the B6.Y(TIR)sex-reversed mouse ovary.

    PubMed

    Alton, Michelle; Lau, Mau Pan; Villemure, Michele; Taketo, Teruko

    2008-02-01

    Sexual differentiation of the germ cells follows gonadal differentiation, which is determined by the presence or the absence of the Y-chromosome. Consequently, oogenesis and spermatogenesis take place in the germ cells with XX and XY sex chromosomal compositions respectively. It is unclear how sexual dimorphic regulation of meiosis is associated with the sex-chromosomal composition. In the present study, we examined the behavior of the sex chromosomes in the oocytes of the B6.Y(TIR) sex-reversed female mouse, in comparison with XO and XX females. As the sex chromosomes fail to pair in both XY and XO oocytes during meiotic prophase, we anticipated that the pairing failure may lead to excessive oocyte loss. However, the total number of germ cells, identified by immunolabeling of germ cell nuclear antigen 1 (GCNA1), did not differ between XY and XX ovaries or XO and XX ovaries up to the day of delivery. The progression of meiotic prophase, assessed by immunolabeling of synaptonemal complex components, was also similar between the two genotypes of ovaries. These observations suggest that the failure in sex-chromosome pairing is not sufficient to cause oocyte loss. On the other hand, labeling of phosphorylated histone gammaH2AX, known to be associated with asynapsis and transcriptional repression, was seen over the X-chromosome but not over the Y-chromosome in the majority of XY oocytes at the pachytene stage. For comparison, gammaH2AX labeling was seen only in the minority of XX oocytes at the same stage. We speculate that the transcriptional activity of sex chromosomes in the XY oocyte may be incompatible with ooplasmic maturation. PMID:18239052

  14. Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro.

    PubMed

    Leonardsen, L; Strömstedt, M; Jacobsen, D; Kristensen, K S; Baltsen, M; Andersen, C Y; Byskov, A G

    2000-01-01

    Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in

  15. Hormonal control of mammalian oocyte meiosis at diplotene stage.

    PubMed

    Zhang, Meijia; Xia, Guoliang

    2012-04-01

    Mammalian oocytes grow and undergo meiosis within ovarian follicles. Fully grown oocytes are arrested at the first meiotic prophase by a mural granulosa origin "arrester" until a surge of luteinizing hormone (LH) from the pituitary at the mid-cycle stimulates the immature oocyte to resume meiosis. Recent evidence indicates that natriuretic peptide precursor type C (NPPC) produced by mural granulosa cells stimulates the generation of cyclic guanosine 3',5'-monophosphate (cGMP) by cumulus cell natriuretic peptide receptor 2 (NPR2), which diffuses into oocyte via gap junctions and inhibits oocyte phosphodiesterase 3A (PDE3A) activity and cyclic adenosine 3',5'-monophosphate (cAMP) hydrolysis and maintains meiotic arrest with a high intraoocyte cAMP level. This cAMP is generated through the activity of the Gs G-protein by the G-protein-coupled receptor, GPR3 and GPR12, and adenylyl cyclases (ADCY) endogenous to the oocyte. Further studies suggest that endocrine hormones, such as follicle-stimulating hormone (FSH), LH, 17β-estradiol (E2) and oocyte-derived paracrine factors (ODPFs), participate in oocyte meiosis possibly by the regulation of NPPC and/or NPR2. A detailed investigation of NPPC and NPR2 expression in follicle cells will elucidate the precise molecular mechanisms of gonadotropins, and control the arrest as well as resumption of meiosis.

  16. Boule gene expression underpins the meiotic arrest in spermatogenesis in male rainbow trout (Oncorhynchus mykiss) exposed to DEHP and butachlor.

    PubMed

    Ahmadivand, Sohrab; Farahmand, Hamid; Teimoori-Toolabi, Ladan; Mirvaghefi, Alireza; Eagderi, Soheil; Geerinckx, Tom; Shokrpoor, Sara; Rahmati-Holasoo, Hooman

    2016-01-01

    Boule, the ancestor of the DAZ (Deleted in AZoospermia) gene family, in most organisms is mainly involved in male meiosis. The present study investigates the effects of the plasticizer DEHP (50mg/kg body weight) and herbicide butachlor (0.39mg/L) on male rainbow trout (Oncorhynchus mykiss) for a 10-day period in two independent experiments. The results showed that plasma testosterone (T) concentrations were significantly lower in fish exposed to either DEHP or butachlor compared to the control fish (P<0.05). Fish showed a significantly elevated hepatosomatic index (HSI) in the butachlor treatment (P<0.05). However, no significant difference was observed in HSI values in the DEHP treatment (P>0.05). In addition, no significant differences were found in the gonadosomatic index (GSI) in both DEHP and butachlor treatments (P>0.05). Histologically, testes of male trout in the control groups were well differentiated and filled with large numbers of cystic structures containing spermatozoa. In contrast, the testes of male trout contained mostly spermatocytes with few spermatozoa in both treated group, suggesting that DEHP and butachlor may inhibit the progression of meiosis. Also, boule gene expression was significantly lower in the testes of male trout affected by DEHP and butachlor in comparison with their control groups (P<0.05), which confirmed the meiotic arrest in affected trout. Based on the results, the present study demonstrated that DEHP and butachlor can inhibit the progression of spermatogenesis in male trout, potentially by causing an arrest of meiosis, maybe due to down-regulation of boule gene expression through T and/or IGF1 via ERK1/2 signaling in T-independent pathways. In addition, these results confirmed that boule can be considered as a predictive marker to assess meiotic efficiency.

  17. Boule gene expression underpins the meiotic arrest in spermatogenesis in male rainbow trout (Oncorhynchus mykiss) exposed to DEHP and butachlor.

    PubMed

    Ahmadivand, Sohrab; Farahmand, Hamid; Teimoori-Toolabi, Ladan; Mirvaghefi, Alireza; Eagderi, Soheil; Geerinckx, Tom; Shokrpoor, Sara; Rahmati-Holasoo, Hooman

    2016-01-01

    Boule, the ancestor of the DAZ (Deleted in AZoospermia) gene family, in most organisms is mainly involved in male meiosis. The present study investigates the effects of the plasticizer DEHP (50mg/kg body weight) and herbicide butachlor (0.39mg/L) on male rainbow trout (Oncorhynchus mykiss) for a 10-day period in two independent experiments. The results showed that plasma testosterone (T) concentrations were significantly lower in fish exposed to either DEHP or butachlor compared to the control fish (P<0.05). Fish showed a significantly elevated hepatosomatic index (HSI) in the butachlor treatment (P<0.05). However, no significant difference was observed in HSI values in the DEHP treatment (P>0.05). In addition, no significant differences were found in the gonadosomatic index (GSI) in both DEHP and butachlor treatments (P>0.05). Histologically, testes of male trout in the control groups were well differentiated and filled with large numbers of cystic structures containing spermatozoa. In contrast, the testes of male trout contained mostly spermatocytes with few spermatozoa in both treated group, suggesting that DEHP and butachlor may inhibit the progression of meiosis. Also, boule gene expression was significantly lower in the testes of male trout affected by DEHP and butachlor in comparison with their control groups (P<0.05), which confirmed the meiotic arrest in affected trout. Based on the results, the present study demonstrated that DEHP and butachlor can inhibit the progression of spermatogenesis in male trout, potentially by causing an arrest of meiosis, maybe due to down-regulation of boule gene expression through T and/or IGF1 via ERK1/2 signaling in T-independent pathways. In addition, these results confirmed that boule can be considered as a predictive marker to assess meiotic efficiency. PMID:26027538

  18. Carrying-over effects of GVBD blocking on post-blocking meiotic progression of oocytes: species difference and the signaling pathway leading to MPF activation.

    PubMed

    Jiao, Guang-Zhong; Lian, Hua-Yu; Gao, Yan; Sun, Ming-Ju; Gong, Shuai; Zheng, Liang-Liang; Zhang, Chuan-Xin; Tan, Jing-He

    2014-01-01

    Efforts to improve the quality of in vitro matured oocytes by blocking germinal vesicle breakdown (GVBD) and allowing more time for ooplasmic maturation have achieved little due to a lack of knowledge on the molecular events during GVBD blocking. Such knowledge is also important for studies aimed at regulating gene expression in maturing oocytes prior to GVBD. We studied species difference and signaling pathways leading to the carrying-over effect of GVBD blocking on post-blocking meiotic progression (PBMP). Overall, GVBD-blocking with roscovitine decelerated PBMP of mouse oocytes but accelerated that of pig oocytes. During blocking culture, whereas cyclin B of pig oocytes increased continuously, that of mouse oocytes declined first and then increased slowly. In both species, (a) whereas active CDC2A showed a dynamics similar to cyclin B, inactive CDC2A decreased continuously; (b) when oocytes were blocked in blocking medium containing cycloheximide, PBMP was decelerated significantly while cyclin B and active CDC2A decreasing to the lowest level; (c) whereas sodium vanadate in blocking medium reduced PBMP, epidermal growth factor (EGF) in blocking medium accelerated PBMP significantly with no effect on cyclin B levels. In conclusion, the EGF signaling cascade accelerated PBMP by promoting the pre-MPF (M-phase-promoting factor) to MPF conversion during GVBD blocking with roscovitine. The significant difference in PBMP observed between mouse and pig oocytes was caused by species difference in cyclin B dynamics during blocking culture as no species difference was observed in either pre-MPF to MPF conversion or the EGF signaling activity. PMID:25078078

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

    PubMed

    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 < 0.05). The viability of oocytes vitrified using open-pulled straws or Cryoloop was significantly higher than that in the CS group (P < 0.05). After IVM, the percentage of oocytes reaching to the metaphase II (MII) stage was significantly higher with Cryoloop and OPS following by CS. However, the in vitro maturing percentage of vitrified oocytes was significantly less than that of unfrozen oocytes (P < 0.05). After PA, the developmental capability of vitrified oocytes was significantly decreased compared to unfrozen oocytes. The cleavage rate of oocytes vitrified using conventional plastic straws was significantly less than those of the other freezing groups (P < 0.05). The cleaving capability of oocytes vitrified using Cryoloop was significantly increased compared to the OPS group. However, there was no significant difference existing amongst the freezing groups as concerning the blastocyst rate. Following IVF, the developmental capability of vitrified oocytes was severely damaged compared to that of unfrozen

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

    PubMed

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

    2015-11-01

    Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation. PMID:26175189

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

    PubMed

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

    2015-11-01

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

  2. Immunohistochemical localization of inducible and endothelial nitric oxide synthase in porcine ovaries and effects of NO on antrum formation and oocyte meiotic maturation.

    PubMed

    Tao, Yong; Fu, Zhuo; Zhang, Meijia; Xia, Guoliang; Yang, Jie; Xie, Huirong

    2004-07-30

    The present study is to investigate the immunolocalization of endothelial and inducible nitric oxide synthase (eNOS, iNOS) in porcine ovary and the effect of nitric oxide (NO) on antrum formation and oocyte meiotic resumption. In Experiment 1, preantral follicles (250-300 microm in diameter) were cultured in 0 (Control), 0.1, 0.3, 0.5 or 1 mM sodium nitroprusside (SNP), a NO donor. In Experiment 2, the cumulus-oocyte complexes (COCs) aspirated from medium follicles (3-6 mm in diameter) were incubated in 0.1mM SNP or two inhibitors for NOS, 10 mM aminoguanidine bicarbonate salt (AG) or 1 mM Nomega-nitro-l-arginine methyl ester (L-NAME), alone or concomitantly. In Experiment 3, ovarian tissues, corpus luteum (CL), corpus albican (CA) and COCs from small (1-2 mm in diameter), medium (3-6 mm) and large follicles (7-10 mm) were isolated, rinsed, fixed, paraffin embedded and stained by the conventional avidin-biotin complex method for the detection of eNOS and iNOS production. The results showed that 0.1mM SNP had no effect on antrum formation (P > 0.05) while 0.3, 0.5 or 1 mM significantly inhibited the antrum formation (P < 0.05). AG markedly inhibited porcine oocyte meiotic resumption (P < 0.05) while L-NAME inhibited first polar body (PB1) extrusion (P < 0.05). The immunoreactivity of eNOS in early antral follicles was restricted to oocyte and it increased from small, medium to large follicle-enclosed oocytes. Cumulus cells from large follicles showed weak eNOS immunoreactivity but those from small or medium follicles not. In CL, eNOS-positive staining was shown in granulosa lutein cells. In CA, it was in some parenchymal cells. In contrast, no immunoreactivity for iNOS was found in primordial, early antral follicle or the COCs aspirated from small and medium follicles. The large follicle-enclosed oocyte showed weak immunoreactivity. In CL, some granulosa lutein cells showed iNOS-positive cytoplasm. Such immunostaining was not found in CA. The results demonstrate

  3. Differences in the Kinetic of the First Meiotic Division and in Active Mitochondrial Distribution between Prepubertal and Adult Oocytes Mirror Differences in their Developmental Competence in a Sheep Model

    PubMed Central

    Leoni, Giovanni Giuseppe; Palmerini, Maria Grazia; Satta, Valentina; Succu, Sara; Pasciu, Valeria; Zinellu, Angelo; Carru, Ciriaco; Macchiarelli, Guido; Nottola, Stefania Annarita; Naitana, Salvatore; Berlinguer, Fiammetta

    2015-01-01

    Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08% respectively in prepubertals and adults; P<0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P<0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P<0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P<0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence

  4. The Anaphase-Promoting Complex/Cyclosome Is Essential for Entry into Meiotic M-Phase.

    PubMed

    Malhotra, Saurav; Vinod, Palakkad Krishnanunni; Mansfeld, Jörg; Stemmann, Olaf; Mayer, Thomas U

    2016-01-11

    Vertebrate immature oocytes are arrested at prophase of meiosis I (MI). Hormonal stimulation breaks this prophase-I arrest and induces re-entry into MI. The mechanism underlying meiotic resumption remains largely elusive. Here, we demonstrate that the anaphase-promoting complex/cyclosome (APC/C) in complex with Cdh1 has an unexpected function in meiosis in that it is essential for meiotic resumption. We identify the catalytic subunit of protein phosphatase 6 (PP6c) as the critical substrate whose APC/C(Cdh1)-mediated destruction is a prerequisite for the re-entry of immature Xenopus laevis oocytes into MI. Preventing PP6c destruction impairs activating autophosphorylation of Aurora A, a cell-cycle kinase critical for meiotic translation. Restoring meiotic translation rescues the meiotic resumption defect of Cdh1-depleted oocytes. Thus, our studies discover that the essential function of the APC/C in triggering cell-cycle transitions is not limited to M-phase exit but also applies to entry into meiotic M-phase, and identify a crucial APC/C-PP6c-Aurora A axis in the resumption of female meiosis.

  5. Aurora Kinase A Is Not Involved in CPEB1 Phosphorylation and cyclin B1 mRNA Polyadenylation during Meiotic Maturation of Porcine Oocytes

    PubMed Central

    Komrskova, Pavla; Susor, Andrej; Malik, Radek; Prochazkova, Barbora; Liskova, Lucie; Supolikova, Jaroslava; Hladky, Stepan; Kubelka, Michal

    2014-01-01

    Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation. PMID:24983972

  6. Aurora kinase A is not involved in CPEB1 phosphorylation and cyclin B1 mRNA polyadenylation during meiotic maturation of porcine oocytes.

    PubMed

    Komrskova, Pavla; Susor, Andrej; Malik, Radek; Prochazkova, Barbora; Liskova, Lucie; Supolikova, Jaroslava; Hladky, Stepan; Kubelka, Michal

    2014-01-01

    Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.

  7. The TRIM-NHL Protein LIN-41 and the OMA RNA-Binding Proteins Antagonistically Control the Prophase-to-Metaphase Transition and Growth of Caenorhabditis elegans Oocytes

    PubMed Central

    Spike, Caroline A.; Coetzee, Donna; Eichten, Carly; Wang, Xin; Hansen, Dave; Greenstein, David

    2014-01-01

    In many animals, oocytes enter meiosis early in their development but arrest in meiotic prophase I. Oocyte growth, which occurs during this arrest period, enables the acquisition of meiotic competence and the capacity to produce healthy progeny. Meiotic resumption, or meiotic maturation, involves the transition to metaphase I (M phase) and is regulated by intercellular signaling and cyclin-dependent kinase activation. Premature meiotic maturation would be predicted to diminish fertility as the timing of this event, which normally occurs after oocyte growth is complete, is crucial. In the accompanying article in this issue, we identify the highly conserved TRIM-NHL protein LIN-41 as a translational repressor that copurifies with OMA-1 and OMA-2, RNA-binding proteins redundantly required for normal oocyte growth and meiotic maturation. In this article, we show that LIN-41 enables the production of high-quality oocytes and plays an essential role in controlling and coordinating oocyte growth and meiotic maturation. lin-41 null mutants display a striking defect that is specific to oogenesis: pachytene-stage cells cellularize prematurely and fail to progress to diplotene. Instead, these cells activate CDK-1, enter M phase, assemble spindles, and attempt to segregate chromosomes. Translational derepression of the CDK-1 activator CDC-25.3 appears to contribute to premature M-phase entry in lin-41 mutant oocytes. Genetic and phenotypic analyses indicate that LIN-41 and OMA-1/2 exhibit an antagonistic relationship, and we suggest that translational regulation by these proteins could be important for controlling and coordinating oocyte growth and meiotic maturation. PMID:25261698

  8. The TRIM-NHL protein LIN-41 and the OMA RNA-binding proteins antagonistically control the prophase-to-metaphase transition and growth of Caenorhabditis elegans oocytes.

    PubMed

    Spike, Caroline A; Coetzee, Donna; Eichten, Carly; Wang, Xin; Hansen, Dave; Greenstein, David

    2014-12-01

    In many animals, oocytes enter meiosis early in their development but arrest in meiotic prophase I. Oocyte growth, which occurs during this arrest period, enables the acquisition of meiotic competence and the capacity to produce healthy progeny. Meiotic resumption, or meiotic maturation, involves the transition to metaphase I (M phase) and is regulated by intercellular signaling and cyclin-dependent kinase activation. Premature meiotic maturation would be predicted to diminish fertility as the timing of this event, which normally occurs after oocyte growth is complete, is crucial. In the accompanying article in this issue, we identify the highly conserved TRIM-NHL protein LIN-41 as a translational repressor that copurifies with OMA-1 and OMA-2, RNA-binding proteins redundantly required for normal oocyte growth and meiotic maturation. In this article, we show that LIN-41 enables the production of high-quality oocytes and plays an essential role in controlling and coordinating oocyte growth and meiotic maturation. lin-41 null mutants display a striking defect that is specific to oogenesis: pachytene-stage cells cellularize prematurely and fail to progress to diplotene. Instead, these cells activate CDK-1, enter M phase, assemble spindles, and attempt to segregate chromosomes. Translational derepression of the CDK-1 activator CDC-25.3 appears to contribute to premature M-phase entry in lin-41 mutant oocytes. Genetic and phenotypic analyses indicate that LIN-41 and OMA-1/2 exhibit an antagonistic relationship, and we suggest that translational regulation by these proteins could be important for controlling and coordinating oocyte growth and meiotic maturation.

  9. The TRIM-NHL protein LIN-41 and the OMA RNA-binding proteins antagonistically control the prophase-to-metaphase transition and growth of Caenorhabditis elegans oocytes.

    PubMed

    Spike, Caroline A; Coetzee, Donna; Eichten, Carly; Wang, Xin; Hansen, Dave; Greenstein, David

    2014-12-01

    In many animals, oocytes enter meiosis early in their development but arrest in meiotic prophase I. Oocyte growth, which occurs during this arrest period, enables the acquisition of meiotic competence and the capacity to produce healthy progeny. Meiotic resumption, or meiotic maturation, involves the transition to metaphase I (M phase) and is regulated by intercellular signaling and cyclin-dependent kinase activation. Premature meiotic maturation would be predicted to diminish fertility as the timing of this event, which normally occurs after oocyte growth is complete, is crucial. In the accompanying article in this issue, we identify the highly conserved TRIM-NHL protein LIN-41 as a translational repressor that copurifies with OMA-1 and OMA-2, RNA-binding proteins redundantly required for normal oocyte growth and meiotic maturation. In this article, we show that LIN-41 enables the production of high-quality oocytes and plays an essential role in controlling and coordinating oocyte growth and meiotic maturation. lin-41 null mutants display a striking defect that is specific to oogenesis: pachytene-stage cells cellularize prematurely and fail to progress to diplotene. Instead, these cells activate CDK-1, enter M phase, assemble spindles, and attempt to segregate chromosomes. Translational derepression of the CDK-1 activator CDC-25.3 appears to contribute to premature M-phase entry in lin-41 mutant oocytes. Genetic and phenotypic analyses indicate that LIN-41 and OMA-1/2 exhibit an antagonistic relationship, and we suggest that translational regulation by these proteins could be important for controlling and coordinating oocyte growth and meiotic maturation. PMID:25261698

  10. Nuclear and cytoplasmic maturation of mouse oocytes after treatment with synthetic meiosis-activating sterol in vitro.

    PubMed

    Hegele-Hartung, C; Kuhnke, J; Lessl, M; Grøndahl, C; Ottesen, J; Beier, H M; Eisner, S; Eichenlaub-Ritter, U

    1999-11-01

    Synthetically produced meiosis-activating sterol, a sterol originally derived from follicular fluid (FF-MAS), induces meiotic maturation of mouse oocytes in vitro. We therefore compared FF-MAS-induced maturation of naked mouse oocytes arrested in prophase I by either hypoxanthine (Hx) or forskolin (Fo) with spontaneous maturation of naked oocytes. FF-MAS-treated oocytes overcame the meiotic block by Hx or Fo, although germinal vesicle breakdown was delayed by 11 h and 7 h, respectively. We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly and by delaying CG release, which is known to contribute to reduced fertilization. PMID:10529286

  11. CDC20 downregulation impairs spindle morphology and causes reduced first polar body emission during bovine oocyte maturation.

    PubMed

    Yang, W L; Li, J; An, P; Lei, A M

    2014-03-01

    The cell division cycle protein 20 (CDC20) is an essential regulator of cell division, encoded by the CDC20 gene. However, the role of CDC20 in bovine oocyte maturation is unknown. In this study, CDC20 morpholino antisense oligonucleotides (MOs) were microinjected into the cytoplasm of bovine oocytes to block the translation of CDC20 mRNA. CDC20 downregulation significantly reduced the rate of first polar body emission (PB1). Further analysis indicated that oocytes treated with CDC20 MO arrested before or at meiotic stage I with abnormal spindles. To further confirm the functions of CDC20 during oocyte meiotic division, CDC20 MOs were microinjected into oocytes together with a supplementary PB1. The results showed that newly synthesized CDC20 was not necessary at the meiosis II-to-anaphase II transition. Our data suggest that CDC20 is required for spindle assembly, chromosomal segregation, and PB1 extrusion during bovine oocyte maturation. PMID:24360405

  12. The nuclear F-actin interactome of Xenopus oocytes reveals an actin-bundling kinesin that is essential for meiotic cytokinesis.

    PubMed

    Samwer, Matthias; Dehne, Heinz-Jürgen; Spira, Felix; Kollmar, Martin; Gerlich, Daniel W; Urlaub, Henning; Görlich, Dirk

    2013-07-01

    Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis. PMID:23727888

  13. Depletion of H2A-H2B dimers in Saccharomyces cerevisiae triggers meiotic arrest by reducing IME1 expression and activating the BUB2-dependent branch of the spindle checkpoint.

    PubMed Central

    Hanlon, Sean E; Norris, David N; Vershon, Andrew K

    2003-01-01

    In the yeast Saccharomyces cerevisiae, diploid strains carrying homozygous hta1-htb1Delta mutations express histone H2A-H2B dimers at a lower level than do wild-type cells. Although this mutation has only minor effects on mitotic growth, it causes an arrest in sporulation prior to the first meiotic division. In this report, we show that the hta1-htb1Delta mutant exhibits reduced expression of early and middle-sporulation-specific genes and that the meiotic arrest of the hta1-htb1Delta mutant can be partially bypassed by overexpression of IME1. Additionally, deletions of BUB2 or BFA1, components of one branch of the spindle checkpoint pathway, bypass the meiotic arrest. Mutations in the other branch of the pathway or in the pachytene checkpoint are unable to suppress the meiotic block. These observations indicate that depletion of the H2A-H2B dimer blocks sporulation by at least two mechanisms: disruption of the expression of meiotic regulatory genes and activation of the spindle checkpoint. Our results show that the failure to progress through the meiotic pathway is not the result of global chromosomal alterations but that specific aspects of meiosis are sensitive to depletion of the H2A-H2B dimer. PMID:12930743

  14. Effects of long-term in vitro exposure to phosphodiesterase type-3 inhibitors on follicle and oocyte development.

    PubMed

    Nogueira, D; Cortvrindt, R; Everaerdt, B; Smitz, J

    2005-08-01

    Germinal vesicle (GV)-stage oocytes retrieved from antral follicles undergo nuclear maturation in vitro, which typically occurs prior to cytoplasmic maturation. Short-term culture with meiotic inhibitors has been applied to arrest oocytes at the GV stage aiming to synchronize nuclear and ooplasmic maturity. However, the results obtained are still far from the in vivo situation. In order to acquire competence, immature oocytes may require meiotic arrest in vitro for a more extended period. The phosphodiesterase type 3-inhibitor (PDE3-I) is a potent meiotic arrester. The effects of a prolonged culture with PDE3-I on oocyte quality prior to and after reversal from the inhibition are not known. This study tested the impact of long-term in vitro exposure of two PDE3-Is, org9935 and cilostamide, on oocytes using a mouse follicle culture model. The results showed that PDE3-I (maximum of 10 microM) during a 12-day culture of follicle-enclosed oocytes did not alter somatic cell proliferation, differentiation or follicle survival. In addition, the steroid production profile was not significantly modified by a 12-day exposure to PDE3-I. The recombinant human chorionic gonadotrophin/recombinant human epidermal growth factor stimulus induced a characteristic normal progesterone peak of luteinization and normal mucification of the cumulus cells, while the enclosed oocyte remained blocked at the GV stage. In vitro maturation of denuded or cumulus-enclosed oocytes derived from org9935- or cilostamide-exposed follicles progressed through meiosis and formed morphologically normal meiotic spindles with chromosomes properly aligned at the equator. In conclusion, long-term culture with PDE3-I was harmless to somatic cell function, differentiation, oocyte growth and maturation. Our results suggested that PDE3-I can be applied when extended oocyte culture is required to improve ooplasmic maturation.

  15. Hormone-induced expression of tumor necrosis factor alpha-converting enzyme/A disintegrin and metalloprotease-17 impacts porcine cumulus cell oocyte complex expansion and meiotic maturation via ligand activation of the epidermal growth factor receptor.

    PubMed

    Yamashita, Yasuhisa; Kawashima, Ikkou; Yanai, Yoshiari; Nishibori, Masahide; Richards, Joanne S; Shimada, Masayuki

    2007-12-01

    The epidermal growth factor (EGF)-like growth factors, amphiregulin (AREG) and epiregulin (EREG), are expressed in murine cumulus oocyte complexes (COCs) where they impact the function of cumulus cells and oocyte maturation during LH-mediated ovulation. Because TNFalpha-converting enzyme (TACE)/a disintegrin and metalloprotease-17 (ADAM17) is essential for ectodomain shedding of AREG and EREG from the surface of other cell types, the expression and function of TACE/ADAM17 was analyzed in a porcine COC culture system in which FSH- and LH-mediated expansion and oocyte meiotic maturation have been well characterized and shown to occur between 20 and 40 h. In this model, Areg, Ereg, and Tace/Adam17 mRNAs increased significantly with maximal levels observed between 5 and 20 h of culture with FSH plus LH. TACE/ADAM17 protein and protease activity were up-regulated markedly at 10 h and maintained to 40 h. Treatment of COCs with the TACE/ADAM17-selective inhibitor TNFalpha-processing inhibitor-2 (TAPI-2) significantly suppressed in a time-dependent manner downstream targets of EGF receptor activation such as ERK1/2 phosphorylation, Ptgs2, Has2, and Tnfaip6 mRNA expression, hormone-induced COC expansion, and meiotic maturation of the oocytes. Addition of EGF to COCs cultured in the presence of FSH/LH reversed the inhibitory effects of TAPI-2 on these ovulation-related processes. Gonadotropin-induced phosphorylation of ERK1/2 was also inhibited in rat granulosa cells treated with TAPI-2 or after transfection with Tace/Adam17 small interfering RNA. Induced expression of Tnfaip6 mRNA was also reduced by Tace/Adam17 small interfering RNA. Thus, TACE/ADAM17 is induced and the activity is involved in porcine COC expansion as well as oocyte meiotic maturation through the activation of EGF receptor in cumulus cells.

  16. Maturation, fertilization, and development of marmoset monkey oocytes in vitro.

    PubMed

    Gilchrist, R B; Nayudu, P L; Hodges, J K

    1997-01-01

    This study was conducted to investigate 1) the capacity of in vitro-matured (IVM) marmoset oocytes to be fertilized and to support embryonic development in vitro and 2) oocyte meiotic maturation in relation to in vivo FSH administration, follicle size, and oocyte-cumulus cell status. Pairs of ovaries were collected on Day 4 of the follicular phase from adult females receiving either 1) human FSH (3 IU; n = 5) or 2) control (saline; n = 5) daily for 4 days. Antral follicles were excised from ovaries and separated into classes according to size: class 1 (660-840 microm), class 2 (> 840-1000 microm), class 3 (> 1000-1400 microm), and class 4 (> 1400 microm). A total of 823 partially naked and cumulus-enclosed oocytes (CEOs) were released from follicles and cultured in vitro. Cumulus cells remaining after 22 h were removed, metaphase II (MII) oocytes were inseminated with epididymal sperm, and resulting embryos were cultured until developmental arrest. Fluorescence microscopy was used to assess oocyte meiotic and embryo developmental progression. Oocyte germinal vesicle breakdown (GVB)- and MII-competencies increased significantly with follicular size (p < 0.01 and p < 0.0001, respectively), although they were independent of oocyte-cumulus cell associations. After 24 and 32 h in vitro, 69% and 93%, respectively, of CEOs with MII competence had completed meiotic maturation, and the rate of nuclear maturation increased progressively with follicle size (p < 0.01) and with the association of cumulus cells (p < 0.01). In vivo FSH priming slightly improved oocyte GVB- and MII-competencies (p < 0.01 and p < 0.05, respectively) and decreased the time required to achieve MII (p < 0.01). IVM oocytes from all follicle sizes fertilized (78-92%) in vitro, with 27% developing to morula- and 4% to blastocyst-stage embryos. This study demonstrates for the first time that IVM New World primate oocytes are able to support advanced preimplantation embryonic development in vitro. Oocyte

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

    PubMed

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

    2016-08-01

    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.

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

    PubMed

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

    2016-08-01

    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. PMID:27260975

  19. Bi-directional communication with the cumulus cells is involved in the deficiency of XY oocytes in the components essential for proper second meiotic spindle assembly.

    PubMed

    Xu, Baozeng; Noohi, Saeid; Shin, Jonghyun S; Tan, Seang Lin; Taketo, Teruko

    2014-01-15

    The oocyte becomes competent for embryonic development by involving mutual communication with cumulus cells (CCs) during folliculogenesis. How this communication takes place under physiological conditions is not fully understood. Current study examined oocyte-CCs communication in the XY sex-revered female mouse. We have previously found that the XY oocyte is defective in its cytoplasm, causing abnormal MII-spindle assembly and a failure in embryonic development. Our present study showed that transcript levels of Pfkp, Pkm2 and Ldh1 involved in glycolysis were lower in the CCs surrounding XY oocytes than in those surrounding XX oocytes. ATP contents in XY oocytes were also lower than those in XX oocytes, suggesting that lower glycolytic gene expression in CCs resulted in lower ATP contents in the enclosed oocyte. Co-culture of oocytectomized CC-oocyte complexes (COCs) with denuded oocytes showed that XY oocytes were less efficient than XX oocytes in promoting glycolytic gene expression in CCs. Furthermore, both glycolytic gene expression levels in CCs and ATP contents in oocytes of XY COCs increased to similar levels to those of XX COCs after culture for 20h in the presence of milrinone (=preincubation), which prevented spontaneous oocyte maturation. By increasing ATP levels in XY oocytes by either COC preincubation or ATP microinjection into oocytes prior to in vitro maturation, an improvement in MII-spindle assembly was observed. We conclude that the XY oocyte produces lesser amounts of paracrine factors that affect its companion CCs, which in turn make the ooplasm deficient in its components, including ATP, essential for MII-spindle assembly. PMID:24247007

  20. Protein synthesis inhibitors prevent both spontaneous and hormone-dependent maturation of isolated mouse oocytes

    SciTech Connect

    Downs, S.M. )

    1990-11-01

    The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors (CX, emetine (EM), and puromycin (PUR)) each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the controls (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP.

  1. Characterization of the effects of metformin on porcine oocyte meiosis and on AMP-activated protein kinase activation in oocytes and cumulus cells.

    PubMed

    Bilodeau-Goeseels, Sylvie; Magyara, Nora; Collignon, Coralie

    2014-05-01

    The adenosine monophosphate-activated protein kinase (AMPK) activators 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) and metformin (MET) inhibit resumption of meiosis in porcine cumulus-enclosed oocytes. The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine cumulus-enclosed oocytes; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells. The AMPK inhibitor compound C (CC; 1 μM) did not reverse the inhibitory effect of AICAR (1 mM) and MET (2 mM) on porcine oocyte meiosis. Additionally, CC had a significant inhibitory effect on its own. eNOS, c-Src and PI-3 kinase pathway inhibitors did not reverse the effect of metformin on porcine oocyte meiosis. The level of acetyl-CoA carboxylase (ACC) phosphorylation in oocytes and cumulus cells did not change in response to culture in the presence of MET, AICAR, CC, the CaMKK inhibitor STO-609 or the Ca2+ chelator BAPTA-AM for 3 h, but STO-609 increased the percentage of porcine cumulus-enclosed oocytes (CEO) that remained at the germinal vesicle (GV) stage after 24 h of culture. These results indicate that the inhibitory effect of MET and AICAR on porcine oocyte meiosis was probably not mediated through activation of AMPK.

  2. Effects of cilostamide and/or forskolin on the meiotic resumption and development competence of growing ovine oocytes selected by brilliant cresyl blue staining.

    PubMed

    Azari-Dolatabad, Nima; Rahmani, H R; Hajian, M; Ostadhosseini, S; Hosseini, S M; Nasr-Esfahani, M H

    2016-05-01

    The relevance of low developmental competence of in vitro-matured oocyte to the incomplete/delayed cytoplasmic maturation, and the heterogeneity of retrieved oocytes is well established in several species. A short phase of prematuration culture was used to allow better oocyte cytoplasmic maturation. The preselection of growing and fully grown oocytes has been proposed to improve developmental competency. This study investigated the effects of phosphodiesterase type 3-specific inhibitor, cilostamide, and adenylate cyclase activator, forskolin, on the resumption of meiosis and developmental competence of growing ovine oocytes selected by brilliant cresyl blue (BCB) staining. Results indicate that cilostamide, forskolin, and their combination significantly (P < 0.05) increased the percentage of growing (BCB-) oocytes maintained at the germinal vesicle stage. However, only forskolin significantly (P < 0.05) increased the yield and quality of blastocysts derived from BCB- oocytes compared with non-BCB-treated oocytes. We conclude that a short prematuration culture with forskolin may improve the in vitro developmental competency of growing oocytes in ovine. PMID:26879998

  3. TFIIB co-localizes and interacts with α-tubulin during oocyte meiosis in the mouse and depletion of TFIIB causes arrest of subsequent embryo development.

    PubMed

    Liu, Hui; Yin, Feng-Xia; Bai, Chun-Ling; Shen, Qi-Yuan; Wei, Zhu-Ying; Li, Xin-Xin; Liang, Hao; Bou, Shorgan; Li, Guang-Peng

    2013-01-01

    TFIIB (transcription factor IIB) is a transcription factor that provides a bridge between promoter-bound TFIID and RNA polymerase II, and it is a target of various transcriptional activator proteins that stimulate the pre-initiation complex assembly. The localization and/or attachment matrix of TFIIB in the cytoplast is not well understood. This study focuses on the function of TFIIB and its interrelationship with α-tubulins in a mouse model. During oocyte maturation TFIIB distributes throughout the entire nucleus of the germinal vesicle (GV). After progression to GV breakdown (GVBD), TFIIB and α-tubulin co-localize and accumulate in the vicinity of the condensed chromosomes. During the MII stage, the TFIIB signals are more concentrated at the equatorial plate and the kinetochores. Colcemid treatment of oocytes disrupts the microtubule (MT) system, although the TFIIB signals are still present with the altered MT state. Injection of oocytes with TFIIB antibodies and siRNAs causes abnormal spindle formation and irregular chromosome alignment. These findings suggest that TFIIB dissociates from the condensed chromatids and then tightly binds to microtubules from GVBD to the MII phase. The assembly and disassembly of TFIIB may very well be associated with and driven by microtubules. TFIIB maintains its contact with the α-tubulins and its co-localization forms a unique distribution pattern. Depletion of Tf2b in oocytes results in a significant decrease in TFIIB expression, although polar body extrusion does not appear to be affected. Knockdown of Tf2b dramatically affects subsequent embryo development with more than 85% of the embryos arrested at the 2-cell stage. These arrested embryos still maintain apparently normal morphology for at least 96h without any obvious degeneration. Analysis of the effects of TFIIB in somatic cells by co-transfection of BiFC plasmids pHA-Tf2b and pFlag-Tuba1α further confirms a direct interaction between TFIIB and α-tubulins.

  4. Transducin-like enhancer of split-6 (TLE6) is a substrate of protein kinase A activity during mouse oocyte maturation.

    PubMed

    Duncan, Francesca E; Padilla-Banks, Elizabeth; Bernhardt, Miranda L; Ord, Teri S; Jefferson, Wendy N; Moss, Stuart B; Williams, Carmen J

    2014-03-01

    Fully grown oocytes in the ovary are arrested at prophase of meiosis I because of high levels of intraoocyte cAMP that maintain increased levels of cAMP-dependent protein kinase (PKA) activity. Following the luteinizing hormone surge at the time of ovulation, cAMP levels drop, resulting in a reduction in PKA activity that triggers meiotic resumption. Although much is known about the molecular mechanisms of how PKA activity fluctuations initiate the oocyte's reentry into meiosis, significantly less is known about the requirement for PKA activity in the oocyte after exit from the prophase I arrest. Here we show that although PKA activity decreases in the oocyte upon meiotic resumption, it increases throughout meiotic progression from the time of germinal vesicle breakdown (GVBD) until the metaphase II (MII) arrest. Blocking this meiotic maturation-associated increase in PKA activity using the pharmacological inhibitor H89 resulted in altered kinetics of GVBD, defects in chromatin and spindle dynamics, and decreased ability of oocytes to reach MII. These effects appear to be largely PKA specific because inhibitors targeting other kinases did not have the same outcomes. To determine potential proteins that may require PKA phosphorylation during meiosis, we separated oocyte protein extracts on an SDS-PAGE gel, extracted regions of the gel that had corresponding immune reactivity towards an anti-PKA substrate antibody, and performed mass spectrometry and microsequencing. Using this approach, we identified transducin-like enhancer of split-6 (TLE6)-a maternal effect gene that is part of the subcortical maternal complex-as a putative PKA substrate. TLE6 localized to the oocyte cortex throughout meiosis in a manner that is spatially and temporally consistent with the localization of critical PKA subunits. Moreover, we demonstrated that TLE6 becomes phosphorylated in a narrow window following meiotic resumption, and H89 treatment can completely block this phosphorylation

  5. Down-regulation of membrana granulosa cell gap junctions is correlated with irreversible commitment to resume meiosis in golden Syrian hamster oocytes.

    PubMed

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

    1989-08-01

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

  6. Phospholipid turnover and ultrastructural correlates during spontaneous germinal vesicle breakdown of the bovine oocyte: Effects of a cyclic AMP phosphodiesterase inhibitor

    SciTech Connect

    Homa, S.T.; Webster, S.D.; Russell, R.K. )

    1991-08-01

    The turnover of (32P)orthophosphate in bovine oocyte phospholipids was studied during the early stages of spontaneous meiotic maturation, and during inhibition of this process by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Radioactive lipids were separated by TLC and the meiotic stage was determined cytogenetically. Ultrastructure of the nuclear membrane was examined using transmission EM. During the commitment period to meiotic resumption, which precedes germinal vesicle breakdown (GVBD), small localized convolutions appeared in the intact nuclear membrane. This was accompanied by a decrease in (32P)phosphatidic acid (PA) and an increase in (32P)-phosphatidylcholine (PC). This was followed by extensive convolutions, and subsequent dissociation, of the nuclear membrane, concomitant with a tremendous surge in (32P)PC and (32P)phosphatidylethanolamine (PE). The cAMP-mediated maintenance of meiotic arrest involved retention of entire nuclear envelope integrity and total inhibition of the surge in (32P)PC and (32P)PE which accompanied GVBD. The increase in (32P)phosphatidylinositol (PI) associated with all stages of early meiotic resumption was unaffected by IBMX. Microinjection of heparin inhibited GVBD, and injection of inositol 1,4,5-trisphosphate (IP3) overrode IBMX-maintained meiotic arrest in almost 40% of the oocytes. The results suggest that there may be several functions for phospholipid turnover in the regulation of spontaneous meiotic resumption in the bovine oocyte. The first precedes the commitment period, and involves IP3 generation to serve as the primary signal for meiotic resumption. The second occurs concomitant with the commitment period, is unaffected by the level of intracellular cAMP, and is associated with the general turnover of phospholipid.

  7. Inhibition of the Binding between RGS2 and β-Tubulin Interferes with Spindle Formation and Chromosome Segregation during Mouse Oocyte Maturation In Vitro

    PubMed Central

    Sun, Zhao-Gui; Zhang, Zhi; Zhu, Yan

    2016-01-01

    RGS2 is a negative regulator of G protein signaling that contains a GTPase-activating domain and a β-tubulin binding region. This study aimed to determine the localization and function of RGS2 during mouse oocyte maturation in vitro. Immunofluorescent staining revealed that RGS2 was widely expressed in the cytoplasm with a greater abundance on both meiotic spindles and first/second polar bodies from the fully-grown germinal vesicle (GV) stage to the MII stages. Co-expression of RGS2 and β-tubulin could also be detected in the spindle and polar body of mouse oocytes at the MI, AI, and MII stages. Inhibition of the binding site between RGS2 and β-tubulin was accomplished by injecting anti-RGS2 antibody into GV-stage oocytes, which could result in oocytes arrest at the MI or AI stage during in vitro maturation, but it did not affect germinal vesicle breakdown. Moreover, injecting anti-RGS2 antibody into oocytes resulted in a significant reduction in the rate of first polar body extrusion and abnormal spindle formation. Additionally, levels of phosphorylated MEK1/2 were significantly reduced in anti-RGS2 antibody injected oocytes compared with control oocytes. These findings suggest that RGS2 might play a critical role in mouse oocyte meiotic maturation by affecting β-tubulin polymerization and chromosome segregation. PMID:27463806

  8. CDC25A phosphatase controls meiosis I progression in mouse oocytes.

    PubMed

    Solc, Petr; Saskova, Adela; Baran, Vladimir; Kubelka, Michal; Schultz, Richard M; Motlik, Jan

    2008-05-01

    CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a -/- mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes.

  9. CDC25A phosphatase controls meiosis I progression in mouse oocytes

    PubMed Central

    Solc, Petr; Saskova, Adela; Baran, Vladimir; Kubelka, Michal; Schultz, Richard M.; Motlik, Jan

    2008-01-01

    CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a −/− mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Last, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes. PMID:18367163

  10. Interactions between oocytes and cumulus cells during in vitro maturation of porcine cumulus-oocyte complexes in a chemically defined medium: effect of denuded oocytes on cumulus expansion and oocyte maturation.

    PubMed

    Appeltant, R; Somfai, T; Nakai, M; Bodó, S; Maes, D; Kikuchi, K; Van Soom, A

    2015-03-01

    The aim of the present study was to clarify interactions between oocytes and cumulus cells (CCs) on the level of cumulus expansion and oocyte maturation during IVM of cumulus-oocyte complexes (COCs) in a chemically defined medium using a system that allows individual tracking of oocytes. Especially, the influence of oocyte-secreted factors was investigated by the aid of addition of denuded oocytes (DOs) as a possible approach to improve the IVM system. The basic maturation medium was porcine oocyte medium with addition of gonadotropins only during the first 20 hours of IVM. During IVM, COCs were kept fixed to the bottom of culture dish by adhesive Cell-Tak coating, which enabled individual tracking of COCs during IVM. Size changes in COCs during IVM were measured by digital image analysis. Cumulus expansion in a porcine oocyte medium of intact COCs increased in a typical manner until 20 hours and decreased in size subsequently until 48 hours of IVM (P < 0.05). Removal of oocytes from COCs by oocytectomy allowed the expansion of CCs to some extent, although their expansion ability was lower than that of COCs (P < 0.05). Addition of DOs (COCs to DOs ratio of 9:16) did not improve cumulus expansion and oocyte maturation rates of intact COCs (P > 0.05) but did enhance cumulus expansion of oocytectomized complexes (P < 0.05). Furthermore, removal of CCs before IVM increased oocyte maturation rates compared with COCs (52.3% and 32.9%, respectively) (P < 0.05) and a similar effect was observed in COCs when the gap junction inhibitor carbenoxolone was added to the IVM medium: carbenoxolone repressed the expansion of COCs at 20 hours of IVM. In conclusion, the porcine oocyte enhances cumulus expansion both by gap junctional communications and presumably by oocyte-secreted factor production. Nevertheless, the presence of oocytes is not a prerequisite for this process. In return, CCs maintain meiotic arrest in cumulus-enclosed oocytes during the initial culture

  11. Interactions between oocytes and cumulus cells during in vitro maturation of porcine cumulus-oocyte complexes in a chemically defined medium: effect of denuded oocytes on cumulus expansion and oocyte maturation.

    PubMed

    Appeltant, R; Somfai, T; Nakai, M; Bodó, S; Maes, D; Kikuchi, K; Van Soom, A

    2015-03-01

    The aim of the present study was to clarify interactions between oocytes and cumulus cells (CCs) on the level of cumulus expansion and oocyte maturation during IVM of cumulus-oocyte complexes (COCs) in a chemically defined medium using a system that allows individual tracking of oocytes. Especially, the influence of oocyte-secreted factors was investigated by the aid of addition of denuded oocytes (DOs) as a possible approach to improve the IVM system. The basic maturation medium was porcine oocyte medium with addition of gonadotropins only during the first 20 hours of IVM. During IVM, COCs were kept fixed to the bottom of culture dish by adhesive Cell-Tak coating, which enabled individual tracking of COCs during IVM. Size changes in COCs during IVM were measured by digital image analysis. Cumulus expansion in a porcine oocyte medium of intact COCs increased in a typical manner until 20 hours and decreased in size subsequently until 48 hours of IVM (P < 0.05). Removal of oocytes from COCs by oocytectomy allowed the expansion of CCs to some extent, although their expansion ability was lower than that of COCs (P < 0.05). Addition of DOs (COCs to DOs ratio of 9:16) did not improve cumulus expansion and oocyte maturation rates of intact COCs (P > 0.05) but did enhance cumulus expansion of oocytectomized complexes (P < 0.05). Furthermore, removal of CCs before IVM increased oocyte maturation rates compared with COCs (52.3% and 32.9%, respectively) (P < 0.05) and a similar effect was observed in COCs when the gap junction inhibitor carbenoxolone was added to the IVM medium: carbenoxolone repressed the expansion of COCs at 20 hours of IVM. In conclusion, the porcine oocyte enhances cumulus expansion both by gap junctional communications and presumably by oocyte-secreted factor production. Nevertheless, the presence of oocytes is not a prerequisite for this process. In return, CCs maintain meiotic arrest in cumulus-enclosed oocytes during the initial culture

  12. Oocytes from small and large follicles exhibit equal development competence following goat cloning despite their differences in meiotic and cytoplasmic maturation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Somatic cell nuclear transfer (SCNT) in animals has been around for nearly 20 years and has been successfully used for cloning of various livestock species. In this study, goat oocytes were collected from large follicles (>3mm) and small follicles (<3mm) to compare the success rate when used in goat...

  13. Abnormal chromosome behavior in human oocytes which remained unfertilized during human in vitro fertilization.

    PubMed

    Spielmann, H; Krüger, C; Stauber, M; Vogel, R

    1985-09-01

    Chromosomal abnormalities and abnormal embryonic development have previously been observed after human in vitro fertilization (IVF). Chromosomal abnormalities may arise not only after fertilization but even earlier during meiotic maturation of human oocytes in culture. Since chromosomal analysis is simple in oocytes during meiotic maturation, the chromosomal status was analyzed in oocytes which remained unfertilized in a human in vitro fertilization program. In 50 fertilization attempts the chromosomes of 62 unfertilized oocytes could be analyzed; 45 of them were in the process of meiotic maturation. In three oocytes two small polar bodies were observed 16-18 hr after insemination in the absence of fertilization. In one oocyte abnormal chromosome behavior was found during the first meiotic division, and in four oocytes during metaphase of the second meiotic division. These data suggest that chromosomal analysis of unfertilized oocytes in human IVF may improve the understanding human oocyte maturation and fertilization.

  14. Antibody microarray analyses of signal transduction protein expression and phosphorylation during porcine oocyte maturation.

    PubMed

    Pelech, Steven; Jelinkova, Lucie; Susor, Andrej; Zhang, Hong; Shi, Xiaoqing; Pavlok, Antonin; Kubelka, Michal; Kovarova, Hana

    2008-07-01

    Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation.

  15. Cell cycle arrest and activation of development in marine invertebrate deuterostomes.

    PubMed

    Costache, Vlad; McDougall, Alex; Dumollard, Rémi

    2014-08-01

    Like most metazoans, eggs of echinoderms and tunicates (marine deuterostomes, there is no data for the cephalochordates) arrest awaiting fertilization due to the activity of the Mos/MEK/MAPK cascade and are released from this cell cycle arrest by sperm-triggered Ca2+ signals. Invertebrate deuterostome eggs display mainly three distinct types of cell cycle arrest before fertilization mediated by potentially different cytostatic factors (CSF): one CSF causes arrest during meiotic metaphase I (MI-CSF in tunicates and some starfishes), another CSF likely causes arrest during meiotic metaphase II (amphioxus), and yet another form of CSF causes arrest to occur after meiotic exit during G1 of the first mitotic cycle (G1-CSF). In tunicates and echinoderms these different CSF activities have been shown to rely on the Mos//MAPK pathway for establishment and on Ca2+ signals for their inactivation. Despite these molecular similarities, release of MI-CSF arrest is caused by APC/C activation (to destroy cyclin B) whereas release from G1-CSF is caused by stimulating S phase and the synthesis of cyclins. Further research is needed to understand how both the Mos//MAPK cascade and Ca2+ achieve these tasks in different marine invertebrate deuterostomes. Another conserved feature of eggs is that protein synthesis of specific mRNAs is necessary to proceed through oocyte maturation and to maintain CSF-induced cell cycle arrest. Then activation of development at fertilization is accompanied by an increase in the rate of protein synthesis but the mechanisms involved are still largely unknown in most of the marine deuterostomes. How the sperm-triggered Ca2+ signals cause an increase in protein synthesis has been studied mainly in sea urchin eggs. Here we review these conserved features of eggs (arrest, activation and protein synthesis) focusing on the non-vertebrate deuterostomes. PMID:24721426

  16. Cell cycle arrest and activation of development in marine invertebrate deuterostomes.

    PubMed

    Costache, Vlad; McDougall, Alex; Dumollard, Rémi

    2014-08-01

    Like most metazoans, eggs of echinoderms and tunicates (marine deuterostomes, there is no data for the cephalochordates) arrest awaiting fertilization due to the activity of the Mos/MEK/MAPK cascade and are released from this cell cycle arrest by sperm-triggered Ca2+ signals. Invertebrate deuterostome eggs display mainly three distinct types of cell cycle arrest before fertilization mediated by potentially different cytostatic factors (CSF): one CSF causes arrest during meiotic metaphase I (MI-CSF in tunicates and some starfishes), another CSF likely causes arrest during meiotic metaphase II (amphioxus), and yet another form of CSF causes arrest to occur after meiotic exit during G1 of the first mitotic cycle (G1-CSF). In tunicates and echinoderms these different CSF activities have been shown to rely on the Mos//MAPK pathway for establishment and on Ca2+ signals for their inactivation. Despite these molecular similarities, release of MI-CSF arrest is caused by APC/C activation (to destroy cyclin B) whereas release from G1-CSF is caused by stimulating S phase and the synthesis of cyclins. Further research is needed to understand how both the Mos//MAPK cascade and Ca2+ achieve these tasks in different marine invertebrate deuterostomes. Another conserved feature of eggs is that protein synthesis of specific mRNAs is necessary to proceed through oocyte maturation and to maintain CSF-induced cell cycle arrest. Then activation of development at fertilization is accompanied by an increase in the rate of protein synthesis but the mechanisms involved are still largely unknown in most of the marine deuterostomes. How the sperm-triggered Ca2+ signals cause an increase in protein synthesis has been studied mainly in sea urchin eggs. Here we review these conserved features of eggs (arrest, activation and protein synthesis) focusing on the non-vertebrate deuterostomes.

  17. Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

    PubMed Central

    Yang, Shang-Wu; Gao, Chen; Chen, Lei; Song, Ya-Li; Zhu, Jin-Liang; Qi, Shu-Tao; Jiang, Zong-Zhe; Wang, Zhong-Wei; Lin, Fei; Huang, Hao; Xing, Fu-Qi; Sun, Qing-Yuan

    2012-01-01

    Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage. PMID:23159858

  18. Post-translational processing of purified human K-ras in Xenopus oocytes.

    PubMed

    Kaplan, J B; Sass, P M

    1991-01-01

    Membrane localization of ras p21 involves a complex series of post-translational processing events, including S-farnesylation of Cys-186, removal of three carboxyl-terminal amino acid residues, and methylation of the carboxyl-terminal farnesylcysteine residue. Palmitoylation of cysteine residues within the hypervariable region (amino acids 165-185) is also required for membrane localization of mammalian H-, N-, and K-ras(A). For K-ras(B), which contains no cysteine residues within the hypervariable region, a polybasic domain substitutes for palmitoylation as a second signal for plasma membrane targeting. In order to investigate the localization of K-ras(B) to the plasma membrane, we purified wild-type and mutant human K-ras(B) proteins from strains of E. coli harboring bacterial expression plasmids and injected them into Xenopus laevis oocytes. Our results show that wild-type and activated K-ras(B) proteins can be post-translationally modified and can induce meiotic maturation in Xenopus oocytes. A mutation at Cys-186 (Cys to Gly) abolished the ability of activated K-ras(B) to induce meiosis. Deprivation of isoprenyl precursors by the addition of lovastatin, a drug that blocks the synthesis of mevalonate, also abolished the ability of activated K-ras(B) to induce meiosis, although this inhibition could be overcome by the addition of exogenous mevalonate. Lovastatin did not block meiotic maturation induced by microinjection of purified mos protein, a component of the cytostatic factor that arrests Xenopus oocytes at the first meiotic prophase. These results indicate that post-translational isoprenylation of K-ras(B) is essential for plasma membrane targeting and induction of meiotic maturation in Xenopus oocytes and that further isoprenyl modification of proteins downstream from mos signal transduction is not essential for this process. PMID:16296004

  19. Cryopreservation of starfish oocytes.

    PubMed

    Hamaratoğlu, Fisun; Eroğlu, Ali; Toner, Mehmet; Sadler, Kirsten C

    2005-02-01

    Research from many laboratories over the past several decades indicates that invertebrate oocytes and eggs are extraordinarily difficult to freeze. Since starfish oocytes, eggs, and embryos are an important cell and developmental biology model system, there is great interest to cryopreserve these cells. Previous starfish oocyte cryopreservation studies using slow cooling protocols revealed that these cells are highly sensitive to osmotic stress and form intracellular ice at very high sub-zero temperatures, suggesting that common freezing methodologies may not prove useful. We report here that a short exposure to 1.5 M Me2SO/1 M trehalose in hypotonic salt solution followed by ultra-rapid cooling to cryogenic temperatures allows starfish oocytes to be cryopreserved with the average survival rate of 34% when normalized to control oocytes that were exposed to CPA, but not frozen. On average, 51% of the oocytes in 77% of the batches of frozen oocytes underwent meiotic maturation in response to the starfish maturation hormone, 1-methyladenine. In one experiment, eggs developing from thawed oocytes were capable of being fertilized and two developed into embryos. These data suggests that successful cryopreservation of starfish oocytes is possible, but will need further refinement to increase the numbers of fully competent embryos. PMID:15710368

  20. Cryopreservation of starfish oocytes.

    PubMed

    Hamaratoğlu, Fisun; Eroğlu, Ali; Toner, Mehmet; Sadler, Kirsten C

    2005-02-01

    Research from many laboratories over the past several decades indicates that invertebrate oocytes and eggs are extraordinarily difficult to freeze. Since starfish oocytes, eggs, and embryos are an important cell and developmental biology model system, there is great interest to cryopreserve these cells. Previous starfish oocyte cryopreservation studies using slow cooling protocols revealed that these cells are highly sensitive to osmotic stress and form intracellular ice at very high sub-zero temperatures, suggesting that common freezing methodologies may not prove useful. We report here that a short exposure to 1.5 M Me2SO/1 M trehalose in hypotonic salt solution followed by ultra-rapid cooling to cryogenic temperatures allows starfish oocytes to be cryopreserved with the average survival rate of 34% when normalized to control oocytes that were exposed to CPA, but not frozen. On average, 51% of the oocytes in 77% of the batches of frozen oocytes underwent meiotic maturation in response to the starfish maturation hormone, 1-methyladenine. In one experiment, eggs developing from thawed oocytes were capable of being fertilized and two developed into embryos. These data suggests that successful cryopreservation of starfish oocytes is possible, but will need further refinement to increase the numbers of fully competent embryos.

  1. The methyltransferase Setdb1 is essential for meiosis and mitosis in mouse oocytes and early embryos.

    PubMed

    Eymery, Angeline; Liu, Zichuan; Ozonov, Evgeniy A; Stadler, Michael B; Peters, Antoine H F M

    2016-08-01

    Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 downregulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a newly discovered meiotic and embryonic competence factor safeguarding genome integrity at the onset of life.

  2. The methyltransferase Setdb1 is essential for meiosis and mitosis in mouse oocytes and early embryos.

    PubMed

    Eymery, Angeline; Liu, Zichuan; Ozonov, Evgeniy A; Stadler, Michael B; Peters, Antoine H F M

    2016-08-01

    Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 downregulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a newly discovered meiotic and embryonic competence factor safeguarding genome integrity at the onset of life. PMID:27317807

  3. Ganglioside GD1a promotes oocyte maturation, furthers preimplantation development, and increases blastocyst quality in pigs

    PubMed Central

    KIM, Jin-Woo; PARK, Hyo-Jin; CHAE, Sung-Kyu; AHN, Jae-Hyun; DO, Geon-Yeop; CHOO, Young-Kug; PARK, Joung Jun; JUNG, Bae Dong; KIM, Sun-Uk; CHANG, Kyu-Tae; KOO, Deog-Bon

    2016-01-01

    Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs. PMID:26860251

  4. Meiotic abnormalities

    SciTech Connect

    1993-12-31

    Chapter 19, describes meiotic abnormalities. These include nondisjunction of autosomes and sex chromosomes, genetic and environmental causes of nondisjunction, misdivision of the centromere, chromosomally abnormal human sperm, male infertility, parental age, and origin of diploid gametes. 57 refs., 2 figs., 1 tab.

  5. Wee1B depletion promotes nuclear maturation of canine oocytes.

    PubMed

    Kim, Yu-Gon; Kim, Dong-Hoon; Song, Seok-Hwan; Lee, Kyeong-Lim; Yang, Byoung-Chul; Oh, Jeong Su; Lee, Sang-Ryeul; Kong, Il-Keun

    2015-03-01

    Most mammalian oocytes are arrested at the germinal vesicle stage by activation of Wee1B. Meiotic resumption is regulated by inactivation of Wee1B and activation of cell division cycle 25B. The aim of this study was to determine whether treatment with Wee1B-targeting small interfering RNA (Wee1B-siRNA) promotes nuclear maturation of canine oocytes from germinal vesicle stage to metaphase II (MII) stage. In experiment 1, the percentage of canine oocytes that matured to MII stage was higher (P < 0.05) among oocytes cultured in vitro for 72 hours than among those cultured for 24 and 48 hours (5.4 ± 2.5% vs. 0.0 ± 0.0% and 1.4 ± 1.0%, respectively). Furthermore, the percentage of oocytes that matured to metaphase I (MI) stage was higher (P < 0.05) among oocytes cultured for 48 and 72 hours than among those cultured for 24 hours (14.9 ± 10.0% and 22.4 ± 8.1%, respectively, vs. 5.7 ± 6.0%). In experiment 2, canine oocytes were intracytoplasmically microinjected with Wee1B-siRNA (50 μM) at various culture time points (0, 24, 48, or 72 hours). The nuclear configuration of the exception of oocytes in the 72-hour group was examined after 84 hours of culture. The percentage of oocytes that matured to the MII stage was higher (P < 0.05) among those treated with Wee1B-siRNA at 0 hours than among control oocytes and those injected at 72 hours (18.0 ± 1.7% vs. 2.1 ± 2.8% and 0.0 ± 0.0%, respectively). Moreover, the percentage of oocytes that matured to the MI stage was higher (P < 0.05) among those injected at 0 hours than among control oocytes and those injected at 24 and 72 hours (45.9 ± 6.8% vs. 22.1 ± 3.5%, 22.8 ± 10.0%, and 10.0 ± 4.4%, respectively). In experiment 3, oocytes were intracytoplasmically microinjected with Wee1B-siRNA at 0 hours of IVM and cultured for 0, 24, 48, or 72 hours. Thereafter, maturation-related gene expression was analyzed by quantitative real-time polymerase chain reaction. Messenger RNA

  6. Nondisjunctional segregations in Drosophila female meiosis I are preceded by homolog malorientation at metaphase arrest.

    PubMed

    Gillies, Shane C; Lane, Fiona M; Paik, Wonbeom; Pyrtel, Khateriaa; Wallace, Nneka T; Gilliland, William D

    2013-02-01

    The model of Drosophila female meiosis I was recently revised by the discovery that chromosome congression precedes metaphase I arrest. Use of the prior framework to interpret data from meiotic mutants led to the conclusion that chromosome segregation errors (nondisjunction, NDJ) occurred when nonexchange chromosomes moved out on the spindle in a maloriented configuration and became trapped there at metaphase arrest. The discovery that congression returns nonexchange chromosomes to the metaphase plate invalidates this interpretation and raises the question of what events actually do lead to NDJ. To address this, we have assayed an allelic series of ald (mps1) meiotic mutants that complete congression at wild-type rates, but have widely varying NDJ rates in an otherwise isogenic background, as well as a nod mutant background that primarily undergoes loss of chromosome 4. Using genetic assays to measure NDJ rates, and FISH assays to measure chromosome malorientation rates in metaphase-arrested oocytes, shows that these two rates are highly correlated across ald mutants, suggesting that malorientation during congression commits these chromosomes to eventually nondisjoin. Likewise, the rate of chromosome loss observed in nod is similar to the rate at which these chromosomes fail to associate with the main chromosome mass. Together these results provide a proximal mechanism for how these meiotic mutants cause NDJ and chromosome loss and improve our understanding of how prometaphase chromosome congression relates to anaphase chromosome segregation. PMID:23222652

  7. Nondisjunctional Segregations in Drosophila Female Meiosis I Are Preceded by Homolog Malorientation at Metaphase Arrest

    PubMed Central

    Gillies, Shane C.; Lane, Fiona M.; Paik, Wonbeom; Pyrtel, Khateriaa; Wallace, Nneka T.; Gilliland, William D.

    2013-01-01

    The model of Drosophila female meiosis I was recently revised by the discovery that chromosome congression precedes metaphase I arrest. Use of the prior framework to interpret data from meiotic mutants led to the conclusion that chromosome segregation errors (nondisjunction, NDJ) occurred when nonexchange chromosomes moved out on the spindle in a maloriented configuration and became trapped there at metaphase arrest. The discovery that congression returns nonexchange chromosomes to the metaphase plate invalidates this interpretation and raises the question of what events actually do lead to NDJ. To address this, we have assayed an allelic series of ald (mps1) meiotic mutants that complete congression at wild-type rates, but have widely varying NDJ rates in an otherwise isogenic background, as well as a nod mutant background that primarily undergoes loss of chromosome 4. Using genetic assays to measure NDJ rates, and FISH assays to measure chromosome malorientation rates in metaphase-arrested oocytes, shows that these two rates are highly correlated across ald mutants, suggesting that malorientation during congression commits these chromosomes to eventually nondisjoin. Likewise, the rate of chromosome loss observed in nod is similar to the rate at which these chromosomes fail to associate with the main chromosome mass. Together these results provide a proximal mechanism for how these meiotic mutants cause NDJ and chromosome loss and improve our understanding of how prometaphase chromosome congression relates to anaphase chromosome segregation. PMID:23222652

  8. Gld-1, a Tumor Suppressor Gene Required for Oocyte Development in Caenorhabditis Elegans

    PubMed Central

    Francis, R.; Barton, M. K.; Kimble, J.; Schedl, T.

    1995-01-01

    We have characterized 31 mutations in the gld-1 (defective in germline development) gene of Caenorhabditis elegans. In gld-1(null) hermaphrodites, oogenesis is abolished and a germline tumor forms where oocyte development would normally occur. By contrast, gld-1(null) males are unaffected. The hermaphrodite germline tumor appears to derive from germ cells that enter the meiotic pathway normally but then exit pachytene and return to the mitotic cycle. Certain gld-1 partial loss-of-function mutations also abolish oogenesis, but germ cells arrest in pachytene rather than returning to mitosis. Our results indicate that gld-1 is a tumor suppressor gene required for oocyte development. The tumorous phenotype suggests that gld-1(+) may function to negatively regulate proliferation during meiotic prophase and/or act to direct progression through meiotic prophase. We also show that gld-1(+) has an additional nonessential role in germline sex determination: promotion of hermaphrodite spermatogenesis. This function of gld-1 is inferred from a haplo-insufficient phenotype and from the properties of gain-of-function gld-1 mutations that cause alterations in the sexual identity of germ cells. PMID:7713419

  9. gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans

    SciTech Connect

    Francis, R.; Schedl, T.; Barton, M.K.; Kimble, J.

    1995-02-01

    We have characterized 31 mutations in the gld-1 (defective in germline development) gene of Caenorhabditis elegans. In gld-1 (null) hermaphrodites, oogenesis is abolished and a germline tumor forms where oocyte development would normally occur. By contrast, gld-1 (null) males are unaffected. The hermaphrodite germline tumor appears to derive from germ cells that enter the meiotic pathway normally but then exit pachytene and return to the mitotic cycle. Certain gld-1 partial loss-of-function mutations also abolish oogenesis, but germ cells arrest in pachytene rather than returning to mitosis. Our results indicate that gld-1 is a tumor suppressor gene required for oocyte development. The tumorous phenotype suggests that gld-1(+) may function to negatively regulate proliferation during meiotic prophase and/or act to direct progression through meiotic prophase. We also show that gld-1(+) has an additional nonessential role in germline sex determination: promotion of hermaphrodite spermatogenesis. This function of gld-1 is inferred from a haplo-insufficient phenotype and from the properties of gain-of-function gld-1 mutations that cause alterations in the sexual identity of germ cells. 69 refs., 10 figs., 8 tabs.

  10. Meiotic process and aneuploidy

    SciTech Connect

    Grell, R.F.

    1985-01-01

    The process of meiosis is analyzed by dissecting it into its component parts using the early oocyte of Drosophila as a model. Entrance of the oocytes into premeiotic interphase signals initiation of DNA replication which continues for 30 h. Coincidentally, extensive synaptonemal complexes appear, averaging 50 ..mu..m (132 h), peaking at 75 ..mu..m (144 h) and continuing into early vitellarial stages. Recombinational response to heat, evidenced by enhancement or induction of exchange, is limited to the S-phase with a peak at 144 h coinciding with maximal extension of the SC. Coincidence of synapsis and recombination response with S at premeiotic interphase is contrary to their conventional localization at meiotic prophase. The interrelationship between exchange and nondisjunction has been clarified by the Distributive Pairing Model of meiosis. Originally revealed through high frequencies of nonrandom assortment of nonhomologous chromosomes, distributive pairing has been shown to follow and to be noncompetitive with exchange, to be based on size-recognition, not homology, and as a raison d'etre, to provide a segregational mechanism for noncrossover homologues. Rearrangements, recombination mutants and aneuploids may contribute noncrossover chromosomes to the distributive pool and so promote the nonhomologous associations responsible for nondisjunction of homologues and regular segregation of nonhomologues. 38 references, 15 figures. (ACR)

  11. Effect of oil overlay on inhibition potential of roscovitine in sheep cumulus-oocyte complexes.

    PubMed

    Crocomo, L F; Marques Filho, W C; Ulian, C M V; Branchini, N S; Silva, D T; Ackermann, C L; Landim-Alvarenga, F C; Bicudo, S D

    2015-06-01

    Inhibitors of cyclin-dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor-free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (-) at 38.5°C and 5% CO2 . At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/-) exhibited total expansion while in both Rosco groups (+/-) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/-). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.

  12. Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

    SciTech Connect

    Bornslaeger, E.A.; Poueymirou, W.T.; Mattei, P.; Schultz, R.M.

    1986-01-01

    Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, oocytes were treated with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4..beta..-phorbol, 12,13-didecanoate (4..beta..-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC/sub 8/). An inactive phorbol ester, 4a-phorbol 12,13-didecanoate (4..cap alpha..-PDD), did not inhibit GVBD. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of a cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC/sub 8/ partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis.

  13. An M-phase-specific protein kinase of Xenopus oocytes: partial purification and possible mechanism of its periodic activation.

    PubMed

    Labbé, J C; Picard, A; Karsenti, E; Dorée, M

    1988-05-01

    The activity of a Ca2+- and cyclic nucleotide-independent protein kinase(s) which catalyzes hyperphosphorylation of a set of endogenous proteins, including a 95-kDa soluble phosphoprotein, is found to fluctuate in both the meiotic and mitotic cell cycles of Xenopus oocytes and activated eggs. The activity is high in M-phase and hardly detectable in interphase. The activity copurifies with a major histone kinase(s) throughout four purification steps: ammonium sulfate precipitation, DEAE-cellulose chromatography, high-performance liquid chromatography on TSK G3000, and CM-Sepharose chromatography. This suggests that a single enzyme shares activity against endogenous proteins and added histones. Changes in the activity of the M-phase-specific protein kinase(s) as assayed in vitro correlate with changes in the extent of protein phosphorylation in oocytes pulse-labeled with 32P-phosphate by microinjection during meiotic maturation and the early embryonic cell cycle. This suggests that the kinase(s) has a broad specificity and plays a key role in the increased protein phosphorylation which occurs at the transition to M-phase. Microinjection of the maturation-promoting factor (MPF) into immature oocytes triggers, after a 10-min lag period, the activation of the M-phase specific kinase(s), even in the absence of protein synthesis. In contrast MPF microinjection does not induce kinase activation in cycloheximide-treated oocytes arrested after completion of the first meiotic cell cycle or in activated eggs arrested in S-phase by incubation in cycloheximide. This suggests that immature oocytes contain an inactive kinase precursor (prokinase) which is synthesized at each of the following cell cycles. In the absence of MPF addition, the prokinase to kinase transition occurs "spontaneously" after a 2-hr lag period in high-speed supernatants prepared from prophase-arrested oocytes if low-molecular-weight metabolites are eliminated by gel filtration. Addition of ATP, but not of

  14. Nitric Oxide Synthase (NOS) Inhibition during Porcine In Vitro Maturation Modifies Oocyte Protein S-Nitrosylation and In Vitro Fertilization

    PubMed Central

    Romero-Aguirregomezcorta, Jon; Santa, Ángela Patricia; García-Vázquez, Francisco Alberto; Coy, Pilar; Matás, Carmen

    2014-01-01

    Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization. PMID:25542028

  15. Luteinizing Hormone Reduces the Activity of the NPR2 Guanylyl Cyclase in Mouse Ovarian Follicles, Contributing to the Cyclic GMP Decrease that Promotes Resumption of Meiosis in Oocytes

    PubMed Central

    Robinson, Jerid W.; Zhang, Meijia; Shuhaibar, Leia C.; Norris, Rachael P.; Geerts, Andreas; Wunder, Frank; Eppig, John J.; Potter, Lincoln R.; Jaffe, Laurinda A.

    2012-01-01

    In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 minutes, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2 hours, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte. PMID:22546688

  16. Cumulus Cell Transcripts Transit to the Bovine Oocyte in Preparation for Maturation.

    PubMed

    Macaulay, Angus D; Gilbert, Isabelle; Scantland, Sara; Fournier, Eric; Ashkar, Fazl; Bastien, Alexandre; Saadi, Habib A Shojaei; Gagné, Dominic; Sirard, Marc-André; Khandjian, Édouard W; Richard, François J; Hyttel, Poul; Robert, Claude

    2016-01-01

    So far, the characteristics of a good quality egg have been elusive, similar to the nature of the physiological, cellular, and molecular cues leading to its production both in vivo and in vitro. Current understanding highlights a strong and complex interdependence between the follicular cells and the gamete. Secreted factors induce cellular responses in the follicular cells, and direct exchange of small molecules from the cumulus cells to the oocyte through gap junctions controls meiotic arrest. Studying the interconnection between the cumulus cells and the oocyte, we previously demonstrated that the somatic cells also contribute transcripts to the gamete. Here, we show that these transcripts can be visualized moving down the transzonal projections (TZPs) to the oocyte, and that a time course analysis revealed progressive RNA accumulation in the TZPs, indicating that RNA transfer occurs before the initiation of meiosis resumption under a timetable fitting with the acquisition of developmental competence. A comparison of the identity of the nascent transcripts trafficking in the TZPs, with those in the oocyte increasing in abundance during maturation, and that are present on the oocyte's polyribosomes, revealed transcripts common to all three fractions, suggesting the use of transferred transcripts for translation. Furthermore, the removal of potential RNA trafficking by stripping the cumulus cells caused a significant reduction in maturation rates, indicating the need for the cumulus cell RNA transfer to the oocyte. These results offer a new perspective to the determinants of oocyte quality and female fertility, as well as provide insight that may eventually be used to improve in vitro maturation conditions.

  17. Cumulus Cell Transcripts Transit to the Bovine Oocyte in Preparation for Maturation.

    PubMed

    Macaulay, Angus D; Gilbert, Isabelle; Scantland, Sara; Fournier, Eric; Ashkar, Fazl; Bastien, Alexandre; Saadi, Habib A Shojaei; Gagné, Dominic; Sirard, Marc-André; Khandjian, Édouard W; Richard, François J; Hyttel, Poul; Robert, Claude

    2016-01-01

    So far, the characteristics of a good quality egg have been elusive, similar to the nature of the physiological, cellular, and molecular cues leading to its production both in vivo and in vitro. Current understanding highlights a strong and complex interdependence between the follicular cells and the gamete. Secreted factors induce cellular responses in the follicular cells, and direct exchange of small molecules from the cumulus cells to the oocyte through gap junctions controls meiotic arrest. Studying the interconnection between the cumulus cells and the oocyte, we previously demonstrated that the somatic cells also contribute transcripts to the gamete. Here, we show that these transcripts can be visualized moving down the transzonal projections (TZPs) to the oocyte, and that a time course analysis revealed progressive RNA accumulation in the TZPs, indicating that RNA transfer occurs before the initiation of meiosis resumption under a timetable fitting with the acquisition of developmental competence. A comparison of the identity of the nascent transcripts trafficking in the TZPs, with those in the oocyte increasing in abundance during maturation, and that are present on the oocyte's polyribosomes, revealed transcripts common to all three fractions, suggesting the use of transferred transcripts for translation. Furthermore, the removal of potential RNA trafficking by stripping the cumulus cells caused a significant reduction in maturation rates, indicating the need for the cumulus cell RNA transfer to the oocyte. These results offer a new perspective to the determinants of oocyte quality and female fertility, as well as provide insight that may eventually be used to improve in vitro maturation conditions. PMID:26586844

  18. Nicotinamide Impairs Entry into and Exit from Meiosis I in Mouse Oocytes

    PubMed Central

    Riepsamen, Angelique; Wu, Lindsay; Lau, Laurin; Listijono, Dave; Ledger, William; Sinclair, David; Homer, Hayden

    2015-01-01

    Following exit from meiosis I, mammalian oocytes immediately enter meiosis II without an intervening interphase, accompanied by rapid reassembly of a bipolar spindle that maintains condensed chromosomes in a metaphase configuration (metaphase II arrest). Here we study the effect of nicotinamide (NAM), a non-competitive pan-sirtuin inhibitor, during meiotic maturation in mouse oocytes. Sirtuins are a family of seven NAD+-dependent deacetylases (Sirt1-7), which are involved in multiple cellular processes and are emerging as important regulators in oocytes and embryos. We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1. GVBD was also inhibited by the Sirt2-specific inhibitor, AGK2, and in a very similar pattern to NAM, supporting the notion that as in somatic cells, NAM inhibits sirtuins in oocytes. NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE). Unexpectedly, however, in the majority of oocytes with a polar body, chromatin was decondensed and a nuclear structure was present. An identical phenotype was observed when flavopiridol was used to induce Cdk1 inactivation during late meiosis I prior to PBE, but not if Cdk1 was inactivated after PBE when metaphase II arrest was already established, altogether indicating that NAM impaired establishment rather than maintenance of metaphase II arrest. During meiosis I exit in NAM-treated medium, we found that cyclin B1 levels were lower and inhibitory Cdk1 phosphorylation was increased compared with controls. Although activation of the anaphase-promoting complex-Cdc20 (APC-Cdc20) occurred on-time in NAM-treated oocytes, Cdc20 levels were higher in very late meiosis I, pointing to exaggerated APC-Cdc20-mediated proteolysis as a reason for lower cyclin B1 levels. Collectively, therefore, our data indicate that by disrupting Cdk1 regulation, NAM impairs entry into meiosis I and

  19. Nicotinamide impairs entry into and exit from meiosis I in mouse oocytes.

    PubMed

    Riepsamen, Angelique; Wu, Lindsay; Lau, Laurin; Listijono, Dave; Ledger, William; Sinclair, David; Homer, Hayden

    2015-01-01

    Following exit from meiosis I, mammalian oocytes immediately enter meiosis II without an intervening interphase, accompanied by rapid reassembly of a bipolar spindle that maintains condensed chromosomes in a metaphase configuration (metaphase II arrest). Here we study the effect of nicotinamide (NAM), a non-competitive pan-sirtuin inhibitor, during meiotic maturation in mouse oocytes. Sirtuins are a family of seven NAD+-dependent deacetylases (Sirt1-7), which are involved in multiple cellular processes and are emerging as important regulators in oocytes and embryos. We found that NAM significantly delayed entry into meiosis I associated with delayed accumulation of the Cdk1 co-activator, cyclin B1. GVBD was also inhibited by the Sirt2-specific inhibitor, AGK2, and in a very similar pattern to NAM, supporting the notion that as in somatic cells, NAM inhibits sirtuins in oocytes. NAM did not affect subsequent spindle assembly, chromosome alignment or the timing of first polar body extrusion (PBE). Unexpectedly, however, in the majority of oocytes with a polar body, chromatin was decondensed and a nuclear structure was present. An identical phenotype was observed when flavopiridol was used to induce Cdk1 inactivation during late meiosis I prior to PBE, but not if Cdk1 was inactivated after PBE when metaphase II arrest was already established, altogether indicating that NAM impaired establishment rather than maintenance of metaphase II arrest. During meiosis I exit in NAM-treated medium, we found that cyclin B1 levels were lower and inhibitory Cdk1 phosphorylation was increased compared with controls. Although activation of the anaphase-promoting complex-Cdc20 (APC-Cdc20) occurred on-time in NAM-treated oocytes, Cdc20 levels were higher in very late meiosis I, pointing to exaggerated APC-Cdc20-mediated proteolysis as a reason for lower cyclin B1 levels. Collectively, therefore, our data indicate that by disrupting Cdk1 regulation, NAM impairs entry into meiosis I and

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

    PubMed Central

    2012-01-01

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

  1. Altered Theca and Cumulus Oocyte Complex Gene Expression, Follicular Arrest and Reduced Fertility in Cows with Dominant Follicle Follicular Fluid Androgen Excess

    PubMed Central

    Summers, Adam F.; Pohlmeier, William E.; Sargent, Kevin M.; Cole, Brizett D.; Vinton, Rebecca J.; Kurz, Scott G.; McFee, Renee M.; Cushman, Robert A.; Cupp, Andrea S.; Wood, Jennifer R.

    2014-01-01

    Aspiration of bovine follicles 12–36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High androstenedione (A4; >40 ng/ml; mean = 102) and another with Low A4 (<20 ng/ml; mean = 9) in follicular fluid. We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance. To test this hypothesis, estrous cycles of cows were synchronized and ovariectomy was performed 36 hours later. HPLC MS/MS analysis of follicular fluid showed increased dehydroepiandrosterone (6-fold), A4 (158-fold) and testosterone (31-fold) in the dominant follicle of High A4 cows. However, estrone (3-fold) and estradiol (2-fold) concentrations were only slightly elevated, suggesting a possible inefficiency in androgen to estrogen conversion in High A4 cows. Theca cell mRNA expression of LHCGR, GATA6, CYP11A1, and CYP17A1 was greater in High A4 cows. Furthermore, abundance of ZAR1 was decreased 10-fold in cumulus oocyte complexes from High A4 cows, whereas NLRP5 abundance tended to be 19.8-fold greater (P = 0.07). There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired. High A4 cows tended (P<0.07) to have a 17% reduction in calving rate compared with Low A4 cows suggesting reduced fertility in the High A4 population. These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development. PMID:25330369

  2. DNA damage response during mouse oocyte maturation.

    PubMed

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

    2016-01-01

    Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II.  Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation. PMID:26745237

  3. Morphological dynamics of cumulus-oocyte complex during oocyte maturation.

    PubMed

    Sato, E

    1998-01-01

    The recent advances on the cytoplasmic regulators of the induction of germinal vesicle break down, maturation and degeneration of oocytes, and glycosaminoglycan composition during cumulus expansion of cumulus-oocyte complexes are discussed. A) Inactive mitogen-activated protein kinases (MAPKs) are present in the oocytes at germinal vesicle (GV) stage, and are activated with germinal vesicle breakdown (GVBD), and remain highly active throughout maturation in porcine oocytes. Inactive MAPKs are localized in the cytoplasm of GV-arrested oocytes and active MAPKs were detected in the GV just before GVBD. B) Cumulus expansion of porcine cumulus-oocyte complexes (COCs) was reduced by oocy tectomy. The profile of total glycosaminoglycan synthesis was attributed to hyaluronic acid rather than chondroitin sulfate in intact COCs and oocytectomy reduced hyaluronic acid synthesis. C) The abnormalities of chromosomes and alpha-tubulin morphology were observed in the oocytes of c-mos deficient mice. MAPK activity of c-mos deficient oocytes did not significantly fluctuate throughout maturation and was clearly lower than that of wild-type oocytes. One of the most drastic abnormalities in c-mos knockout mouse oocytes was their entrance into the interphase instead of second meiosis after first polar body emission. D) Reverse transcriptase/polymerase chain reaction-Southern blot hybridization demonstrated positive expression of Fas in intraovarian mouse oocytes. In contrast, expression of Fas ligand was detected in granulosa cells. These findings were histologically confirmed by in situ hybridization with Fas- and FasL-specific probes. Co-culture of intact and zona-free eggs and granulosa cells demonstrated positive TUNEL staining only zona-free eggs.

  4. Caffeine treatment prevents age-related changes in ovine oocytes and increases cell numbers in blastocysts produced by somatic cell nuclear transfer.

    PubMed

    Lee, Joon-Hee; Campbell, Keith H S

    2008-09-01

    Maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are key regulators of both meiotic and mitotic cycles. Oocytes arrested at metaphase of the second meiotic division (MII) contain high levels of both kinases; however, these activities decline with age. Caffeine (an inhibitor of Myt1/Wee1 activity) can increase MPF and MAPK activities in ovine oocytes; however, the effects of caffeine treatment on the activation, nuclear configuration and developmental potential of ovine SC nuclear transfer (SCNT) embryos were unknown. We examined the effects of aging and caffeine treatment on MPF and MAPK activities, activation, development, and nuclear remodeling of SCNT embryos. Both kinases reached maximum activities at 24-h postonset of maturation (hpm) and then decreased with time. The decline in MPF activity occurred rapidly, whereas MAPK activity declined more slowly. Caffeine treatment (10.0 mM) of aging oocytes prevented the decline in activities associated with both kinases and prevented the acquisition of activation competence by a single activation stimulus. However, treatment of aged oocytes with caffeine could not increase kinase activities or reverse the acquisition of activation competence. Enucleation did not affect kinase activities, but caffeine treatment significantly increased both. Caffeine treatment did not affect the decline in MPF or MAPK activities following activation or significantly affect development of parthenogenetically activated oocytes. When SCNT reconstructed embryos were treated with caffeine following fusion, no increase in the frequency of development to blastocyst was observed; however, a significant increase in the occurrence of nuclear envelope break-down (NEBD) and an increase in total cell numbers occurred. PMID:18673075

  5. Calcium signals and oocyte maturation in marine invertebrates.

    PubMed

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

    2015-01-01

    In various oocytes and eggs of animals, transient elevations in cytoplasmic calcium ion concentrations are known to regulate key processes during fertilization and the completion of meiosis. However, whether or not calcium transients also help to reinitiate meiotic progression at the onset of oocyte maturation remains controversial. This article summarizes reports of calcium signals playing essential roles during maturation onset (=germinal vesicle breakdown, GVBD) in several kinds of marine invertebrate oocytes. Conversely, other data from the literature, as well as previously unpublished findings for jellyfish oocytes, fail to support the view that calcium signals are required for GVBD. In addition to assessing the effects of calcium transients on GVBD in marine invertebrate oocytes, the ability of maturing oocytes to enhance their calcium-releasing capabilities after GVBD is also reviewed. Furthermore, possible explanations are proposed for the contradictory results that have been obtained regarding calcium signals during oocyte maturation in marine invertebrates. PMID:26679945

  6. Calcium signals and oocyte maturation in marine invertebrates.

    PubMed

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

    2015-01-01

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

  7. Effects of clinostat rotation on mouse meiotic maturation in vitro

    NASA Technical Reports Server (NTRS)

    Wolgemuth, D. J.; Grills, G. S.

    1984-01-01

    The effects of microgravity on meiosis, fertilization, and early embryonic development in mammals are being examined by using a clinostat to reorient the cells with respect to the gravity vector. A clinostat capable of supporting mammalian cells in tissue culture has been developed. Initial studies have focused on examining the effects of clinostat rotation on meiotic maturation in mouse oocytes. Oocytes recovered from ovarian follicles were subjected to clinostat rotation on a horizontal or vertical axis or to static conditions for a 16 hr period. No gross morphological changes and no effects on germinal vesicle breakdown were observed under any rotation conditions (1/4, 1, 10, 30, 100 RPM). Success of meiotic progression to Metaphase II was comparable among experimental and control groups except at 100 RPM, where a slight inhibition was observed.

  8. Oocyte maturation and quality: role of cyclic nucleotides.

    PubMed

    Gilchrist, R B; Luciano, A M; Richani, D; Zeng, H T; Wang, X; Vos, M De; Sugimura, S; Smitz, J; Richard, F J; Thompson, J G

    2016-11-01

    The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP - inhibiting the hydrolysis of intra-oocyte cAMP - and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options. PMID:27422885

  9. Differing roles of pyruvate dehydrogenase kinases during mouse oocyte maturation

    PubMed Central

    Hou, Xiaojing; Zhang, Liang; Han, Longsen; Ge, Juan; Ma, Rujun; Zhang, Xuesen; Moley, Kelle; Schedl, Tim; Wang, Qiang

    2015-01-01

    ABSTRACT Pyruvate dehydrogenase kinases (PDKs) modulate energy homeostasis in multiple tissues and cell types, under various nutrient conditions, through phosphorylation of the α subunit (PDHE1α, also known as PDHA1) of the pyruvate dehydrogenase (PDH) complex. However, the roles of PDKs in meiotic maturation are currently unknown. Here, by undertaking knockdown and overexpression analysis of PDK paralogs (PDK1–PDK4) in mouse oocytes, we established the site-specificity of PDKs towards the phosphorylation of three serine residues (Ser232, Ser293 and Ser300) on PDHE1α. We found that PDK3-mediated phosphorylation of Ser293-PDHE1α results in disruption of meiotic spindle morphology and chromosome alignment and decreased total ATP levels, probably through inhibition of PDH activity. Unexpectedly, we discovered that PDK1 and PDK2 promote meiotic maturation, as their knockdown disturbs the assembly of the meiotic apparatus, without significantly altering ATP content. Moreover, phosphorylation of Ser232-PDHE1α was demonstrated to mediate PDK1 and PDK2 action in meiotic maturation, possibly through a mechanism that is distinct from PDH inactivation. These findings reveal that there are divergent roles of PDKs during oocyte maturation and indicate a new mechanism controlling meiotic structure. PMID:25991547

  10. Differing roles of pyruvate dehydrogenase kinases during mouse oocyte maturation.

    PubMed

    Hou, Xiaojing; Zhang, Liang; Han, Longsen; Ge, Juan; Ma, Rujun; Zhang, Xuesen; Moley, Kelle; Schedl, Tim; Wang, Qiang

    2015-07-01

    Pyruvate dehydrogenase kinases (PDKs) modulate energy homeostasis in multiple tissues and cell types, under various nutrient conditions, through phosphorylation of the α subunit (PDHE1α, also known as PDHA1) of the pyruvate dehydrogenase (PDH) complex. However, the roles of PDKs in meiotic maturation are currently unknown. Here, by undertaking knockdown and overexpression analysis of PDK paralogs (PDK1-PDK4) in mouse oocytes, we established the site-specificity of PDKs towards the phosphorylation of three serine residues (Ser232, Ser293 and Ser300) on PDHE1α. We found that PDK3-mediated phosphorylation of Ser293-PDHE1α results in disruption of meiotic spindle morphology and chromosome alignment and decreased total ATP levels, probably through inhibition of PDH activity. Unexpectedly, we discovered that PDK1 and PDK2 promote meiotic maturation, as their knockdown disturbs the assembly of the meiotic apparatus, without significantly altering ATP content. Moreover, phosphorylation of Ser232-PDHE1α was demonstrated to mediate PDK1 and PDK2 action in meiotic maturation, possibly through a mechanism that is distinct from PDH inactivation. These findings reveal that there are divergent roles of PDKs during oocyte maturation and indicate a new mechanism controlling meiotic structure. PMID:25991547

  11. RAD51C deficiency in mice results in early prophase I arrest in males and sister chromatid separation at metaphase II in females

    PubMed Central

    Kuznetsov, Sergey; Pellegrini, Manuela; Shuda, Kristy; Fernandez-Capetillo, Oscar; Liu, Yilun; Martin, Betty K.; Burkett, Sandra; Southon, Eileen; Pati, Debananda; Tessarollo, Lino; West, Stephen C.; Donovan, Peter J.; Nussenzweig, Andre; Sharan, Shyam K.

    2007-01-01

    RAD51C is a member of the RecA/RAD51 protein family, which is known to play an important role in DNA repair by homologous recombination. In mice, it is essential for viability. Therefore, we have generated a hypomorphic allele of Rad51c in addition to a null allele. A subset of mice expressing the hypomorphic allele is infertile. This infertility is caused by sexually dimorphic defects in meiotic recombination, revealing its two distinct functions. Spermatocytes undergo a developmental arrest during the early stages of meiotic prophase I, providing evidence for the role of RAD51C in early stages of RAD51-mediated recombination. In contrast, oocytes can progress normally to metaphase I after superovulation but display precocious separation of sister chromatids, aneuploidy, and broken chromosomes at metaphase II. These defects suggest a possible late role of RAD51C in meiotic recombination. Based on the marked reduction in Holliday junction (HJ) resolution activity in Rad51c-null mouse embryonic fibroblasts, we propose that this late function may be associated with HJ resolution. PMID:17312021

  12. Mitochondrial dynamics and their intracellular traffic in porcine oocytes.

    PubMed

    Yamochi, T; Hashimoto, S; Amo, A; Goto, H; Yamanaka, M; Inoue, M; Nakaoka, Y; Morimoto, Y

    2016-08-01

    Meiotic maturation of oocytes requires a variety of ATP-dependent reactions, such as germinal vesicle breakdown, spindle formation, and rearrangement of plasma membrane structure, which is required for fertilization. Mitochondria are accordingly expected be localized to subcellular sites of energy utilization. Although microtubule-dependent cellular traffic for mitochondria has been studied extensively in cultured neuronal (and some other somatic) cells, the molecular mechanism of their dynamics in mammalian oocytes at different stages of maturation remains obscure. The present work describes dynamic aspects of mitochondria in porcine oocytes at the germinal vesicle stage. After incubation of oocytes with MitoTracker Orange followed by centrifugation, mitochondria-enriched ooplasm was obtained using a glass needle and transferred into a recipient oocyte. The intracellular distribution of the fluorescent mitochondria was then observed over time using a laser scanning confocal microscopy equipped with an incubator. Kinetic analysis revealed that fluorescent mitochondria moved from central to subcortical areas of oocytes and were dispersed along plasma membranes. Such movement of mitochondria was inhibited by either cytochalasin B or cytochalasin D but not by colcemid, suggesting the involvement of microfilaments. This method of visualizing mitochondrial dynamics in live cells permits study of the pathophysiology of cytoskeleton-dependent intracellular traffic of mitochondria and associated energy metabolism during meiotic maturation of oocytes.

  13. A mammalian KASH domain protein coupling meiotic chromosomes to the cytoskeleton

    PubMed Central

    Horn, Henning F.; Kim, Dae In; Wright, Graham D.; Wong, Esther Sook Miin; Roux, Kyle J.

    2013-01-01

    Chromosome pairing is an essential meiotic event that ensures faithful haploidization and recombination of the genome. Pairing of homologous chromosomes is facilitated by telomere-led chromosome movements and formation of a meiotic bouquet, where telomeres cluster to one pole of the nucleus. In metazoans, telomere clustering is dynein and microtubule dependent and requires Sun1, an inner nuclear membrane protein. Here we provide a functional analysis of KASH5, a mammalian dynein-binding protein of the outer nuclear membrane that forms a meiotic complex with Sun1. This protein is related to zebrafish futile cycle (Fue), a nuclear envelope (NE) constituent required for pronuclear migration. Mice deficient in this Fue homologue are infertile. Males display meiotic arrest in which pairing of homologous chromosomes fails. These findings demonstrate that telomere attachment to the NE is insufficient to promote pairing and that telomere attachment sites must be coupled to cytoplasmic dynein and the microtubule system to ensure meiotic progression. PMID:24062341

  14. Investigations of oocyte in vitro maturation within a mouse model.

    PubMed

    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. PMID:15214575

  15. Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes.

    PubMed

    Egbert, Jeremy R; Shuhaibar, Leia C; Edmund, Aaron B; Van Helden, Dusty A; Robinson, Jerid W; Uliasz, Tracy F; Baena, Valentina; Geerts, Andreas; Wunder, Frank; Potter, Lincoln R; Jaffe, Laurinda A

    2014-09-01

    In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte.

  16. Translation in the mammalian oocyte in space and time.

    PubMed

    Susor, Andrej; Jansova, Denisa; Anger, Martin; Kubelka, Michal

    2016-01-01

    A hallmark of oocyte development in mammals is the dependence on the translation and utilization of stored RNA and proteins rather than the de novo transcription of genes in order to sustain meiotic progression and early embryo development. In the absence of transcription, the completion of meiosis and early embryo development in mammals relies significantly on maternally synthesized RNAs. Post-transcriptional control of gene expression at the translational level has emerged as an important cellular function in normal development. Therefore, the regulation of gene expression in oocytes is controlled almost exclusively at the level of mRNA and protein stabilization and protein synthesis. This current review is focused on the recently emerged findings on RNA distribution related to the temporal and spatial translational control of the meiotic progression of the mammalian oocyte.

  17. Increasing the cAMP concentration during in vitro maturation of pig oocytes improves cumulus maturation and subsequent fertilization in vitro.

    PubMed

    Appeltant, R; Beek, J; Vandenberghe, L; Maes, D; Van Soom, A

    2015-02-01

    Porcine IVF faces various problems such as incomplete cytoplasmic maturation of the oocyte and polyspermy. Previous studies proved the importance of cAMP in regulating nuclear and cytoplasmic maturation of oocytes. This study investigated the effect of the cAMP-modulating agents 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cAMP sodium salt (dbcAMP) on several parameters during in vitro production of porcine embryos. First, we wanted to see if oocyte collection in IBMX could meiotically arrest oocytes and, as such, improve synchronization of nuclear and cytoplasmic maturation. To this end, cumulus-oocyte complexes (COCs) were collected from gilts in HEPES-buffered Tyrode balanced salt solution medium with 0.5-mM IBMX or without IBMX. At the end of oocyte collection, the effect of IBMX on chromatin configuration was evaluated. However, no differences could be observed in nuclear configuration between IBMX- and IBMX+ oocytes (P > 0.05). Second, we added dbcAMP during IVM to improve cytoplasmic maturation and evaluated cumulus expansion (lack of adhesion), a disintegrin and metalloproteinase with thrombospondin-like repeats (ADAMTS-1) levels in cumulus cells, fertilization, and blastocyst rates. Cumulus-oocyte complexes were matured in modified North Carolina State University medium 37 with or without 1-mM dbcAMP. Frozen-thawed, epididymal, boar spermatozoa were used for IVF. After IVF, presumed zygotes were cultured for 7 days in North Carolina State University medium 23. Penetration rate decreased in dbcAMP+ (57.3%) compared with dbcAMP- (67.8%), but the polyspermy rate also decreased (43.3% vs. 53.4%, respectively) leading to an increased normal fertilization rate (56.7% vs. 46.6%, respectively; P < 0.05). Only 7.2% of the COCs showed adhesion in dbcAMP+ which was lower than 15.7% in dbcAMP- (P < 0.05) probably because of an upregulation of the ADAMTS-1 protein by dbcAMP. When the adherent oocytes were removed during maturation, no difference could be

  18. Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes

    PubMed Central

    Tischer, Thomas; Santhanam, Balaji; Schuh, Melina

    2015-01-01

    During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone1,2, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down’s syndrome3-5. Which genes safeguard accurate progression through meiosis is largely unclear. Here, we developed high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNAi within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated dataset of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and now allows systematic studies of meiosis in mammals. PMID:26147080

  19. Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes.

    PubMed

    Pfender, Sybille; Kuznetsov, Vitaliy; Pasternak, Michał; Tischer, Thomas; Santhanam, Balaji; Schuh, Melina

    2015-08-13

    During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone, and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down's syndrome. Which genes safeguard accurate progression through meiosis is largely unclear. Here we develop high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNA interference within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated data set of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and allows systematic studies of meiosis in mammals.

  20. Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level.

    PubMed

    Coutandin, Daniel; Osterburg, Christian; Srivastav, Ratnesh Kumar; Sumyk, Manuela; Kehrloesser, Sebastian; Gebel, Jakob; Tuppi, Marcel; Hannewald, Jens; Schäfer, Birgit; Salah, Eidarus; Mathea, Sebastian; Müller-Kuller, Uta; Doutch, James; Grez, Manuel; Knapp, Stefan; Dötsch, Volker

    2016-01-01

    Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction. PMID:27021569

  1. Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level

    PubMed Central

    Coutandin, Daniel; Osterburg, Christian; Srivastav, Ratnesh Kumar; Sumyk, Manuela; Kehrloesser, Sebastian; Gebel, Jakob; Tuppi, Marcel; Hannewald, Jens; Schäfer, Birgit; Salah, Eidarus; Mathea, Sebastian; Müller-Kuller, Uta; Doutch, James; Grez, Manuel; Knapp, Stefan; Dötsch, Volker

    2016-01-01

    Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction. DOI: http://dx.doi.org/10.7554/eLife.13909.001 PMID:27021569

  2. Control of Oocyte Reawakening by Kit.

    PubMed

    Saatcioglu, Hatice Duygu; Cuevas, Ileana; Castrillon, Diego H

    2016-08-01

    In mammals, females are born with finite numbers of oocytes stockpiled as primordial follicles. Oocytes are "reawakened" via an ovarian-intrinsic process that initiates their growth. The forkhead transcription factor Foxo3 controls reawakening downstream of PI3K-AKT signaling. However, the identity of the presumptive upstream cell surface receptor controlling the PI3K-AKT-Foxo3 axis has been questioned. Here we show that the receptor tyrosine kinase Kit controls reawakening. Oocyte-specific expression of a novel constitutively-active KitD818V allele resulted in female sterility and ovarian failure due to global oocyte reawakening. To confirm this result, we engineered a novel loss-of-function allele, KitL. Kit inactivation within oocytes also led to premature ovarian failure, albeit via a contrasting phenotype. Despite normal initial complements of primordial follicles, oocytes remained dormant with arrested oocyte maturation. Foxo3 protein localization in the nucleus versus cytoplasm explained both mutant phenotypes. These genetic studies provide formal genetic proof that Kit controls oocyte reawakening, focusing future investigations into the causes of primary ovarian insufficiency and ovarian aging. PMID:27500836

  3. Control of Oocyte Reawakening by Kit

    PubMed Central

    Castrillon, Diego H.

    2016-01-01

    In mammals, females are born with finite numbers of oocytes stockpiled as primordial follicles. Oocytes are “reawakened” via an ovarian-intrinsic process that initiates their growth. The forkhead transcription factor Foxo3 controls reawakening downstream of PI3K-AKT signaling. However, the identity of the presumptive upstream cell surface receptor controlling the PI3K-AKT-Foxo3 axis has been questioned. Here we show that the receptor tyrosine kinase Kit controls reawakening. Oocyte-specific expression of a novel constitutively-active KitD818V allele resulted in female sterility and ovarian failure due to global oocyte reawakening. To confirm this result, we engineered a novel loss-of-function allele, KitL. Kit inactivation within oocytes also led to premature ovarian failure, albeit via a contrasting phenotype. Despite normal initial complements of primordial follicles, oocytes remained dormant with arrested oocyte maturation. Foxo3 protein localization in the nucleus versus cytoplasm explained both mutant phenotypes. These genetic studies provide formal genetic proof that Kit controls oocyte reawakening, focusing future investigations into the causes of primary ovarian insufficiency and ovarian aging. PMID:27500836

  4. Mos limits the number of meiotic divisions in urochordate eggs.

    PubMed

    Dumollard, Rémi; Levasseur, Mark; Hebras, Céline; Huitorel, Philippe; Carroll, Michael; Chambon, Jean-Philippe; McDougall, Alex

    2011-03-01

    Mos kinase is a universal mediator of oocyte meiotic maturation and is produced during oogenesis and destroyed after fertilization. The hallmark of maternal meiosis is that two successive M phases (meiosis I and II) drive two rounds of asymmetric cell division (ACD). However, how the egg limits the number of meioses to just two, thereby preventing gross aneuploidy, is poorly characterized. Here, in urochordate eggs, we show that loss of Mos/MAPK activity is necessary to prevent entry into meiosis III. Remarkably, maintaining the Mos/MAPK pathway active after fertilization at near physiological levels induces additional rounds of meiotic M phase (meiosis III, IV and V). During these additional rounds of meiosis, the spindle is positioned asymmetrically resulting in further rounds of ACD. In addition, inhibiting meiotic exit with Mos prevents pronuclear formation, cyclin A accumulation and maintains sperm-triggered Ca(2+) oscillations, all of which are hallmarks of the meiotic cell cycle in ascidians. It will be interesting to determine whether Mos availability in mammals can also control the number of meioses as it does in the urochordates. Our results demonstrate the power of urochordate eggs as a model to dissect the egg-to-embryo transition.

  5. Meiotic recombination initiated by a double-strand break in rad50{Delta} yeast cells otherwise unable to initiate meiotic recombination

    SciTech Connect

    Malkova, A.; Haber, J.E.; Dawson, D.

    1996-06-01

    Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast`s 16 chromosomes. In Rad{sup +} strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad{sup +} and in rad50{Delta} cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50{Delta} cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad50{Delta} diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination. 57 refs., 5 figs., 3 tabs.

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Mitochondria Synthesize Melatonin to Ameliorate Its Function and Improve Mice Oocyte's Quality under in Vitro Conditions.

    PubMed

    He, Changjiu; Wang, Jing; Zhang, Zhenzhen; Yang, Minghui; Li, Yu; Tian, Xiuzhi; Ma, Teng; Tao, Jingli; Zhu, Kuanfeng; Song, Yukun; Ji, Pengyun; Liu, Guoshi

    2016-01-01

    The physiology of oocyte in vitro maturation remains elusive. Generally, the oocytes have a very low maturation rate under in vitro conditions. In the current study, we found that melatonin promotes the maturation of oocytes in which mitochondria play a pivotal role. It was identified that; (1) mitochondria are the major sites for melatonin synthesis in oocytes and they synthesize large amounts of melatonin during their maturation; (2) melatonin improves mitochondrial function by increased mtDNA copy, mitochondrial membrane potential (ΔΨm) and mitochondrial distribution and ATP production in oocytes; (3) the meiotic spindle assembly is enhanced; (4) melatonin reduces ROS production and inhibits 8-oxodG formation, thereby protecting potential DNA mutation from oxidative damage. As a result, melatonin improves the quality of oocytes, significantly accelerates the developmental ability of IVF embryo. The results provide novel knowledge on the physiology of oocyte's maturation, especially under in vitro conditions. PMID:27314334

  8. Cardiac arrest

    MedlinePlus

    ... Article.jsp. Accessed June 16, 2014. Myerburg RJ, Castellanos A. Approach to cardiac arrest and life-threatening ... PA: Elsevier Saunders; 2011:chap 63. Myerburg RJ, Castellanos A. Cardiac arrest and audden aardiac death. In: ...

  9. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  10. Maternal factors required for oocyte developmental competence in mice: transcriptome analysis of non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) oocytes.

    PubMed

    Ma, Jun-Yu; Li, Mo; Luo, Yi-Bo; Song, Shuhui; Tian, Dongmei; Yang, Jin; Zhang, Bing; Hou, Yi; Schatten, Heide; Liu, Zhonghua; Sun, Qing-Yuan

    2013-06-15

    During mouse antral follicle development, the oocyte chromatin gradually transforms from a less condensed state with no Hoechst-positive rim surrounding the nucleolus (NSN) to a fully condensed chromatin state with a Hoechst-positive rim surrounding the nucleolus (SN). Compared with SN oocytes, NSN oocytes display a higher gene transcription activity and a lower rate of meiosis resumption (G2/M transition), and they are mostly arrested at the two-cell stage after in vitro fertilization. To explore the differences between NSN and SN oocytes, and the maternal factors required for oocyte developmental competence, we compared the whole-transcriptome profiles between NSN and SN oocytes. First, we found that the NSN and SN oocytes were different in their metabolic pathways. In the phosphatidylinositol signaling pathway, the SN oocytes tend to produce diacylglycerol, whereas the NSN oocytes tend to produce phosphatidylinositol (3,4,5)-trisphosphate. For energy production, the SN oocytes and NSN oocytes differed in the gluconeogenesis and in the synthesis processes. Second, we also found that the key genes associated with oocyte meiosis and/or preimplantation embryo development were differently expressed in the NSN and SN oocytes. Our results illustrate that during the NSN-SN transition, the oocytes change their metabolic activities and accumulate maternal factors for further oocyte maturation and post-fertilization embryo development.

  11. Meiotic recombination mechanisms.

    PubMed

    Grelon, Mathilde

    2016-01-01

    Meiosis is a specialized cell division at the origin of the haploid cells that eventually develop into the gametes. It therefore lies at the heart of Mendelian heredity. Recombination and redistribution of the homologous chromosomes arising during meiosis constitute an important source of genetic diversity, conferring to meiosis a particularly important place in the evolution and the diversification of the species. Our understanding of the molecular mechanisms governing meiotic recombination has considerably progressed these last decades, benefiting from complementary approaches led on various model species. An overview of these mechanisms will be provided as well as a discussion on the implications of these recent discoveries. PMID:27180110

  12. Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa.

    PubMed

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

    1996-10-01

    Cumulus oocyte complexes (COCs) and cumulus oocyte complexes connected to a piece of the membrane granulosa (COCGs) were isolated from bovine antral follicles with a diameter of 2 to 8 mm. After culture of COCGs without gonadotrophic hormones for 22 hr approximately 50% of the oocytes were still in the germinal vesicle (GV) stage. Histology of the COCGs showed that the pieces of the membrana granulosa were free of thecal cells and parts of the basal membrane. This indicates that the membrana granulosa solely inhibits the progression of meiosis. To investigate the effect of gonadotropins on the resumption of meiosis of oocytes from small and medium sized antral follicles, COCs and COCGs were cultured with or without rec-hFSH or hCG. Addition of 0.05 IU rec-hFSH to the culture medium of COCGs resulted in germinal vesicle breakdown in 97.8% of the oocytes compared to 46% in the control group, and an increase of the diameter of the COCs (479 microns vs. 240 microns in the control group). Addition of 0.05 IU hCG to the culture medium had no effect on nuclear maturation (47.2% GV vs. 48.5% GV in the control group) nor on cumulus expansion (246 microns vs. 240 microns in the control group). RT-PCR on cDNA of the follicular wall, cumulus cells, granulosa cells, COCs, and oocytes revealed that mRNA for FSH receptor was present in all cell types except oocytes. mRNA of the LH receptor was detected exclusively in thecal cells. Nucleotide sequence analysis and alignment of the cloned PCR products showed the presence of two isoforms of the FSH receptor mRNA and two isoforms of the LH receptor mRNA. It is concluded that, in vitro, resumption of meiosis of oocytes, originating from small and medium sized antral follicles and meiotically arrested by the membrana granulosa, is triggered by FSH and not by LH. This is supported by the fact that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of these follicles. PMID:8914080

  13. LIMK1/2 inhibitor LIMKi 3 suppresses porcine oocyte maturation

    PubMed Central

    Jia, Ru-Xia; Duan, Xing; Song, Si-Jing

    2016-01-01

    LIMKi 3 is a specific selective LIMK inhibitor against LIMK1 and LIMK2, while LIMK1 and LIMK2 are the main regulators of actin cytoskeleton to participate in many cell activities. However, the effect of LIMKi 3 in porcine oocyte meiosis is still unclear. The present study was designed to investigate the effects of LIMKi 3 and potential regulatory role of LIMK1/2 on porcine oocyte meiotic maturation. Immunofluorescent staining of p-LIMK1/2 antibody showed that LIMK1/2 was localized mainly to the cortex of porcine oocyte, which co-localized with actin. After LIMKi 3 treatment, the diffusion of COCs became weak and the rate of polar body extrusion was decreased. This could be rescued by moving oocytes to fresh medium. After prolonging the culture time of oocytes, the maturation rate of porcine oocyte increased in LIMKi 3 groups, indicating that LIMKi 3 may suppress the cell cycle during porcine oocyte maturation. We also found that after LIMKi 3 treatment actin distribution was significantly disturbed at porcine oocyte membranes and cytoplasm, indicating the conserved roles of LIMK1/2 on actin dynamics. Next we examined the meiotic spindle positioning in porcine oocyte, and the results showed that a majority of spindles were not attached to the cortex of porcine oocyte, indicating that LIMKi 3 may affect actin-mediated spindle positioning. Taken together, these results showed that LIMK1/2 inhibitor LIMKi 3 had a repressive role on porcine oocyte meiotic maturation. PMID:27761340

  14. Nuclear structures in Tribolium castaneum oocytes.

    PubMed

    Bogolyubov, Dmitry S; Batalova, Florina M; Kiselyov, Artyom M; Stepanova, Irina S

    2013-10-01

    The first ultrastructural and immunomorphological characteristics of the karyosphere (karyosome) and extrachromosomal nuclear bodies in the red flour beetle, Tribolium castaneum, are presented. The karyosphere forms early in the diplotene stage of meiotic prophase by the gathering of all oocyte chromosomes in a limited nuclear volume. Using the BrUTP assay, T. castaneum oocyte chromosomes united in the karyosphere maintain their transcriptional activity until the end of oocyte growth. Hyperphosphorylated RNA polymerase II and basal transcription factors (TFIID and TFIIH) were detected in the perichromatin region of the karyosphere. The T. castaneum karyosphere has an extrachromosomal capsule that separates chromosomes from the rest of the nucleoplasm. Certain structural proteins (F-actin, lamin B) were found in the capsule. Unexpectedly, the karyosphere capsule in T. castaneum oocytes was found to be enriched in TMG-capped snRNAs, which suggests that the capsule is not only a structural support for the karyosphere, but may be involved in biogenesis of snRNPs. We also identified the counterparts of 'universal' extrachromosomal nuclear domains, Cajal bodies (CBs) and interchromatin granule clusters (IGCs). Nuclear bodies containing IGC marker protein SC35 display some features unusual for typical IGCs. SC35 domains in T. castaneum oocytes are predominantly fibrillar complex bodies that do not contain trimethyl guanosine (TMG)-capped small nuclear (sn) RNAs. Microinjections of 2'-O-methyl (U)22 probes into the oocytes allowed revealing poly(A)+ RNAs in these nuclear domains. Several proteins related to mRNA export (heterogeneous ribonucleoprotein core protein A1, export adapters Y14 and Aly and export receptor NXF1) were also detected there. We believe that unusual SC35 nuclear domains of T. castaneum oocytes are possibly involved in mRNP but not snRNP biogenesis.

  15. The type and extent of injuries in vitrified mouse oocytes.

    PubMed

    Liang, Yang; Ning, Fang-Yong; Du, Wen-Jing; Wang, Chun-Sheng; Piao, Shan-Hua; An, Tie-Zhu

    2012-04-01

    To improve the vitrification of mouse oocytes using straws, we attempted to estimate the type and extent of injuries during vitrification with a vitrification solution EAFS10/10. Injuries in oocytes were assessed based on cellular viability, the integrity of the plasma membrane, the status of the meiotic spindle/chromosomes, and morphological appearance. For morphologically normal oocytes, the ability to be fertilized and to develop into blastocysts was examined. Morphological assessment revealed 15% of oocytes to be injured by intracellular ice formed during vitrification, and 10% by osmotic swelling during removal of the cryoprotectant. When assessed by the status of spindles/chromosomes, the most sensitive criterion, damage was found in 16% of oocytes without any treatment. This value was similar to the proportion of fresh oocytes that did not cleave after insemination (13%). On exposure to EAFS10/10, the spindles/chromosomes were affected in 33% of oocytes. The exposure reduced the rate of cleavage by 18% points and the rate of development into blastocysts by 19 points. Vitrification reduced these rates by 15% and 36% points, respectively. Although the mechanism responsible for this moderate toxic effect on developmental ability is not known, information obtained in the present study will be useful to develop a practical method for the vitrification of mouse oocytes using straws.

  16. The type and extent of injuries in vitrified mouse oocytes.

    PubMed

    Liang, Yang; Ning, Fang-Yong; Du, Wen-Jing; Wang, Chun-Sheng; Piao, Shan-Hua; An, Tie-Zhu

    2012-04-01

    To improve the vitrification of mouse oocytes using straws, we attempted to estimate the type and extent of injuries during vitrification with a vitrification solution EAFS10/10. Injuries in oocytes were assessed based on cellular viability, the integrity of the plasma membrane, the status of the meiotic spindle/chromosomes, and morphological appearance. For morphologically normal oocytes, the ability to be fertilized and to develop into blastocysts was examined. Morphological assessment revealed 15% of oocytes to be injured by intracellular ice formed during vitrification, and 10% by osmotic swelling during removal of the cryoprotectant. When assessed by the status of spindles/chromosomes, the most sensitive criterion, damage was found in 16% of oocytes without any treatment. This value was similar to the proportion of fresh oocytes that did not cleave after insemination (13%). On exposure to EAFS10/10, the spindles/chromosomes were affected in 33% of oocytes. The exposure reduced the rate of cleavage by 18% points and the rate of development into blastocysts by 19 points. Vitrification reduced these rates by 15% and 36% points, respectively. Although the mechanism responsible for this moderate toxic effect on developmental ability is not known, information obtained in the present study will be useful to develop a practical method for the vitrification of mouse oocytes using straws. PMID:22202671

  17. ATM controls meiotic double-strand break formation

    PubMed Central

    Lange, Julian; Pan, Jing; Cole, Francesca; Thelen, Michael P.; Jasin, Maria; Keeney, Scott

    2011-01-01

    In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes1. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation2. However, improperly repaired DSBs can cause meiotic arrest or mutation3,4, thus having too many DSBs is likely as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse5, and SPO11 and its accessory factors remain abundant long after most DSB formation ceases1, implying the existence of mechanisms that restrain SPO11 activity to limit DSB numbers. Here we report that the number of meiotic DSBs in mouse is controlled by ATM, a kinase activated by DNA damage to trigger checkpoint signaling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least ten-fold in spermatocytes lacking ATM. Moreover, Atm mutation renders SPO11-oligonucleotide levels sensitive to genetic manipulations that modulate SPO11 protein levels. We propose that ATM restrains SPO11 via a negative feedback loop in which kinase activation by DSBs suppresses further DSB formation. Our findings explain previously puzzling phenotypes of Atm-null mice and provide a molecular basis for the gonadal dysgenesis observed in ataxia telangiectasia, the human syndrome caused by ATM deficiency. PMID:22002603

  18. In vitro effects of cilostazol, a phosphodiesterase 3A inhibitor, on mouse oocyte maturation and morphology.

    PubMed

    Taiyeb, Ahmed M; Dees, William L; Ridha-Albarzanchi, Mundhir T; Sayes, Christie M; Kraemer, Duane C

    2014-02-01

    Inhibition of phosphodiesterase 3A (PDE3A) in oocytes has been reported to arrest oocyte maturation and to increase intra-oocyte cyclic adenosine monophosphate levels. Although many PDE3A inhibitors have been found to arrest oocyte maturation in different species, including humans, the most commonly prescribed PDE3A inhibitor named cilostazol (CLZ) has not yet been fully evaluated in reproduction. The present study was designed to investigate the potential inhibitory effects of CLZ on oocyte maturation and morphology in vitro. Antral oocytes were recovered from hyperstimulated mice and allocated to 10 different CLZ concentrations (0.00-67.66 μmol/L). Oocytes were then assessed after 24 and 48 h of incubation for maturation and morphology. Some of the evaluated CLZ concentrations (1.06-4.23 μmol/L) were made similar to those observed in human clinical trials. CLZ arrested oocyte maturation at the germinal vesicle (GV) stage at concentrations as low as 1.06 μmol/L (P < 0.0001). A selective degenerative impact of CLZ targeting arrested oocytes at the GV stage was observed during 24 h of incubation (r = -0.781, P < 0.0001). This was not the case with non-arrested oocytes (r = -0.082, P = 0.64). Such degenerative impact was dose-dependent (P < 0.0001), suggesting a role for cyclic adenosine monophosphate in this degenerative process. The degenerated oocytes were of distorted oolema or fragmented cytoplasm. Based on the experiments, it is concluded that CLZ can inhibit oocyte maturation in vitro, at concentrations similar to those observed in humans taking CLZ, and under such conditions the prolonged maintenance of oocytes at the GV stage is harmful. PMID:24341287

  19. MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice[OPEN

    PubMed Central

    Wang, Chong; Yu, Junping; Zong, Jie; Lu, Pingli

    2016-01-01

    F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression. PMID:27436711

  20. MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice.

    PubMed

    He, Yi; Wang, Chong; Higgins, James D; Yu, Junping; Zong, Jie; Lu, Pingli; Zhang, Dabing; Liang, Wanqi

    2016-08-01

    F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression. PMID:27436711

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

    PubMed

    Touati, Sandra A; Wassmann, Katja

    2016-06-01

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

  2. Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1.

    PubMed

    Susor, Andrej; Ellederova, Zdenka; Jelinkova, Lucie; Halada, Petr; Kavan, Daniel; Kubelka, Michal; Kovarova, Hana

    2007-10-01

    In this study, we performed proteomic analysis of porcine oocytes during in vitro maturation. Comparison of oocytes at the initial and final stages of meiotic division characterized candidate proteins that were differentially synthesized during in vitro maturation. While the biosynthesis of many of these proteins was significantly decreased, we found four proteins with increased biosynthetic rate, which are supposed to play an essential role in meiosis. Among them, the ubiquitin C-terminal hydrolase-L1 (UCH-L1) was identified by mass spectrometry. To study the regulatory role of UCH-L1 in the process of meiosis in pig model, we used a specific inhibitor of this enzyme, marked C30, belonging to the class of isatin O-acyl oximes. When germinal vesicle (GV) stage cumulus-enclosed oocytes were treated with C30, GV breakdown was inhibited after 28 h of culture, and most of the oocytes were arrested at the first meiosis after 44 h. The block of metaphase I-anaphase transition was not completely reversible. In addition, the inhibition of UCH-L1 resulted in elevated histone H1 kinase activity, corresponding to cyclin-dependent kinase(CDK1)-cyclin B1 complex, and a low level of monoubiquitin. These results supported the hypothesis that UCH-L1 might play a role in metaphase I-anaphase transition by regulating ubiquitin-dependent proteasome mechanisms. In summary, a proteomic approach coupled with protein verification study revealed an essential role of UCH-L1 in the completion of the first meiosis and its transition to anaphase.

  3. Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos.

    PubMed

    Ding, Li-Jun; Tian, Hai-Bin; Wang, Jing-Jun; Chen, Juan; Sha, Hong-Ying; Chen, Jian-Quan; Cheng, Guo-Xiang

    2008-12-01

    The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used

  4. Inhibition of Rac1 GTPase activity affects porcine oocyte maturation and early embryo development

    PubMed Central

    Song, Si-Jing; Wang, Qiao-Chu; Jia, Ru-Xia; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2016-01-01

    Mammalian oocyte asymmetric division relies on the eccentric positioning of the spindle, resulting in the polar body formation. Small signaling G protein Rac1 is a member of GTPases, which regulates a diverse array of cellular events, including the control of cell growth, cytoskeletal reorganization, and the activation of protein kinases. However, effects of Rac1 on the porcine oocyte maturation and early embryo development are not fully understood. In present study we investigated the role of Rac1 in oocyte maturation and embryo cleavage. We first found that Rac1 localized at the cortex of the porcine oocytes, and disrupting the Rac1 activities by treating with NSC 23766 led to the failure of polar body emission. In addition, a majority of treated oocytes exhibited abnormal spindle morphology, indicating that Rac1 may involve into porcine oocyte spindle formation. This might be due to the regulation of Rac1 on MAPK, since p-MAPK expression decreased after NSC 23766 treatments. Moreover, we found that the position of most meiotic spindles in treated oocytes were away from the cortex, indicating the roles of Rac1 on meiotic spindle positioning. Our results also showed that inhibition of Rac1 activity caused the failure of early embryo development. Therefore, our study showed the critical roles of Rac1 GTPase on porcine oocyte maturation and early embryo cleavage. PMID:27694954

  5. Localisation of the SMC loading complex Nipbl/Mau2 during mammalian meiotic prophase I.

    PubMed

    Visnes, T; Giordano, F; Kuznetsova, A; Suja, J A; Lander, A D; Calof, A L; Ström, L

    2014-06-01

    Evidence from lower eukaryotes suggests that the chromosomal associations of all the structural maintenance of chromosome (SMC) complexes, cohesin, condensin and Smc5/6, are influenced by the Nipbl/Mau2 heterodimer. Whether this function is conserved in mammals is currently not known. During mammalian meiosis, very different localisation patterns have been reported for the SMC complexes, and the localisation of Nipbl/Mau2 has just recently started to be investigated. Here, we show that Nipbl/Mau2 binds on chromosomal axes from zygotene to mid-pachytene in germ cells of both sexes. In spermatocytes, Nipbl/Mau2 then relocalises to chromocenters, whereas in oocytes it remains bound to chromosomal axes throughout prophase to dictyate arrest. The localisation pattern of Nipbl/Mau2, together with those seen for cohesin, condensin and Smc5/6 subunits, is consistent with a role as a loading factor for cohesin and condensin I, but not for Smc5/6. We also demonstrate that Nipbl/Mau2 localises next to Rad51 and γH2AX foci. NIPBL gene deficiencies are associated with the Cornelia de Lange syndrome in humans, and we find that haploinsufficiency of the orthologous mouse gene results in an altered distribution of double-strand breaks marked by γH2AX during prophase I. However, this is insufficient to result in major meiotic malfunctions, and the chromosomal associations of the synaptonemal complex proteins and the three SMC complexes appear cytologically indistinguishable in wild-type and Nipbl (+/-) spermatocytes. PMID:24287868

  6. Optimized Protocols for In Vitro Maturation of Rat Oocytes Dramatically Improve Their Developmental Competence to a Level Similar to That of Ovulated Oocytes.

    PubMed

    Jiao, Guang-Zhong; Cui, Wei; Yang, Rui; Lin, Juan; Gong, Shuai; Lian, Hua-Yu; Sun, Ming-Ju; Tan, Jing-He

    2016-02-01

    The developmental capacity of in vitro-matured (IVM) oocytes is markedly lower than that of their in vivo-matured (IVO) counterparts, suggesting the need for optimization of IVM protocols in different species. There are few studies on IVM of rat oocytes, and there are even fewer attempts to improve ooplasmic maturation compared to those reported in other species. Furthermore, rat oocytes are well known to undergo spontaneous activation (SA) after leaving the oviduct; however, whether IVM rat oocytes have lower SA rates than IVO oocytes and can potentially be used for nuclear transfer is unknown. In this study, we investigated the effects of maturation protocols on cytoplasmic maturation of IVM rat oocytes and observed the possibility to reduce SA by using IVM rat oocytes. Ooplasmic maturation was assessed using multiple markers, including pre- and postimplantation development, meiotic progression, CG redistribution, redox state, and the expression of developmental potential- and apoptosis-related genes. The results showed that the best protocol consisting of modified Tissue Culture Medium-199 (TCM-199) supplemented with cysteamine/cystine and the cumulus cell monolayer dramatically improved the developmental competence of rat oocytes and supported both pre- and postimplantation development and other ooplasmic maturation makers to levels similar to that observed in ovulated oocytes. Rates of SA were significantly lower in IVM oocytes than in IVO oocytes when observed at the same intervals after nuclear maturation. In conclusion, we have optimized protocols for IVM of rat oocytes that sustain ooplasmic maturation to a level similar to ovulated oocytes. The results suggest that IVM rat oocytes might be used to reduce SA for rat cloning. PMID:26679437

  7. Structural basis for recognition of Emi2 by Polo-like kinase 1 and development of peptidomimetics blocking oocyte maturation and fertilization

    PubMed Central

    Jia, Jia-Lin; Han, Young-Hyun; Kim, Hak-Cheol; Ahn, Mija; Kwon, Jeong-Woo; Luo, Yibo; Gunasekaran, Pethaiah; Lee, Soo-Jae; Lee, Kyung S.; Kyu Bang, Jeong; Kim, Nam-Hyung; Namgoong, Suk

    2015-01-01

    In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr152 and Thr176) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization. PMID:26459104

  8. Heat stress and antioxidant enzyme activity in bubaline ( Bubalus bubalis) oocytes during in vitro maturation

    NASA Astrophysics Data System (ADS)

    Waiz, Syma Ashraf; Raies-ul-Haq, Mohammad; Dhanda, Suman; Kumar, Anil; Goud, T. Sridhar; Chauhan, M. S.; Upadhyay, R. C.

    2016-09-01

    In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly ( P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5 °C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5 °C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly ( P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

  9. Heat stress and antioxidant enzyme activity in bubaline (Bubalus bubalis) oocytes during in vitro maturation

    NASA Astrophysics Data System (ADS)

    Waiz, Syma Ashraf; Raies-ul-Haq, Mohammad; Dhanda, Suman; Kumar, Anil; Goud, T. Sridhar; Chauhan, M. S.; Upadhyay, R. C.

    2016-01-01

    In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly (P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5 °C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5 °C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly (P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

  10. DNA damage-induced metaphase I arrest is mediated by the spindle assembly checkpoint and maternal age

    PubMed Central

    Marangos, Petros; Stevense, Michelle; Niaka, Konstantina; Lagoudaki, Michaela; Nabti, Ibtissem; Jessberger, Rolf; Carroll, John

    2015-01-01

    In mammalian oocytes DNA damage can cause chromosomal abnormalities that potentially lead to infertility and developmental disorders. However, there is little known about the response of oocytes to DNA damage. Here we find that oocytes with DNA damage arrest at metaphase of the first meiosis (MI). The MI arrest is induced by the spindle assembly checkpoint (SAC) because inhibiting the SAC overrides the DNA damage-induced MI arrest. Furthermore, this MI checkpoint is compromised in oocytes from aged mice. These data lead us to propose that the SAC is a major gatekeeper preventing the progression of oocytes harbouring DNA damage. The SAC therefore acts to integrate protection against both aneuploidy and DNA damage by preventing production of abnormal mature oocytes and subsequent embryos. Finally, we suggest escaping this DNA damage checkpoint in maternal ageing may be one of the causes of increased chromosome anomalies in oocytes and embryos from older mothers. PMID:26522734

  11. DNA damage-induced metaphase I arrest is mediated by the spindle assembly checkpoint and maternal age.

    PubMed

    Marangos, Petros; Stevense, Michelle; Niaka, Konstantina; Lagoudaki, Michaela; Nabti, Ibtissem; Jessberger, Rolf; Carroll, John

    2015-01-01

    In mammalian oocytes DNA damage can cause chromosomal abnormalities that potentially lead to infertility and developmental disorders. However, there is little known about the response of oocytes to DNA damage. Here we find that oocytes with DNA damage arrest at metaphase of the first meiosis (MI). The MI arrest is induced by the spindle assembly checkpoint (SAC) because inhibiting the SAC overrides the DNA damage-induced MI arrest. Furthermore, this MI checkpoint is compromised in oocytes from aged mice. These data lead us to propose that the SAC is a major gatekeeper preventing the progression of oocytes harbouring DNA damage. The SAC therefore acts to integrate protection against both aneuploidy and DNA damage by preventing production of abnormal mature oocytes and subsequent embryos. Finally, we suggest escaping this DNA damage checkpoint in maternal ageing may be one of the causes of increased chromosome anomalies in oocytes and embryos from older mothers. PMID:26522734

  12. Xenopus oocyte meiosis lacks spindle assembly checkpoint control

    PubMed Central

    Shao, Hua; Ma, Chunqi; Chen, Eric

    2013-01-01

    The spindle assembly checkpoint (SAC) functions as a surveillance mechanism to detect chromosome misalignment and to delay anaphase until the errors are corrected. The SAC is thought to control mitosis and meiosis, including meiosis in mammalian eggs. However, it remains unknown if meiosis in the eggs of nonmammalian vertebrate species is also regulated by SAC. Using a novel karyotyping technique, we demonstrate that complete disruption of spindle microtubules in Xenopus laevis oocytes did not affect the bivalent-to-dyad transition at the time oocytes are undergoing anaphase I. These oocytes also acquired the ability to respond to parthenogenetic activation, which indicates proper metaphase II arrest. Similarly, oocytes exhibiting monopolar spindles, via inhibition of aurora B or Eg5 kinesin, underwent monopolar anaphase on time and without additional intervention. Therefore, the metaphase-to-anaphase transition in frog oocytes is not regulated by SAC. PMID:23569212

  13. TACC3 Is Important for Correct Progression of Meiosis in Bovine Oocytes

    PubMed Central

    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 during meiosis using qRT-PCR, and while it was also expressed in cleavage stage embryos, its expression was down-regulated at the morula and blastocyst stages. Immunofluorescence was used to demonstrate that TACC3 co-localized with tubulin in the metaphase I and II spindles. However, TACC3 was not detected at anaphase or telophase of the first meiotic division. Aurora A, which is known to phosphorylate and activate TACC3 in mitotic cells, showed a similar pattern of gene expression to that of TACC3 in meiotic oocytes and preimplantation embryos. Aurora A protein was however only very transiently associated to the meiotic spindle. Pharmaceutical inhibition of Aurora A activity inhibited TACC3 phosphorylation but did not prevent TACC3 appearance in the spindle. Inhibiting Aurora A activity did however lead to abnormal meiotic spindle formation and impaired maturation of bovine oocytes. Similar results were obtained by knock-down of TACC3 expression using siRNA injection. These results suggest that TACC3 is important for stabilizing the meiotic spindle, but phosphorylation of TACC3 by Aurora A is not required for its recruitment to the meiotic spindle although phosphorylation of TACC3 by other kinases cannot be excluded. PMID:26168150

  14. TACC3 Is Important for Correct Progression of Meiosis in Bovine Oocytes.

    PubMed

    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 during meiosis using qRT-PCR, and while it was also expressed in cleavage stage embryos, its expression was down-regulated at the morula and blastocyst stages. Immunofluorescence was used to demonstrate that TACC3 co-localized with tubulin in the metaphase I and II spindles. However, TACC3 was not detected at anaphase or telophase of the first meiotic division. Aurora A, which is known to phosphorylate and activate TACC3 in mitotic cells, showed a similar pattern of gene expression to that of TACC3 in meiotic oocytes and preimplantation embryos. Aurora A protein was however only very transiently associated to the meiotic spindle. Pharmaceutical inhibition of Aurora A activity inhibited TACC3 phosphorylation but did not prevent TACC3 appearance in the spindle. Inhibiting Aurora A activity did however lead to abnormal meiotic spindle formation and impaired maturation of bovine oocytes. Similar results were obtained by knock-down of TACC3 expression using siRNA injection. These results suggest that TACC3 is important for stabilizing the meiotic spindle, but phosphorylation of TACC3 by Aurora A is not required for its recruitment to the meiotic spindle although phosphorylation of TACC3 by other kinases cannot be excluded.

  15. Chromosome choreography: the meiotic ballet.

    PubMed

    Page, Scott L; Hawley, R Scott

    2003-08-01

    The separation of homologous chromosomes during meiosis in eukaryotes is the physical basis of Mendelian inheritance. The core of the meiotic process is a specialized nuclear division (meiosis I) in which homologs pair with each other, recombine, and then segregate from each other. The processes of chromosome alignment and pairing allow for homolog recognition. Reciprocal meiotic recombination ensures meiotic chromosome segregation by converting sister chromatid cohesion into mechanisms that hold homologous chromosomes together. Finally, the ability of sister kinetochores to orient to a single pole at metaphase I allows the separation of homologs to two different daughter cells. Failures to properly accomplish this elegant chromosome dance result in aneuploidy, a major cause of miscarriage and birth defects in human beings. PMID:12907787

  16. Granulosa cell-oocyte interactions: the phosphorylation of specific proteins in mouse oocytes at the germinal vesicle stage is dependent upon the differentiative state of companion somatic cells

    SciTech Connect

    Cecconi, S.; Tatone, C.; Buccione, R.; Mangia, F.; Colonna, R. )

    1991-05-01

    The role of granulosa cells in the regulation of mouse ovarian oocyte metabolism was investigated. Fully grown antral oocytes, isolated from surrounding cumulus cells, were cultured on monolayers of preantral granulosa cells in the presence of dbcAMP to prevent the resumption of meiosis. Under these conditions metabolic cooperativity was established between the two cell types as early as 1 hr after seeding. Moreover, cocultured oocytes phosphorylated two polypeptides of 74 and 21 kDa which are normally phosphorylated in follicle-enclosed growing oocytes but not in cumulus cell-enclosed fully grown oocytes at the germinal vesicle stage. When cocultured oocytes were allowed to resume meiosis, the 74 and 21 kDa proteins were synthesized but no longer phosphorylated even though intercellular coupling between the two cell types was maintained during radiolabeling. It appears therefore: (a) that the different protein kinase activity of growing and fully grown germinal vesicle-stage mouse oocytes is related to the differentiative state of granulosa cells, and (b) that the regulation of oocyte protein phosphorylation activity by granulosa cells is dependent on the meiotic stage of the oocyte.

  17. Metabolic control of oocyte development: linking maternal nutrition and reproductive outcomes

    PubMed Central

    Liu, Honglin; Gu, Xi; Boots, Christina; Moley, Kelle H.

    2015-01-01

    Obesity, diabetes, and related metabolic disorders are major health issues worldwide. As the epidemic of metabolic disorders continues, the associated medical comorbidities, including the detrimental impact on reproduction, increase as well. Emerging evidence suggests that the effects of maternal nutrition on reproductive outcomes are likely to be mediated, at least in part, by oocyte metabolism. Well-balanced and timed energy metabolism is critical for optimal development of oocytes. To date, much of our understanding of oocyte metabolism comes from the effects of extrinsic nutrients on oocyte maturation. In contrast, intrinsic regulation of oocyte development by metabolic enzymes, intracellular mediators, and transport systems is less characterized. Specifically, decreased acid transport proteins levels, increased glucose/lipid content and elevated reactive oxygen species in oocytes have been implicated in meiotic defects, organelle dysfunction and epigenetic alteration. Therefore, metabolic disturbances in oocytes may contribute to the diminished reproductive potential experienced by women with metabolic disorders. In-depth research is needed to further explore the underlying mechanisms. This review also discusses several approaches for metabolic analysis. Metabolomic profiling of oocytes, the surrounding granulosa cells, and follicular fluid will uncover the metabolic networks regulating oocyte development, potentially leading to the identification of oocyte quality markers and prevention of reproductive disease and poor outcomes in offspring. PMID:25280482

  18. Both diet and gene mutation induced obesity affect oocyte quality in mice

    PubMed Central

    Hou, Yan-Jun; Zhu, Cheng-Cheng; Duan, Xing; Liu, Hong-Lin; Wang, Qiang; Sun, Shao-Chen

    2016-01-01

    Obesity was shown to cause reproductive dysfunctions such as reduced conception, infertility and early pregnancy loss. However, the possible effects of obesity on oocyte quality are still not fully understood. In this study we investigated the effects of both diet and gene mutation induced obesity on impairments in mouse oocyte polarization, oxidative stress, and epigenetic modifications. Our results showed that high-fat diet induced obesity (HFD) and gene mutation induced obesity (ob/ob) could both impair oocyte meiotic maturation, disrupt spindle morphology, and reduce oocyte polarity. Oocytes from obese mice underwent oxidative stress, as shown by high DHE and ROS levels. Abnormal mitochondrial distributions and structures were observed in oocytes from obese groups of mice and early apoptosis signals were detected, which suggesting that oxidative stress had impaired mitochondrial function and resulted in oocyte apoptosis. Our results also showed that 5 mC levels and H3K9 and H3K27 methylation levels were altered in oocytes from obese mice, which indicated that DNA methylation and histone methylation had been affected. Our results showed that both HFD and ob/ob induced obesity affected oocyte maturation and that oxidative stress-induced early apoptosis and altered epigenetic modifications may be the reasons for reduced oocyte quality in obese mice. PMID:26732298

  19. Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes

    PubMed Central

    Dehapiot, Benoit; Carrière, Virginie; Carroll, John; Halet, Guillaume

    2013-01-01

    Asymmetric meiotic divisions in mammalian oocytes rely on the eccentric positioning of the spindle and the remodeling of the overlying cortex, resulting in the formation of small polar bodies. The mechanism of this cortical polarization, exemplified by the formation of a thick F-actin cap, is poorly understood. Cdc42 is a major player in cell polarization in many systems; however, the spatio-temporal dynamics of Cdc42 activation during oocyte meiosis, and its contribution to mammalian oocyte polarization, have remained elusive. In this study, we investigated Cdc42 activation (Cdc42–GTP), dynamics and role during mouse oocyte meiotic divisions. We show that Cdc42–GTP accumulates in restricted cortical regions overlying meiotic chromosomes or chromatids, in a Ran–GTP-dependent manner. This polarized activation of Cdc42 is required for the recruitment of N-WASP and the formation of F-actin-rich protrusions during polar body formation. Cdc42 inhibition in MII oocytes resulted in the release of N-WASP into the cytosol, a loss of the polarized F-actin cap, and a failure to protrude the second polar body. Cdc42 inhibition also resulted in central spindle defects in activated MII oocytes. In contrast, emission of the first polar body during oocyte maturation could occur in the absence of a functional Cdc42/N-WASP pathway. Therefore, Cdc42 is a new protagonist in chromatin-induced cortical polarization in mammalian oocytes, with an essential role in meiosis II completion, through the recruitment and activation of N-WASP, downstream of the chromatin-centered Ran–GTP gradient. PMID:23384564

  20. Age-Dependent Susceptibility of Chromosome Cohesion to Premature Separase Activation in Mouse Oocytes1

    PubMed Central

    Chiang, Teresa; Schultz, Richard M.; Lampson, Michael A.

    2011-01-01

    ABSTRACT A hypothesis to explain the maternal age-dependent increase in formation of aneuploid eggs is deterioration of chromosome cohesion. Although several lines of evidence are consistent with this hypothesis, whether cohesion is actually reduced in naturally aged oocytes has not been directly tested by any experimental perturbation. To directly target cohesion, we increased the activity of separase, the protease that cleaves the meiotic cohesin REC8, in oocytes. We show that cohesion is more susceptible to premature separase activation in old oocytes than in young oocytes, demonstrating that cohesion is significantly reduced. Furthermore, cohesion is protected by two independent mechanisms that inhibit separase, securin and an inhibitory phosphorylation of separase by CDK1; both mechanisms must be disrupted to prematurely activate separase. With the continual loss of cohesins from chromosomes that occurs throughout the natural reproductive lifespan, tight regulation of separase in oocytes may be particularly important to maintain cohesion and prevent aneuploidy. PMID:21865557

  1. Oocyte Polarization Is Coupled to the Chromosomal Bouquet, a Conserved Polarized Nuclear Configuration in Meiosis.

    PubMed

    Elkouby, Yaniv M; Jamieson-Lucy, Allison; Mullins, Mary C

    2016-01-01

    The source of symmetry breaking in vertebrate oocytes is unknown. Animal-vegetal oocyte polarity is established by the Balbiani body (Bb), a conserved structure found in all animals examined that contains an aggregate of specific mRNAs, proteins, and organelles. The Bb specifies the oocyte vegetal pole, which is key to forming the embryonic body axes as well as the germline in most vertebrates. How Bb formation is regulated and how its asymmetric position is established are unknown. Using quantitative image analysis, we trace oocyte symmetry breaking in zebrafish to a nuclear asymmetry at the onset of meiosis called the chromosomal bouquet. The bouquet is a universal feature of meiosis where all telomeres cluster to one pole on the nuclear envelope, facilitating chromosomal pairing and meiotic recombination. We show that Bb precursor components first localize with the centrosome to the cytoplasm adjacent to the telomere cluster of the bouquet. They then aggregate around the centrosome in a specialized nuclear cleft that we identified, assembling the early Bb. We show that the bouquet nuclear events and the cytoplasmic Bb precursor localization are mechanistically coordinated by microtubules. Thus the animal-vegetal axis of the oocyte is aligned to the nuclear axis of the bouquet. We further show that the symmetry breaking events lay upstream to the only known regulator of Bb formation, the Bucky ball protein. Our findings link two universal features of oogenesis, the Bb and the chromosomal bouquet, to oocyte polarization. We propose that a meiotic-vegetal center couples meiosis and oocyte patterning. Our findings reveal a novel mode of cellular polarization in meiotic cells whereby cellular and nuclear polarity are aligned. We further reveal that in zygotene nests, intercellular cytoplasmic bridges remain between oocytes and that the position of the cytoplasmic bridge coincides with the location of the centrosome meiotic-vegetal organizing center. These results

  2. Fatty acid synthesis and oxidation in cumulus cells support oocyte maturation in bovine.

    PubMed

    Sanchez-Lazo, Laura; Brisard, Daphné; Elis, Sébastien; Maillard, Virginie; Uzbekov, Rustem; Labas, Valérie; Desmarchais, Alice; Papillier, Pascal; Monget, Philippe; Uzbekova, Svetlana

    2014-09-01

    Oocyte meiotic maturation requires energy from various substrates including glucose, amino acids, and lipids. Mitochondrial fatty acid (FA) β-oxidation (FAO) in the oocyte is required for meiotic maturation, which is accompanied by differential expression of numerous genes involved in FAs metabolism in surrounding cumulus cells (CCs) in vivo. The objective was to elucidate components involved in FAs metabolism in CCs during oocyte maturation. Twenty-seven genes related to lipogenesis, lipolysis, FA transport, and FAO were chosen from comparative transcriptome analysis of bovine CCs before and after maturation in vivo. Using real-time PCR, 22 were significantly upregulated at different times of in vitro maturation (IVM) in relation to oocyte meiosis progression from germinal vesicle breakdown to metaphase-II. Proteins FA synthase, acetyl-coenzyme-A carboxylase, carnitine palmitoyltransferase, perilipin 2, and FA binding protein 3 were detected by Western blot and immunolocalized to CCs and oocyte cytoplasm, with FA binding protein 3 concentrated around oocyte chromatin. By mass spectrometry, CCs lipid profiling was shown to be different before and after IVM. FAO inhibitors etomoxir and mildronate dose-dependently decreased the oocyte maturation rate in vitro. In terms of viability, cumulus enclosed oocytes were more sensitive to etomoxir than denuded oocytes. In CCs, etomoxir (150 μM) led to downregulation of lipogenesis genes and upregulated lipolysis and FAO genes. Moreover, the number of lipid droplets decreased, whereas several lipid species were more abundant compared with nontreated CCs after IVM. In conclusion, FAs metabolism in CCs is important to maintain metabolic homeostasis and may influence meiosis progression and survival of enclosed oocytes. PMID:25058602

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

    USGS Publications Warehouse

    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.

  4. Improvement in in vitro fertilization outcome following in vivo synchronization of oocyte maturation in mice.

    PubMed

    Taiyeb, Ahmed M; Muhsen-Alanssari, Saeeda A; Dees, W L; Ridha-Albarzanchi, Mundhir T; Kraemer, Duane C

    2015-04-01

    Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2-4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.

  5. Mnd1p: an evolutionarily conserved protein required for meiotic recombination.

    PubMed

    Gerton, Jennifer L; DeRisi, Joseph L

    2002-05-14

    We used a functional genomics approach to identify a gene required for meiotic recombination, YGL183c or MND1. MND1 was spliced in meiotic cells, extending the annotated YGL183c ORF N terminus by 45 aa. Saccharomyces cerevisiae mnd1-1 mutants, in which the majority of the MND1 coding sequence was removed, arrested before the first meiotic division with a phenotype reminiscent of dmc1 mutants. Physical and genetic analysis showed that these cells initiated recombination, but did not form heteroduplex DNA or double Holliday junctions, suggesting that Mnd1p is involved in strand invasion. Orthologs of MND1 were identified in protists, several yeasts, plants, and mammals, suggesting that its function has been conserved throughout evolution.

  6. Dual effects of hydrogen sulfide donor on meiosis and cumulus expansion of porcine cumulus-oocyte complexes.

    PubMed

    Nevoral, Jan; Petr, Jaroslav; Gelaude, Armance; Bodart, Jean-Francois; Kucerova-Chrpova, Veronika; Sedmikova, Marketa; Krejcova, Tereza; Kolbabova, Tereza; Dvorakova, Marketa; Vyskocilova, Alena; Weingartova, Ivona; Krivohlavkova, Lenka; Zalmanova, Tereza; Jilek, Frantisek

    2014-01-01

    Hydrogen sulfide (H2S) has been revealed to be a signal molecule with second messenger action in the somatic cells of many tissues, including the reproductive tract. The aim of this study was to address how exogenous H2S acts on the meiotic maturation of porcine oocytes, including key maturation factors such as MPF and MAPK, and cumulus expansion intensity of cumulus-oocyte complexes. We observed that the H2S donor, Na2S, accelerated oocyte in vitro maturation in a dose-dependent manner, following an increase of MPF activity around germinal vesicle breakdown. Concurrently, the H2S donor affected cumulus expansion, monitored by hyaluronic acid production. Our results suggest that the H2S donor influences oocyte maturation and thus also participates in the regulation of cumulus expansion. The exogenous H2S donor apparently affects key signal pathways of oocyte maturation and cumulus expansion, resulting in faster oocyte maturation with little need of cumulus expansion.

  7. Effects of maternal ageing on ICSI outcomes and embryo development in relation to oocytes morphological characteristics of birefringent structures.

    PubMed

    Korkmaz, Cem; Tekin, Yesim Bayoglu; Sakinci, Mehmet; Ercan, Cihangir Mutlu

    2015-08-01

    The aim of this study was to determine the morphological characteristics of the older reproductive aged women's oocytes and to reveal the influence of these characteristics on intra-cytoplasmic sperm injection (ICSI) outcomes. The oocytes of women older than 35 years of age were evaluated retrospectively. Non-invasive polarization microscopy (PolScope) examinations of mature oocytes were performed by measurement of meiotic spindles' length, area and retardance and zona pellucida thickness and retardance. Fertilization and conception competence and the correlation with the birefringent structures were assessed. Two hundred and thirteen mature oocytes from 54 women were evaluated with a PolScope. Length of the meiotic spindle was shown to be related to fertilization success of women with advanced maternal age. In conclusion, the PolScope is a useful device used to identify the oocyte quality. Quantitative measurements of meiotic spindle parameters may be valuable for the selection of high-quality oocytes that have the potential for embryo development in the in vitro fertilization (IVF) laboratory of women older than 35 years of age who are mostly poor responders.

  8. Changes in the reciprocal position of the first polar body and oocyte chromosome set in golden hamsters.

    PubMed

    Wang, Lingyan; Li, Dexue; Li, Ziyi

    2009-10-01

    The golden hamster is an attractive model organism for studying reproductive physiology, oncology, genetics and virology. In an effort to establish experimental protocols necessary for cloning golden hamsters, we examined changes in the reciprocal position of the FPB (first polar body) and chromosome set of MII (the second meiotic metaphase) oocytes of golden hamsters. Oocytes were collected under three different conditions: (i) oocyte direct recovery from the oviduct of hormonally treated donor; (ii) oocyte recovery from the oviduct of hormonally treated donor followed by 5 h/10 h in vitro culture; and (iii) oocyte recovery from ovaries of hormonally treated donors and in vitro maturation. Then oocyte recovery was performed from the oviduct of hormonally treated donors, followed by 5 h in vitro culture with colchicine and/or CB (cytochalasin B). Denuded oocytes were stained with Hoechst 33342 and propidium iodide and evaluated under a microscope. Our results demonstrate that the change in FPB position in relation to the MII oocyte chromosome set increases with age of in vivo-matured oocytes. Cumulus cells can protect the FPB of in vitro-cultured oocytes from degeneration but do not significantly affect its repositioning, and in vitro-matured oocytes age slower. The colchicine has a stronger effect on cytoplasmic protrusions of golden hamster oocytes when compared with CB. These results define conditions for changes in FPB position relative to the MII oocyte chromosome set. Early ovulated oocytes, in vitro-matured oocytes and oocytes treated with colchicine should improve the effectiveness of the cloning procedure in golden hamsters as an animal model for human diseases. PMID:18980577

  9. The role of phosphatidylinositol signaling pathway in regulating serotonin-induced oocyte maturation in Mercenaria mercenaria

    NASA Astrophysics Data System (ADS)

    Wang, Qing; Zhang, Tao

    2011-05-01

    Serotonin (5-HT) has been found to stimulate meiotic maturation of oocytes in many molluscs. During maturation, several signaling pathways are involved, especially the phosphatidylinositol and cAMP pathways. In order to examine the possible role of the phosphatidylinositol signaling pathway in regulating oocyte maturation in Mercenaria mercenaria, the effects of the activator/inhibitor of phospholipase (PLC) and protein kinase C (PKC) on serotonin-induced maturation were studied. Results show that high-concentrations of neomycin (inhibitor of PLC) blocked oocyte maturation, while 9, 10-dimethyl-1, 2-benzanthracene (DMBA, activator of PLC) promoted oocyte maturation in the presence of serotonin. It was also found that in the presence of serotonin, phorbol 12-myristate 13-acetate (PMA, activator of PKC) inhibited oocyte maturation, while sphingosine (inhibitor of PKC) stimulated oocyte maturation. These results indicate that serotonin-induced oocyte maturation requires the activation of the phosphatidylinositol pathway. Decrease of PLC inhibited serotonin-induced oocyte maturation, whereas a decrease of PKC stimulated the maturation. Thus, our study indicates that serotonin promotes maturation of M. mercenaria oocytes through PLC stimulated increase in calcium ion concentration via inositol-1, 4, 5-trisphosphate (IP3) but not PKC.

  10. The growth of the oocyte and follicle in the ovaries of monotremes and marsupials.

    PubMed

    Lintern-Moore, S; Moore, G P; Tyndale-Biscoe, C H; Poole, W E

    1976-07-01

    Oocyte growth in relation to follicular growth in marsupials conforms to the uniform biphasic pattern characteristic of eutherian mammals. However, the oocyte of the marsupial is larger and completes growth in a correspondingly larger follicle. At the completion of oocyte growth the follicle, like that of eutherians, shows no appreciable accumulation of follicular fluid. The ratio of oocyte/follicular diameter in marsupials at this stage is 0.59, a value close to that of 0.55 for eutherian mammals. The oocyte nucleus of the marsupial is also significantly larger than that of the eutherian, but the oocyte nucleolus is approximately the same diameter. By contrast, the growth of the oocyte in relation to the follicle in monotremes in linear throughout the whole period of follicular growth. However, like eutherians and marsupials oocyte nuclear and oocyte nucleolar growth are arrested in the early stages of follicular growth. The diameters of the oocyte nucleus and nucleolus at the completion of growth are approximately twice those found in marsupials. It is concluded that the growth patterns of the oocyte nucleus and nucleolus are common to all mammals but that the biphasic pattern of oocyte and follicular growth which is characteristic of eutherian and marsupial ovaries is not found in the monotremes.

  11. Sirt6 depletion causes spindle defects and chromosome misalignment during meiosis of mouse oocyte

    PubMed Central

    Han, Longsen; Ge, Juan; Zhang, Liang; Ma, Rujun; Hou, Xiaojing; Li, Bin; Moley, Kelle; Wang, Qiang

    2015-01-01

    Sirt6, a member of the sirtuin family of NAD-dependent protein deacetylases, has been implicated in multiple biological processes. However, the roles of Sirt6 in meiosis have not been addressed. In the present study, by employing knockdown analysis in mouse oocytes, we evaluated the effects of Sirt6 on meiotic apparatus. We found that specific depletion of Sirt6 results in disruption of spindle morphology and chromosome alignment in oocytes. Consistent with this observation, incidence of aneuploidy is also markedly increased in Sirt6-depleted oocytes. Furthermore, confocal scanning showed that kinetochore-microtubule interaction, an important mechanism controlling chromosome segregation, is severely impaired in metaphase oocytes following Sirt6 knockdown. Unexpectedly, we discovered that Sirt6 modulates the acetylation status of histone H4K16 as their knockdown specifically induces the hyperacetylation of H4K16 in oocytes, which may be associated with the defective phenotypes described above via altering kinetochore function. Altogether, our data reveal a novel function of Sirt6 during oocyte meiosis and indicate a pathway regulating meiotic apparatus. PMID:26481302

  12. Sirt6 depletion causes spindle defects and chromosome misalignment during meiosis of mouse oocyte.

    PubMed

    Han, Longsen; Ge, Juan; Zhang, Liang; Ma, Rujun; Hou, Xiaojing; Li, Bin; Moley, Kelle; Wang, Qiang

    2015-10-20

    Sirt6, a member of the sirtuin family of NAD-dependent protein deacetylases, has been implicated in multiple biological processes. However, the roles of Sirt6 in meiosis have not been addressed. In the present study, by employing knockdown analysis in mouse oocytes, we evaluated the effects of Sirt6 on meiotic apparatus. We found that specific depletion of Sirt6 results in disruption of spindle morphology and chromosome alignment in oocytes. Consistent with this observation, incidence of aneuploidy is also markedly increased in Sirt6-depleted oocytes. Furthermore, confocal scanning showed that kinetochore-microtubule interaction, an important mechanism controlling chromosome segregation, is severely impaired in metaphase oocytes following Sirt6 knockdown. Unexpectedly, we discovered that Sirt6 modulates the acetylation status of histone H4K16 as their knockdown specifically induces the hyperacetylation of H4K16 in oocytes, which may be associated with the defective phenotypes described above via altering kinetochore function. Altogether, our data reveal a novel function of Sirt6 during oocyte meiosis and indicate a pathway regulating meiotic apparatus.

  13. Protein kinase C (PKC) role in bovine oocyte maturation and early embryo development.

    PubMed

    Mondadori, R G; Neves, J P; Gonçalves, P B D

    2008-08-01

    The aims of the present study were to determine the role of protein kinase C (PKC) on meiotic resumption and its effects on pronuclear formation and cleavage in the bovine. Oocytes were matured in the presence of 0, 1, 10 and 100 nM of phorbol 12-myristate 13-acetate (PMA), to evaluate the percentage of germinal vesicle breakdown. To study pronuclear formation and cleavage, oocytes were randomly distributed in four groups and matured in modified TCM-199 with LH and FSH (negative control); 10% of estrous cow serum (positive control); 100 nM of PMA (treatment); 100 nM of 4alpha-PDD (phorbol ester control). Oocytes were also matured in positive control medium, fertilized and transferred to KSOM with increasing concentrations of a PKC inhibitor. The protein profile and the presence of PKC at the end of maturation period were determined by SDS-PAGE followed by Silver Stain and Western blot, respectively. PMA stimulated meiotic resumption in a concentration-dependent manner. PKC stimulation during oocyte maturation caused an increase in pronuclear formation and did not cause parthenogenetic activation. Inhibitor of PKC (MyrPKC) inhibited cleavage in a dose-dependent and irreversible manner. A protein band around 74 kDa was not detected in PMA-treated oocytes and PKC was not detected by Western blot at the end of the maturation period. In conclusion, meiotic resumption was accelerated and the rate of oocytes with two pronuclei was increased when PKC was activated during oocyte maturation. Moreover, cleavage was inhibited in the presence of PMA. PMID:17646065

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

    PubMed Central

    Lee, Seung Eun; Kim, Eun Young; Choi, Hyun Yong; Moon, Jeremiah Jiman; Park, Min Jee; Lee, Jun Beom; Jeong, Chang Jin; Park, Se Pill

    2014-01-01

    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. PMID:25049998

  15. HORMAD2 is essential for synapsis surveillance during meiotic prophase via the recruitment of ATR activity.

    PubMed

    Kogo, Hiroshi; Tsutsumi, Makiko; Inagaki, Hidehito; Ohye, Tamae; Kiyonari, Hiroshi; Kurahashi, Hiroki

    2012-11-01

    Meiotic chromosome segregation requires homologous pairing, synapsis and crossover recombination during meiotic prophase. The checkpoint kinase ATR has been proposed to be involved in the quality surveillance of these processes, although the underlying mechanisms remain largely unknown. In our present study, we generated mice lacking HORMAD2, a protein that localizes to unsynapsed meiotic chromosomes. We show that this Hormad2 deficiency hampers the proper recruitment of ATR activity to unsynapsed chromosomes. Male Hormad2-deficient mice are infertile due to spermatocyte loss as a result of characteristic impairment of sex body formation; an ATR- and γH2AX-enriched repressive chromatin domain is formed, but is partially dissociated from the elongated sex chromosome axes. In contrast to males, Hormad2-deficient females are fertile. However, our analysis of Hormad2/Spo11 double-mutant females shows that the oocyte number is negatively correlated with the frequency of pseudo-sex body formation in a Hormad2 gene dosage-dependent manner. This result suggests that the elimination of Spo11-deficient asynaptic oocytes is associated with the HORMAD2-dependent pseudo-sex body formation that is likely initiated by local concentration of ATR activity in the absence of double-strand breaks. Our results thus show a HORMAD2-dependent quality control mechanism that recognizes unsynapsis and recruits ATR activity during mammalian meiosis. PMID:23039116

  16. Detection of condensin I and II in maturing pig oocytes.

    PubMed

    Lisková, Lucie; Susor, Andrej; Pivonková, Katerina; Sasková, Adéla; Karabínová, Pavla; Kubelka, Michal

    2010-01-01

    The multiprotein complexes known as condensins (I and II) are major players in chromosome dynamics in mitotic and meiotic cells. Here, we report for the first time the detection of different condensin subunits from both complexes in mammalian oocytes. Using immunoblotting analysis we examined expression levels of condensin subunits during meiotic maturation of porcine oocytes. The expression of the core subunit structural maintenance of chromosomes 2 (SMC2), identical in both condensin complexes, did not change significantly during maturation. Similarly, there was no significant change in the expression of the chromosome associated protein (CAP)-H and CAP-H2 subunits, components of condensin I and II, respectively. Conversely, the expression profiles of CAP-G, CAP-D2 (condensin I) and CAP-D3 (condensin II) were more interesting. At least two isoforms of the CAP-D2 subunit were detected, along with three isoforms of the CAP-D3 and CAP-G subunits. We suggest that this diverse migration of subunit isoforms is due to post-translational modification. Earlier, it was reported that non-SMC proteins are phosphorylated by cyclin-dependent kinase 1. In the present study, we analysed the phosphorylation status of the three subunits in oocyte extracts using alkaline phosphatase treatment and we found that at least the fastest migrating form of CAP-D3 was likely to be phosphorylated in maturing porcine oocytes. In addition, the localisation of CAP-H and CAP-H2 subunits was examined using immunofluorescence staining with specific antibodies, as well as following microinjection of their enhanced green fluorescent protein-tagged mRNA into germinal vesicle-stage oocytes. CAP-H was found in the cytoplasm, whereas CAP-H2 was localised within the nucleus.

  17. Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

    PubMed Central

    Bernal-Ulloa, Sandra Milena; Heinzmann, Julia; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Winkler, Sylke; Pache, Dorit; Baulain, Ulrich; Lucas-Hahn, Andrea; Niemann, Heiner

    2016-01-01

    High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency. PMID:26926596

  18. Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes.

    PubMed

    Das, Arunika; Shah, Shital J; Fan, Bensen; Paik, Daniel; DiSanto, Daniel J; Hinman, Anna Maria; Cesario, Jeffry M; Battaglia, Rachel A; Demos, Nicole; McKim, Kim S

    2016-01-01

    Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule-chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation.

  19. Meiotic Recombination in Drosophila Females Depends on Chromosome Continuity Between Genetically Defined Boundaries

    PubMed Central

    Sherizen, Dalia; Jang, Janet K.; Bhagat, Rajal; Kato, Naohiro; McKim, Kim S.

    2005-01-01

    In the pairing-site model, specialized regions on each chromosome function to establish meiotic homolog pairing. Analysis of these sites could provide insights into the mechanism used by Drosophila females to form a synaptonemal complex (SC) in the absence of meiotic recombination. These specialized sites were first established on the X chromosome by noting that there were barriers to crossover suppression caused by translocation heterozygotes. These sites were genetically mapped and proposed to be pairing sites. By comparing the cytological breakpoints of third chromosome translocations to their patterns of crossover suppression, we have mapped two sites on chromosome 3R. We have performed experiments to determine if these sites have a role in meiotic homolog pairing and the initiation of recombination. Translocation heterozygotes exhibit reduced gene conversion within the crossover-suppressed region, consistent with an effect on the initiation of meiotic recombination. To determine if homolog pairing is disrupted in translocation heterozygotes, we used fluorescent in situ hybridization to measure the extent of homolog pairing. In wild-type oocytes, homologs are paired along their entire lengths prior to accumulation of the SC protein C(3)G. Surprisingly, translocation heterozygotes exhibited homolog pairing similar to wild type within the crossover-suppressed regions. This result contrasted with our observations of c(3)G mutant females, which were found to be defective in pairing. We propose that each Drosophila chromosome is divided into several domains by specialized sites. These sites are not required for homolog pairing. Instead, the initiation of meiotic recombination requires continuity of the meiotic chromosome structure within each of these domains. PMID:15545646

  20. Coordination of Recombination with Meiotic Progression in the Caenorhabditis elegans Germline by KIN-18, a TAO Kinase That Regulates the Timing of MPK-1 Signaling.

    PubMed

    Yin, Yizhi; Donlevy, Sean; Smolikove, Sarit

    2016-01-01

    Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase's role in germline development in higher eukaryotes.

  1. Exposure to HT-2 toxin causes oxidative stress induced apoptosis/autophagy in porcine oocytes

    PubMed Central

    Zhang, Yue; Han, Jun; Zhu, Cheng-Cheng; Tang, Feng; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2016-01-01

    T-2 toxin is a main type A trichothecene mycotoxin which is the most toxic trichothecence. T-2 toxin has posed various toxic effects on human and animals in vigorous cell proliferation tissues like lymphoid, hematopoietic and gastrointestinal tissues, while HT-2 toxin is the major metabolite which is deacetylated by T-2 toxin. In this study, we focused on the toxic effects of HT-2 on porcine oocyte maturation. We treated the porcine oocyte with HT-2 toxin in vitro, and we first found that HT-2 treatment inhibited porcine oocyte polar body extrusion and cumulus cell expansion. We observed the disrupted meiotic spindle morphology after treatment, which might be due to the reduced p-MAPK protein level. Actin distribution was also disturbed, indicating that HT-2 affects cytoskeleton of porcine oocytes. We next explored the causes for the failure of oocyte maturation after HT-2 treatment. We found that HT-2 treated oocytes showed the increased ROS level, which indicated that oxidative stress had occurred. We also detected autophagy as well as early apoptosis in the treatment oocytes. Due to the fact that oxidative stress could induced apoptosis, our results indicated that HT-2 toxin caused oxidative stress induced apoptosis and autophagy, which further affected porcine oocyte maturation. PMID:27658477

  2. Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop.

    PubMed

    Su, You-Qiang; Wu, Xuemei; O'Brien, Marilyn J; Pendola, Frank L; Denegre, James N; Matzuk, Martin M; Eppig, John J

    2004-12-01

    Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9+/-Bmp15-/- [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte

  3. The polyubiquitin gene Ubi-p63E is essential for male meiotic cell cycle progression and germ cell differentiation in Drosophila.

    PubMed

    Lu, Chenggang; Kim, Jongmin; Fuller, Margaret T

    2013-09-01

    The ubiquitin proteasome system (UPS) regulates many biological pathways by post-translationally ubiquitylating proteins for degradation. Although maintaining a dynamic balance between free ubiquitin and ubiquitylated proteins is key to UPS function, the mechanisms that regulate ubiquitin homeostasis in different tissues through development are not clear. Here we show, via analysis of the magellan (magn) complementation group, that loss of function of the Drosophila polyubiquitin Ubi-p63E results specifically in meiotic arrest sterility in males. Ubi-p63E contributes predominantly to maintaining the free ubiquitin pool in testes. The function of Ubi-p63E is required cell-autonomously for proper meiotic chromatin condensation, cell cycle progression and spermatid differentiation. magn mutant germ cells develop normally to the spermatocyte stage but arrest at the G2/M transition of meiosis I, with lack of protein expression of the key meiotic cell cycle regulators Boule and Cyclin B. Loss of Ubi-p63E function did not strongly affect the spermatocyte transcription program regulated by the testis TBP-associated factor (tTAF) or meiosis arrest complex (tMAC) genes. Knocking down proteasome function specifically in spermatocytes caused a different meiotic arrest phenotype, suggesting that the magn phenotype might not result from general defects in protein degradation. Our results suggest a conserved role of polyubiquitin genes in male meiosis and a potential mechanism leading to meiosis I maturation arrest.

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

    PubMed Central

    Ezoe, Kenji; Yabuuchi, Akiko; Tani, Tetsuya; Mori, Chiemi; Miki, Tetsuya; Takayama, Yuko; Beyhan, Zeki; Kato, Yoko; Okuno, Takashi; Kobayashi, Tamotsu; Kato, Keiichi

    2015-01-01

    Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming. PMID:25965267

  5. Essential Role for endogenous siRNAs during meiosis in mouse oocytes.

    PubMed

    Stein, Paula; Rozhkov, Nikolay V; Li, Fan; Cárdenas, Fabián L; Davydenko, Olga; Davydenk, Olga; Vandivier, Lee E; Gregory, Brian D; Hannon, Gregory J; Schultz, Richard M

    2015-02-01

    The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO) family of proteins. In mammals, there are four AGO proteins (AGO1-4), of which only AGO2 possesses endonucleolytic activity. siRNAs trigger endonucleolytic cleavage of target mRNAs, mediated by AGO2, whereas miRNAs cause translational repression and mRNA decay through association with any of the four AGO proteins. Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because mouse oocytes express both miRNAs and endo-siRNAs, this phenotype could be due to the absence of either class of small RNA, or both. However, we and others demonstrated that miRNA function is suppressed in mouse oocytes, which suggested that endo-siRNAs, not miRNAs, are essential for female meiosis. To determine if this was the case we generated mice that express a catalytically inactive knock-in allele of Ago2 (Ago2ADH) exclusively in oocytes and thereby disrupted the function of siRNAs. Oogenesis and hormonal response are normal in Ago2ADH oocytes, but meiotic maturation is impaired, with severe defects in spindle formation and chromosome alignment that lead to meiotic catastrophe. The transcriptome of these oocytes is widely perturbed and shows a highly significant correlation with the transcriptome of Dicer null and Ago2 null oocytes. Expression of the mouse transcript (MT), the most abundant transposable element in mouse oocytes, is increased. This study reveals that endo-siRNAs are essential during meiosis I in mouse females, demonstrating a role for endo-siRNAs in mammals.

  6. Backcrossing to increase meiotic stability in triticale.

    PubMed

    Giacomin, R M; Assis, R; Brammer, S P; Nascimento Junior, A; Da-Silva, P R

    2015-01-01

    Triticale (X Triticosecale Wittmack) is an intergeneric hybrid derived from a cross between wheat and rye. As a newly created allopolyploid, the plant shows instabilities during the meiotic process, which may result in the loss of fertility. This genomic instability has hindered the success of triticale-breeding programs. Therefore, strategies should be developed to obtain stable triticale lines for use in breeding. In some species, backcrossing has been effective in increasing the meiotic stability of lineages. To assess whether backcrossing has the same effect in triticale, indices of meiotic abnormalities, meiotic index, and pollen viability were determined in genotypes from multiple generations of triticale (P1, P2, F1, F2, BC1a, and BC1b). All analyzed genotypes exhibited instability during meiosis, and their meiotic index values were all lower than normal. However, the backcrosses BC1a and BC1b showed the lowest mean meiotic abnormalities and the highest meiotic indices, demonstrating higher stability. All genotypes showed a high rate of pollen viability, with the backcrosses BC1a and BC1b again exhibiting the best values. Statistical analyses confirmed that backcrossing positively affects the meiotic stability of triticale. Our results show that backcrossing should be considered by breeders aiming to obtain triticale lines with improved genomic stability. PMID:26400358

  7. CPEB and miR-15/16 Co-Regulate Translation of Cyclin E1 mRNA during Xenopus Oocyte Maturation

    PubMed Central

    Wilczynska, Ania; Git, Anna; Argasinska, Joanna; Belloc, Eulàlia; Standart, Nancy

    2016-01-01

    Cell cycle transitions spanning meiotic maturation of the Xenopus oocyte and early embryogenesis are tightly regulated at the level of stored inactive maternal mRNA. We investigated here the translational control of cyclin E1, required for metaphase II arrest of the unfertilised egg and the initiation of S phase in the early embryo. We show that the cyclin E1 mRNA is regulated by both cytoplasmic polyadenylation elements (CPEs) and two miR-15/16 target sites within its 3’UTR. Moreover, we provide evidence that maternal miR-15/16 microRNAs co-immunoprecipitate with CPE-binding protein (CPEB), and that CPEB interacts with the RISC component Ago2. Experiments using competitor RNA and mutated cyclin E1 3’UTRs suggest cooperation of the regulatory elements to sustain repression of the cyclin E1 mRNA during early stages of maturation when CPEB becomes limiting and cytoplasmic polyadenylation of repressed mRNAs begins. Importantly, injection of anti-miR-15/16 LNA results in the early polyadenylation of endogenous cyclin E1 mRNA during meiotic maturation, and an acceleration of GVBD, altogether strongly suggesting that the proximal CPEB and miRNP complexes act to mutually stabilise each other. We conclude that miR-15/16 and CPEB co-regulate cyclin E1 mRNA. This is the first demonstration of the co-operation of these two pathways. PMID:26829217

  8. Meiotic Recombination: The Essence of Heredity.

    PubMed

    Hunter, Neil

    2015-10-28

    The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles. Thus, meiotic recombination underlies both the independent assortment of parental chromosomes and genetic linkage. This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotic recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase. A more in-depth review presents our understanding of how crossover and noncrossover pathways of meiotic recombination are differentiated and regulated. The final section of this review summarizes the studies that have defined defective recombination as a leading cause of pregnancy loss and congenital disease in humans.

  9. Improvement in in vitro fertilization outcome following in vivo synchronization of oocyte maturation in mice

    PubMed Central

    Muhsen-Alanssari, Saeeda A; Dees, WL; Ridha-Albarzanchi, Mundhir T; Kraemer, Duane C

    2015-01-01

    Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients. PMID:25245076

  10. Cryopreservation of embryos and oocytes: an update.

    PubMed

    Gelety, T; Surrey, E

    1993-10-01

    Widespread incorporation of human embryo cryopreservation into IVF programs may reduce the risk of multiple gestation and severe ovarian hyperstimulation syndrome as well as contributing to an overall increase in pregnancy rates. Slow cooling techniques appropriate to the stage of embryo development are most commonly employed. Growing experience with ultrarapid freezing suggests that this technique may offer similar success rates and has the advantages of simplicity and economy. The developmental stage at cryopreservation has not been conclusively shown to influence successful pregnancy outcome, although several retrospective studies suggest improved embryo survival and pregnancy rate following freezing at the pronuclear stage. Endometrial preparation using a number of different regimens before thawed-embryo transfer appears to confer no advantage over the natural cycle, but may be necessary with ovulatory dysfunction. The use of gonadotropin-releasing hormone (GnRH) analogs during ovarian stimulation for IVF has been suggested to decrease subsequent embryo freeze-thaw survival rates. However, increased pregnancy rates have been reported following transfer of previously cryopreserved embryos originating in cycles using GnRH analog (GnRHa), possibly reflecting increased numbers of embryos available for freezing. Cryopreservation of embryos originating from fertile donor oocytes has been very successful and may be used to facilitate synchronization between a donor and one or more recipients. Recent work in oocyte cryopreservation has addressed the problems of meiotic spindle disruption, the risks of aneuploidy, and decreased fertilization associated with zona hardening and polyspermy. Further refinements in technique will be required before widespread oocyte banking becomes feasible. PMID:8241436

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

    PubMed

    Lai, Fang-Nong; Ma, Jun-Yu; Liu, Jing-Cai; Wang, Jun-Jie; Cheng, Shun-Feng; Sun, Xiao-Feng; Li, Lan; Li, Bo; Nyachoti, Charles Martin; Shen, Wei

    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. PMID:26386189

  12. [Heart arrest].

    PubMed

    Chiarella, F; Giovannini, E; Bozzano, A; Caristo, G; Delise, P; Fedele, F; Fera, M S; Lavalle, C; Roghi, A; Valagussa, F

    2001-03-01

    Cardiac arrest is one of the leading causes of mortality in industrialized countries and is mainly due to ischemic heart disease. According to ISTAT estimates, approximately 45,000 sudden deaths occur annually in Italy whereas according to the World Health Organization, its incidence is 1 per 1000 persons. The most common cause of cardiac arrest is ventricular fibrillation due to an acute ischemic episode. During acute ischemia the onset of a ventricular tachyarrhythmia is sudden, unpredictable and often irreversible and lethal. Each minute that passes, the probability that the patient survives decreases by 10%. For this reason, the first 10 min are considered to be priceless for an efficacious first aid. The possibility of survival depends on the presence of witnesses, on the heart rhythm and on the resolution of the arrhythmia. In the majority of cases, the latter is possible by means of electrical defibrillation followed by the reestablishment of systolic function. An increase in equipment alone does not suffice for efficacious handling of cardiac arrest occurring outside the hospital premises. Above all, an adequate intervention strategy is required. Ambulance personnel must be well trained and capable of intervening rapidly, possibly within the first 5 min. The key to success lies in the diffusion and proper use of defibrillators. The availability of new generation instruments, the external automatic defibrillators, encourages their widespread use. On the territory, these emergencies are the responsibility of the 118 organization based, according to the characteristics specific to each country, on the regulated coordination between the operative command, the crews and the first-aid means. Strategies for the handling of these emergencies within hospitals have been proposed by the Conference of Bethesda and tend to guarantee an efficacious resuscitation with a maximum latency of 2 min between cardiac arrest and the first electric shock. The diffusion of external

  13. DAZL and CPEB1 regulate mRNA translation synergistically during oocyte maturation.

    PubMed

    Sousa Martins, Joao P; Liu, Xueqing; Oke, Ashwini; Arora, Ripla; Franciosi, Federica; Viville, Stephan; Laird, Diana J; Fung, Jennifer C; Conti, Marco

    2016-03-15

    Meiotic progression requires exquisitely coordinated translation of maternal messenger (m)RNA that has accumulated during oocyte growth. A major regulator of this program is the cytoplasmic polyadenylation element binding protein 1 (CPEB1). However, the temporal pattern of translation at different meiotic stages indicates the function of additional RNA binding proteins (RBPs). Here, we report that deleted in azoospermia-like (DAZL) cooperates with CPEB1 to regulate maternal mRNA translation. Using a strategy that monitors ribosome loading onto endogenous mRNAs and a prototypic translation target, we show that ribosome loading is induced in a DAZL- and CPEB1-dependent manner, as the oocyte reenters meiosis. Depletion of the two RBPs from oocytes and mutagenesis of the 3' untranslated regions (UTRs) demonstrate that both RBPs interact with the Tex19.1 3' UTR and cooperate in translation activation of this mRNA. We observed a synergism between DAZL and cytoplasmic polyadenylation elements (CPEs) in the translation pattern of maternal mRNAs when using a genome-wide analysis. Mechanistically, the number of DAZL proteins loaded onto the mRNA and the characteristics of the CPE might define the degree of cooperation between the two RBPs in activating translation and meiotic progression. PMID:26826184

  14. TRAIP is involved in chromosome alignment and SAC regulation in mouse oocyte meiosis

    PubMed Central

    Yuan, Yi-Feng; Ren, Yi-Xin; Yuan, Peng; Yan, Li-Ying; Qiao, Jie

    2016-01-01

    Recent whole-exome sequencing (WES) studies demonstrated that TRAIP is associated with primordial dwarfism. Although TRAIP was partially studied in mitosis, its function in oocyte meiosis remained unknown. In this study, we investigated the roles of TRAIP during mouse oocyte meiosis. TRAIP was stably expressed during oocytes meiosis and co-localized with CREST at the centromere region. Knockdown of TRAIP led to DNA damage, as revealed by the appearance of γH2AX. Although oocytes meiotic maturation was not affected, the proportions of misaligned chromosomes and aneuploidy were elevated after TRAIP knockdown, suggesting TRAIP is required for stable kinetochore–microtubule (K-MT) attachment. TRAIP knockdown decreased the accumulation of Mad2 on centromeres, potentially explaining why oocyte maturation was not affected following formation of DNA lesions. Securin, a protein which was prevent from precocious degradation by Mad2, was down-regulated after TRAIP knockdown. Inhibition of TRAIP by microinjection of antibody into pro-metaphase I (pro-MI) stage oocytes resulted in precocious first polar body (PB1) extrusion, and live-cell imaging clearly revealed misaligned chromosomes after TRAIP knockdown. Taken together, these data indicate that TRAIP plays important roles in oocyte meiosis regulation. PMID:27405720

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

    PubMed

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

    2016-06-01

    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.

  16. TRAIP is involved in chromosome alignment and SAC regulation in mouse oocyte meiosis.

    PubMed

    Yuan, Yi-Feng; Ren, Yi-Xin; Yuan, Peng; Yan, Li-Ying; Qiao, Jie

    2016-07-11

    Recent whole-exome sequencing (WES) studies demonstrated that TRAIP is associated with primordial dwarfism. Although TRAIP was partially studied in mitosis, its function in oocyte meiosis remained unknown. In this study, we investigated the roles of TRAIP during mouse oocyte meiosis. TRAIP was stably expressed during oocytes meiosis and co-localized with CREST at the centromere region. Knockdown of TRAIP led to DNA damage, as revealed by the appearance of γH2AX. Although oocytes meiotic maturation was not affected, the proportions of misaligned chromosomes and aneuploidy were elevated after TRAIP knockdown, suggesting TRAIP is required for stable kinetochore-microtubule (K-MT) attachment. TRAIP knockdown decreased the accumulation of Mad2 on centromeres, potentially explaining why oocyte maturation was not affected following formation of DNA lesions. Securin, a protein which was prevent from precocious degradation by Mad2, was down-regulated after TRAIP knockdown. Inhibition of TRAIP by microinjection of antibody into pro-metaphase I (pro-MI) stage oocytes resulted in precocious first polar body (PB1) extrusion, and live-cell imaging clearly revealed misaligned chromosomes after TRAIP knockdown. Taken together, these data indicate that TRAIP plays important roles in oocyte meiosis regulation.

  17. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  18. TRAIP is involved in chromosome alignment and SAC regulation in mouse oocyte meiosis.

    PubMed

    Yuan, Yi-Feng; Ren, Yi-Xin; Yuan, Peng; Yan, Li-Ying; Qiao, Jie

    2016-01-01

    Recent whole-exome sequencing (WES) studies demonstrated that TRAIP is associated with primordial dwarfism. Although TRAIP was partially studied in mitosis, its function in oocyte meiosis remained unknown. In this study, we investigated the roles of TRAIP during mouse oocyte meiosis. TRAIP was stably expressed during oocytes meiosis and co-localized with CREST at the centromere region. Knockdown of TRAIP led to DNA damage, as revealed by the appearance of γH2AX. Although oocytes meiotic maturation was not affected, the proportions of misaligned chromosomes and aneuploidy were elevated after TRAIP knockdown, suggesting TRAIP is required for stable kinetochore-microtubule (K-MT) attachment. TRAIP knockdown decreased the accumulation of Mad2 on centromeres, potentially explaining why oocyte maturation was not affected following formation of DNA lesions. Securin, a protein which was prevent from precocious degradation by Mad2, was down-regulated after TRAIP knockdown. Inhibition of TRAIP by microinjection of antibody into pro-metaphase I (pro-MI) stage oocytes resulted in precocious first polar body (PB1) extrusion, and live-cell imaging clearly revealed misaligned chromosomes after TRAIP knockdown. Taken together, these data indicate that TRAIP plays important roles in oocyte meiosis regulation. PMID:27405720

  19. Mitochondrial fission factor Drp1 maintains oocyte quality via dynamic rearrangement of multiple organelles.

    PubMed

    Udagawa, Osamu; Ishihara, Takaya; Maeda, Maki; Matsunaga, Yui; Tsukamoto, Satoshi; Kawano, Natsuko; Miyado, Kenji; Shitara, Hiroshi; Yokota, Sadaki; Nomura, Masatoshi; Mihara, Katsuyoshi; Mizushima, Noboru; Ishihara, Naotada

    2014-10-20

    Mitochondria are dynamic organelles that change their morphology by active fusion and fission in response to cellular signaling and differentiation. The in vivo role of mitochondrial fission in mammals has been examined by using tissue-specific knockout (KO) mice of the mitochondria fission-regulating GTPase Drp1, as well as analyzing a human patient harboring a point mutation in Drp1, showing that Drp1 is essential for embryonic and neonatal development and neuronal function. During oocyte maturation and aging, structures of various membrane organelles including mitochondria and the endoplasmic reticulum (ER) are changed dynamically, and their organelle aggregation is related to germ cell formation and epigenetic regulation. However, the underlying molecular mechanisms of organelle dynamics during the development and aging of oocytes have not been well understood. Here, we analyzed oocyte-specific mitochondrial fission factor Drp1-deficient mice and found that mitochondrial fission is essential for follicular maturation and ovulation in an age-dependent manner. Mitochondria were highly aggregated with other organelles, such as the ER and secretory vesicles, in KO oocyte, which resulted in impaired Ca(2+) signaling, intercellular communication via secretion, and meiotic resumption. We further found that oocytes from aged mice displayed reduced Drp1-dependent mitochondrial fission and defective organelle morphogenesis, similar to Drp1 KO oocytes. On the basis of these findings, it appears that mitochondrial fission maintains the competency of oocytes via multiorganelle rearrangement. PMID:25264261

  20. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  1. Coenzyme Q10 restores oocyte mitochondrial function and fertility during reproductive aging

    PubMed Central

    Ben-Meir, Assaf; Burstein, Eliezer; Borrego-Alvarez, Aluet; Chong, Jasmine; Wong, Ellen; Yavorska, Tetyana; Naranian, Taline; Chi, Maggie; Wang, Ying; Bentov, Yaakov; Alexis, Jennifer; Meriano, James; Sung, Hoon-Ki; Gasser, David L; Moley, Kelle H; Hekimi, Siegfried; Casper, Robert F; Jurisicova, Andrea

    2015-01-01

    Female reproductive capacity declines dramatically in the fourth decade of life as a result of an age-related decrease in oocyte quality and quantity. The primary causes of reproductive aging and the molecular factors responsible for decreased oocyte quality remain elusive. Here, we show that aging of the female germ line is accompanied by mitochondrial dysfunction associated with decreased oxidative phosphorylation and reduced Adenosine tri-phosphate (ATP) level. Diminished expression of the enzymes responsible for CoQ production, Pdss2 and Coq6, was observed in oocytes of older females in both mouse and human. The age-related decline in oocyte quality and quantity could be reversed by the administration of CoQ10. Oocyte-specific disruption of Pdss2 recapitulated many of the mitochondrial and reproductive phenotypes observed in the old females including reduced ATP production and increased meiotic spindle abnormalities, resulting in infertility. Ovarian reserve in the oocyte-specific Pdss2-deficient animals was diminished, leading to premature ovarian failure which could be prevented by maternal dietary administration of CoQ10. We conclude that impaired mitochondrial performance created by suboptimal CoQ10 availability can drive age-associated oocyte deficits causing infertility. PMID:26111777

  2. Oocyte maturation and fertilization in marine nemertean worms: using similar sorts of signaling pathways as in mammals, but often with differing results.

    PubMed

    Stricker, Stephen A; Cline, Cory; Goodrich, David

    2013-08-01

    In marine worms belonging to the phylum Nemertea, oocyte maturation and fertilization are regulated by the same general kinds of signals that control such processes in mammals. However, unlike mammalian oocytes that develop within follicles, nemertean oocytes characteristically lack a surrounding sheath of follicle cells and often respond differently to maturation-related cues than do mammalian oocytes. For example, elevators of cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP) levels promote the resumption of meiotic maturation (=germinal vesicle breakdown, GVBD) in nemertean oocytes, whereas increasing intraoocytic cAMP and cGMP typically blocks GVBD in mammals. Similarly, AMP-activated kinase (AMPK) signaling keeps nemertean oocytes from maturing, but in mouse oocytes, AMPK activation triggers GVBD. In addition, protein kinase C (PKC) activity is required for seawater-induced GVBD in nemerteans, whereas some PKCs have been shown to inhibit GVBD in mammals. Furthermore, although fertilization causes both types of oocytes to reorganize their endoplasmic reticulum and generate calcium oscillations that can involve soluble sperm factor activity and inositol 1,4,5-trisphosphate signaling, some discrepancies in the spatiotemporal patterns and underlying mechanisms of fertilization are also evident in nemerteans versus mammals. Thus, to characterize differences and similarities in gamete biology more fully, aspects of oocyte maturation and fertilization in marine nemertean worms are reviewed and briefly compared with related findings that have been published for mammalian oocytes. In addition, possible causes of the alternative responses displayed by oocytes in these two animal groups are addressed.

  3. Meiotic cohesin STAG3 is required for chromosome axis formation and sister chromatid cohesion.

    PubMed

    Winters, Tristan; McNicoll, Francois; Jessberger, Rolf

    2014-06-01

    The cohesin complex is essential for mitosis and meiosis. The specific meiotic roles of individual cohesin proteins are incompletely understood. We report in vivo functions of the only meiosis-specific STAG component of cohesin, STAG3. Newly generated STAG3-deficient mice of both sexes are sterile with meiotic arrest. In these mice, meiotic chromosome architecture is severely disrupted as no bona fide axial elements (AE) form and homologous chromosomes do not synapse. Axial element protein SYCP3 forms dot-like structures, many partially overlapping with centromeres. Asynapsis marker HORMAD1 is diffusely distributed throughout the chromatin, and SYCP1, which normally marks synapsed axes, is largely absent. Centromeric and telomeric sister chromatid cohesion are impaired. Centromere and telomere clustering occurs in the absence of STAG3, and telomere structure is not severely affected. Other cohesin proteins are present, localize throughout the STAG3-devoid chromatin, and form complexes with cohesin SMC1β. No other deficiency in a single meiosis-specific cohesin causes a phenotype as drastic as STAG3 deficiency. STAG3 emerges as the key STAG cohesin involved in major functions of meiotic cohesin. PMID:24797474

  4. Effects of cumulus cells on rabbit oocyte in vitro maturation.

    PubMed

    Tao, Y; Cao, C; Zhang, M; Fang, F; Liu, Y; Zhang, Y; Ding, J; Zhang, X

    2008-08-01

    , but the ooplasmic maturation was not. Few NOs incubated with or without CCs accomplished nuclear maturation (approximately 2% both), even when the culture time was prolonged from 24 to 30 h. The oocyte degeneration rates were significantly different for each culture model after both 24 and 30 h incubation (p < or = 0.05). There was no significant difference in oocyte degeneration in the same groups between 24 and 30 h incubation (p > 0.05). The results suggest that rabbit CCs affect oocyte nuclear and ooplasmic maturation, and their survival. The prolongation of the culture time of rabbit oocyte from 24 to 30 h improves the nuclear and ooplasmic maturation differently in the present system. Rabbit oocytes free of CCs, especially NOs, show weak meiotic resumption potential and compromised viability, which cannot be improved by co-culture with dispersed CCs. The degeneration mostly happens at early time of IVM. PMID:18662353

  5. Targeted disruption of Nrg1 in granulosa cells alters the temporal progression of oocyte maturation.

    PubMed

    Kawashima, Ikko; Umehara, Takashi; Noma, Noritaka; Kawai, Tomoko; Shitanaka, Manami; Richards, Joanne S; Shimada, Masayuki

    2014-05-01

    Neuregulin 1 (NRG1) is induced in granulosa cells by LH and acts on granulosa and cumulus cells during ovulation. In this study, we sought to determine the role of NRG1 in oocyte maturation by generating a granulosa cell-specific Nrg1 knockout mouse (Nrg1(flox/flox);Cyp19a1Cre mice [gcNrg1KO]). In the gcNrg1KO mice, meiosis was induced 2 hours earlier than in control mice. More than 60% of the oocytes in the mutant mice spontaneously re-resumed meiosis beyond the MII stage. The percentage of successful fertilization was comparable in oocytes of both genotypes collected at 14 or 16 hours after human chorionic gonadotropin injection but was significantly lower in oocytes of the gcNrg1KO mice at 18 or 20 hours. The number of pups per litter was significantly decreased in gcNrg1KO mice. To determine the molecular events associated with the abnormal progression of meiosis in the gcNrg1KO mouse oocytes, the defects of cumulus/granulosa cell functions were analyzed. The expression of genes involved in luteinization and cumulus expansion was significantly higher at 2 hours after human chorionic gonadotropin injection in the gcNrg1KO mice; this was related to abnormal activation of protein kinase C (PKC) and phosphorylation of connexin-43 in cumulus cells. Changes in connexin-43 by PKC might lead to early meiotic resumption of oocytes in gcNrg1KO mice. We conclude that NRG1 is induced by LH in mural granulosa cells and exerts an important regulatory role in oocyte meiotic maturation and competence by reducing PKC activation in cumulus cells and preventing premature progression to the MII stage that leads to abnormal fertilization and fertility. PMID:24650175

  6. Targeted Disruption of Nrg1 in Granulosa Cells Alters the Temporal Progression of Oocyte Maturation

    PubMed Central

    Kawashima, Ikko; Umehara, Takashi; Noma, Noritaka; Kawai, Tomoko; Shitanaka, Manami

    2014-01-01

    Neuregulin 1 (NRG1) is induced in granulosa cells by LH and acts on granulosa and cumulus cells during ovulation. In this study, we sought to determine the role of NRG1 in oocyte maturation by generating a granulosa cell–specific Nrg1 knockout mouse (Nrg1flox/flox;Cyp19a1Cre mice [gcNrg1KO]). In the gcNrg1KO mice, meiosis was induced 2 hours earlier than in control mice. More than 60% of the oocytes in the mutant mice spontaneously re-resumed meiosis beyond the MII stage. The percentage of successful fertilization was comparable in oocytes of both genotypes collected at 14 or 16 hours after human chorionic gonadotropin injection but was significantly lower in oocytes of the gcNrg1KO mice at 18 or 20 hours. The number of pups per litter was significantly decreased in gcNrg1KO mice. To determine the molecular events associated with the abnormal progression of meiosis in the gcNrg1KO mouse oocytes, the defects of cumulus/granulosa cell functions were analyzed. The expression of genes involved in luteinization and cumulus expansion was significantly higher at 2 hours after human chorionic gonadotropin injection in the gcNrg1KO mice; this was related to abnormal activation of protein kinase C (PKC) and phosphorylation of connexin-43 in cumulus cells. Changes in connexin-43 by PKC might lead to early meiotic resumption of oocytes in gcNrg1KO mice. We conclude that NRG1 is induced by LH in mural granulosa cells and exerts an important regulatory role in oocyte meiotic maturation and competence by reducing PKC activation in cumulus cells and preventing premature progression to the MII stage that leads to abnormal fertilization and fertility. PMID:24650175

  7. Absence of cumulus cells during in vitro maturation affects lipid metabolism in bovine oocytes.

    PubMed

    Auclair, Sylvain; Uzbekov, Rustem; Elis, Sébastien; Sanchez, Laura; Kireev, Igor; Lardic, Lionel; Dalbies-Tran, Rozenn; Uzbekova, Svetlana

    2013-03-15

    Cumulus cells (CC) surround the oocyte and are coupled metabolically through regulation of nutrient intake. CC removal before in vitro maturation (IVM) decreases bovine oocyte developmental competence without affecting nuclear meiotic maturation. The objective was to investigate the influence of CC on oocyte cytoplasmic maturation in relation to energy metabolism. IVM with either cumulus-enclosed (CEO) or -denuded (DO) oocytes was performed in serum-free metabolically optimized medium. Transmission electron microscopy revealed different distribution of membrane-bound vesicles and lipid droplets between metaphase II DO and CEO. By Nile Red staining, a significant reduction in total lipid level was evidenced in DO. Global transcriptomic analysis revealed differential expression of genes regulating energy metabolism, transcription, and translation between CEO and DO. By Western blot, fatty acid synthase (FAS) and hormone-sensitive phospholipase (HSL) proteins were detected in oocytes and in CC, indicating a local lipogenesis and lypolysis. FAS protein was significantly less abundant in DO that in CEO and more highly expressed in CC than in the oocytes. On the contrary, HSL protein was more abundant in oocytes than in CC. In addition, active Ser⁵⁶³-phosphorylated HSL was detected in the oocytes only after IVM, and its level was similar in CEO and DO. In conclusion, absence of CC during IVM affected lipid metabolism in the oocyte and led to suboptimal cytoplasmic maturation. Thus, CC may influence the oocyte by orienting the consumption of nutritive storage via regulation of local fatty acid synthesis and lipolysis to provide energy for maturation. PMID:23321473

  8. Rex Rabbit Somatic Cell Nuclear Transfer with In Vitro-Matured Oocytes.

    PubMed

    Liu, Yong; Wang, Huili; Lu, Jinhua; Miao, Yiliang; Cao, Xinyan; Zhang, Ling; Wu, Xiaoqing; Wu, Fengrui; Ding, Biao; Wang, Rong; Luo, Mingjiu; Li, Wenyong; Tan, Jinghe

    2016-06-01

    Somatic cell nuclear transfer (SCNT) requires large numbers of matured oocytes. In vitro-matured (IVM) oocytes have been used in SCNT in many animals. We investigated the use of IVM oocytes in Rex rabbit SCNT using Rex rabbit ovaries obtained from a local abattoir. The meiotic ability of oocytes isolated from follicles of different diameters was studied. Rex rabbit SCNT was optimized for denucleation, activation, and donor cell synchronization. Rex rabbit oocytes grew to the largest diameter (110 μm) when the follicle diameter was 1.0 mm. Oocytes isolated from <0.5-mm follicles lacked the ability to resume meiosis. More than 90% of these oocytes remained in the germinal vesicle (GV) stage after in vitro culture (IVC) for 18 h. Oocytes isolated from >0.7-mm follicles acquired maturation ability. More than 90% of these oocytes matured after IVC for 18 h. The developmental potential of oocytes isolated from >1-mm follicles was greater than that of oocytes isolated from 0.7- to 1.0-mm follicles. The highest activation rates for IVM Rex rabbit oocytes were seen after treatment with 2.5 μM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) and 5 μg/mL cycloheximide (CHX) for 1 h. Ionomycin induced the chromatin of IVM oocytes to protrude from the oocyte surface, promoting denucleation. Fetal fibroblast cells (FFCs) and cumulus cells (CCs) were more suitable for Rex rabbit SCNT than skin fibroblast cells (SFCs) (blastocyst rate was 35.6 ± 2.2% and 38.0 ± 6.0% vs. 19.7 ± 3.1%). The best fusion condition was a 2DC interval for 1 sec, 1.6 kV/cm voltages, and 40 μsec duration in 0.28 M mannitol. In conclusion, the in vitro maturation of Rex rabbit oocytes and SCNT procedures were studied systematically and optimized in this study. PMID:27159389

  9. Meiotic recombination and male infertility: from basic science to clinical reality?

    PubMed

    Hann, Michael C; Lau, Patricio E; Tempest, Helen G

    2011-03-01

    Infertility is a common problem that affects approximately 15% of the population. Although many advances have been made in the treatment of infertility, the molecular and genetic causes of male infertility remain largely elusive. This review will present a summary of our current knowledge on the genetic origin of male infertility and the key events of male meiosis. It focuses on chromosome synapsis and meiotic recombination and the problems that arise when errors in these processes occur, specifically meiotic arrest and chromosome aneuploidy, the leading cause of pregnancy loss in humans. In addition, meiosis-specific candidate genes will be discussed, including a discussion on why we have been largely unsuccessful at identifying disease-causing mutations in infertile men. Finally clinical applications of sperm aneuploidy screening will be touched upon along with future prospective clinical tests to better characterize male infertility in a move towards personalized medicine. PMID:21297654

  10. Spo13 Negatively Regulates the Progression of Mitotic and Meiotic Nuclear Division in Saccharomyces Cerevisiae

    PubMed Central

    McCarroll, R. M.; Esposito, R. E.

    1994-01-01

    The meiosis-specific yeast gene SPO13 has been previously shown to be required to obtain two successive divisions in meiosis. We report here that vegetative expression of this gene causes a CDC28-dependent cell-cycle arrest at mitosis. Overexpression of SPO13 during meiosis causes a transient block to completion of the meiosis I division and suppresses the inability of cdc28(ts) strains to execute meiosis II. The spo13 defect can be partially suppressed by conditions that slow progression of the first meiotic division. Based on the results presented below, we propose that SPO13 acts as a meiotic timing function by transiently blocking progression through the meiosis I division, thereby allowing (1) coordination of the first division with assembly of the reductional segregation apparatus, and (2) subsequent entry into a second round of segregation to separate replicated sister chromatids without an intervening S-phase. PMID:8001793

  11. Mechanism and regulation of meiotic recombination initiation

    PubMed Central

    Lam, Isabel; Keeney, Scott

    2015-01-01

    Meiotic recombination involves the formation and repair of programmed DNA double-strand breaks (DSBs) catalyzed by the conserved Spo11 protein. This review summarizes recent studies pertaining to the formation of meiotic DSBs, including the mechanism of DNA cleavage by Spo11, proteins required for break formation, and mechanisms that control the location, timing, and number of DSBs. Where appropriate, findings in different organisms are discussed to highlight evolutionary conservation or divergence. PMID:25324213

  12. Cytological analysis of Arabidopsis thaliana meiotic chromosomes.

    PubMed

    Armstrong, Susan J; Sanchez-Moran, Eugenio; Franklin, F Chris H

    2009-01-01

    Advances in molecular biology and in the genetics of Arabidopsis thaliana have led to this organism becoming an important model for the analysis of meiosis in plants. Cytogenetic investigations are pivotal to meiotic studies and a number of technological improvements for Arabidopsis cytology have provided a range of tools to investigate chromosome behaviour during meiosis. This chapter includes protocols on basic cytology, FISH analysis, immunocytology, a procedure for a meiotic time course and electron microscopy.

  13. Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency.

    PubMed

    Cagnone, Gael L M; Tsai, Te-Sha; Makanji, Yogeshwar; Matthews, Pamela; Gould, Jodee; Bonkowski, Michael S; Elgass, Kirstin D; Wong, Ashley S A; Wu, Lindsay E; McKenzie, Matthew; Sinclair, David A; St John, Justin C

    2016-01-01

    An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte's mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes. PMID:26987907

  14. Kdm5/Lid Regulates Chromosome Architecture in Meiotic Prophase I Independently of Its Histone Demethylase Activity

    PubMed Central

    Zhaunova, Liudmila; Ohkura, Hiroyuki; Breuer, Manuel

    2016-01-01

    During prophase of the first meiotic division (prophase I), chromatin dynamically reorganises to recombine and prepare for chromosome segregation. Histone modifying enzymes are major regulators of chromatin structure, but our knowledge of their roles in prophase I is still limited. Here we report on crucial roles of Kdm5/Lid, one of two histone demethylases in Drosophila that remove one of the trimethyl groups at Lys4 of Histone 3 (H3K4me3). In the absence of Kdm5/Lid, the synaptonemal complex was only partially formed and failed to be maintained along chromosome arms, while localisation of its components at centromeres was unaffected. Kdm5/Lid was also required for karyosome formation and homologous centromere pairing in prophase I. Although loss of Kdm5/Lid dramatically increased the level of H3K4me3 in oocytes, catalytically inactive Kdm5/Lid can rescue the above cytological defects. Therefore Kdm5/Lid controls chromatin architecture in meiotic prophase I oocytes independently of its demethylase activity. PMID:27494704

  15. Maternal diabetes and oocyte quality

    PubMed Central

    Wang, Qiang; Moley, Kelle H.

    2016-01-01

    Maternal diabetes has been demonstrated to adversely affect preimplantation embryo development and pregnancy outcomes. Emerging evidence has implicated that these effects are associated with compromised oocyte competence. Several developmental defects during oocyte maturation in diabetic mice have been reported over past decades. Most recently, we further identified the structural, spatial and metabolic dysfunction of mitochondria in oocytes from diabetic mice, suggesting the impaired oocyte quality. These defects in the oocyte may be maternally transmitted to the embryo and then manifested later as developmental abnormalities in preimplantation embryo, congenital malformations, and even metabolic disease in the offspring. In this paper, we briefly review the effects of maternal diabetes on oocyte quality, with a particular emphasis on the mitochondrial dysfunction. The possible connection between dysfunctional oocyte mitochondria and reproductive failure of diabetic females, and the mechanism(s) by which maternal diabetes exerts its effects on the oocyte are also discussed. PMID:20226883

  16. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects.

    PubMed

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A

    2016-09-19

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. PMID:27257060

  17. MS5 Mediates Early Meiotic Progression and Its Natural Variants May Have Applications for Hybrid Production in Brassica napus.

    PubMed

    Xin, Qiang; Shen, Yi; Li, Xi; Lu, Wei; Wang, Xiang; Han, Xue; Dong, Faming; Wan, Lili; Yang, Guangsheng; Hong, Dengfeng; Cheng, Zhukuan

    2016-06-01

    During meiotic prophase I, chromatin undergoes dynamic changes to establish a structural basis for essential meiotic events. However, the mechanism that coordinates chromosome structure and meiotic progression remains poorly understood in plants. Here, we characterized a spontaneous sterile mutant MS5(b)MS5(b) in oilseed rape (Brassica napus) and found its meiotic chromosomes were arrested at leptotene. MS5 is preferentially expressed in reproductive organs and encodes a Brassica-specific protein carrying conserved coiled-coil and DUF626 domains with unknown function. MS5 is essential for pairing of homologs in meiosis, but not necessary for the initiation of DNA double-strand breaks. The distribution of the axis element-associated protein ASY1 occurs independently of MS5, but localization of the meiotic cohesion subunit SYN1 requires functional MS5. Furthermore, both the central element of the synaptonemal complex and the recombination element do not properly form in MS5(b)MS5(b) mutants. Our results demonstrate that MS5 participates in progression of meiosis during early prophase I and its allelic variants lead to differences in fertility, which may provide a promising strategy for pollination control for heterosis breeding. PMID:27194707

  18. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects

    PubMed Central

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A.

    2016-01-01

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. PMID:27257060

  19. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects.

    PubMed

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A

    2016-09-19

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes.

  20. Meiotic segregation of a homeologous chromosome pair.

    PubMed

    Maxfield Boumil, R; Kemp, B; Angelichio, M; Nilsson-Tillgren, T; Dawson, D S

    2003-03-01

    During meiosis, the alignment of homologous chromosomes facilitates their subsequent migration away from one another to opposite spindle poles at anaphase I. Recombination is part of the mechanism by which chromosomes identify their homologous partners, and serves to link the homologs in a way that, in some organisms, has been shown to promote proper attachment to the meiotic spindle. We have built a diploid strain that contains a pair of homeologous chromosomes V': one is derived from Saccharomyces cerevisiae and one originates from S. carlsbergensis. Sequence analysis reveals that these chromosomes share 71% sequence identity. The homeologs experience high levels of meiotic double-stranded breaks. Despite their relatedness and their competence to initiate recombination, the meiotic segregation behavior of the homeologous chromosomes suggests that, in most meioses, they are partitioned by a meiotic segregation system that has been shown previously to partition non-exchange chromosomes and pairs with no homology. Though the homeologous chromosomes show a degree of meiotic segregation fidelity similar to that of other non-exchange pairs, our data provide evidence that their limited sequence homology may provide some bias in meiotic partner choice. PMID:12655401

  1. Brca2-Pds5 complexes mobilize persistent meiotic recombination sites to the nuclear envelope.

    PubMed

    Kusch, Thomas

    2015-02-15

    Homologous recombination is required for reciprocal exchange between homologous chromosome arms during meiosis. Only select meiotic recombination events become chromosomal crossovers; the majority of recombination outcomes are noncrossovers. Growing evidence suggests that crossovers are repaired after noncrossovers. Here, I report that persisting recombination sites are mobilized to the nuclear envelope of Drosophila pro-oocytes during mid-pachytene. Their number correlates with the average crossover rate per meiosis. Proteomic and interaction studies reveal that the recombination mediator Brca2 associates with lamin and the cohesion factor Pds5 to secure persistent recombination sites at the nuclear envelope. In Rad51(-/-) females, all persistent DNA breaks are directed to the nuclear envelope. By contrast, a reduction of Pds5 or Brca2 levels abolishes the movement and has a negative impact on crossover rates. The data suggest that persistent meiotic DNA double-strand breaks might correspond to crossovers, which are mobilized to the nuclear envelope for their repair. The identification of Brca2-Pds5 complexes as key mediators of this process provides a first mechanistic explanation for the contribution of lamins and cohesins to meiotic recombination.

  2. Oocyte Maturation and Development

    PubMed Central

    Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Sexual reproduction is essential for many organisms to propagate themselves. It requires the formation of haploid female and male gametes: oocytes and sperms. These specialized cells are generated through meiosis, a particular type of cell division that produces cells with recombined genomes that differ from their parental origin. In this review, we highlight the end process of female meiosis, the divisions per se, and how they can give rise to a functional female gamete preparing itself for the ensuing zygotic development. In particular, we discuss why such an essential process in the propagation of species is so poorly controlled, producing a strong percentage of abnormal female gametes in the end. Eventually, we examine aspects related to the lack of centrosomes in female oocytes, the asymmetry in size of the mammalian oocyte upon division, and in mammals the direct consequences of these long-lived cells in the ovary. PMID:26998245

  3. A morphologic study of unfertilized oocytes and abnormal embryos in human in vitro fertilization.

    PubMed

    Bałakier, H; Casper, R F

    1991-04-01

    The morphology of human, unfertilized oocytes and abnormal embryos cultured in vitro for 48-72 hr was examined in an attempt to learn more about oocyte maturation and reproductive failure in in vitro fertilization (IVF). About 21% of the unfertilized oocytes were totally degenerated. The majority (56%) of the remaining oocytes was arrested at the metaphase II stage. They contained coherent chromosomal plates and had extruded the first polar body with nuclear material. About 13% of oocytes underwent spontaneous activation. In most of these cases the second polar body was retained and many subnuclei or one big nucleus was formed. Five percent of metaphase II oocytes penetrated by sperm were not activated, likely as a result of oocyte immaturity. The developmental ability of abnormal embryos was poor. Several one-cell-stage zygotes were arrested at the pronuclear stage or at mitosis of the first mitotic division. Polyspermic embryos, especially those which contained four or more pronuclei, did not divide or formed uneven, multinucleated blastomeres. However, some triploid and tetraploid embryos often appeared normal morphologically despite their lethal chromosomal abnormalities.

  4. Microsensors and image processing for single oocyte qualification: toward multiparametric determination of the best time for fertilization

    NASA Astrophysics Data System (ADS)

    Wacogne, B.; Ivascu, I.; Zeggari, R.; Pieralli, C.; Amiot, C.; Pazart, L.; Roux, C.

    2013-10-01

    During intracytoplasmic sperm injection (ICSI) attempts, oocytes reaching metaphase II are microinjected. A morphological examination under a microscope is the usual method for determining oocyte maturity. The level of oocyte maturity is based on the meiotic status (Germinal Vesicle, metaphase I and metaphase II) of the oocytes with respect to their increasing maturity. In this letter, we summarize the studies conducted to analyze cytoplasm maturity using various microsystems and image processing. Optical microsystems are used to measure the transmission spectra and refractive index of the oocytes. We compared the transmission spectra measurements to the transmission electron microscopy results. Karhunen-Loeve transform is also used to evaluate the maturity of the oocytes. To summarize, optical analysis techniques are a minimally invasive technology allowing cytoplasm maturity to be assessed. Oocytes should not only be qualified in terms of GV, MI or MII, but also regarding their temporal evolution over the course of these maturation stages. The ultimate aim of this work is to describe the maturation of the oocytes by a trajectory in a multidimensional space and to determine when would be the best time for successful fertilization.

  5. Php4 Is a Key Player for Iron Economy in Meiotic and Sporulating Cells

    PubMed Central

    Brault, Ariane; Rallis, Charalampos; Normant, Vincent; Garant, Jean-Michel; Bähler, Jürg; Labbé, Simon

    2016-01-01

    Meiosis is essential for sexually reproducing organisms, including the fission yeast Schizosaccharomyces pombe. In meiosis, chromosomes replicate once in a diploid precursor cell (zygote), and then segregate twice to generate four haploid meiotic products, named spores in yeast. In S. pombe, Php4 is responsible for the transcriptional repression capability of the heteromeric CCAAT-binding factor to negatively regulate genes encoding iron-using proteins under low-iron conditions. Here, we show that the CCAAT-regulatory subunit Php4 is required for normal progression of meiosis under iron-limiting conditions. Cells lacking Php4 exhibit a meiotic arrest at metaphase I. Microscopic analyses of cells expressing functional GFP-Php4 show that it colocalizes with chromosomal material at every stage of meiosis under low concentrations of iron. In contrast, GFP-Php4 fluorescence signal is lost when cells undergo meiosis under iron-replete conditions. Global gene expression analysis of meiotic cells using DNA microarrays identified 137 genes that are regulated in an iron- and Php4-dependent manner. Among them, 18 genes are expressed exclusively during meiosis and constitute new putative Php4 target genes, which include hry1+ and mug14+. Further analysis validates that Php4 is required for maximal and timely repression of hry1+ and mug14+ genes. Using a chromatin immunoprecipitation approach, we show that Php4 specifically associates with hry1+ and mug14+ promoters in vivo. Taken together, the results reveal that in iron-starved meiotic cells, Php4 is essential for completion of the meiotic program since it participates in global gene expression reprogramming to optimize the use of limited available iron. PMID:27466270

  6. Naringenin (NAR) and 8-prenylnaringenin (8-PN) reduce the developmental competence of porcine oocytes in vitro.

    PubMed

    Solak, Kamila A; Santos, Regiane R; van den Berg, Martin; Blaauboer, Bas J; Roelen, Bernard A J; van Duursen, Majorie B M

    2014-11-01

    Flavanones such as naringenin (NAR) and 8-prenylnaringenin (8-PN) are increasingly used as dietary supplements despite scientific concern regarding adverse effects on female reproduction upon excessive intake. In the present study, NAR and 8-PN (0.3-1μM) significantly affected porcine oocyte maturation in vitro by decreasing cumulus expansion. In addition, NAR and 8-PN decreased percentages of meiotic spindle formation, oocyte cleavage and blastocyst formation. The effects of NAR and 8-PN were different from estradiol (3.12μM)-induced effects. Still, the flavanone-induced effects were observed at concentrations that can be found in human plasma upon supplement intake and that resemble physiological estrogen equivalence levels in follicular fluids. Considering that abnormal oocyte maturation can cause subfertility, our study warrants that precautions are in place and excessive intake of NAR and 8-PN e.g. via dietary supplements should be avoided by women.

  7. Effect of organochlorine pesticides on maturation of starfish and mouse oocytes.

    PubMed

    Picard, André; Pahlavan, Golbahar; Palavan, Golbahar; Robert, Stéphanie; Pesando, Danielle; Ciapa, Brigitte

    2003-05-01

    Methoxychlor, lindane, and dieldrin are organochlorine pesticides that have been described as altering different reproductive functions in mammals and in invertebrates. However, few data have been published concerning the effects these pesticides have on oocyte maturation and fertilization. The aim of this study was to determine whether these compounds could affect maturation of mouse and starfish oocytes. We observed that germinal vesicle breakdown (GVBD) in starfish oocytes was significantly inhibited by the pesticides. Furthermore, formation of the first meiotic spindle and extrusion of the first polar body were also altered in mouse as well as in starfish. Our results suggest that the three pesticides act on common intracellular targets in invertebrates as well as in vertebrates.

  8. Geminin deletion in mouse oocytes results in impaired embryo development and reduced fertility

    PubMed Central

    Ma, Xue-Shan; Lin, Fei; Wang, Zhong-Wei; Hu, Meng-Wen; Huang, Lin; Meng, Tie-Gang; Jiang, Zong-Zhe; Schatten, Heide; Wang, Zhen-Bo; Sun, Qing-Yuan

    2016-01-01

    Geminin controls proper centrosome duplication, cell division, and differentiation. We investigated the function of geminin in oogenesis, fertilization, and early embryo development by deleting the geminin gene in oocytes from the primordial follicle stage. Oocyte-specific disruption of geminin results in low fertility in mice. Even though there was no evident anomaly of oogenesis, oocyte meiotic maturation, natural ovulation, or fertilization, early embryo development and implantation were impaired. The fertilized eggs derived from mutant mice showed developmental delay, and many were blocked at the late zygote stage. Cdt1 protein was decreased, whereas Chk1 and H2AX phosphorylation was increased, in fertilized eggs after geminin depletion. Our results suggest that disruption of maternal geminin may decrease Cdt1 expression and cause DNA rereplication, which then activates the cell cycle checkpoint and DNA damage repair and thus impairs early embryo development. PMID:26764091

  9. Aurora kinase A controls meiosis I progression in mouse oocytes

    PubMed Central

    Saskova, Adela; Solc, Petr; Baran, Vladimir; Kubelka, Michal; Schultz, Richard M.; Motlik, Jan

    2011-01-01

    Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G2 and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G2 to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6-treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Overexpression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition. PMID:18677115

  10. Aurora kinase A controls meiosis I progression in mouse oocytes.

    PubMed

    Saskova, Adela; Solc, Petr; Baran, Vladimir; Kubelka, Michal; Schultz, Richard M; Motlik, Jan

    2008-08-01

    Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G(2) and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G(2) to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6-treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Overexpression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.

  11. Correlation of meiotic events in testis sections and microspreads of mouse spermatocytes relative to the mid-pachytene checkpoint.

    PubMed

    Ashley, Terry; Gaeth, Ann P; Creemers, Laura B; Hack, Adelle M; de Rooij, Dirk G

    2004-09-01

    In mouse, asynaptic meiotic mutants arrest at Testis Epithelial Stage IV. This arrest is 4.5 days after homologous chromosomes begin to synapse and approximately 2.5 days after synapsis is usually completed. To correlate cytological events with meiotic progression in testis and to determine which meiotic events are normally completed by Stage IV, we induced spermatogenic arrest by placing mice on a vitamin A deficient diet. Subsequent injection of retinoic acid and a return to a normal diet resulted in resumption of spermatogenesis with all spermatocytes proceeding through meiosis in a highly synchronous cohort. Between Days 11 and 16 post-injection we prepared one testis for immunocytological and the other for histological evaluation, then used antibodies to SCP3 and either RPA, or MLH1 to follow quantitative changes in synapsis and recombination. RPA was found at sites along the synaptonemal complex as soon as homologs synapsed, and most, but not all, RPA disappeared by Stage IV. MLH1 foci appeared between Stage II and IV and remained through Stage VII, the end point of the study. The data suggest that the earliest the mid-pachytene checkpoint can be activated is Epithelial Stage IV, but that activities monitored by the checkpoint may not be completed by this time.

  12. Distribution of cortical granules in bovine oocytes classified by cumulus complex.

    PubMed

    Hosoe, M; Shioya, Y

    1997-11-01

    The present study was conducted to examine distributional changes of cortical granules (CGs) during meiotic maturation and fertilisation in vitro and the developmental ability in bovine oocytes classified by cumulus cells. The oocytes were classified by the morphology of their cumulus cell layers as follows: class A, compact and thick; class B, compact but thin; class C, naked; and class D, expanded. Some of the oocytes were stained with Lens curinalis agglutinin (LCA) before and after maturation in vitro and after insemination, and then stained with orcein to observe their nuclear stages. The others were left in culture. Distributional patterns of the CGs were classified into four types: type I, CGs distributed in clusters; type II, CGs dispersed and partly clustered; type III, all CGs dispersed; and type IV, no CGs. Most of the oocytes before culture showed a type I pattern, but this decreased after maturation culture, whereas type III increased in class A. The oocytes of class B showed similar changes while the oocytes of class C did not. In class C, many oocytes showed type I after culture, indicating that cytoplasmic maturation was not completed. In class D, 80.4% of the oocytes exhibited type III before maturation culture, indicating that their cytoplasmic maturation was different from classes A-C. And about 70% of the class D oocytes were at the nuclear stage of germinal vesicle breakdown (GVBD) before culture. The developmental rates to blastocysts in classes A-D were 28.7%, 23.1%, 0.5% and 3.4% respectively. PMID:9563685

  13. Oocyte recovery and maturation in the American black bear (Ursus americanus): a model for endangered ursids.

    PubMed

    Johnston, L A; Donoghue, A M; Igo, W; Simmons, L G; Wildt, D E; Rieffenberger, J

    1994-05-15

    A study was conducted to determine if meiotic maturation could be induced in ovarian oocytes of the American black bear (Ursus americanus), a model for gamete "rescue" techniques for endangered ursids. Ovaries obtained from 48 black bears yielded 2,403 oocytes (51.1 +/- 4.9/female), of which 777 (32.3%) were morphologically classified as excellent quality. More total oocytes were recovered from donors that were anestrous compared to luteal/pregnant (P < 0.05) at the time of ovarian excision. Delaying the recovery of oocytes from antral follicles within excised ovaries from 12-24 hr to 25-36 hr had no effect (P > 0.05) on the overall number of high quality oocytes recovered or subsequent maturational ability. The highest incidence of metaphase II was reached between 48 and 60 hr of in vitro incubation. Donor status (anestrous vs. luteal/pregnant) had no influence on the oocyte maturation rate by 24 or 48 hr, but by 60 hr, more (P < 0.05) oocytes recovered from anestrous females (43.9%) had achieved metaphase II compared to luteal/pregnant counterparts (23.1%). In preliminary trials involving endangered ursids, 54 ovarian oocytes were recovered from three aged sun bears (Helarctos malayanus), of which 72.2% were excellent quality and 15.4% matured in vitro to metaphase II. Similarly, 119 antral oocytes were recovered from two aged sloth bears (Melursus ursinus), of which 41.2% were excellent and 17.5% matured in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8207381

  14. Does women’s age influence zona pellucida birefringence of metaphase ΙΙ oocytes in in-vitro maturation program?

    PubMed Central

    Omidi, Marjan; Khalili, Mohammad Ali; Nahangi, Hossein; Ashourzadeh, Sareh; Rahimipour, Marzieh

    2013-01-01

    Background: In vitro maturation (IVM) is a promising treatment option for certain infertile women. Nowadays, with the aid of PolScope, it has become possible to evaluate zona pellucida (ZP) characteristics as a parameter of oocyte quality. Moreover, quality of oocytes can be influenced by many factors, such as patient’s age. The PolScope system is a non-invasive technique to assess birefringent structures such as the meiotic spindle and ZP in living oocytes. Objective: The aim was to determine the influence of the woman's age on ZP birefringence, a sign of oocyte quality, and morphology of in-vitro matured human oocytes using non-invasive polarized light (PolScope) microscopy. Materials and Methods: ZP birefringence and morphology were determined in 105 retrieved oocytes from 58 women undergoing ICSI in two age groups (≥30 years and <30 years). The immature oocytes were selected and after IVM, the quality of metaphase ΙΙ (MII) oocytes was assessed. The oocytes abnormalities were classified as intracytoplasmic and extracytoplasmic abnormalities. Results: Oocyte maturation rates were significantly reduced in ≥30 year’s women (56%) in comparison with other age group (80.7%). In addition, the ZP birefringence was significantly higher in MII oocytes in the younger group compared with the older group (76.2% vs. 38.1%; p=0.00). Following morphologic assessment, the rates of oocytes with extracytoplasmic (p=0.02) and both abnormalities (extra- and intracytoplasmic) (p=0.01) were higher in aged versus the younger women. Conclusion: There was a positive relationship between advanced maternal age with decreased ZP birefringence and oocyte morphological quality in in-vitro matured human oocytes. PMID:24639703

  15. Exceptional material requirement for reproduction in mouse oocytes.

    PubMed

    Yu, L; Wang, S F; Zhai, Q Z; Yao, Y Q; Jiang, F; Lu, Y X

    2015-01-01

    Limited information on oocytes and fertilization prevents the efficient therapy of patients with infertility. The most important reason for this lack of understanding is a deficiency in research dedicated to oocytes and fertilization. Currently, we are concerned with the role of nutrition in the process of oocyte development to better understand the relationship between nutrition and infertility. The aim of this study was to explore the relationship between some exceptional materials and infertility to elucidate the role of these materials in oocyte development. We used proteomic analysis to identify numerous nutrition-related proteins in three developmental stages: the germinal vesicle stage, the metaphase II-arrested stage, and the fertilized oocyte-zygote stage. Specific proteins were abundantly expressed during the three stages. These proteins included astacin-like metalloendopeptidase, selenium-binding proteins, and other proteins involved in metabolic and signaling pathways. Other proteins were involved in the citrate cycle, the electron transport chain, the urea cycle, fatty acid metabolism, and the insulin signaling pathway. Almost all these proteins exhibited different expression levels in the three stages. The results of the present study provide a better understanding of the molecular mechanisms of early embryonic development and suggest new treatment methods for infertility. PMID:26600495

  16. Expression Patterns of CREBs in Oocyte Growth and Maturation of Fish

    PubMed Central

    Wang, De-Shou; Sudhakumari, Cheni-Chery; Kobayashi, Tohru; Nagahama, Yoshitaka

    2015-01-01

    In fish, oocyte meiotic maturation is regulated by 17α, 20β-dihydroxy-progesterone through cAMP. To study the role of cAMP response element binding protein (CREB) in meiotic maturation, we cloned and characterized the expression pattern of CREBs from two fish models, the Nile tilapia and catfish. In the Nile tilapia three different CREBs were identified where in CREB1 was found in many tissues including gonads with abundant expression in testis. CREB2, few amino acids shorter than CREB1, was expressed in several tissues with abundant expression in ovary. In addition, a 3’UTR variant form, CREB3 was exclusively found in ovary. During natural 14-day ovarian cycle of the Nile tilapia, CREB1 expression was stable throughout vitellogenesis with a sharp decrease on the day of spawning. In contrast, CREB2 remain unchanged throughout the ovarian cycle, however elevated in 11-day full-grown immature ovarian follicle and after hCG-induction. Interestingly, CREB3 expression was induced three folds on the day of spawning as well as during hCG-induced oocyte maturation. Based on the synergistic expression pattern, CREB1 is likely to control oocyte growth, whereas CREB 2 and 3 contribute to oocyte maturation in tilapia and the latter seems to be critical. In catfish, a single form of CREB showed a maximum expression during spawning phase and hCG-induced maturation both in vivo and in vitro augmented CREB expression. These results suggest that spatial and temporal expression of CREBs seems to be important for final oocyte maturation and may also regulate oocyte growth in fish. PMID:26700177

  17. Expression Patterns of CREBs in Oocyte Growth and Maturation of Fish.

    PubMed

    Senthilkumaran, Balasubramanian; Sreenivasulu, Gunti; Wang, De-Shou; Sudhakumari, Cheni-Chery; Kobayashi, Tohru; Nagahama, Yoshitaka

    2015-01-01

    In fish, oocyte meiotic maturation is regulated by 17α, 20β-dihydroxy-progesterone through cAMP. To study the role of cAMP response element binding protein (CREB) in meiotic maturation, we cloned and characterized the expression pattern of CREBs from two fish models, the Nile tilapia and catfish. In the Nile tilapia three different CREBs were identified where in CREB1 was found in many tissues including gonads with abundant expression in testis. CREB2, few amino acids shorter than CREB1, was expressed in several tissues with abundant expression in ovary. In addition, a 3'UTR variant form, CREB3 was exclusively found in ovary. During natural 14-day ovarian cycle of the Nile tilapia, CREB1 expression was stable throughout vitellogenesis with a sharp decrease on the day of spawning. In contrast, CREB2 remain unchanged throughout the ovarian cycle, however elevated in 11-day full-grown immature ovarian follicle and after hCG-induction. Interestingly, CREB3 expression was induced three folds on the day of spawning as well as during hCG-induced oocyte maturation. Based on the synergistic expression pattern, CREB1 is likely to control oocyte growth, whereas CREB 2 and 3 contribute to oocyte maturation in tilapia and the latter seems to be critical. In catfish, a single form of CREB showed a maximum expression during spawning phase and hCG-induced maturation both in vivo and in vitro augmented CREB expression. These results suggest that spatial and temporal expression of CREBs seems to be important for final oocyte maturation and may also regulate oocyte growth in fish. PMID:26700177

  18. Female meiotic sex chromosome inactivation in chicken.

    PubMed

    Schoenmakers, Sam; Wassenaar, Evelyne; Hoogerbrugge, Jos W; Laven, Joop S E; Grootegoed, J Anton; Baarends, Willy M

    2009-05-01

    During meiotic prophase in male mammals, the heterologous X and Y chromosomes remain largely unsynapsed, and meiotic sex chromosome inactivation (MSCI) leads to formation of the transcriptionally silenced XY body. In birds, the heterogametic sex is female, carrying Z and W chromosomes (ZW), whereas males have the homogametic ZZ constitution. During chicken oogenesis, the heterologous ZW pair reaches a state of complete heterologous synapsis, and this might enable maintenance of transcription of Z- and W chromosomal genes during meiotic prophase. Herein, we show that the ZW pair is transiently silenced, from early pachytene to early diplotene using immunocytochemistry and gene expression analyses. We propose that ZW inactivation is most likely achieved via spreading of heterochromatin from the W on the Z chromosome. Also, persistent meiotic DNA double-strand breaks (DSBs) may contribute to silencing of Z. Surprisingly, gammaH2AX, a marker of DSBs, and also the earliest histone modification that is associated with XY body formation in mammalian and marsupial spermatocytes, does not cover the ZW during the synapsed stage. However, when the ZW pair starts to desynapse, a second wave of gammaH2AX accumulates on the unsynapsed regions of Z, which also show a reappearance of the DSB repair protein RAD51. This indicates that repair of meiotic DSBs on the heterologous part of Z is postponed until late pachytene/diplotene, possibly to avoid recombination with regions on the heterologously synapsed W chromosome. Two days after entering diplotene, the Z looses gammaH2AX and shows reactivation. This is the first report of meiotic sex chromosome inactivation in a species with female heterogamety, providing evidence that this mechanism is not specific to spermatogenesis. It also indicates the presence of an evolutionary force that drives meiotic sex chromosome inactivation independent of the final achievement of synapsis. PMID:19461881

  19. Female Meiotic Sex Chromosome Inactivation in Chicken

    PubMed Central

    Schoenmakers, Sam; Wassenaar, Evelyne; Hoogerbrugge, Jos W.; Laven, Joop S. E.; Grootegoed, J. Anton; Baarends, Willy M.

    2009-01-01

    During meiotic prophase in male mammals, the heterologous X and Y chromosomes remain largely unsynapsed, and meiotic sex chromosome inactivation (MSCI) leads to formation of the transcriptionally silenced XY body. In birds, the heterogametic sex is female, carrying Z and W chromosomes (ZW), whereas males have the homogametic ZZ constitution. During chicken oogenesis, the heterologous ZW pair reaches a state of complete heterologous synapsis, and this might enable maintenance of transcription of Z- and W chromosomal genes during meiotic prophase. Herein, we show that the ZW pair is transiently silenced, from early pachytene to early diplotene using immunocytochemistry and gene expression analyses. We propose that ZW inactivation is most likely achieved via spreading of heterochromatin from the W on the Z chromosome. Also, persistent meiotic DNA double-strand breaks (DSBs) may contribute to silencing of Z. Surprisingly, γH2AX, a marker of DSBs, and also the earliest histone modification that is associated with XY body formation in mammalian and marsupial spermatocytes, does not cover the ZW during the synapsed stage. However, when the ZW pair starts to desynapse, a second wave of γH2AX accumulates on the unsynapsed regions of Z, which also show a reappearance of the DSB repair protein RAD51. This indicates that repair of meiotic DSBs on the heterologous part of Z is postponed until late pachytene/diplotene, possibly to avoid recombination with regions on the heterologously synapsed W chromosome. Two days after entering diplotene, the Z looses γH2AX and shows reactivation. This is the first report of meiotic sex chromosome inactivation in a species with female heterogamety, providing evidence that this mechanism is not specific to spermatogenesis. It also indicates the presence of an evolutionary force that drives meiotic sex chromosome inactivation independent of the final achievement of synapsis. PMID:19461881

  20. Transient exposure to sodium butyrate after germinal vesicle breakdown improves meiosis but not developmental competence in pig oocytes.

    PubMed

    Liu, Limei; Song, Guangqi; Gao, Fei; Guan, Jiyu; Tang, Bo; Li, Ziyi

    2012-05-01

    Oocyte maturation is a complex process during which epigenetic modifications are dramatically changed, especially histone acetylation and phosphorylation. We have investigated the effects of NaBu (sodium butyrate), a natural HDAC (histone deacetylase) inhibitor, on porcine oocyte maturation at different stages and subsequent embryonic development to improve IVF (in vitro fertilization) and embryo production. COCs (cumulus oocyte complexes) were cultured, IVM (in vitro maturation) supplemented with 1 mM NaBu before or after GVBD [GV (germinal vesicle) breakdown] during maturation. NaBu delayed oocyte meiosis in the GV and GVBD stages in an exposure-dependent manner. However, the short treatment with 1 mM NaBu after GVBD significantly improved the meiotic competence. No positive effects of NaBu on GSH levels and subsequent embryonic development following IVF were seen. Transient exposure to NaBu after GVBD improves meiotic competence, but not subsequently, probably by having an effect on histone acetylation during oocyte maturation.

  1. Association between Maternal Age and Meiotic Recombination for Trisomy 21

    PubMed Central

    Lamb, Neil E.; Yu, Kai; Shaffer, John; Feingold, Eleanor; Sherman, Stephanie L.

    2005-01-01

    Altered genetic recombination has been identified as the first molecular correlate of chromosome nondisjunction in both humans and model organisms. Little evidence has emerged to link maternal age—long recognized as the primary risk factor for nondisjunction—with altered recombination, although some studies have provided hints of such a relationship. To determine whether an association does exist, chromosome 21 recombination patterns were examined in 400 trisomy 21 cases of maternal meiosis I origin, grouped by maternal age. These recombination patterns were used to predict the chromosome 21 exchange patterns established during meiosis I. There was no statistically significant association between age and overall rate of exchange. The placement of meiotic exchange, however, differed significantly among the age groups. Susceptible patterns (pericentromeric and telomeric exchanges) accounted for 34% of all exchanges among the youngest class of women but only 10% of those among the oldest class. The pattern of exchanges among the oldest age group mimicked the pattern observed among normally disjoining chromosomes 21. These results suggest that the greatest risk factor for nondisjunction among younger women is the presence of a susceptible exchange pattern. We hypothesize that environmental and age-related insults accumulate in the ovary as a woman ages, leading to malsegregation of oocytes with stable exchange patterns. It is this risk, due to recombination-independent factors, that would be most influenced by increasing age, leading to the observed maternal age effect. PMID:15551222

  2. Temporal and spatial regulation of translation in the mammalian oocyte via the mTOR-eIF4F pathway.

    PubMed

    Susor, Andrej; Jansova, Denisa; Cerna, Renata; Danylevska, Anna; Anger, Martin; Toralova, Tereza; Malik, Radek; Supolikova, Jaroslava; Cook, Matthew S; Oh, Jeong Su; Kubelka, Michal

    2015-01-28

    The fully grown mammalian oocyte is transcriptionally quiescent and utilizes only transcripts synthesized and stored during early development. However, we find that an abundant RNA population is retained in the oocyte nucleus and contains specific mRNAs important for meiotic progression. Here we show that during the first meiotic division, shortly after nuclear envelope breakdown, translational hotspots develop in the chromosomal area and in a region that was previously surrounded the nucleus. These distinct translational hotspots are separated by endoplasmic reticulum and Lamin, and disappear following polar body extrusion. Chromosomal translational hotspots are controlled by the activity of the mTOR-eIF4F pathway. Here we reveal a mechanism that-following the resumption of meiosis-controls the temporal and spatial translation of a specific set of transcripts required for normal spindle assembly, chromosome alignment and segregation.

  3. Temporal and spatial regulation of translation in the mammalian oocyte via the mTOR–eIF4F pathway

    PubMed Central

    Susor, Andrej; Jansova, Denisa; Cerna, Renata; Danylevska, Anna; Anger, Martin; Toralova, Tereza; Malik, Radek; Supolikova, Jaroslava; Cook, Matthew S.; Oh, Jeong Su; Kubelka, Michal

    2015-01-01

    The fully grown mammalian oocyte is transcriptionally quiescent and utilizes only transcripts synthesized and stored during early development. However, we find that an abundant RNA population is retained in the oocyte nucleus and contains specific mRNAs important for meiotic progression. Here we show that during the first meiotic division, shortly after nuclear envelope breakdown, translational hotspots develop in the chromosomal area and in a region that was previously surrounded the nucleus. These distinct translational hotspots are separated by endoplasmic reticulum and Lamin, and disappear following polar body extrusion. Chromosomal translational hotspots are controlled by the activity of the mTOR–eIF4F pathway. Here we reveal a mechanism that—following the resumption of meiosis—controls the temporal and spatial translation of a specific set of transcripts required for normal spindle assembly, chromosome alignment and segregation. PMID:25629602

  4. Evidence that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary.

    PubMed

    Sui, Xu-Xia; Luo, Li-Li; Xu, Jin-Jie; Fu, Yu-Cai

    2010-08-01

    Previous studies have proposed that the forkhead transcription factor FOXO3a is involved in cell cycle arrest and apoptosis and that it may also repress follicular development by inducing cell cycle arrest in ovaries. We have recently demonstrated that FOXO3a induces oocyte apoptosis of neonatal rat ovaries under in vitro conditions. In the present study, we evaluated the role of FOXO3a in oocyte apoptosis under in vivo conditions. Ovaries from rats were obtained from newborns on postnatal day (PD) 1, 2, 3, and 4. TUNEL assay results showed that oocyte apoptosis occurred mainly on PD 1 and 2. Immunohistochemical staining of FOXO3a, Bim, Fas ligand (FasL), p27KIP1, caspase-8, and caspase-3 showed that they were all expressed mainly in naked oocytes on PD 1 and 2. The percentage of positive FOXO3a staining of oocytes reached peak levels in the ovaries of 2-day-old rats, which was consistent with the rate of the apoptotic profiles determined by TUNEL. The percentage between TUNEL-positive and FOXO3a-positive oocytes in the nucleus showed no statistical differences within the 4-day-old rat ovaries. Furthermore, the positive oocyte percentage of the target factors of FOXO3a (Bim, p27KIP1, and FasL) and pro-apoptotic proteins (caspase-3 and caspase-8) also reached peak levels in the ovaries of 2-day-old rats, which was similar to the rate of FOXO3a-positive oocytes. These results suggest that FOXO3a in the oocyte nucleus is involved in oocyte apoptosis; that is, FOXO3a-positive oocytes may be the apoptotic cells. To verify this, rat oocytes were subjected to TUNEL and immunofluorescent double-labeling assays. We found that TUNEL-positive cells were also FOXO3a-, Bim-, or FasL-positive. To identify the downstream target of FOXO3a, double immunofluorescent staining with antibodies to Bim and FasL was performed. We found that FOXO3a-positive cells were also Bim- and FasL-positive. We conclude that the overexpression of FOXO3a in the oocyte nucleus of neonatal rat ovaries

  5. Meiotic chromosome structure and function in plants.

    PubMed

    Mainiero, Samantha; Pawlowski, Wojciech P

    2014-01-01

    Chromosome structure is important for many meiotic processes. Here, we outline 3 main determinants of chromosome structure and their effects on meiotic processes in plants. Cohesins are necessary to hold sister chromatids together until the first meiotic division, ensuring that homologous chromosomes and not sister chromatids separate during anaphase I. During meiosis in maize, Arabidopsis, and rice, cohesins are needed for establishing early prophase chromosome structure and recombination and for aligning bivalents at the metaphase plate. Condensin complexes play pivotal roles in controlling the packaging of chromatin into chromosomes through chromatin compaction and chromosome individualization. In animals and fungi, these complexes establish a meiotic chromosome structure that allows for proper recombination, pairing, and synapsis of homologous chromosomes. In plants, information on the role of condensins in meiosis is limited, but they are known to be required for successful completion of reproductive development. Therefore, we speculate that they play roles similar to animal and fungal condensins during meiosis. Plants generally have large and complex genomes due to frequent polyploidy events, and likely, condensins and cohesins organize chromosomes in such a way as to ensure genome stability. Hexaploid wheat has evolved a unique mechanism using a Ph1 locus-controlled chromosome organization to ensure proper chromosome pairing in meiosis. Altogether, studies on meiotic chromosome structure indicate that chromosome organization is not only important for chromatin packaging but also fulfills specific functions in facilitating chromosome interactions during meiosis, including pairing and recombination. PMID:25096046

  6. Oocyte-specific inactivation of Omcg1 leads to DNA damage and c-Abl/TAp63-dependent oocyte death associated with dramatic remodeling of ovarian somatic cells

    PubMed Central

    Vandormael-Pournin, S; Guigon, C J; Ishaq, M; Coudouel, N; Avé, P; Huerre, M; Magre, S; Cohen-Tannoudji, J; Cohen-Tannoudji, M

    2015-01-01

    Aberrant loss of oocytes following cancer treatments or genetic mutations leads to premature ovarian insufficiency (POI) associated with endocrine-related disorders in 1% of women. Therefore, understanding the mechanisms governing oocyte death is crucial for the preservation of female fertility. Here, we report the striking reproductive features of a novel mouse model of POI obtained through oocyte-specific inactivation (ocKO) of Omcg1/Zfp830 encoding a nuclear zinc finger protein involved in pre-mRNA processing. Genetic ablation of OMCG1 in early growing oocytes leads to reduced transcription, accumulation of DNA double-strand breaks and subsequent c-Abl/TAp63-dependent oocyte death, thus uncovering the key role of OMCG1 for oocyte genomic integrity. All adult Omcg1ocKO females displayed complete elimination of early growing oocytes and sterility. Unexpectedly, mutant females exhibited a normal onset of puberty and sexual receptivity. Detailed studies of Omcg1ocKO ovaries revealed that the ovarian somatic compartment underwent a dramatic structural and functional remodeling. This allowed the cooperation between oocyte-depleted follicles and interstitial tissue to produce estradiol. Moreover, despite early folliculogenesis arrest, mutant mice exhibited sexual cyclicity as shown by cyclical changes in estrogen secretion, vaginal epithelium cytology and genital tract weight. Collectively, our findings demonstrate the key role of Omcg1 for oocyte survival and highlight the contribution of p63 pathway in damaged oocyte elimination in adulthood. Moreover, our findings challenge the prevailing view that sexual cyclicity is tightly dependent upon the pace of folliculogenesis and luteal differentiation. PMID:25168238

  7. Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency

    PubMed Central

    Cagnone, Gael L. M.; Tsai, Te-Sha; Makanji, Yogeshwar; Matthews, Pamela; Gould, Jodee; Bonkowski, Michael S.; Elgass, Kirstin D.; Wong, Ashley S. A.; Wu, Lindsay E.; McKenzie, Matthew; Sinclair, David A.; John, Justin C. St.

    2016-01-01

    An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte’s mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes. PMID:26987907

  8. Differential post-transcriptional regulations of wnt mRNAs upon axolotl meiotic maturation.

    PubMed

    Vaur, Sabine; Montreau, Nicole; Dautry, François; Andéol, Yannick

    2002-08-01

    The products of the Wntgene family play an essential role in several aspects of embryo patterning. We have investigated the post-transcriptional regulation of three of these genes: Awnt-1, Awnt-5A and Awnt-5B during axolotl (Ambystoma mexicanum) oogenesis, oocyte maturation and early development. We show that Awnt-1, Awnt-5A and Awnt-5B mRNAs are maternally expressed. The three transcripts are tightly regulated at specific times and display differential mRNA stability, poly(A) tail length and localization. In contrastto Awnt-5Bwhich is restricted to the animal hemisphere, Awnt-1 and Awnt-5A have no particular localization in stage VI oocytes. Interestingly, these two mRNAs exhibit a polyadenylation gradient along the animal-vegetal axis. Moreover, after meiotic maturation, Awnt-1 and 5A mRNAs become exclusively localized to the animal pole. This isthe first evidence of a complete mRNA re-localization to the animal hemisphere during oocyte maturation. PMID:12216985

  9. Oocyte Polarization Is Coupled to the Chromosomal Bouquet, a Conserved Polarized Nuclear Configuration in Meiosis

    PubMed Central

    Elkouby, Yaniv M.; Jamieson-Lucy, Allison; Mullins, Mary C.

    2016-01-01

    The source of symmetry breaking in vertebrate oocytes is unknown. Animal—vegetal oocyte polarity is established by the Balbiani body (Bb), a conserved structure found in all animals examined that contains an aggregate of specific mRNAs, proteins, and organelles. The Bb specifies the oocyte vegetal pole, which is key to forming the embryonic body axes as well as the germline in most vertebrates. How Bb formation is regulated and how its asymmetric position is established are unknown. Using quantitative image analysis, we trace oocyte symmetry breaking in zebrafish to a nuclear asymmetry at the onset of meiosis called the chromosomal bouquet. The bouquet is a universal feature of meiosis where all telomeres cluster to one pole on the nuclear envelope, facilitating chromosomal pairing and meiotic recombination. We show that Bb precursor components first localize with the centrosome to the cytoplasm adjacent to the telomere cluster of the bouquet. They then aggregate around the centrosome in a specialized nuclear cleft that we identified, assembling the early Bb. We show that the bouquet nuclear events and the cytoplasmic Bb precursor localization are mechanistically coordinated by microtubules. Thus the animal—vegetal axis of the oocyte is aligned to the nuclear axis of the bouquet. We further show that the symmetry breaking events lay upstream to the only known regulator of Bb formation, the Bucky ball protein. Our findings link two universal features of oogenesis, the Bb and the chromosomal bouquet, to oocyte polarization. We propose that a meiotic—vegetal center couples meiosis and oocyte patterning. Our findings reveal a novel mode of cellular polarization in meiotic cells whereby cellular and nuclear polarity are aligned. We further reveal that in zygotene nests, intercellular cytoplasmic bridges remain between oocytes and that the position of the cytoplasmic bridge coincides with the location of the centrosome meiotic—vegetal organizing center. These

  10. Influence of caffeine pretreatment on biphasic in vitro maturation of dog oocytes.

    PubMed

    Salavati, Mazdak; Ghafari, Fataneh; Zhang, Tiantian; Fouladi-Nashta, Ali A

    2013-10-15

    Phosphodiesterase (PDE) inhibitors have been utilized for in vitro maturation (IVM) of oocytes to manipulate the meiotic resumption and progression. Premature chromatin condensation and DNA replication of the oocytes, immediately after the decrease in the cAMP level, are the difficulties in canine IVM. Caffeine, a nonselective competitive PDE inhibitor, due to its structural similarity to adenosine molecule maintains the cAMP level by occupying PDE enzymes such as PDE-3A inside the oocyte and PDE-4 and PDE-5 in the cumulus cells. In this study, the effects of 12-hour caffeine pretreatment in a biphasic IVM protocol were assessed on maturation rates of canine oocytes. Sixty hours of culture after a 12-hour of 10 mM caffeine pretreatment resulted in 16.9% ± 2.4 of the oocytes reaching metaphase II stage (MII) and 25.9% ± 5.2 degeneration rate compared with the control group with 2.2% ± 2.2 MII and 37.6% ± 4.3 degeneration rates (P < 0.05). Caffeine pretreatment induced higher mitogen-activated protein kinases (MAPK1 and MAPK3) phosphorylation and maturation-promoting factor activity at 12 hours and activated MAPK1 and maturation-promoting factor at 48 hours after culture in cumulus-oocyte complexes (COCs) compared with the control group (P < 0.05). Fresh canine COCs were also analyzed before IVM using brilliant cresyl blue (BCB) staining. Oocytes showed difference in meiotic resumption (MI-MII) (BCB+ = 16.11% ± 5.5, BCB- = 9.86% ± 5.0; P < 0.05) after 60 hours of culture following 12-hour caffeine pretreatment. The BCB+ canine oocytes had higher MII rate than the BCB- group under caffeine pretreatment (10.2% ± 2.9 vs. 1.1% ± 1.1, respectively; P < 0.05). Results indicated that 12-hour caffeine pretreatment of canine COCs improves the MII maturation rates at 72 hours and BCB+ oocytes have higher competency in vitro for nuclear maturation. PMID:23921184

  11. Neem (Azadirachta indica L.) leaf extract deteriorates oocyte quality by inducing ROS-mediated apoptosis in mammals.

    PubMed

    Chaube, Shail K; Shrivastav, Tulsidas G; Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ajai K

    2014-01-01

    Neem (Azadirachta indica L.) leaf has been widely used in ayurvedic system of medicine for fertility regulation for a long time. The molecular mechanism by which neem leaf regulates female fertility remains poorly understood. Animal studies suggest that aqueous neem leaf extract (NLE) induces reactive oxygen species (ROS) - mediated granulosa cell apoptosis. Granulosa cell apoptosis deprives oocytes from nutrients, survival factors and cell cycle proteins required for the achievement of meiotic competency of follicular oocytes prior to ovulation. Under this situation, follicular oocyte becomes more susceptible towards apoptosis after ovulation. The increased level of hydrogen peroxide (H2O2) inside the follicular fluid results in the transfer of H2O2 from follicular fluid to the oocyte. The increased level of H2O2 induces p53 activation and over expression of Bax protein that modulates mitochondrial membrane potential and trigger cytochrome c release. The increased cytosolic cytochrome c level induces caspase-9 and caspase-3 activities that trigger destruction of structural and specific proteins leading to DNA fragmentation and thereby oocyte apoptosis. Based on these animal studies, we propose that NLE induces generation of ROS and mitochondria-mediated apoptosis both in granulosa cells as well as in follicular oocyte. The induction of apoptosis deteriorates oocyte quality and thereby limits reproductive outcome in mammals.

  12. Factors affecting the survival, fertilization, and embryonic development of mouse oocytes after vitrification using glass capillaries.

    PubMed

    Tan, Xiuwen; Song, Enliang; Liu, Xiaomu; You, Wei; Wan, Fachun

    2009-09-01

    Cryopreservation of mammalian oocytes is an important way to provide a steady source of materials for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. However, oocytes cryopreservation has not been common used, as there still are some problems waiting to be solved on the repeatability, safety, and validity. Then, it is necessary to investigate the damage occurred from vitrification and find a way to avoid or repair it. In this study, mouse mature oocytes were firstly pretreated in different equilibrium media, such as 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO), 10% EG + 10% DMSO, and 15% EG + 15% DMSO in TCM199 supplemented with 20% fetal calf serum (FCS), for 1, 3, and 5 min, respectively, and then oocytes were transferred into vitrification solution (20% EG, 20% DMSO, 0.3 M sucrose, and 20% FCS in TCM199, M2, Dulbecco's phosphate buffered saline, and 0.9% saline medium, respectively) and immediately loaded into glass capillaries to be plunged into liquid nitrogen. After storage from 1 h to 1 wk, they were diluted in stepwise sucrose solutions. The surviving oocytes were stained for cortical granule, meiotic spindles, and chromosomes. Oocytes without treatments were used as controls. The results showed that oocytes pretreated in 5% EG +5% DMSO group for 3-5 min or in 10% EG + 10% DMSO group for 1-3 min were better than other treatments. Oocytes vitrified in TCM199 as basic medium showed higher survival and better subsequent embryonic development than other groups. When the concentration of FCS in vitrification solution reduced below 15%, the rates of survival, fertilization, and developing to blastocyst declined dramatically. The inner diameter (0.6 mm) of glass capillaries and amount of vitrification solution (1-3 microl) achieved more rapid cooling and warming and so reduce the injury to oocytes. Cropreservation led to the exocytosis of cortical granule of oocytes (about 10%) and serious disturbance of

  13. Factors affecting the survival, fertilization, and embryonic development of mouse oocytes after vitrification using glass capillaries.

    PubMed

    Tan, Xiuwen; Song, Enliang; Liu, Xiaomu; You, Wei; Wan, Fachun

    2009-09-01

    Cryopreservation of mammalian oocytes is an important way to provide a steady source of materials for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. However, oocytes cryopreservation has not been common used, as there still are some problems waiting to be solved on the repeatability, safety, and validity. Then, it is necessary to investigate the damage occurred from vitrification and find a way to avoid or repair it. In this study, mouse mature oocytes were firstly pretreated in different equilibrium media, such as 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO), 10% EG + 10% DMSO, and 15% EG + 15% DMSO in TCM199 supplemented with 20% fetal calf serum (FCS), for 1, 3, and 5 min, respectively, and then oocytes were transferred into vitrification solution (20% EG, 20% DMSO, 0.3 M sucrose, and 20% FCS in TCM199, M2, Dulbecco's phosphate buffered saline, and 0.9% saline medium, respectively) and immediately loaded into glass capillaries to be plunged into liquid nitrogen. After storage from 1 h to 1 wk, they were diluted in stepwise sucrose solutions. The surviving oocytes were stained for cortical granule, meiotic spindles, and chromosomes. Oocytes without treatments were used as controls. The results showed that oocytes pretreated in 5% EG +5% DMSO group for 3-5 min or in 10% EG + 10% DMSO group for 1-3 min were better than other treatments. Oocytes vitrified in TCM199 as basic medium showed higher survival and better subsequent embryonic development than other groups. When the concentration of FCS in vitrification solution reduced below 15%, the rates of survival, fertilization, and developing to blastocyst declined dramatically. The inner diameter (0.6 mm) of glass capillaries and amount of vitrification solution (1-3 microl) achieved more rapid cooling and warming and so reduce the injury to oocytes. Cropreservation led to the exocytosis of cortical granule of oocytes (about 10%) and serious disturbance of

  14. In vitro maturation of oocytes derived from the brown bear (Ursus arctos).

    PubMed

    Yin, Xi-Jun; Lee, Hyo-Sang; Choi, Eu-Gene; Yu, Xian-Feng; Park, Gye-Young; Bae, Inhyu; Yang, Chul-Ju; Oh, Dong-Hwan; Kim, Nam-Hung; Kong, Il-Keun

    2007-06-01

    This study was conducted to determine whether meiotic maturation could be induced in ovarian oocytes from the American brown bear (Ursus arctos), a model for gamete "rescue" techniques for endangered ursids. The bears were euthanized, and their ovaries were transported to the laboratory within 4 h. The mean ovarian size was 2.4 x 1.8 cm (range: 2.0-3.3 x 1.5-2.2 cm). The ovaries obtained from the 2 brown bears yielded 97 oocytes (48.5/female), and 88 (90.7%) of them were morphologically classified as normal quality. Oocytes were in vitro matured at 38.5 C in 5% CO2 for 24 or 48 h in TCM-199 supplemented with 10% FBS, 1 microg/ml estradiol-17beta, and 10 microg/ml FSH. In Exp. 1, morphologic evaluation of matured oocytes was conducted by measuring the diameters of oocytes with a zona pellucida (ZP) or cytoplasm without a ZP. In Exp. 2, activation was induced by applying two 20 microsec DC pulses of 2.0 kV/cm delivered by an Electro Cell Fusion Generator. The activated oocytes were cultured in TCM-199 containing 2 mM of 6-dimethylaminopurine for 4 h, in Charles Rosenkrans (CR) 1 for 3 days and the in CR2 for another 4 days. The diameters of the matured bear oocytes with a ZP and with cytoplasm without a ZP (161.8 +/- 6.0 and 135.3 +/- 7.5 microm, respectively) were significantly (P<0.05) larger than those of bovine oocytes (150.7 +/- 4.9 and 118.7 +/- 7.5 microm). The maturation rates of the bear oocytes were 17.6 and 59.4% at 24 and 48 h of in vitro maturation, the percentage of activated oocytes that developed to the 2 or 4-cell stage was 31.6%; however, no blastocysts were observed. These results indicate that bear oocytes can develop to metaphase II in an in vitro culture system and that activated oocytes can develop to the 2 or 4-cell stages. PMID:17310081

  15. Cortical Granule Exocytosis Is Mediated by Alpha-SNAP and N-Ethilmaleimide Sensitive Factor in Mouse Oocytes

    PubMed Central

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

    2015-01-01

    Cortical granule exocytosis (CGE), also known as cortical reaction, is a calcium- regulated secretion that represents a membrane fusion process during meiotic cell division of oocytes. The molecular mechanism of membrane fusion during CGE is still poorly understood and is thought to be mediated by the SNARE pathway; nevertheless, it is unkown if SNAP (acronym for soluble NSF attachment protein) and NSF (acronym for N-ethilmaleimide sensitive factor), two key proteins in the SNARE pathway, mediate CGE in any oocyte model. In this paper, we documented the gene expression of α-SNAP, γ-SNAP and NSF in mouse oocytes. Western blot analysis showed that the expression of these proteins maintains a similar level during oocyte maturation and early activation. Their localization was mainly observed at the cortical region of metaphase II oocytes, which is enriched in cortical granules. To evaluate the function of these proteins in CGE we set up a functional assay based on the quantification of cortical granules metaphase II oocytes activated parthenogenetically with strontium. Endogenous α-SNAP and NSF proteins were perturbed by microinjection of recombinant proteins or antibodies prior to CGE activation. The microinjection of wild type α-SNAP and the negative mutant of α-SNAP L294A in metaphase II oocytes inhibited CGE stimulated by strontium. NEM, an irreversibly inhibitor of NSF, and the microinjection of the negative mutant NSF D1EQ inhibited cortical reaction. The microinjection of anti-α-SNAP and anti-NSF antibodies was able to abolish CGE in activated metaphase II oocytes. The microinjection of anti-γ SNAP antibody had no effect on CGE. Our findings indicate, for the first time in any oocyte model, that α-SNAP, γ-SNAP, and NSF are expressed in mouse oocytes. We demonstrate that α-SNAP and NSF have an active role in CGE and propose a working model. PMID:26267363

  16. MicroRNA Profile of Granulosa Cells after Ovarian Stimulation Differs According to Maturity of Retrieved Oocytes

    PubMed Central

    Kim, Y. J.; Ku, S.-Y.; Kim, Y. Y.; Suh, C. S.; Kim, S. H.; Choi, Y. M.

    2016-01-01

    Background: Recent animal studies demonstrated that regulating the microRNA (miRNA) in granulosa cells (GCs) modulates the meiotic competence of oocytes. However, the difference in expression profiles of miRNAs in human GCs according to the maturity of the oocyte still remains to be elucidated. Objective: This observational study investigated whether the miRNA profile of human GCs differs according to the maturity of the retrieved oocyte after controlled ovarian stimulation for in vitro fertilization (IVF). Methods: Ten women who underwent ovarian stimulation cycles with GnRH agonist long protocols were recruited. The follicular fluid (FF) from dominant follicles was individually aspirated at oocyte retrieval. Oocytes were divided into two groups according to oocyte maturity (“mature group” vs. “immature group”). GCs were collected from the FF and miRNA was analyzed using real-time PCR. Results: Mean number of MII oocytes in the mature group was 1.6 ± 0.9 with none in the immature group (p = 0.008). Mean number of MI oocytes was 5.6 ± 2.1 in the mature group and 1.0 ± 0.0 in the immature group (p = 0.008). The total number of retrieved oocytes was 8.8 ± 1.9 in the mature group and 2.0 ± 1.2 in the immature group (p = 0.008). The GCs of the mature group showed a significantly lower expression of hsa-let-7b compared to the GCs of the immature group (p < 0.001). Conclusion: Taken together, the miRNA expression profiles of human GCs obtained from dominant follicles are associated with maturity of the adjacent oocyte and may be useful as a prognosticator of IVF outcome. PMID:27365541

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

    PubMed

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

    2015-01-01

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

  18. Calcium and actin in the saga of awakening oocytes

    SciTech Connect

    Santella, Luigia 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

  19. Effects of Activation on Functional Aster Formation, Microtubule Assembly, and Blastocyst Development of Goat Oocytes Injected with Round Spermatids

    PubMed Central

    Liu, Xin-Yong; Miao, Yi-Long; Zhang, Jie; Qiu, Jian-Hua; Cui, Xiang-Zhong; Gao, Wei-Qiang; Luo, Ming-Jiu

    2012-01-01

    Abstract A systematic study was conducted on round spermatids (ROS) injection (ROSI) using the goat model. After ROSI, the oocytes were treated or not with ionomycin (ROSI+I and ROSI−I, respectively) and compared with intracytoplasmic sperm injection (ICSI). After ROSI−I, most oocytes were arrested with premature chromatin condensation and few oocytes formed pronuclei. In contrast, most oocytes formed pronuclei after ROSI+I. Some ROS were observed to form asters that organized a dense microtubule network after ROSI+I, but after ROSI−I, no ROS asters were observed. Whereas most of the oocytes showed Ca2+ rises and a significant decline in maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities after ROSI+I, no such changes were observed after ROSI−I. Due to the lack of Ca2+ oscillations after ROSI−I, oocytes were injected with more ROS. Interestingly, different from the results observed in a single ROS injection, injection with four ROS effectively activated oocytes by inducing typical Ca2+ oscillations. Whereas ROSI+I oocytes and ICSI oocytes both showed extensive microtubule networks, no such a network was observed in parthenogenetic oocytes. Together, the results suggest that goat ROS is not able to trigger intracellular Ca2+ rises and thus to inhibit MPF and MAPK activities, but artificial activation improved fertilization and development of ROSI goat oocytes. Goat ROS can organize functional microtubular asters in activated oocytes. A ROS-derived factor(s) may be essential for organization of a functional microtubule network to unite pronuclei. Goat centrosome is of paternal origin because both ROS and sperm asters organized an extensive microtubule network after intra-oocyte injection. PMID:22908906

  20. Next Generation Sequencing-Based Comprehensive Chromosome Screening in Mouse Polar Bodies, Oocytes, and Embryos.

    PubMed

    Treff, Nathan R; Krisher, Rebecca L; Tao, Xin; Garnsey, Heather; Bohrer, Chelsea; Silva, Elena; Landis, Jessica; Taylor, Deanne; Scott, Richard T; Woodruff, Teresa K; Duncan, Francesca E

    2016-04-01

    Advanced reproductive age is unequivocally associated with increased aneuploidy in human oocytes, which contributes to infertility, miscarriages, and birth defects. The frequency of meiotic chromosome segregation errors in oocytes derived from reproductively aged mice appears to be similar to that observed in humans, but a limitation of this important model system is our inability to accurately identify chromosome-specific aneuploidy. Here we report the validation and application of a new low-pass whole-genome sequencing approach to comprehensively screen chromosome aneuploidy in individual mouse oocytes and blastocysts. First, we validated this approach by using single mouse embryonic fibroblasts engineered to have stable trisomy 16. We further validated this method by identifying reciprocal chromosome segregation errors in the products of meiosis I (gamete and polar body) in oocytes from reproductively aged mice. Finally, we applied this technology to investigate the incidence of aneuploidy in blastocysts derived from in vitro- and in vivo-matured oocytes in both young and reproductively aged mice. Using this next generation sequencing approach, we quantitatively assessed meiotic and mitotic segregation errors at the single chromosome level, distinguished between errors due to premature separation of sister chromatids and classical nondisjunction of homologous chromosomes, and quantified mitochondrial DNA (mtDNA) segregation in individual cells. This whole-genome sequencing technique, therefore, greatly improves the utility of the mouse model system for the study of aneuploidy and is a powerful quantitative tool with which to examine the molecular underpinnings of mammalian gamete and early embryo chromosome segregation in the context of reproductive aging and beyond. PMID:26911429

  1. TCTP regulates spindle microtubule dynamics by stabilizing polar microtubules during mouse oocyte meiosis.

    PubMed

    Jeon, Hyuk-Joon; You, Seung Yeop; Park, Yong Seok; Chang, Jong Wook; Kim, Jae-Sung; Oh, Jeong Su

    2016-04-01

    Dynamic changes in spindle structure and function are essential for maintaining genomic integrity during the cell cycle. Spindle dynamics are highly dependent on several microtubule-associated proteins that coordinate the dynamic behavior of microtubules, including microtubule assembly, stability and organization. Here, we show that translationally controlled tumor protein (TCTP) is a novel microtubule-associated protein that regulates spindle dynamics during meiotic maturation. TCTP was expressed and widely distributed in the cytoplasm with strong enrichment at the spindle microtubules during meiosis. TCTP was found to be phosphorylated during meiotic maturation, and was exclusively localized to the spindle poles. Knockdown of TCTP impaired spindle organization without affecting chromosome alignment. These spindle defects were mostly due to the destabilization of the polar microtubules. However, the stability of kinetochore microtubules attached to chromosomes was not affected by TCTP knockdown. Overexpression of a nonphosphorylable mutant of TCTP disturbed meiotic maturation, stabilizing the spindle microtubules. In addition, Plk1 was decreased by TCTP knockdown. Taken together, our results demonstrate that TCTP is a microtubule-associating protein required to regulate spindle microtubule dynamics during meiotic maturation in mouse oocytes.

  2. Musashi regulates the temporal order of mRNA translation during Xenopus oocyte maturation.

    PubMed

    Charlesworth, Amanda; Wilczynska, Anna; Thampi, Prajitha; Cox, Linda L; MacNicol, Angus M

    2006-06-21

    A strict temporal order of maternal mRNA translation is essential for meiotic cell cycle progression in oocytes of the frog Xenopus laevis. The molecular mechanisms controlling the ordered pattern of mRNA translational activation have not been elucidated. We report a novel role for the neural stem cell regulatory protein, Musashi, in controlling the translational activation of the mRNA encoding the Mos proto-oncogene during meiotic cell cycle progression. We demonstrate that Musashi interacts specifically with the polyadenylation response element in the 3' untranslated region of the Mos mRNA and that this interaction is necessary for early Mos mRNA translational activation. A dominant inhibitory form of Musashi blocks maternal mRNA cytoplasmic polyadenylation and meiotic cell cycle progression. Our data suggest that Musashi is a target of the initiating progesterone signaling pathway and reveal that late cytoplasmic polyadenylation element-directed mRNA translation requires early, Musashi-dependent mRNA translation. These findings indicate that Musashi function is necessary to establish the temporal order of maternal mRNA translation during Xenopus meiotic cell cycle progression.

  3. Proteasomal degradation of ubiquitinated proteins in oocyte meiosis and fertilization in mammals.

    PubMed

    Karabinova, Pavla; Kubelka, Michal; Susor, Andrej

    2011-10-01

    Gametogenesis and fertilization are the key events in sexual reproduction. In the female, meiosis results in a large oocyte that is competent for fertilization and fundamental for the success of early embryonic development. Progression through meiosis is monitored by fine regulatory mechanisms. In this review, we focus on one of the most well-known regulatory elements, the E3 ligase APC/C, which mediates proteolytic degradation of a number of important substrates via the ubiquitin proteasome pathway (UPP). The UPP also indirectly regulates protein synthesis by affecting proteins involved in RNA metabolism, a process that is paramount for the transcriptionally silent oocyte. During the past few years, more evidence has accumulated to suggest that the UPP has an important role in zona pellucida penetration and gamete fusion in mammals. This review focuses on the function of the UPP in regulating oocyte meiotic maturation in mammals, with special attention to its role in chromosome segregation and polar body extrusion, its role in the acquisition of meiotic/developmental competence and recent advances in our understanding of the UPP role in fertilization.

  4. Aflatoxin B1 is toxic to porcine oocyte maturation.

    PubMed

    Liu, Jun; Wang, Qiao-Chu; Han, Jun; Xiong, Bo; Sun, Shao-Chen

    2015-07-01

    As a toxic secondary metabolite of Aspergillus species, Aflatoxin B1 (AFB1) is a major food and feed contaminant in tropical and sub-tropical regions with high temperature and humidity. It has been reported to be toxic to the female reproductive system in laboratory and domestic animals. In the present study, the influence of acute exposure to AFB1 (10 and 50 μM, 44h) on porcine oocyte maturation and its possible mechanism were investigated. The maturation rates of oocytes decreased significantly in the presence of 50 μM of AFB1. Cell cycle analysis showed that most oocytes were arrested at germinal vesicle breakdown or meosis I stage. However, actin assembly, spindle structure and chromosome alignment were not disrupted after exposure to 50 μM AFB1. Further study showed that DNA methylation levels increased in treated oocytes (50 μM). Histone methylation levels were also analysed after treatment (50 μM): H3K27me3 and H3K4me2 levels decreased, whereas H3K9me3 level increased, indicating that epigenetic modification was affected. AFB1 treatment (50 μM) also induced oxidative stress and further led to autophagy, as shown by accumulation of reactive oxygen species, up-regulated LC3 protein expression and increased mRNA levels of ATG3, ATG5 and ATG7. Annexin V-FITC staining assay revealed that AFB1 treatment (50 μM) resulted in oocyte early apoptosis, which was confirmed by increased Bak, Bax, Bcl-xl mRNA levels. Collectively, our results suggest that AFB1 disrupts porcine oocyte maturation through changing epigenetic modifications as well as inducing oxidative stress, excessive autophagy and apoptosis. PMID:25778688

  5. Aflatoxin B1 is toxic to porcine oocyte maturation.

    PubMed

    Liu, Jun; Wang, Qiao-Chu; Han, Jun; Xiong, Bo; Sun, Shao-Chen

    2015-07-01

    As a toxic secondary metabolite of Aspergillus species, Aflatoxin B1 (AFB1) is a major food and feed contaminant in tropical and sub-tropical regions with high temperature and humidity. It has been reported to be toxic to the female reproductive system in laboratory and domestic animals. In the present study, the influence of acute exposure to AFB1 (10 and 50 μM, 44h) on porcine oocyte maturation and its possible mechanism were investigated. The maturation rates of oocytes decreased significantly in the presence of 50 μM of AFB1. Cell cycle analysis showed that most oocytes were arrested at germinal vesicle breakdown or meosis I stage. However, actin assembly, spindle structure and chromosome alignment were not disrupted after exposure to 50 μM AFB1. Further study showed that DNA methylation levels increased in treated oocytes (50 μM). Histone methylation levels were also analysed after treatment (50 μM): H3K27me3 and H3K4me2 levels decreased, whereas H3K9me3 level increased, indicating that epigenetic modification was affected. AFB1 treatment (50 μM) also induced oxidative stress and further led to autophagy, as shown by accumulation of reactive oxygen species, up-regulated LC3 protein expression and increased mRNA levels of ATG3, ATG5 and ATG7. Annexin V-FITC staining assay revealed that AFB1 treatment (50 μM) resulted in oocyte early apoptosis, which was confirmed by increased Bak, Bax, Bcl-xl mRNA levels. Collectively, our results suggest that AFB1 disrupts porcine oocyte maturation through changing epigenetic modifications as well as inducing oxidative stress, excessive autophagy and apoptosis.

  6. Initiation of meiotic recombination in Ustilago maydis.

    PubMed

    Kojic, Milorad; Sutherland, Jeanette H; Pérez-Martín, José; Holloman, William K

    2013-12-01

    A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar(+) recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.

  7. Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis.

    PubMed

    Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen

    2014-02-01

    During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

  8. Human oocyte developmental potential is predicted by mechanical properties within hours after fertilization

    PubMed Central

    Yanez, Livia Z.; Han, Jinnuo; Behr, Barry B.; Pera, Renee A. Reijo; Camarillo, David B.

    2016-01-01

    The causes of embryonic arrest during pre-implantation development are poorly understood. Attempts to correlate patterns of oocyte gene expression with successful embryo development have been hampered by the lack of reliable and nondestructive predictors of viability at such an early stage. Here we report that zygote viscoelastic properties can predict blastocyst formation in humans and mice within hours after fertilization, with >90% precision, 95% specificity and 75% sensitivity. We demonstrate that there are significant differences between the transcriptomes of viable and non-viable zygotes, especially in expression of genes important for oocyte maturation. In addition, we show that low-quality oocytes may undergo insufficient cortical granule release and zona-hardening, causing altered mechanics after fertilization. Our results suggest that embryo potential is largely determined by the quality and maturation of the oocyte before fertilization, and can be predicted through a minimally invasive mechanical measurement at the zygote stage. PMID:26904963

  9. Human oocyte developmental potential is predicted by mechanical properties within hours after fertilization.

    PubMed

    Yanez, Livia Z; Han, Jinnuo; Behr, Barry B; Reijo Pera, Renee A; Camarillo, David B

    2016-01-01

    The causes of embryonic arrest during pre-implantation development are poorly understood. Attempts to correlate patterns of oocyte gene expression with successful embryo development have been hampered by the lack of reliable and nondestructive predictors of viability at such an early stage. Here we report that zygote viscoelastic properties can predict blastocyst formation in humans and mice within hours after fertilization, with >90% precision, 95% specificity and 75% sensitivity. We demonstrate that there are significant differences between the transcriptomes of viable and non-viable zygotes, especially in expression of genes important for oocyte maturation. In addition, we show that low-quality oocytes may undergo insufficient cortical granule release and zona-hardening, causing altered mechanics after fertilization. Our results suggest that embryo potential is largely determined by the quality and maturation of the oocyte before fertilization, and can be predicted through a minimally invasive mechanical measurement at the zygote stage. PMID:26904963

  10. Premeiotic events and meiotic chromosome pairing.

    PubMed

    Bennett, M D

    1984-01-01

    There is practical difficulty in identifying when meiosis begins. Moreover, because of contradictory definitions there is ambiguity and some confusion as to when, in terms of the cell cycle, premeiosis ends and meiosis begins. Nevertheless, results for several organisms show clearly that meiotic chromosome behaviour is affected by premeiotic events and especially by events during the final premeiotic mitosis and/or premeiotic interphase. This review considers only premeiotic events which do (or might) affect meiotic chromosome pairing by their effect on genomic characters, such as: chromosome number, homology, condition and position, with particular emphasis on the last. Interpreted in its widest sense 'premeiotic events affecting meiotic chromosome pairing' must include karyogamy. Moreover, while karyogamy is the normal means of achieving the diploid chromosome number and pairs of homologues essential for normal chromosome pairing, it is not the only way, as illustrated by the remarkable premeiotic adaptations seen in the apogamous ferns and the frog Rana esculenta. Little is known about the condition (including the molecular organization) of chromosomes during their approach and switch to meiosis. However, completion during premeiosis of some DNA synthesis may be essential for normal meiotic chromosome pairing. Various results (including different effects of colchicine given first at different premeiotic stages) have been claimed as evidence of one or other type of premeiotic spatial ordering of chromosomes which might favour, or be essential for, meiotic chromosome pairing. Chromosome placement has been studied recently using the electron microscope, serial thin-section, reconstruction technique. This has revealed clear evidence of non-random spatial placement of chromosomes in non-meiotic and premeiotic cells. For example, in root-tip cells of barley, Hordeum vulgare L. cv. Tuleen 346 (2n = 2x = 14), it showed: a significant spatial separation of two haploid

  11. Pentose phosphate pathway activity: effect on in vitro maturation and oxidative status of bovine oocytes.

    PubMed

    Gutnisky, Cynthia; Dalvit, Gabriel C; Thompson, Jeremy G; Cetica, Pablo D

    2014-08-01

    The relationship between pentose phosphate pathway (PPP) activity in cumulus-oocyte complexes (COCs) and oxidative and mitochondrial activity in bovine oocytes was evaluated with the aim of analysing the impact of two inhibitors (NADPH and 6-aminonicotinamide (6-AN)) and a stimulator (NADP) of the key enzymes of the PPP on the maturation rate, oxidative and mitochondrial activity and the mitochondrial distribution in oocytes. The proportion of COCs with measurable PPP activity (assessed using brilliant cresyl blue staining), glucose uptake, lactate production and meiotic maturation rate diminished when 6-AN (0.1, 1, 5 and 10mM for 22h) was added to the maturation medium (P<0.05). The addition of NADPH did not modify glucose uptake or lactate production, but reduced PPP activity in COCs and meiotic maturation rates (P<0.05). The presence of NADP (0.0125, 0.125, 1.25 and 12.5mM for 22h of culture) in the maturation medium had no effect on PPP activity in COCs, glucose uptake, lactate production and meiotic maturation rate. However, in the absence of gonadotropin supplementation, NADP stimulated both glucose uptake and lactate production at 12.5mM (the highest concentration tested; P<0.05). NADP did not modify cleavage rate, but decreased blastocyst production (P<0.05). During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22h maturation, which was also related to progressive mitochondrial migration. Inhibiting the PPP with 6-AN or NADPH led to reduced oxidative and mitochondrial activity compared with the respective control groups and inhibition of mitochondrial migration (P<0.05). Stimulation of the PPP with NADP increased oxidative and mitochondrial activity at 9h maturation (P<0.05) and delayed mitochondrial migration. The present study shows the significance of altering PPP activity during bovine oocyte IVM, revealing that there is a link between the activity of the PPP and the oxidative status of the oocyte.

  12. Modulation of glycolysis and the pentose phosphate pathway influences porcine oocyte in vitro maturation.

    PubMed

    Alvarez, G M; Ferretti, E L; Gutnisky, C; Dalvit, G C; Cetica, P D

    2013-08-01

    Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose-dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p < 0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6-AN) and the physiological (NADPH) inhibitors of PPP induced a dose-dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p < 0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.

  13. In vitro Developmental Competence of Adult Sheep Oocytes Treated with Roscovitine.

    PubMed

    Crocomo, L F; Ariu, F; Bogliolo, L; Bebbere, D; Ledda, S; Bicudo, S D

    2016-04-01

    The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus-oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 μm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos. PMID:26890275

  14. Maturation of Oocytes in Vitro.

    PubMed

    Lonergan, Patrick; Fair, Trudee

    2016-01-01

    Only a fraction of oocytes present in the ovaries at birth are ever ovulated during the lifetime of a female mammal. In vitro maturation (IVM) offers the possibility to exploit what is a largely untapped biological resource. Although IVM is used routinely for the in vitro production of embryos in domestic species, especially cattle, its clinical use in human-assisted reproduction is still evolving. The successful recapitulation in vitro of the events associated with successful oocyte maturation is not always achieved, with the majority of immature oocytes typically failing to develop to the blastocyst stage. Evidence suggests that although culture conditions throughout in vitro embryo production may have a modest influence on the developmental potential of the early embryo, the quality of the oocyte at the start of the process is the key factor determining the proportion of oocytes developing to the blastocyst stage.

  15. Rat oocyte tissue plasminogen activator is a catalytically efficient enzyme in the absence of fibrin. Endogenous potentiation of enzyme activity.

    PubMed

    Bicsak, T A; Hsueh, A J

    1989-01-01

    Rat oocytes synthesize tissue plasminogen activator (tPA) in response to stimuli which initiate meiotic maturation. Purified tPA exhibits optimal activity only in the presence of fibrin or fibrin substitutes. Because oocytes are not exposed to fibrin in situ, we investigated the possible stimulation of rat oocyte tPA activity by other endogenous factor(s). Oocytes were obtained from immature female rats which were induced to ovulate with gonadotropins. tPA activity was measured by the plasminogen-dependent cleavage of a chromogenic substrate. Measurements of kinetic parameters with Glu- or Lys-plasminogen revealed a Km for the rat oocyte enzyme of 1.3-2.1 microM compared with 23-24 microM for purified human tPA. Inclusion of the soluble fibrin substitute polylysine lowered the Km of human tPA by 30-fold (0.8 microM) but had no effect on the oocyte tPA Km. Polylysine had no significant effect on the Vmax values. The rate of plasminogen activation catalyzed by oocyte tPA was increased only 4.3-fold by fibrin while fibrin stimulated purified human tPA activity by 15.2-fold. After fractionation of oocyte extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polylysine enhanced oocyte tPA activity as seen by casein zymography. tPA activity in the conditioned medium of a rat insulinoma cell line was also not stimulated with polylysine prior to fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that extravascular cells which elaborate tPA may produce stimulatory factor(s) which allow for full tPA activity at physiological concentrations of plasminogen in the absence of fibrin. PMID:2491854

  16. Essential CDK1 inhibitory role for separase during meiosis I in vertebrate oocytes

    PubMed Central

    Gorr, Ingo H.; Reis, Alexandra; Boos, Dominik; Wühr, Martin; Madgwick, Suzanne; Jones, Keith T.; Stemmann, Olaf

    2008-01-01

    Separase not only triggers anaphase of meiosis I by proteolytic cleavage of cohesin on chromosome arms; in vitro vertebrate separase also acts as a direct inhibitor of cyclin-dependent kinase 1 (Cdk1) upon liberation from inhibitory securin. Blocking separase-Cdk1 complex formation by microinjection of anti-separase antibodies prevents polar body extrusion in vertebrate oocytes. Importantly, proper meiotic maturation is rescued by chemical inhibition of Cdk1 or expression of Cdk1-binding separase fragments lacking cohesin-cleaving activity. PMID:16906143

  17. Meiotic HORMA domain proteins prevent untimely centriole disengagement during Caenorhabditis elegans spermatocyte meiosis.

    PubMed

    Schvarzstein, Mara; Pattabiraman, Divya; Bembenek, Joshua N; Villeneuve, Anne M

    2013-03-01

    In many species where oocytes lack centrosomes, sperm contribute both genetic material and centriole(s) to the zygote. Correct centriole organization during male meiosis is critical to guarantee a normal bipolar mitotic spindle in the zygote. During Caenorhabditis elegans male meiosis, centrioles normally undergo two rounds of duplication, resulting in haploid sperm each containing a single tightly engaged centriole pair. Here we identify an unanticipated role for C. elegans HORMA (Hop1/Rev7/Mad2) domain proteins HTP-1/2 and HIM-3 in regulating centriole disengagement during spermatocyte meiosis. In him-3 and htp-1 htp-2 mutants, centrioles separate inappropriately during meiosis II, resulting in spermatids with disengaged centrioles. Moreover, extra centrosomes are detected in a subset of zygotes. Together, these data implicate HIM-3 and HTP-1/2 in preventing centriole disengagement during meiosis II. We showed previously that HTP-1/2 prevents premature loss of sister chromatid cohesion during the meiotic divisions by inhibiting removal of meiotic cohesin complexes containing the REC-8 subunit. Worms lacking REC-8, or expressing a mutant separase protein with elevated local concentration at centrosomes and in sperm, likewise exhibit inappropriate centriole separation during spermatocyte meiosis. These observations are consistent with HIM-3 and HTP-1/2 preventing centriole disengagement by inhibiting separase-dependent cohesin removal. Our data suggest that the same specialized meiotic mechanisms that function to prevent premature release of sister chromatid cohesion during meiosis I in C. elegans also function to inhibit centriole separation at meiosis II, thereby ensuring that the zygote inherits the appropriate complement of chromosomes and centrioles.

  18. The Chromosomal Passenger Complex Is Required for Meiotic Acentrosomal Spindle Assembly and Chromosome Biorientation

    PubMed Central

    Radford, Sarah J.; Jang, Janet K.; McKim, Kim S.

    2012-01-01

    DURING meiosis in the females of many species, spindle assembly occurs in the absence of the microtubule-organizing centers called centrosomes. In the absence of centrosomes, the nature of the chromosome-based signal that recruits microtubules to promote spindle assembly as well as how spindle bipolarity is established and the chromosomes orient correctly toward the poles is not known. To address these questions, we focused on the chromosomal passenger complex (CPC). We have found that the CPC localizes in a ring around the meiotic chromosomes that is aligned with the axis of the spindle at all stages. Using new methods that dramatically increase the effectiveness of RNA interference in the germline, we show that the CPC interacts with Drosophila oocyte chromosomes and is required for the assembly of spindle microtubules. Furthermore, chromosome biorientation and the localization of the central spindle kinesin-6 protein Subito, which is required for spindle bipolarity, depend on the CPC components Aurora B and Incenp. Based on these data we propose that the ring of CPC around the chromosomes regulates multiple aspects of meiotic cell division including spindle assembly, the establishment of bipolarity, the recruitment of important spindle organization factors, and the biorientation of homologous chromosomes. PMID:22865736

  19. Triploid planarian reproduces truly bisexually with euploid gametes produced through a different meiotic system between sex.

    PubMed

    Chinone, Ayako; Nodono, Hanae; Matsumoto, Midori

    2014-06-01

    Although polyploids are common among plants and some animals, polyploidization often causes reproductive failure. Triploids, in particular, are characterized by the problems of chromosomal pairing and segregation during meiosis, which may cause aneuploid gametes and results in sterility. Thus, they are generally considered to reproduce only asexually. In the case of the Platyhelminthes Dugesia ryukyuensis, populations with triploid karyotypes are normally found in nature as both fissiparous and oviparous triploids. Fissiparous triploids can also be experimentally sexualized if they are fed sexual planarians, developing both gonads and other reproductive organs. Fully sexualized worms begin reproducing by copulation rather than fission. In this study, we examined the genotypes of the offspring obtained by breeding sexualized triploids and found that the offspring inherited genes from both parents, i.e., they reproduced truly bisexually. Furthermore, meiotic chromosome behavior in triploid sexualized planarians differed significantly between male and female germ lines, in that female germ line cells remained triploid until prophase I, whereas male germ line cells appeared to become diploid before entry into meiosis. Oocytes at the late diplotene stage contained not only paired bivalents but also unpaired univalents that were suggested to produce diploid eggs if they remained in subsequent processes. Triploid planarians may therefore form euploid gametes by different meiotic systems in female and male germ lines and thus are be able to reproduce sexually in contrast to many other triploid organisms. PMID:24402417

  20. Development of the follicle complex and oocyte staging in red drum, Sciaenops ocellatus Linnaeus, 1776 (Perciformes, Sciaenidae).

    PubMed

    Grier, Harry J

    2012-08-01

    Pelagic egg development in red drum, Sciaenops ocellatus, is described using tiered staging. Based on mitosis and meiosis, there are five periods: Mitosis of Oogonia, Active Meiosis I, Arrested Meiosis I, Active Meiosis II, and Arrested Meiosis II. The Periods are divided into six stages: Mitotic Division of Oogonia, Chromatin Nucleolus, Primary Growth, Secondary Growth, Oocyte Maturation and Ovulation. The Chromatin Nucleolus Stage is divided into four steps: Leptotene, Zygotene, Pachytene, and Early Diplotene. Oocytes in the last step possess one nucleolus, dispersed chromatin with forming lampbrush chromosomes and lack basophilic ooplasm. The Primary Growth Stage, characterized by basophilic ooplasm and absence of yolk in oocytes, is divided into five steps: One-Nucleolus, Multiple Nucleoli, Perinucleolar, Oil Droplets, and Cortical Alveolar. During primary growth, the Balbiani body develops from nuage, enlarges and disperses throughout the ooplasm as both endoplasmic reticulum and Golgi develop within it. Secondary growth or vitellogenesis has three steps: Early Secondary Growth, Late Secondary Growth and Full-Grown. The Oocyte Maturation Stage, including ooplasmic and germinal vesicle maturation, has four steps: Eccentric Germinal Vesicle, Germinal Vesicle Migration, Germinal Vesicle Breakdown and Resumption of Meiosis when complete yolk hydration occurs. The period is Arrested Meiosis II. When folliculogenesis is completed, the ovarian follicle, an oocyte and encompassing follicle cells, is surrounded by a basement membrane and developing theca, all forming a follicle complex. After ovulation, a newly defined postovulatory follicle complex remains attached to the germinal epithelium. It is composed of a basement membrane that separates the postovulatory follicle from the postovulatory theca. Arrested Meiosis I encompasses primary and secondary growth (vitellogenesis) and includes most of oocyte maturation until the resumption of meiosis (Active Meiosis II

  1. The plant-specific protein FEHLSTART controls male meiotic entry, initializing meiotic synchronization in Arabidopsis.

    PubMed

    Li, Junhua; Dukowic-Schulze, Stefanie; Lindquist, Ingrid E; Farmer, Andrew D; Kelly, Bridget; Li, Tao; Smith, Alan G; Retzel, Ernest F; Mudge, Joann; Chen, Changbin

    2015-11-01

    Meiosis marks the transition from the sporophyte to the gametophyte generation in the life cycle of flowering plants, and creates genetic variations through homologous recombination. In most flowering plants, meiosis is highly synchronized within each anther, which is significant for efficient fertilization. To date, little is known about the molecular mechanisms of entry into meiosis and exit from it, and only a few genes in Arabidopsis have been characterized with a role in regulating meiotic progression. In this study, we report the functional characterization of a plant-specific basic helix-loop-helix (bHLH) protein, FEHLSTART (FST), a defect in which leads to premature meiotic entry and asynchronous meiosis, and results in decreased seed yield. Investigation of the time course of meiosis showed that the onset of leptotene, the first stage of prophase I, frequently occurred earlier in fst-1 than in the wild type. Asynchronous meiosis followed, which could manifest in the disruption of regular spindle structures and symmetric cell divisions in fst-1 mutants during the meiosis I/II transition. In accordance with frequently accelerated meiotic entry, whole-transcriptome analysis of fst-1 anthers undergoing meiosis revealed that 19 circadian rhythm genes were affected and 47 pollen-related genes were prematurely expressed at a higher level. Taken together, we propose that FST is required for normal meiotic entry and the establishment of meiotic synchrony. PMID:26382719

  2. Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation

    PubMed Central

    Handyside, Alan H; Montag, Markus; Magli, M Cristina; Repping, Sjoerd; Harper, Joyce; Schmutzler, Andreas; Vesela, Katerina; Gianaroli, Luca; Geraedts, Joep

    2012-01-01

    Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age in the decade preceding the menopause. Molecular genetic analysis following natural conception and spontaneous miscarriage demonstrates that trisomies arise mainly in female meiosis and particularly in the first meiotic division. Here, we studied copy number gains and losses for all chromosomes in the two by-products of female meiosis, the first and second polar bodies, and the corresponding zygotes in women of advanced maternal age undergoing IVF, using microarray comparative genomic hybridisation (array CGH). Analysis of the segregation patterns underlying the copy number changes reveals that premature predivision of chromatids rather than non-disjunction of whole chromosomes causes almost all errors in the first meiotic division and unlike natural conception, over half of aneuploidies result from errors in the second meiotic division. Furthermore, most abnormal zygotes had multiple aneuploidies. These differences in the aetiology of aneuploidy in IVF compared with natural conception may indicate a role for ovarian stimulation in perturbing meiosis in ageing oocytes. PMID:22317970

  3. A single bivalent efficiently inhibits cyclin B1 degradation and polar body extrusion in mouse oocytes indicating robust SAC during female meiosis I.

    PubMed

    Hoffmann, Steffen; Maro, Bernard; Kubiak, Jacek Z; Polanski, Zbigniew

    2011-01-01

    The Spindle Assembly Checkpoint (SAC) inhibits anaphase until microtubule-to-kinetochore attachments are formed, thus securing correct chromosome separation and preventing aneuploidy. Whereas in mitosis even a single unattached chromosome keeps the SAC active, the high incidence of aneuploidy related to maternal meiotic errors raises a concern about the lower efficiency of SAC in oocytes. Recently it was suggested that in mouse oocytes, contrary to somatic cells, not a single chromosome but a critical mass of chromosomes triggers efficient SAC pointing to the necessity of evaluating the robustness of SAC in oocytes. Two types of errors in chromosome segregation upon meiosis I related to SAC were envisaged: (1) SAC escape, when kinetochores emit SAC-activating signal unable to stop anaphase I; and (2) SAC deceive, when kinetochores do not emit the signal. Using micromanipulations and live imaging of the first polar body extrusion, as well as the dynamics of cyclin B1 degradation, here we show that in mouse oocytes a single bivalent keeps the SAC active. This is the first direct evaluation of SAC efficiency in mouse oocytes, which provides strong evidence that the robustness of SAC in mammalian oocytes is comparable to other cell types. Our data do not contradict the hypothesis of the critical mass of chromosomes necessary for SAC activation, but suggest that the same rule may govern SAC activity also in other cell types. We postulate that the innate susceptibility of oocytes to errors in chromosome segregation during the first meiotic division may not be caused by lower efficiency of SAC itself, but could be linked to high critical chromosome mass necessary to keep SAC active in oocyte of large size.

  4. Topoisomerase II Mediates Meiotic Crossover Interference

    PubMed Central

    Zhang, Liangran; Wang, Shunxin; Yin, Shen; Hong, Soogil; Kim, Keun P.; Kleckner, Nancy

    2014-01-01

    Summary Spatial patterning is a ubiquitous feature of biological systems. Meiotic crossovers provide an interesting example, defined by the classical phenomenon of crossover interference. Here, analysis of crossover patterns in budding yeast identifies a molecular pathway for interference. Topoisomerase II (Topo II) plays a central role, thus identifying a new function for this critical molecule. SUMOylation [of TopoII and axis component Red1] and ubiquitin-mediated removal of SUMOylated proteins are also required. These and other findings support the hypothesis that crossover interference involves accumulation, relief and redistribution of mechanical stress along the protein/DNA meshwork of meiotic chromosome axes, with TopoII required to adjust spatial relationships among DNA segments. PMID:25043020

  5. Drosophila PCH2 Is Required for a Pachytene Checkpoint That Monitors Double-Strand-Break-Independent Events Leading to Meiotic Crossover Formation

    PubMed Central

    Joyce, Eric F.; McKim, Kim S.

    2009-01-01

    During meiosis, programmed DNA double-strand breaks (DSBs) are repaired to create at least one crossover per chromosome arm. Crossovers mature into chiasmata, which hold and orient the homologous chromosomes on the meiotic spindle to ensure proper segregation at meiosis I. This process is usually monitored by one or more checkpoints that ensure that DSBs are repaired prior to the meiotic divisions. We show here that mutations in Drosophila genes required to process DSBs into crossovers delay two important steps in meiotic progression: a chromatin-remodeling process associated with DSB formation and the final steps of oocyte selection. Consistent with the hypothesis that a checkpoint has been activated, the delays in meiotic progression are suppressed by a mutation in the Drosophila homolog of pch2. The PCH2-dependent delays also require proteins thought to regulate the number and distribution of crossovers, suggesting that this checkpoint monitors events leading to crossover formation. Surprisingly, two lines of evidence suggest that the PCH2-dependent checkpoint does not reflect the accumulation of unprocessed recombination intermediates: the delays in meiotic progression do not depend on DSB formation or on mei-41, the Drosophila ATR homolog, which is required for the checkpoint response to unrepaired DSBs. We propose that the sites and/or conditions required to promote crossovers are established independently of DSB formation early in meiotic prophase. Furthermore, the PCH2-dependent checkpoint is activated by these events and pachytene progression is delayed until the DSB repair complexes required to generate crossovers are assembled. Interestingly, PCH2-dependent delays in prophase may allow additional crossovers to form. PMID:18957704

  6. A DNA topoisomerase VI-like complex initiates meiotic recombination.

    PubMed

    Vrielynck, Nathalie; Chambon, Aurélie; Vezon, Daniel; Pereira, Lucie; Chelysheva, Liudmila; De Muyt, Arnaud; Mézard, Christine; Mayer, Claudine; Grelon, Mathilde

    2016-02-26

    The SPO11 protein catalyzes the formation of meiotic DNA double strand breaks (DSBs) and is homologous to the A subunit of an archaeal topoisomerase (topo VI). Topo VI are heterotetrameric enzymes comprising two A and two B subunits; however, no topo VIB involved in meiotic recombination had been identified. We characterized a structural homolog of the archaeal topo VIB subunit [meiotic topoisomerase VIB-like (MTOPVIB)], which is essential for meiotic DSB formation. It forms a complex with the two Arabidopsis thaliana SPO11 orthologs required for meiotic DSB formation (SPO11-1 and SPO11-2) and is absolutely required for the formation of the SPO11-1/SPO11-2 heterodimer. These findings suggest that the catalytic core complex responsible for meiotic DSB formation in eukaryotes adopts a topo VI-like structure.

  7. The ATM Signaling Cascade Promotes Recombination-Dependent Pachytene Arrest in Mouse Spermatocytes

    PubMed Central

    Lange, Julian; Jasin, Maria; Keeney, Scott; Roig, Ignasi

    2015-01-01

    Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest

  8. Comparative importance of fatty acid beta-oxidation to nuclear maturation, gene expression, and glucose metabolism in mouse, bovine, and porcine cumulus oocyte complexes.

    PubMed

    Paczkowski, Melissa; Silva, Elena; Schoolcraft, William B; Krisher, Rebecca L

    2013-05-01

    The objective of these experiments was to evaluate the importance of fatty acid beta-oxidation (FAO) in the cumulus oocyte complex (COC) during in vitro maturation (IVM) to oocyte nuclear maturation and gene expression in both the oocyte and cumulus cells in three species with differing amounts of oocyte intracellular lipids (mouse, low; bovine, moderate; porcine, high). We inhibited FAO using etomoxir at 0, 10, 25, 100, or 250 μM. Completion of oocyte nuclear maturation was inhibited after COC exposure to 250 μM etomoxir in mouse oocytes, 100 μM etomoxir in bovine oocytes, and as little as 10 μM etomoxir in porcine oocytes (P < 0.05). When FAO was inhibited in mouse and porcine COCs resulting in inhibition of meiosis, the abundance of FAO, glycolytic, and oxidative stress gene transcripts were decreased in oocytes and cumulus cells (P < 0.05), although to a much greater extent in the pig. In bovine oocytes and cumulus cells, FAO gene transcripts were increased and glycolytic gene expression altered following meiotic inhibition due to etomoxir. Etomoxir, at doses that did not inhibit nuclear maturation in bovine and murine COCs, increased glucose consumption (P < 0.05), suggesting glucose metabolism is increased to meet the metabolic demands of the COCs when fatty acid metabolism is compromised. Our data demonstrates that FAO is essential to oocyte nuclear maturation in all three species. Sensitivity of nuclear maturation to FAO inhibition reflects the amount of lipid present in the ooplasm and may suggest a relative reliance on this metabolic pathway.

  9. Microinjection of follicle-enclosed mouse oocytes

    PubMed Central

    Jaffe, Laurinda A.; Norris, Rachael P.; Freudzon, Marina; Ratzan, William J.; Mehlmann, Lisa M.

    2011-01-01

    Summary The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment. PMID:19085139

  10. Microinjection of Follicle-Enclosed Mouse Oocytes

    NASA Astrophysics Data System (ADS)

    Jaffe, Laurinda A.; Norris, Rachael P.; Freudzon, Marina; Ratzan, William J.; Mehlmann, Lisa M.

    The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment.

  11. The meiotic transcriptome architecture of plants

    PubMed Central

    Dukowic-Schulze, Stefanie; Chen, Changbin

    2014-01-01

    Although a number of genes that play key roles during the meiotic process have been characterized in great detail, the whole process of meiosis is still not completely unraveled. To gain insight into the bigger picture, large-scale approaches like RNA-seq and microarray can help to elucidate the transcriptome landscape during plant meiosis, discover co-regulated genes, enriched processes, and highly expressed known and unknown genes which might be important for meiosis. These high-throughput studies are gaining more and more popularity, but their beginnings in plant systems reach back as far as the 1960's. Frequently, whole anthers or post-meiotic pollen were investigated, while less data is available on isolated cells during meiosis, and only few studies addressed the transcriptome of female meiosis. For this review, we compiled meiotic transcriptome studies covering different plant species, and summarized and compared their key findings. Besides pointing to consistent as well as unique discoveries, we finally draw conclusions what can be learned from these studies so far and what should be addressed next. PMID:24926296

  12. Developmental competence and gene expression of immature oocytes following liquid helium vitrification in bovine.

    PubMed

    Chen, Jun-Yi; Li, Xiao-Xia; Xu, Ya-Kun; Wu, Hua; Zheng, Jun-Jun; Yu, Xue-Li

    2014-12-01

    The objective of this study was to develop an effective ultra-rapid vitrification method and evaluate its effect on maturation, developmental competence and development-related gene expression in bovine immature oocytes. Bovine cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) liquid nitrogen vitrification, and (3) liquid helium vitrification. Oocytes were vitrified and then warmed, the percentage of morphologically normal oocytes in liquid helium group (89.0%) was significantly higher (P<0.05) than that of the liquid nitrogen group (81.1%). When the vitrified-thawed oocytes were matured in vitro for 24h, the maturation rate in liquid helium group (50.6%) was higher (P<0.05) than liquid nitrogen group (42.6%). Oocytes of liquid helium vitrification had higher cleavage and blastocyst rates (41.1% and 10.0%) than that of liquid nitrogen vitrification (33.0% and 4.5%; P<0.05) after in vitro fertilization. Moreover, the expression of GDF9 (growth/differentiation factor-9), BAX (apoptosis factor) and ZAR1 (zygote arrest 1) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) when the vitrified-thawed oocytes were matured 24h. The expression of these genes was altered after vitrification. Expression of GDF9 and BAX in the liquid helium vitrification group was not significantly different from that of the control, however there were significant differences between the liquid nitrogen vitrification group and control. In conclusion, it was feasible to use liquid helium for vitrifying bovine immature oocytes. There existed an association between the compromised developmental competence and the altered expression levels of these genes for the vitrified oocytes.

  13. Growth and fertilization of porcine fetal oocytes grafted under the renal capsules of nude mice.

    PubMed

    Kaneko, Hiroyuki; Kikuchi, Kazuhiro; Noguchi, Junko

    2016-10-15

    The fetal ovary contains a larger pool of oocytes than the adult ovary, but utilization of the fetal oocytes of large animals has hardly been examined. In this study, we investigated the developmental competence of oocytes grown in host mice harboring ovarian grafts obtained from fetal pigs. Ovarian fragments from fetuses at 55, 70, and 90 days postartificial insemination (dpi) were grafted into ovariectomized nude mice (Crlj:CD1-Foxn1(nu); 55-, 70- and 90-dpi groups, respectively). For comparison, ovarian fragments from 20-day postpartum (dpp) piglets were also grafted (20-dpp group). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 13 days by infusion to enhance their follicular development. In the fetal ovaries before grafting, the percentage of germ cells in primordial follicles (termed primordial oocytes) relative to the total number of germ cells was 0.06% at 55 dpi, 2.4% at 70 dpi, and 7.2% at 90 dpi, but the majority was contained within egg nests. At 20 dpp, primordial oocytes accounted for 91.7% of the total number of germ cells and the rest were mostly in primary follicles. After FSH stimulation of host mice, formation of antral follicles was promoted in the grafts of the 70- and 90-dpi groups as well as the 20-dpp group, but a very small number of antral follicles developed in the 55-dpi group consistent with the lowest (P < 0.05) levels of circulating inhibin in that group. The mean number of full-sized oocytes with meiotic competence recovered per mouse was 6.0 in the 70-dpi, 18.0 in the 90-dpi, and 21.2 in the 20-dpp groups, whereas virtually no oocytes were recovered from mice in the 55-dpi group. Moreover, the mature oocytes in the 70- and 90-dpi groups were fertilized in vitro, as shown by formation of male and female pronuclei, but the percentage of oocytes penetrated by sperm was low in the 70- (49%) and 90-dpi (29%) groups as compared with the 20-dpp group (88%). These results clearly

  14. Meiotic drive of chromosomal knobs reshaped the maize genome.

    PubMed Central

    Buckler, E S; Phelps-Durr, T L; Buckler, C S; Dawe, R K; Doebley, J F; Holtsford, T P

    1999-01-01

    Meiotic drive is the subversion of meiosis so that particular genes are preferentially transmitted to the progeny. Meiotic drive generally causes the preferential segregation of small regions of the genome; however, in maize we propose that meiotic drive is responsible for the evolution of large repetitive DNA arrays on all chromosomes. A maize meiotic drive locus found on an uncommon form of chromosome 10 [abnormal 10 (Ab10)] may be largely responsible for the evolution of heterochromatic chromosomal knobs, which can confer meiotic drive potential to every maize chromosome. Simulations were used to illustrate the dynamics of this meiotic drive model and suggest knobs might be deleterious in the absence of Ab10. Chromosomal knob data from maize's wild relatives (Zea mays ssp. parviglumis and mexicana) and phylogenetic comparisons demonstrated that the evolution of knob size, frequency, and chromosomal position agreed with the meiotic drive hypothesis. Knob chromosomal position was incompatible with the hypothesis that knob repetitive DNA is neutral or slightly deleterious to the genome. We also show that environmental factors and transposition may play a role in the evolution of knobs. Because knobs occur at multiple locations on all maize chromosomes, the combined effects of meiotic drive and genetic linkage may have reshaped genetic diversity throughout the maize genome in response to the presence of Ab10. Meiotic drive may be a major force of genome evolution, allowing revolutionary changes in genome structure and diversity over short evolutionary periods. PMID:10471723

  15. The relationship between chemically-induced meiotic delay and aneuploidy in mouse oocytes and zygotes

    SciTech Connect

    Mailhes, J.B.; Marchetti, F.

    1993-12-31

    Aneuploidy is a relatively common genetic disorder that results in human morbidity and mortality. Approximately 30% of embryonic and fetal deaths and 3.45 per thousand livebirths are associated with an abnormal number of chromosomes. Unfortunately, very little is known about the etiology and mechanism of chromosome missegregation. This situation dictates that considerable research be directed toward understanding the causes of aneuploidy. Although several hypotheses have been advanced for the etiology of aneuploidy, there still exists a paucity of information about the direct cuases and mechanisms of aneuploidy production. Without such specific knowledge, there is little hope of reducing the incidence of aneuploidy in humans. Some progress has been made. We now know that various chemicals can induce aneuploidy by interacting with certain cellular organelles, especially components of the spindle apparatus. These results have been demonstrated in various organisms and cell types both in vivo and in vitro. Since the ultimate objective of aneuploidy research is to obtain information that can be used to reduce the aneuploidy burden in humans, we have concentrated our research efforts on studying chemically-induced aneuploidy in mammalian germ cells and zygotes.

  16. Drosophila mus301/spindle-C Encodes a Helicase With an Essential Role in Double-Strand DNA Break Repair and Meiotic Progression

    PubMed Central

    McCaffrey, Ruth; St Johnston, Daniel; González-Reyes, Acaimo

    2006-01-01

    mus301 was identified independently in two genetic screens, one for mutants hypersensitive to chemical mutagens and another for maternal mutants with eggshell defects. mus301 is required for the proper specification of the oocyte and for progression through meiosis in the Drosophila ovary. We have cloned mus301 and show that it is a member of the Mus308 subfamily of ATP-dependent helicases and the closest homolog of human and mouse HEL308. Functional analyses demonstrate that Mus301 is involved in chromosome segregation in meiosis and in the repair of double-strand-DNA breaks in both meiotic and mitotic cells. Most of the oogenesis defects of mus301 mutants are suppressed by mutants in the checkpoint kinase Mei41 and in MeiW68, the Spo11 homolog that is thought to generate the dsDNA breaks that initiate recombination, indicating that these phenotypes are caused by activation of the DNA damage checkpoint in response to unrepaired Mei-W68-induced dsDNA breaks. However, neither mei-W68 nor mei-41 rescue the defects in oocyte specification of mus301 mutants, suggesting that this helicase has another function in oocyte selection that is independent from its role in meiotic recombination. PMID:16888338

  17. Membrane associated cancer-oocyte neoantigen SAS1B/ovastacin is a candidate immunotherapeutic target for uterine tumors.

    PubMed

    Pires, Eusebio S; D'Souza, Ryan S; Needham, Marisa A; Herr, Austin K; Jazaeri, Amir A; Li, Hui; Stoler, Mark H; Anderson-Knapp, Kiley L; Thomas, Theodore; Mandal, Arabinda; Gougeon, Alain; Flickinger, Charles J; Bruns, David E; Pollok, Brian A; Herr, John C

    2015-10-01

    The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathway, and tumor cell growth arrest induced by antibody-toxin conjugates suggest therapeutic approaches that would selectively target tumors while limiting adverse drug effects in healthy cells. The SAS1B metalloproteinase is proposed as a prototype cancer-oocyte tumor surface neoantigen for development of targeted immunotherapeutics with limited on-target/off tumor effects predicted to be restricted to the population of growing oocytes.

  18. Membrane associated cancer-oocyte neoantigen SAS1B/ovastacin is a candidate immunotherapeutic target for uterine tumors

    PubMed Central

    Pires, Eusebio S.; D'Souza, Ryan S.; Needham, Marisa A.; Herr, Austin K.; Jazaeri, Amir A.; Li, Hui; Stoler, Mark H.; Anderson-Knapp, Kiley L.; Thomas, Theodore; Mandal, Arabinda; Gougeon, Alain; Flickinger, Charles J.; Bruns, David E.; Pollok, Brian A.; Herr, John C.

    2015-01-01

    The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathway, and tumor cell growth arrest induced by antibody-toxin conjugates suggest therapeutic approaches that would selectively target tumors while limiting adverse drug effects in healthy cells. The SAS1B metalloproteinase is proposed as a prototype cancer-oocyte tumor surface neoantigen for development of targeted immunotherapeutics with limited on-target/off tumor effects predicted to be restricted to the population of growing oocytes. PMID:26327203

  19. Cardiac Arrest Resuscitation.

    PubMed

    Guyette, Francis X; Reynolds, Joshua C; Frisch, Adam

    2015-08-01

    Cardiac arrest is a dynamic disease that tests the multitasking and leadership abilities of emergency physicians. Providers must simultaneously manage the logistics of resuscitation while searching for the cause of cardiac arrest. The astute clinician will also realize that he or she is orchestrating only one portion of a larger series of events, each of which directly affects patient outcomes. Resuscitation science is rapidly evolving, and emergency providers must be familiar with the latest evidence and controversies surrounding resuscitative techniques. This article reviews evidence, discusses controversies, and offers strategies to provide quality cardiac arrest resuscitation.

  20. Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice.

    PubMed

    Bernstein, Lori R; Mackenzie, Amelia C L; Lee, Se-Jin; Chaffin, Charles L; Merchenthaler, István

    2016-03-01

    Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called "egg infertility." A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women. PMID:26713784

  1. Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice.

    PubMed

    Bernstein, Lori R; Mackenzie, Amelia C L; Lee, Se-Jin; Chaffin, Charles L; Merchenthaler, István

    2016-03-01

    Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called "egg infertility." A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women.

  2. Comparison of the effects of BPA and BPAF on oocyte spindle assembly and polar body release in mice.

    PubMed

    Nakano, Kei; Nishio, Manami; Kobayashi, Norio; Hiradate, Yuuki; Hoshino, Yumi; Sato, Eimei; Tanemura, Kentaro

    2016-04-01

    Bisphenol AF (BPAF), a homolog of bisphenol A (BPA), is a widely used environmental chemical that has adverse effects on reproduction. The aim of this study was to analyse the effects of BPA and BPAF exposure on oocyte maturation in vitro. Oocytes were cultured in the presence of BPA or BPAF (2, 20, 50 or 100 μg/ml) for 18 h. At concentrations of 50 and 100 μg/ml, BPA and BPAF inhibited oocyte maturation, with BPAF treatment causing a sharp decrease in the number of oocytes reaching maturity. Oocytes were exposed to BPA or BPAF at 2 μg/ml and cultured for different durations (6, 9, 12, 15 or 18 h). Both BPAF and BPA caused a cell cycle delay under these conditions. Oocytes cultured in the presence of BPA or BPAF (50 μg/ml) for 21 h were tested for the localization of α-tubulin and MAD2 using immunofluorescence. High concentrations of BPAF induced cell cycle arrest through the activation of the spindle assembly checkpoint. After 12 h of culture in BPAF (50 μg/ml), oocytes were transferred to control medium for 9 h. Only 63.3% oocytes treated in this manner progressed to metaphase II (MII). Oocytes exposed to high doses of BPA experienced a cell cycle delay, but managed to progress to MII when the culture period was prolonged. In addition, MAD2 was localized in the cytoplasm of these oocytes. In conclusion, both BPAF and BPA exposure affected oocyte maturation, however BPAF and BPA have differential effects on SAC activity.

  3. Making ICSI Safer and More Effective: A Review of the Human Oocyte and ICSI Practice.

    PubMed

    Simopoulou, Mara; Giannelou, Polina; Bakas, Panagiotis; Gkoles, Laertis; Kalampokas, Theodoros; Pantos, Konstantinos; Koutsilieris, Michael

    2016-01-01

    Intracytoplasmic sperm injection (ICSI) has become an indispensable procedure of every assisted reproduction unit. This has created as much controversy as it has awe. As this is a multistep invasive technique, every part of the procedure has become subject to investigation. We contribute this review aspiring to offer the embryologist insight into all available approaches of securing an effective ICSI practice. Herein we present all the different approaches with respect to handling of the human oocyte, taking into consideration the important steps of the technique such as the oocyte positioning, timing of performing ICSI, the option of viewing the meiotic spindle and further individual action such as artificial oocyte activation, rescue ICSI and in vitro maturation. We enrich this by including our view based on our collective experience and current practice. Published studies have led to various options for performing ICSI, resulting in in vitro fertilization units around the world adopting different approaches. The steps that ICSI technique entails discussed from the oocyte perspective are still a long way from being organized into one secure and optimal protocol. Our stressing of the need to secure an efficient ICSI protocol could be the trigger for further well-designed larger scale studies with all the latest technological advantages. We aim to approach this subject in categories and assess them separately. However, ICSI is a multifaceted procedure involving several consecutive steps and when evaluating one we cannot exclude the end effect of the previous, or the overall effect of the different practitioners involved form beginning to end.

  4. [Interchromatin granule clusters of mouse preovulatory oocytes, organization, molecular composition and possible functions].

    PubMed

    Pochukalina, G N; Bogoliubov, D S; Parfenov, V N

    2010-01-01

    At the diplotene stage of meiotic prophase, the nucleus of mouse preovulatory oocytes contains multiple interchromatin granule clusters (IGC). These nuclear compartments are universal and evolutionary conserved and enriched in pre-mRNA splicing factors. Nowadays, IGCs are believed to play an important role in gene expression events and contain different molecular components that allow coupling of many processes from transcription to mRNA export. We obtained the data on the distributions of poly(A)+RNA, hnRNPS A/B, and NXF1/TAP factor of mRNA export. These factors were found to associate with IGCs of mouse preovulatory oocytes. In the present study, we have demonstrated for the first time the dynamics of large IGCs after specific phosphorilation of SR-proteins with okadaic acid, an inhibitor of protein phosphatases. Using electron microscopy, conventional fluorescent and confocal microscopies, as well as microinjections of olygonucleotide probes in mouse oocytes, some features of structural organization and molecular compositions of IGCs in the nuclei of mouse oocyte from antral follicles were established. Possible roles of IGCs in pre-mRNA metabolism and the participation of these structures in mRNA export are discussed. PMID:20302015

  5. The roles of vitamin A for cytoplasmic maturation of bovine oocytes.

    PubMed

    Ikeda, Shuntaro; Kitagawa, Masayuki; Imai, Hiroshi; Yamada, Masayasu

    2005-02-01

    Vitamin A is one of the micronutrients which have been implicated in cattle reproduction. In cattle, ingested vitamin A, mainly as beta-carotene (BC) from forages and retinol ester from formula feed, is metabolized and transported to the oocytes and cumulus-granulosa cells in ovarian follicles through binding to various interacting molecules. The active form of vitamin A, retinoic acid (RA), functions as a regulator of gene expression in these targets. Early research showed the positive effects of vitamin A supplementation on bovine fertility in artificial insemination, and several studies on effects of vitamin A metabolites used in other artificial reproductive techniques (ART), including superovulation, ovum pick up, and in vitro maturation culture have provided evidence for the specific roles of vitamin A in oocyte cytoplasmic maturation (acquisition of developmental competence of oocytes during their meiotic maturation period for the embryonic development after fertilization). BC may enhance cytoplasmic maturation by its antioxidant properties which cannot be replaced by RA. Furthermore, RA may promote cytoplasmic maturation of bovine oocytes via its modulatory effects on the gene expression of gonadotrophin receptors, midkine, cyclooxygenase-2, and nitric oxide synthase in cumulus-granulosa cells. PMID:15750294

  6. The rat oocyte synthesises melatonin.

    PubMed

    Sakaguchi, Kenichiro; Itoh, Masanori T; Takahashi, Noriyuki; Tarumi, Wataru; Ishizuka, Bunpei

    2013-01-01

    Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine originally identified in the pineal gland, where it is synthesised enzymatically from serotonin (5-hydroxytryptamine) by the sequential action of arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT; also known as hydroxyindole O-methyltransferase). Melatonin directly affects ovarian functions and previous studies have suggested that melatonin is synthesised in the ovary. In the present study, we examined whether AANAT and ASMT are expressed in the adult rat ovary. Reverse transcription-polymerase chain reaction analyses demonstrated that both AANAT and ASMT mRNAs are expressed in the ovary. Western blotting for AANAT protein showed that the ovary, like the pineal gland, contains this enzymatic protein with a molecular mass of 24kDa. Immunohistochemistry revealed that the AANAT protein is localised to the oocyte, corpus luteum and medulla, including mast cells. AANAT protein was found in oocytes at all stages of follicular development, and its levels in oocytes increased progressively throughout follicular development. Furthermore, isolated oocytes metabolised exogenous serotonin to melatonin. These findings demonstrate that melatonin is synthesised from serotonin in oocytes. Melatonin synthesised in the oocyte may be implicated in its own growth or maturation, for example, by acting as a calmodulin antagonist or an antioxidant.

  7. Intrinsic quality of goat oocytes already found denuded at collection for in vitro embryo production.

    PubMed

    Souza-Fabjan, Joanna Maria Gonçalves; Locatelli, Yann; Duffard, Nicolas; Corbin, Emilie; Batista, Ribrio Ivan Tavares Pereira; de Figueirêdo Freitas, Vicente José; Beckers, Jean-François; Mermillod, Pascal

    2016-11-01

    Although cumulus cells are essential for efficient oocyte maturation, the establishment of protocols that support IVD of embryos obtained from denuded oocytes (DOCs) is important for optimizing the use of reproductive biotechnologies. Thus, this study aimed to establish a protocol for IVD of goat DOC using different strategies of IVM and methods of oocyte activation. Four experiments were performed. Similar developmental competence of slaughterhouse DOC was obtained, regardless of maturation media (complex, semidefined or simplified). However, the ability to reach the blastocyst stage was affected by the activation method. Denuded oocytes subjected to parthenogenetic activation had greater (P < 0.05) development capacity, compared with those undergoing IVF with average cleavage rate of 83% and 75%, blastocyst rate of 49% and 28%, and blastocysts in relation to the cleaved embryos of 59% and 38, respectively. In addition, the quality of embryos evaluated after vitrification/warming was similar between parthenogenetic activation and IVF. Finally, we demonstrated that the coculture of cumulus-oocyte complex (COC) with DOC increased the competence of DOC at a ratio of 1:1 and 1:9 (DOC:COC). We believe that presence of cumulus cells (CCs) is not essential to the meiotic maturation, if at the time of removal of the oocyte from follicular environment, they already acquired competence to development. However, when the oocytes still need to acquire competence, the presence of CC may significantly contribute in their developmental capacity acquisition during IVM. Thus, regardless of the source, these oocytes will require longer time in IVM, contrary to what happens in the absence of CC. In conclusion, although DOC had a lower developmental potential, especially after IVF, they were able to produce blastocysts and the coculture of DOC with COC increased this developmental capacity. PMID:27453560

  8. Intrinsic quality of goat oocytes already found denuded at collection for in vitro embryo production.

    PubMed

    Souza-Fabjan, Joanna Maria Gonçalves; Locatelli, Yann; Duffard, Nicolas; Corbin, Emilie; Batista, Ribrio Ivan Tavares Pereira; de Figueirêdo Freitas, Vicente José; Beckers, Jean-François; Mermillod, Pascal

    2016-11-01

    Although cumulus cells are essential for efficient oocyte maturation, the establishment of protocols that support IVD of embryos obtained from denuded oocytes (DOCs) is important for optimizing the use of reproductive biotechnologies. Thus, this study aimed to establish a protocol for IVD of goat DOC using different strategies of IVM and methods of oocyte activation. Four experiments were performed. Similar developmental competence of slaughterhouse DOC was obtained, regardless of maturation media (complex, semidefined or simplified). However, the ability to reach the blastocyst stage was affected by the activation method. Denuded oocytes subjected to parthenogenetic activation had greater (P < 0.05) development capacity, compared with those undergoing IVF with average cleavage rate of 83% and 75%, blastocyst rate of 49% and 28%, and blastocysts in relation to the cleaved embryos of 59% and 38, respectively. In addition, the quality of embryos evaluated after vitrification/warming was similar between parthenogenetic activation and IVF. Finally, we demonstrated that the coculture of cumulus-oocyte complex (COC) with DOC increased the competence of DOC at a ratio of 1:1 and 1:9 (DOC:COC). We believe that presence of cumulus cells (CCs) is not essential to the meiotic maturation, if at the time of removal of the oocyte from follicular environment, they already acquired competence to development. However, when the oocytes still need to acquire competence, the presence of CC may significantly contribute in their developmental capacity acquisition during IVM. Thus, regardless of the source, these oocytes will require longer time in IVM, contrary to what happens in the absence of CC. In conclusion, although DOC had a lower developmental potential, especially after IVF, they were able to produce blastocysts and the coculture of DOC with COC increased this developmental capacity.

  9. Cytoskeletal and mitochondrial properties of bovine oocytes obtained by Ovum Pick-Up: the effects of follicle stimulation and in vitro maturation.

    PubMed

    Somfai, Tamás; Matoba, Satoko; Inaba, Yasushi; Nakai, Michiko; Imai, Kei; Nagai, Takashi; Geshi, Masaya

    2015-12-01

    Follicle stimulation by follicular stimulating hormone (FSH) is known to improve developmental competence of bovine oocytes obtained by Ovum Pick-Up (OPU); however, the exact factors in oocytes affected by this treatment have remained unclear. We compared in vitro matured (IVM) oocytes obtained at the immature stage from cows by OPU either without or with stimulation with FSH (non-stimulated and stimulated OPU, respectively) to those obtained by superstimulation and in vivo maturation in terms of cytoskeleton morphology, mitochondrial distribution, intracellular adenosine triphosphate (ATP) content and H2 O2 levels at the metaphase-II stage and intracellular Ca(2+) levels after in vitro fertilization (IVF). Confocal microscopy after immunostaining revealed reduced size of the meiotic spindle, associated with increased tendencies of microfilament degradation and insufficient mitochondrial re-distribution in non-stimulated OPU-derived IVM oocytes compared with those collected by stimulated OPU, which in turn resembled in vivo matured oocytes. However, there was no difference in mitochondrial functions between oocytes obtained by stimulated or non-stimulated OPU in terms of ATP content, cytoplasmic H2 O2 levels, base Ca(2+) levels and the frequencies and amplitudes of Ca(2+) oscillations after IVF. Larger size of metaphase spindles in oocytes obtained by stimulated OPU may reflect and potentially contribute to their high developmental competence.

  10. Nanoliter droplet vitrification for oocyte cryopreservation

    PubMed Central

    Zhang, Xiaohui; Khimji, Imran; Shao, Lei; Safaee, Hooman; Desai, Khanjan; Keles, Hasan Onur; Gurkan, Umut Atakan; Kayaalp, Emre; Nureddin, Aida; Anchan, Raymond M; Maas, Richard L; Demirci, Utkan

    2011-01-01

    Aim Oocyte cryopreservation remains largely experimental, with live birth rates of only 2–4% per thawed oocyte. In this study, we present a nanoliter droplet technology for oocyte vitrification. Materials & methods An ejector-based droplet vitrification system was designed to continuously cryopreserve oocytes in nanoliter droplets. Oocyte survival rates, morphologies and parthenogenetic development after each vitrification step were assessed in comparison with fresh oocytes. Results Oocytes were retrieved after cryoprotectant agent loading/unloading, and nanoliter droplet encapsulation showed comparable survival rates to fresh oocytes after 24 h in culture. Also, oocytes recovered after vitrification/thawing showed similar morphologies to those of fresh oocytes. Additionally, the rate of oocyte parthenogenetic activation after nanoliter droplet encapsulation was comparable with that observed for fresh oocytes. This nanoliter droplet technology enables the vitrification of oocytes at higher cooling and warming rates using lower cryoprotectant agent levels (i.e., 1.4 M ethylene glycol, 1.1 M dimethyl sulfoxide and 1 M sucrose), thus making it a potential technology to improve oocyte cryopreservation outcomes. PMID:22188180

  11. Spatiotemporal regulation of meiotic recombination by Liaisonin.

    PubMed

    Miyoshi, Tomoichiro; Ito, Masaru; Ohta, Kunihiro

    2013-01-01

    Sexual reproduction involves diversification of genetic information in successive generations. Meiotic recombination, which substantially contributes to the increase in genetic diversity, is initiated by programmed DNA double-strand breaks (DSBs) catalyzed by the evolutionarily conserved Spo11 protein. Spo11 requires additional partner proteins for its DNA cleavage reaction. DSBs are preferentially introduced at defined chromosomal sites called "recombination hotspots." Recent studies have revealed that meiotically established higher-order chromosome structures, such as chromosome axes and loops, are also crucial in the control of DSB formation. Most of the DSB sites are located within chromatin loop regions, while many of the proteins involved in DSB formation reside on chromosomal axes. Hence, DSB proteins and DSB sites seem to be distantly located. To resolve this paradox, we conducted comprehensive proteomics and ChIP-chip analyses on Spo11 partners in Schizosaccharomyces pombe, in combination with mutant studies. We identified two distinct DSB complexes, the "DSBC (DSB Catalytic core)" and "SFT (Seven-Fifteen-Twenty four; Rec7-Rec15-Rec24)" subcomplexes. The DSBC subcomplex contains Spo11 and functions as the catalytic core for the DNA cleavage reaction. The SFT subcomplex is assumed to execute regulatory functions. To activate the DSBC subcomplex, the SFT subcomplex tethers hotspots to axes via its interaction with Mde2, which can interact with proteins in both DSBC and SFT subcomplexes. Thus, Mde2 is likely to bridge these two subcomplexes, forming a "tethered loop-axis complex." It should be noted that Mde2 expression is strictly regulated by S phase checkpoint monitoring of the completion of DNA replication. From these observations, we proposed that Mde2 is a central coupler for meiotic recombination initiation to establish a tethered loop-axis complex in liaison with the S phase checkpoint.

  12. Activin A accelerates the progression of fetal oocytes throughout meiosis and early oogenesis in the mouse.

    PubMed

    Liang, Gui-Jin; Zhang, Xi-Feng; Wang, Jun-Jie; Sun, Yuan-Chao; Sun, Xiao-Feng; Cheng, Shun-Feng; Li, Lan; De Felici, Massimo; Shen, Wei

    2015-10-15

    Activins can exert several roles in ovary development. However, little is known about their involvement in early mammalian oogenesis. In this study, we reported that activin receptors (including ActRIA, ActRIB, ActRIIA, and ActRIIB) are expressed throughout the development of the mouse ovaries from 12.5 days postcoitum (dpc) to 21 days postparturition (dpp). Moreover, we found that in vitro, the addition of activin A (ActA) to the culture medium of 12.5 dpc ovarian tissues accelerated the progression of oocytes throughout meiotic prophase I stages. This result was reproduced in vivo following administration of ActA to pregnant mice. The in vitro effect of ActA was associated with increased expression of premeiotic and meiotic genes (including Dazl, Spo11, Stra8, Scp3, and Rec8) in the ovarian tissues. Mechanistically, ActA-dependent SMAD3 signaling modulated the expression of members of the retinoic acid (RA) system, including the RA degradation CYP26B1 enzyme and the RA receptors. Finally, ActA promoted the survival and growth of fetal and early postnatal oocytes and primordial follicle assembly both in vitro and in vivo. In conclusion, the present study identifies new roles of ActA in early oogenesis and suggested that ActA and RA might cooperate in promoting meiosis in female germ cells.

  13. Oocyte aging-induced Neuronatin (NNAT) hypermethylation affects oocyte quality by impairing glucose transport in porcine

    PubMed Central

    Gao, Ying-Ying; Chen, Li; Wang, Tao; Nie, Zheng-Wen; Zhang, Xia; Miao, Yi-Liang

    2016-01-01

    DNA methylation plays important roles in regulating many physiological behaviors; however, few studies were focused on the changes of DNA methylation during oocyte aging. Early studies showed that some imprinted genes’ DNA methylation had been changed in aged mouse oocytes. In this study, we used porcine oocytes to test the hypothesis that oocyte aging would alter DNA methylation pattern of genes and disturb their expression in age oocytes, which affected the developmental potential of oocytes. We compared several different types of genes and found that the expression and DNA methylation of Neuronatin (NNAT) were disturbed in aged oocytes significantly. Additional experiments demonstrated that glucose transport was impaired in aged oocytes and injection of NNAT antibody into fresh oocytes led to the same effects on glucose transport. These results suggest that the expression of NNAT was declined by elevating DNA methylation, which affected oocyte quality by decreasing the ability of glucose transport in aged oocytes. PMID:27782163

  14. Recent progress in reproduction of whale oocytes.

    PubMed

    Zheng, Yue-Liang

    2013-08-01

    Whale oocytes recovered from follicles can be matured in vitro. Whale sperm and mature oocytes can be used for in vitro fertilization (IVF), and IVF embryos have the ability to develop to morula stage. Whale sperm injected into bovine or mouse oocytes can activate the oocytes and form pronucleus. Interspecies somatic cell nuclear transfer embryos have been reconstructed with whale somatic cell nucleus and enucleated bovine or porcine oocytes, and interspecies cloned embryos can develop in vitro. This paper reviews recent progress in maturation, fertilization and development of whale oocytes.

  15. Patients with polycystic ovary syndrome have successful embryo arrest

    PubMed Central

    Yin, Baoli; Hao, Haoying; Wei, Duo; Song, Xiaobing; Xie, Juanke; Zhang, Cuilian

    2015-01-01

    In this retrospective study, we investigate the relationship between embryo arrest and polycystic ovary syndrome (PCOS) during in vitro fertilization-embryo transfer (IVF-ET). In this study, 667 subjects were enrolled, including 330 patients with PCOS and 337 subjects without PCOS. The subjects underwent in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET) cycles at the Reproductive Medical Centre of Henan Provincial Hospital from January 2009 to December 2012. Four protocols were used to stimulate the ovaries, including long protocol, super-long down-regulation protocol, short protocol and antagonist protocol. Oocytes were retrieved using transvaginal ultrasound guidance. Pronuclei were checked on the next morning after IVF/ICSI. Cleavage stage embryo was assessed after 62-66 hours. Women with PCOS had significantly elevated body mass index, basal luteinizing hormone, estradiol and testosterone compared with normal women. Basal Follicle stimulating hormone level in PCOS patients was lower compared with that in control group. After IVF-ET, PCOS patients had more available oocytes than subjects in control group. PCOS patients had slightly lower fertilization rate than the controls in IVF cycles, but in ICSI cycles, fertilization rate in PCOS patients was significantly higher than that in controls. For either IVF or ICSI, the embryo arrest rate was not changed by PCOS. Moreover, there was no significant difference in embryo arrest rate between both groups adopting different stimulation protocols. Interestingly, embryo arrest rate was not correlated with testosterone for patients in PCOS group. The data indicated that patients with PCOS had successful early embryo arrest during IVF-ET. PMID:26131233

  16. Prometaphase APCcdh1 activity prevents non-disjunction in mammalian oocytes

    PubMed Central

    Reis, Alexandra; Madgwick, Suzanne; Chang, Heng-Yu; Nabti, Ibtissem; Levasseur, Mark; Jones, Keith T

    2008-01-01

    Summary The first female meiotic division (MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy loss1. Here, we show a fundamental difference in the control of mammalian meiosis which may underlie such susceptibility. It involved a reversal in the well-established timing of activation of the Anaphase-Promoting Complex (APC)2, 3 by its co-activators cdc20 and cdh1. APCcdh1 was active first, during prometaphase I, and was needed in order to allow homologue congression, since loss of cdh1 speeded up MI, leading to premature chromosome segregation and a non-disjunction phenotype. APCcdh1 targeted cdc20 for degradation but not securin and cyclin B1. These were degraded later in MI through APCcdc20, making cdc20 re-synthesis essential for successful meiotic progression. The switch from APCcdh1 to APCcdc20 activity was controlled by increasing CDK1 and cdh1 loss. These findings demonstrate a fundamentally different mechanism of control for the first meiotic division in mammalian oocytes not observed in meioses of other species. PMID:17891138

  17. Prometaphase APCcdh1 activity prevents non-disjunction in mammalian oocytes.

    PubMed

    Reis, Alexandra; Madgwick, Suzanne; Chang, Heng-Yu; Nabti, Ibtissem; Levasseur, Mark; Jones, Keith T

    2007-10-01

    The first female meiotic division (meiosis I, MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy loss. Here, we show a fundamental difference in the control of mammalian meiosis that may underlie such susceptibility. It involves a reversal in the well-established timing of activation of the anaphase-promoting complex (APC) by its co-activators cdc20 and cdh1. APC(cdh1) was active first, during prometaphase I, and was needed in order to allow homologue congression, as loss of cdh1 speeded up MI, leading to premature chromosome segregation and a non-disjunction phenotype. APC(cdh1) targeted cdc20 for degradation, but did not target securin or cyclin B1. These were degraded later in MI through APC(cdc20), making cdc20 re-synthesis essential for successful meiotic progression. The switch from APC(cdh1) to APC(cdc20) activity was controlled by increasing CDK1 and cdh1 loss. These findings demonstrate a fundamentally different mechanism of control for the first meiotic division in mammalian oocytes that is not observed in meioses of other species. PMID:17891138

  18. Meiotic wave adds extra asymmetry to the development of female chicken gonads.

    PubMed

    de Melo Bernardo, Ana; Heeren, A Marijne; van Iperen, Liesbeth; Fernandes, Maria Gomes; He, Nannan; Anjie, Stafford; Noce, Toshiaki; Ramos, Ester Silveira; de Sousa Lopes, Susana M Chuva

    2015-10-01

    Development of female gonads in the chicken is asymmetric. This asymmetry affects gene expression, morphology, and germ cell development; consequently only the left ovary develops into a functional organ, whereas the right ovary remains vestigial. In males, on the other hand, both gonads develop into functional testes. Here, we revisited the development of asymmetric traits in female (and male) chicken gonads between Hamburger Hamilton stage 16 (HH16) and hatching. At HH16, primordial germ cells migrated preferentially to the left gonad, accumulating in the left coelomic hinge between the gut mesentery and developing gonad in both males and females. Using the meiotic markers SYCP3 and phosphorylated H2AFX, we identified a previously undescribed, pronounced asymmetryc meiotic progression in the germ cells located in the central, lateral, and extreme cortical regions of the left female gonad from HH38 until hatching. Moreover, we observed that--in contrast to the current view--medullary germ cells are not apoptotic, but remain arrested in pre-leptotene until hatching. In addition to the systematic analysis of the asymmetric distribution of germ cells in female chicken gonads, we propose an updated model suggesting that the localization of germ cells--in the left or right gonad; in the cortex or medulla of the left gonad; and in the central part or the extremities of the left cortex--has direct consequences for their development and participation in adult reproduction. PMID:26096940

  19. CDK-1 inhibits meiotic spindle shortening and dynein-dependent spindle rotation in C. elegans.

    PubMed

    Ellefson, Marina L; McNally, Francis J

    2011-06-27

    In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B-CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I-arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5-ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.

  20. Mitoguardin-1 and -2 promote maturation and the developmental potential of mouse oocytes by maintaining mitochondrial dynamics and functions.

    PubMed

    Liu, Xiao-Man; Zhang, Yong-Ping; Ji, Shu-Yan; Li, Bo-Tai; Tian, Xuejun; Li, Dali; Tong, Chao; Fan, Heng-Yu

    2016-01-12

    Mitochondrial dynamics change mitochondrial morphological features and numbers as a part of adaptive cellular metabolism, which is vital for most eukaryotic cells and organisms. A disease or even death of an animal can occur if these dynamics are disrupted. Using large-scale genetic screening in fruit flies, we previously found the gene mitoguardin (Miga), which encodes a mitochondrial outer-membrane protein and promotes mitochondrial fusion. Knockout mouse strains were generated for the mammalian Miga homologs Miga1 and Miga2. Miga1/2-/- females show greatly reduced quality of oocytes and early embryos and are subfertile. Mitochondria became clustered in the cytoplasm of oocytes from the germinal-vesicle stage to meiosis II; production of reactive oxygen species increased in mitochondria and caused damage to mitochondrial ultrastructures. Additionally, reduced ATP production, a decreased mitochondrial-DNA copy number, and lower mitochondrial membrane potential were detected in Miga1/2-/- oocytes during meiotic maturation. These changes resulted in low rates of polar-body extrusion during oocyte maturation, reduced developmental potential of the resulting early embryos, and consequently female subfertility. We provide direct evidence that MIGA1/2-regulated mitochondrial dynamics is crucial for mitochondrial functions, ensure oocyte maturation, and maintain the developmental potential. PMID:26716412

  1. Mitoguardin-1 and -2 promote maturation and the developmental potential of mouse oocytes by maintaining mitochondrial dynamics and functions

    PubMed Central

    Liu, Xiao-Man; Zhang, Yong-Ping; Ji, Shu-Yan; Li, Bo-Tai; Tian, Xuejun; Li, Dali; Tong, Chao; Fan, Heng-Yu

    2016-01-01

    Mitochondrial dynamics change mitochondrial morphological features and numbers as a part of adaptive cellular metabolism, which is vital for most eukaryotic cells and organisms. A disease or even death of an animal can occur if these dynamics are disrupted. Using large-scale genetic screening in fruit flies, we previously found the gene mitoguardin (Miga), which encodes a mitochondrial outer-membrane protein and promotes mitochondrial fusion. Knockout mouse strains were generated for the mammalian Miga homologs Miga1 and Miga2. Miga1/2−/− females show greatly reduced quality of oocytes and early embryos and are subfertile. Mitochondria became clustered in the cytoplasm of oocytes from the germinal-vesicle stage to meiosis II; production of reactive oxygen species increased in mitochondria and caused damage to mitochondrial ultrastructures. Additionally, reduced ATP production, a decreased mitochondrial-DNA copy number, and lower mitochondrial membrane potential were detected in Miga1/2−/− oocytes during meiotic maturation. These changes resulted in low rates of polar-body extrusion during oocyte maturation, reduced developmental potential of the resulting early embryos, and consequently female subfertility. We provide direct evidence that MIGA1/2-regulated mitochondrial dynamics is crucial for mitochondrial functions, ensure oocyte maturation, and maintain the developmental potential. PMID:26716412

  2. Combining Microinjection and Immunoblotting to Analyze MAP Kinase Phosphorylation in Single Starfish Oocytes and Eggs

    NASA Astrophysics Data System (ADS)

    Carroll, David J.; Hua, Wei

    The starfish oocyte has proven useful for studies involving microinjection because it is relatively large (190 μm) and optically clear. These oocytes are easily obtained from the ovary arrested at prophase of meiosis I, making them useful as a model system for the study of cell cycle-related events. In this chapter, a method for combining microinjection with immunoblotting of single cells is described. Individual starfish oocytes are injected, removed from the microinjection chamber, and analyzed by immunoblotting for the dual-phosphorylated form of mitogen-activated protein kinase (MAPK). This method will allow for experiments testing the regulation of MAPK in single cells and for the manipulation of these cells by a quantitative microinjection technique.

  3. Meiotic drive and evolution of female choice.

    PubMed

    Reinhold, K; Engqvist, L; Misof, B; Kurtz, J

    1999-07-01

    As a special version of the good-genes hypothesis, it was recently proposed that females could benefit from choosing drive-resistant males in a meiotic drive system. Here, we examine with a three-locus, six-allele population genetic model whether female choice for drive resistance can evolve. An allele leading to female preference for drive-resistant males was introduced at low frequency into a population polymorphic for meiotic drive and drive resistance. Our simulations show that female choice of drive-resistant males is disadvantageous when resistance is Y-linked. This disadvantage occurs because, at equilibrium, drive-resistant males have lower reproductive success than drive-susceptible males. Thus, female choice of drive-susceptible males can evolve when resistance is Y-linked. When resistance is autosomal, selection on female choice for drive resistance is less strong and depends on the frequency of choice: female preference of resistant males is favoured when choice is rare and disadvantageous when choice is frequent, leading to a stable equilibrium at a low frequency of the choice allele. Independent of the location of drive resistance alleles, males with the non-driving allele always have above average reproductive success. Female choice is therefore beneficial when choosy females prefer males with the non-driving allele.

  4. Meiotic drive and evolution of female choice.

    PubMed Central

    Reinhold, K; Engqvist, L; Misof, B; Kurtz, J

    1999-01-01

    As a special version of the good-genes hypothesis, it was recently proposed that females could benefit from choosing drive-resistant males in a meiotic drive system. Here, we examine with a three-locus, six-allele population genetic model whether female choice for drive resistance can evolve. An allele leading to female preference for drive-resistant males was introduced at low frequency into a population polymorphic for meiotic drive and drive resistance. Our simulations show that female choice of drive-resistant males is disadvantageous when resistance is Y-linked. This disadvantage occurs because, at equilibrium, drive-resistant males have lower reproductive success than drive-susceptible males. Thus, female choice of drive-susceptible males can evolve when resistance is Y-linked. When resistance is autosomal, selection on female choice for drive resistance is less strong and depends on the frequency of choice: female preference of resistant males is favoured when choice is rare and disadvantageous when choice is frequent, leading to a stable equilibrium at a low frequency of the choice allele. Independent of the location of drive resistance alleles, males with the non-driving allele always have above average reproductive success. Female choice is therefore beneficial when choosy females prefer males with the non-driving allele. PMID:10445289

  5. Transgenic cattle produced by reverse-transcribed gene transfer in oocytes

    PubMed Central

    Chan, Anthony W. S.; Homan, E. Jane; Ballou, Linda U.; Burns, Jane C.; Bremel, Robert D.

    1998-01-01

    A critical requirement for integration of retroviruses, other than HIV and possibly related lentiviruses, is the breakdown of the nuclear envelope during mitosis. Nuclear envelope breakdown occurs during mitotic M-phase, the envelope reforming immediately after cell division, thereby permitting the translocation of the retroviral preintegration complex into the nucleus and enabling integration to proceed. In the oocyte, during metaphase II (MII) of the second meiosis, the nuclear envelope is also absent and the oocyte remains in MII arrest for a much longer period of time compared with M-phase in a somatic cell. Pseudotyped replication-defective retroviral vector was injected into the perivitelline space of bovine oocytes during MII. We show that reverse-transcribed gene transfer can take place in an oocyte in MII arrest of meiosis, leading to production of offspring, the majority of which are transgenic. We discuss the implications of this mechanism both as a means of production of transgenic livestock and as a model for naturally occurring recursive transgenesis. PMID:9826647

  6. Unique geometry of sister kinetochores in human oocytes during meiosis I may explain maternal age-associated increases in chromosomal abnormalities

    PubMed Central

    Patel, Jessica; Tan, Seang Lin; Hartshorne, Geraldine M.; McAinsh, Andrew D.

    2016-01-01

    ABSTRACT The first meiotic division in human oocytes is highly error-prone and contributes to the uniquely high incidence of aneuploidy observed in human pregnancies. A successful meiosis I (MI) division entails separation of homologous chromosome pairs and co-segregation of sister chromatids. For this to happen, sister kinetochores must form attachments to spindle kinetochore-fibres emanating from the same pole. In mouse and budding yeast, sister kinetochores remain closely associated with each other during MI, enabling them to act as a single unified structure. However, whether this arrangement also applies in human meiosis I oocytes was unclear. In this study, we perform high-resolution imaging of over 1900 kinetochores in human oocytes, to examine the geometry and architecture of the human meiotic kinetochore. We reveal that sister kinetochores in MI are not physically fused, and instead individual kinetochores within a pair are capable of forming independent attachments to spindle k-fibres. Notably, with increasing female age, the separation between kinetochores increases, suggesting a degradation of centromeric cohesion and/or changes in kinetochore architecture. Our data suggest that the differential arrangement of sister kinetochores and dual k-fibre attachments may explain the high proportion of unstable attachments that form in MI and thus indicate why human oocytes are prone to aneuploidy, particularly with increasing maternal age. PMID:26718930

  7. Unique geometry of sister kinetochores in human oocytes during meiosis I may explain maternal age-associated increases in chromosomal abnormalities.

    PubMed

    Patel, Jessica; Tan, Seang Lin; Hartshorne, Geraldine M; McAinsh, Andrew D

    2015-12-30

    The first meiotic division in human oocytes is highly error-prone and contributes to the uniquely high incidence of aneuploidy observed in human pregnancies. A successful meiosis I (MI) division entails separation of homologous chromosome pairs and co-segregation of sister chromatids. For this to happen, sister kinetochores must form attachments to spindle kinetochore-fibres emanating from the same pole. In mouse and budding yeast, sister kinetochores remain closely associated with each other during MI, enabling them to act as a single unified structure. However, whether this arrangement also applies in human meiosis I oocytes was unclear. In this study, we perform high-resolution imaging of over 1900 kinetochores in human oocytes, to examine the geometry and architecture of the human meiotic kinetochore. We reveal that sister kinetochores in MI are not physically fused, and instead individual kinetochores within a pair are capable of forming independent attachments to spindle k-fibres. Notably, with increasing female age, the separation between kinetochores increases, suggesting a degradation of centromeric cohesion and/or changes in kinetochore architecture. Our data suggest that the differential arrangement of sister kinetochores and dual k-fibre attachments may explain the high proportion of unstable attachments that form in MI and thus indicate why human oocytes are prone to aneuploidy, particularly with increasing maternal age.

  8. The Dynamics of Connexin Expression, Degradation and Localisation Are Regulated by Gonadotropins during the Early Stages of In Vitro Maturation of Swine Oocytes

    PubMed Central

    Santiquet, Nicolas; Robert, Claude; Richard, François J.

    2013-01-01

    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. PMID:23861906

  9. A new light on the meiotic DSB catalytic complex.

    PubMed

    Robert, Thomas; Vrielynck, Nathalie; Mézard, Christine; de Massy, Bernard; Grelon, Mathilde

    2016-06-01

    Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs). More than 15 years ago, Spo11 was identified as the protein responsible for meiotic DSB formation, notably because of its striking similarities with the A subunit of topoisomerase VI (TopoVI). TopoVI are enzymes that modify DNA topology by generating transient DSBs and are active as heterotetramers, composed of two A and two B subunits. A2 dimers catalyse the DNA cleavage reaction, whereas the B subunits regulate A2 conformation, DNA capture, cleavage and re-ligation. The recent identification in plants and mammals of a B-like TopoVI subunit that interacts with SPO11 and is required for meiotic DSB formation makes us to reconsider our understanding of the meiotic DSB catalytic complex. We provide here an overview of the knowledge on TopoVI structure and mode of action and we compare them with their meiotic counterparts. This allows us to discuss the nature, structure and functions of the meiotic TopoVI-like complex during meiotic DSB formation.

  10. Effect of collection-maturation interval time and pregnancy status of donor mares on oocyte developmental competence in horse cloning.

    PubMed

    Gambini, A; Andrés, G; Jarazo, J; Javier, J; Karlanian, F; Florencia, K; De Stéfano, A; Salamone, D F

    2014-02-01

    The current limitations for obtaining ovaries from slaughterhouses and the low efficiency of in vivo follicular aspiration necessitate a complete understanding of the variables that affect oocyte developmental competence in the equine. For this reason, we assessed the effect on equine oocyte meiotic competence and the subsequent in vitro cloned embryo development of 1) the time interval between ovary collection and the onset of oocyte in vitro maturation (collection-maturation interval time) and 2) the pregnancy status of the donor mares. To define the collection-maturation interval time, collected oocytes were classified according to the slaughtering time and the pregnancy status of the mare. Maturation rate was recorded and some matured oocytes of each group were used to reconstruct zona free cloned embryos. Nuclear maturation rates were lower when the collection-maturation interval time exceeded 10 h as compared to 4 h (32/83 vs. 76/136, respectively; P = 0.0128) and when the donor mare was pregnant as compared to nonpregnant (53/146 vs. 177/329, respectively; P = 0.0004). Low rates of cleaved embryos were observed when the collection-maturation interval time exceeded 10 h as compared to 6 to 10 h (11/27 vs. 33/44, respectively; P = 0.0056), but the pregnancy status of donor mares did not affect cloned equine blastocyst development (3/49 vs. 1/27 for blastocyst rates of nonpregnant and pregnant groups, respectively; P = 1.00). These results indicate that, to apply assisted reproductive technologies in horses, oocytes should be harvested within approximately 10 h after ovary collection. Also, even though ovaries from pregnant mares are a potential source of oocytes, they should be processed at the end of the collection routine due to the lower collection and maturation rate in this group.

  11. Improvement of transgenic cloning efficiencies by culturing recipient oocytes and donor cells with antioxidant vitamins in cattle.

    PubMed

    Wongsrikeao, Pimprapar; Nagai, Takashi; Agung, Budiyanto; Taniguchi, Masayasu; Kunishi, Miho; Suto, Shizuyo; Otoi, Takeshige

    2007-06-01

    The present study was conducted to investigate effects of antioxidants during maturation culture of recipient oocytes and/or culture of gene-transfected donor cells on the meiotic competence of recipient oocytes, and the developmental competence and quality of the reconstructed embryos after nuclear transfer (NT) in cattle. Gene-transfected donor cells had negative effects on the proportions of blastocyst formation, total cell numbers, and DNA fragmentation indices of reconstructed embryos. Supplementation of either vitamin E (alpha-tocopherol: 100 microM) or vitamin C (ascorbic acid: 100 microM) during maturation culture significantly enhanced the cytoplasmic maturation of oocytes and subsequent development of embryos reconstructed with the oocytes and gene-transfected donor cells, but did not have synergistic effects. The supplementation of vitamin E during maturation culture of recipient oocytes increased the proportions of fusion and blastocyst formation of gene-transfected NT embryos, in which the proportions were similar to those of nontransfected NT embryos. When the gene-transfected donor cells that had been cultured with 0, 50, or 100 microM of vitamin E were transferred into recipient oocytes matured with vitamin E (100 microM), 50 microM of vitamin E increased the proportion of blastocyst formation and reduced the index of DNA fragmentation of blastocysts. In conclusion, gene-transfected donor cells have negatively influenced the NT outcome. Supplementation of vitamin E during both recipient oocyte maturation and donor cell culture enhanced the blastocyst formation and efficiently blocked DNA damage in transgenic NT embryos.

  12. Identification of PDE9 as a cGMP-specific phosphodiesterase in germinal vesicle oocytes: A proposed role in the resumption of meiosis

    PubMed Central

    Hanna, Carol B.; Yao, Shan; Wu, Xuemei; Jensen, Jeffrey T.

    2012-01-01

    Objective To identify a cGMP-specific phosphodiesterase (PDE) in non-human primate germinal vesicle (GV) oocytes and establish a proposed effect on oocyte maturation through preliminary experiments in mouse GV oocytes. Design Controlled non-human primate and rodent experiments. Setting Academic research institution. Animals Rhesus macaques and B6/129F1 mice. Interventions Rhesus macaques were stimulated with FSH to collect GV oocytes and cumulus for gene expression analysis. Female mice were stimulated with PMSG to collect GV oocytes. Main Outcome Measures PDE transcript expression in primate GV oocytes and cumulus cells. Fluorescence polarization (FP) measurements of PDE3A activity. Spontaneous resumption of meiosis in mouse GV oocytes. Results Five PDE transcripts were detected in Rhesus GV oocytes, only PDE9A was cGMP-specific. FP assays indicated cGMP has an inhibitory effect on PDE3A while the PDE9 inhibitor, BAY73-6691, did not. Similarly, BAY73-6691, had little effect on preventing spontaneous maturation in oocytes, but did augment the inhibitory effects of cGMP. Inclusion of 0µM (control), 10µM, 100µM, and 1 mM BAY73-6691 significantly increased the proportion of mouse oocytes maintaining GV arrest in the presence of the cGMP analog 8-Br-cGMP at: 100µM (8.8%, 11.4%, 18.8%, and 28%), 500µM (21.1%, 38.1%, 74.5%,and 66.5%), and 1 mM (57.8%, 74.5%, 93.9%, and 94.0%) respectively, when P<0.05. Conclusions PDE9 is a cGMP-specific hydrolyzing enzyme present in primate oocytes, and PDE9 antagonists augment the inhibitory effect of cGMP during spontaneous in vitro maturation of GV mouse oocytes. PMID:22704629

  13. Meiotic recombination, synapsis, meiotic inactivation and sperm aneuploidy in a chromosome 1 inversion carrier.

    PubMed

    Kirkpatrick, Gordon; Chow, Victor; Ma, Sai

    2012-01-01

    Disrupted meiotic behaviour of inversion carriers may be responsible for suboptimal sperm parameters in these carriers. This study investigated meiotic recombination, synapsis, transcriptional silencing and chromosome segregation effects in a pericentric inv(1) carrier. Recombination (MLH1), synapsis (SYCP1, SYCP3) and transcriptional inactivation (γH2AX, BRCA1) were examined by fluorescence immunostaining. Chromosome specific rates of recombination were determined by fluorescence in-situ hybridization. Furthermore, testicular sperm was examined for aneuploidy and segregation of the inv(1). Our findings showed that global recombination rates were similar to controls. Recombination on the inv(1) and the sex chromosomes were reduced. The inv(1) associated with the XY body in 43.4% of cells, in which XY recombination was disproportionately absent, and 94.3% of cells displayed asynapsed regions which displayed meiotic silencing regardless of their association with the XY body. Furthermore, a low frequency of chromosomal imbalance was observed in spermatozoa (3.4%). Our results suggest that certain inversion carriers may display unimpaired global recombination and impaired recombination on the involved and the sex chromosomes during meiosis. Asynapsis or inversion-loop formation in the inverted region may be responsible for impaired spermatogenesis and may prevent sperm-chromosome imbalance.

  14. In vitro development of vitrified buffalo oocytes following parthenogenetic activation and intracytoplasmic sperm injection.

    PubMed

    Liang, Y Y; Phermthai, T; Nagai, T; Somfai, T; Parnpai, R

    2011-06-01

    The objective of this study was to investigate the potential of swamp buffalo oocytes vitrified-warmed at the metaphase of the second meiotic cell division (M-II) stage to develop to the blastocyst stage after parthenogenetic activation (PA) or intracytoplasmic sperm injection (ICSI). In Experiment 1, we examined the effects of exposure time of oocytes to cryoprotectants (CPA) on their in vitro development after PA. In vitro matured (IVM) oocytes were placed in 10% dimethylsulfoxide (DMSO) + 10% ethylene glycol (EG) for 1 min and then exposed to 20% DMSO + 20% EG + 0.5 M sucrose for 30 s, 45 s or 60 s (1 min + 30 s, 1 min + 45 s and 1 min + 60 s groups, respectively). The oocytes were then exposed to warming solution (TCM199 HEPES + 20% FBS and 0.5M sucrose) for 5 min and then washed in TCM199 HEPES + 20% FBS for 5 min. IVM oocytes without CPA treatments served as a control group. The viability assessed by fluorescein diacetate (FDA) staining was 100% in all groups. The developmental rates after PA to the blastocyst stage between 1min+30s (16%) and control (26%) groups did not differ significantly, but they were significantly higher than those in 1 min + 45 s (10%) and 1 min + 60 s (2%) groups. In Experiment 2, we examined the effect of two CPA exposure times, 1 min + 30 s and 1 min + 45 s on the in vitro development after PA of oocytes vitrified by the microdrop method. The viabilities in vitrified 1 min + 30 s, 1 min + 45 s and the control (without CPA treatments) groups were not different (97%, 95% and 100%, respectively). The development of surviving oocytes to the blastocyst stage in the vitrified 1 min + 30 s group (8%) was significantly higher than that in the vitrified 1 min + 45 s group (4%) and significantly lower than those in control group (26%). In Experiment 3, we examined the effect of two CPA exposure times, 1 min + 30 s and 1 min + 45 s on in vitro development after ICSI of vitrified oocytes. Viabilities in vitrified oocytes among 1 min + 30 s, 1

  15. Endoplasmic reticulum reorganizations and Ca2+ signaling in maturing and fertilized oocytes of marine protostome worms: the roles of MAPKs and MPF.

    PubMed

    Stricker, Stephen A; Smythe, Toni L

    2003-07-01

    Before a proper Ca(2+) response is produced at fertilization, oocytes typically undergo a maturation process during which their endoplasmic reticulum (ER) is restructured. In marine protostome worms belonging to the phylum Nemertea, the ER of maturing oocytes forms numerous distinct clusters that are about 5 micro m in diameter. After fertilization, mature oocytes with such aggregates generate a normal series of Ca(2+) oscillations and eventually disassemble their ER clusters at around the time that the oscillations cease. Immature oocytes, however, lack prominent ER clusters and fail to exhibit repetitive Ca(2+) oscillations upon insemination, collectively suggesting that cell cycle-related changes in ER structure may play a role in Ca(2+) signaling. To assess the effects of meiotic regulators on the morphology of the ER and the type of Ca(2+) response that is produced at fertilization, nemertean oocytes were treated with pharmacological modulators of mitogen-activated protein kinases (MAPKs) or maturation-promoting factor (MPF) prior to confocal microscopic analyses. Based on such imaging studies and correlative assays of kinase activities, MAPKs of the ERK1/2 type (extracellular signal regulated kinases 1/2) do not seem to be essential for either structural reorganizations of the ER or repetitive Ca(2+) signaling at fertilization. Conversely, MPF levels appear to modulate both ER structure and the capacity to produce normal Ca(2+) oscillations. The significance of these findings is discussed with respect to other reports on ER structure, MPF cycling and Ca(2+) signaling in oocytes of deuterostome animals.

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

    PubMed

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

    2016-05-15

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

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

    PubMed

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

    2016-05-15

    The ultrastructure of the ovary of Fasciola hepatica collected from field-infected sheep, was compared with that of flukes from laboratory-infected rats harbouring the Oberon or the Cullompton fluke isolate. At the periphery of the ovarian tubules, in all flukes, interstitial tissue was identified that appears to provide physical support and facilitate the metabolism of the germinal-line cells. Oogonia undergo mitotic division to maintain the cell population and to produce oocytes. Early oocytes feature conspicuous synaptonemal complexes in the nucleoplasm, and these become less evident as the oocytes grow in size, move towards the core of the ovarian tubule, and synthesise osmiophilic bodies. The latter may represent cortical granules, and serve to block polyspermy. The identity of the synaptonemal complexes was confirmed by immunocytochemical labelling of synaptonemal proteins. The occurrence of synaptonemal complexes in the oocytes of all fluke types examined indicates that pairing of bivalent chromosomes, with the potential for genetic recombination and chiasmata formation, is a feature of the triploid aspermic parthenogenetic Cullompton flukes, as well as of the wild-type out-breeding field-derived and Oberon isolate flukes. In oocytes within shelled eggs in the proximal uterus of all flukes, condensed chromosomes align at meiotic metaphase plates. Following the reduction division, two equal pronuclei appear in each oocyte in the distal uterus. On the basis of these observations, a mechanism of facultative parthenogenesis for F. hepatica is proposed that accommodates the survival and clonal expansion of triploid aspermic isolates. PMID:27084479

  18. Repression of Gurken translation by a meiotic checkpoint in Drosophila oogenesis is suppressed by a reduction in the dose of eIF1A

    PubMed Central

    Li, Wei; Klovstad, Martha; Schüpbach, Trudi

    2014-01-01

    In Drosophila melanogaster, the anteroposterior (AP) and dorsoventral (DV) axes of the oocyte and future embryo are established through the localization and translational regulation of gurken (grk) mRNA. This process involves binding of specific factors to the RNA during transport and a dynamic remodeling of the grk-containing ribonucleoprotein (RNP) complexes once they have reached their destination within the oocyte. In ovaries of spindle-class females, an activated DNA damage checkpoint causes inefficient Grk translation and ventralization of the oocyte. In a screen for modifiers of the oocyte DV patterning defects, we identified a mutation in the eIF1A gene as a dominant suppressor. We show that reducing the function of eIF1A in spnB ovaries suppresses the ventralized eggshell phenotype by restoring Grk expression. This suppression is not the result of more efficient DNA damage repair or of disrupted checkpoint activation, but is coupled to an increase in the amount of grk mRNA associated with polysomes. In spnB ovaries, the activated meiotic checkpoint blocks Grk translation by disrupting the accumulation of grk mRNA in a translationally competent RNP complex that contains the translational activator Oo18 RNA-binding protein (Orb); this regulation involves the translational repressor Squid (Sqd). We further propose that reduction of eIF1A allows more efficient Grk translation possibly because of the presence of specific structural features in the grk 5′UTR. PMID:25231760

  19. Global transcriptome analysis of two ameiotic1 alleles in maize anthers: defining steps in meiotic entry and progression through prophase I

    PubMed Central

    2011-01-01

    Background Developmental cues to start meiosis occur late in plants. Ameiotic1 (Am1) encodes a plant-specific nuclear protein (AM1) required for meiotic entry and progression through early prophase I. Pollen mother cells (PMCs) remain mitotic in most am1 mutants including am1-489, while am1-praI permits meiotic entry but PMCs arrest at the leptotene/zygotene (L/Z) transition, defining the roles of AM1 protein in two distinct steps of meiosis. To gain more insights into the roles of AM1 in the transcriptional pre-meiotic and meiotic programs, we report here an in depth analysis of gene expression alterations in carefully staged anthers at 1 mm (meiotic entry) and 1.5 mm (L/Z) caused by each of these am1 alleles. Results 1.0 mm and 1.5 mm anthers of am1-489 and am1-praI were profiled in comparison to fertile siblings on Agilent® 4 × 44 K microarrays. Both am1-489 and am1-praI anthers are cytologically normal at 1.0 mm and show moderate transcriptome alterations. At the 1.5-mm stage both mutants are aberrant cytologically, and show more drastic transcriptome changes. There are substantially more absolute On/Off and twice as many differentially expressed genes (sterile versus fertile) in am1-489 than in am1-praI. At 1.5 mm a total of 4,418 genes are up- or down-regulated in either am1-489 or am1-praI anthers. These are predominantly stage-specific transcripts. Many putative meiosis-related genes were found among them including a small subset of allele-specific, mis-regulated genes specific to the PMCs. Nearly 60% of transcriptome changes in the set of transcripts mis-regulated in both mutants (N = 530) are enriched in PMCs, and only 1% are enriched in the tapetal cell transcriptome. All array data reported herein will be deposited and accessible at MaizeGDB http://www.maizegdb.org/. Conclusions Our analysis of anther transcriptome modulations by two distinct am1 alleles, am1-489 and am1-praI, redefines the role of AM1 as a modulator of expression of a subset of

  20. A Whole-Chromosome Analysis of Meiotic Recombination in Drosophila melanogaster.

    PubMed

    Miller, Danny E; Takeo, Satomi; Nandanan, Kavyasree; Paulson, Ariel; Gogol, Madelaine M; Noll, Aaron C; Perera, Anoja G; Walton, Kendra N; Gilliland, William D; Li, Hua; Staehling, Karen K; Blumenstiel, Justin P; Hawley, R Scott

    2012-02-01

    Although traditional genetic assays have characterized the pattern of crossing over across the genome in Drosophila melanogaster, these assays could not precisely define the location of crossovers. Even less is known about the frequency and distribution of noncrossover gene conversion events. To assess the specific number and positions of both meiotic gene conversion and crossover events, we sequenced the genomes of male progeny from females heterozygous for 93,538 X chromosomal single-nucleotide and InDel polymorphisms. From the analysis of the 30 F1 hemizygous X chromosomes, we detected 15 crossover and 5 noncrossover gene conversion events. Taking into account the nonuniform distribution of polymorphism along the chromosome arm, we estimate that most oocytes experience 1 crossover event and 1.6 gene conversion events per X chromosome pair per meiosis. An extrapolation to the entire genome would predict approximately 5 crossover events and 8.6 conversion events per meiosis. Mean gene conversion tract lengths were estimated to be 476 base pairs, yielding a per nucleotide conversion rate of 0.86 × 10(-5) per meiosis. Both of these values are consistent with estimates of conversion frequency and tract length obtained from studies of rosy, the only gene for which gene conversion has been studied extensively in Drosophila. Motif-enrichment analysis revealed a GTGGAAA motif that was enriched near crossovers but not near gene conversions. The low-complexity and frequent occurrence of this motif may in part explain why, in contrast to mammalian systems, no meiotic crossover hotspots have been found in Drosophila. PMID:22384403

  1. A Whole-Chromosome Analysis of Meiotic Recombination in Drosophila melanogaster

    PubMed Central

    Miller, Danny E.; Takeo, Satomi; Nandanan, Kavyasree; Paulson, Ariel; Gogol, Madelaine M.; Noll, Aaron C.; Perera, Anoja G.; Walton, Kendra N.; Gilliland, William D.; Li, Hua; Staehling, Karen K.; Blumenstiel, Justin P.; Hawley, R. Scott

    2012-01-01

    Although traditional genetic assays have characterized the pattern of crossing over across the genome in Drosophila melanogaster, these assays could not precisely define the location of crossovers. Even less is known about the frequency and distribution of noncrossover gene conversion events. To assess the specific number and positions of both meiotic gene conversion and crossover events, we sequenced the genomes of male progeny from females heterozygous for 93,538 X chromosomal single-nucleotide and InDel polymorphisms. From the analysis of the 30 F1 hemizygous X chromosomes, we detected 15 crossover and 5 noncrossover gene conversion events. Taking into account the nonuniform distribution of polymorphism along the chromosome arm, we estimate that most oocytes experience 1 crossover event and 1.6 gene conversion events per X chromosome pair per meiosis. An extrapolation to the entire genome would predict approximately 5 crossover events and 8.6 conversion events per meiosis. Mean gene conversion tract lengths were estimated to be 476 base pairs, yielding a per nucleotide conversion rate of 0.86 × 10−5 per meiosis. Both of these values are consistent with estimates of conversion frequency and tract length obtained from studies of rosy, the only gene for which gene conversion has been studied extensively in Drosophila. Motif-enrichment analysis revealed a GTGGAAA motif that was enriched near crossovers but not near gene conversions. The low-complexity and frequent occurrence of this motif may in part explain why, in contrast to mammalian systems, no meiotic crossover hotspots have been found in Drosophila. PMID:22384403

  2. Evidence for meiotic sex in bdelloid rotifers.

    PubMed

    Signorovitch, Ana; Hur, Jae; Gladyshev, Eugene; Meselson, Matthew

    2016-08-22

    In their study of genetic exchange in the bdelloid rotifer Adineta vaga, Debortoli et al. [1] conclude that the patchwork pattern of allele sharing among three individuals in the genomic regions they examined is "…unlikely to arise in cases of PTH (Oenothera-like) meiosis since haplotypes are transferred as entire blocks…" and therefore that "Genetic exchange among bdelloid rotifers is more likely due to horizontal gene transfer than to meiotic sex." This assumes without justification that horizontal gene transfer (HGT) in bdelloids precludes the sexual transmission of entire haplotypes, for which we have reported evidence in the bdelloid Macrotrachela quadricornifera[2]. And it does not consider the contribution to such a patchwork pattern that would result from conversion and subsequent outcrossing, even in Oenothera-like systems. PMID:27554650

  3. Cardiac arrest in the skies.

    PubMed

    Charles, R A

    2011-08-01

    Cardiac arrest occurring on board aeroplanes is rare, but remains a common cause of inflight incidents. This review examines some of the management problems unique to inflight cardiac arrests, and emphasises the use of cardiopulmonary resuscitation and automated external defibrillators.

  4. Meiotic behavior of Brachiaria decumbens hybrids.

    PubMed

    Souza, V F; Pagliarini, M S; Valle, C B; Bione, N C P; Menon, M U; Mendes-Bonato, A B

    2015-01-01

    Brachiaria decumbens is a forage grass of inestimable value for livestock in Brazil due to its production of good quality forage even when planted on acid and poor soils, although it is susceptible to pasture spittlebugs. Only one cultivar, cv. Basilisk, has been used as the pollen donor in crosses with Brachiaria ruziziensis since 1988 at Embrapa Gado de Corte Research Center. Breeding within the species only became possible from 2009 when sexual accessions were successfully tetraploidized using colchicine. Three sexual genotypes were obtained and hybridization within B. decumbens was finally achieved. Here, we evaluated microspore tetrads using conventional cytology and found meiotic indexes above 78% for all three female genitors (cD24-2, cD24-27, cD24-45), but a low meiotic index (<22%) in the natural apomictic genitor D62 (cv. Basilisk) and in 49 hybrids. Analysis of the relationship between abnormal tetrad frequency and non-viable pollen grains yielded a highly significant Pearson correlation coefficient. The t-test proved significant for the progeny of cD24-45 x D62, with lower abnormalities and pollen sterility when compared to the other two progenies resulting from cD24-2 and cD24-27 crossed to D62, but these two did not differ. Apomictic hybrids such as S036 and X030 with low pollen sterility have the potential for use in cultivar development, whereas the sexual hybrids T012, X072, and X078 might be of use as female genitors in polycross blocks if they display good agronomic traits. PMID:26505437

  5. Transcriptome analysis of bovine oocytes from distinct follicle sizes: Insights from correlation network analysis.

    PubMed

    Labrecque, Rémi; Fournier, Eric; Sirard, Marc-André

    2016-06-01

    Follicle size is recognized as a predictor of the potential for the enclosed oocyte to yield an embryo following in vitro maturation and in vitro fertilization. Oocytes from larger follicles are more likely to reach the blastocyst stage than those from smaller follicles. A growing oocyte accumulates all the transcripts needed to ensure development until the maternal embryonic transition, and this accumulation must be completed before the period of transcriptional arrest. Accordingly, the transcriptomes of bovine germinal-vesicle-stage oocytes collected from follicles of increasing sizes (<3, 3-5, >5-8, and >8 mm) were evaluated, using the EmbryoGENE bovine transcriptomic platform (custom Agilent 4 × 44 K), to better understand transcriptional modulation in the oocyte as the follicle becomes larger. Microarray analyses revealed very few differences between oocytes from small follicles (<3 vs. 3-5 mm), whereas an important number of differences were detected at the mRNA level between oocytes from larger follicles. Weighted gene correlation network analysis allowed for the identification of several hub genes involved in crucial functions such as transcriptional regulation (TAF2), chromatin remodeling (PPP1CB), energy production (SLC25A31), as well as transport of key molecules within the cell (NAGPA, CYHR1, and SLC3A12). The results presented here thus reinforce the hypothesis that developmental competence acquisition cannot be seen as a simple one-step process, especially in regards to the modulation of mRNA. Mol. Reprod. Dev. 83: 558-569, 2016. © 2016 Wiley Periodicals, Inc. PMID:27127921

  6. Impact of gonadotropins on oocyte maturation, fertilisation and developmental competence in vitro.

    PubMed

    Wang, Xuemei; Tsai, Tony; Qiao, Jie; Zhang, Zhan; Feng, Huai L

    2014-06-01

    The aim of the present study was to evaluate the dose-dependent effects of gonadotropins, either singly (Bravelle (B), Luveris (L), Menupur (M), Repronex (R), Gonal-F (G), Follism (F) and Norvarel (N)) or in combination (Menupur+Bravelle; Repronext+Bravelle; and Bravelle+Norvarel), on rates of oocyte maturation, fertilisation and early embryo development in vitro in an animal model. Bovine cumulus-oocyte complexes (COCs) were purchased commercially and cultured in TCM-199 with 10% fetal bovine serum supplemented with varying concentrations of gonadotropin (0, 5, 10, 20, 40IU or United States Pharmacopoeia (USP) mL-1) for 24 and 48h according to current IVF clinical stimulation protocols. All gonadotropins enhanced oocyte maturation in vitro in a dose-dependent manner. Individually, Gonal-F (Merck KGaA, Darmstadt, Germany), Follism (Merck Co, Whitehouse Station, NJ, USA) and Repronext (Ferring, Parsippany, NJ, USA) promoted oocyte maturation; in combination, they effectively enhanced COC expansion and increased the maturation competence of MII oocytes. However, high concentrations of gonadotropins may result in maturation arrest. Specific combinations of gonadotropins may change the rate of early embryonic development (8-16-cells) and morula-blastocyst formation. These data provide support for the responsiveness of bovine oocytes to gonadotropins in vitro and the need to consider variations in the relative concentrations and ratio of combinations (FSH/LH or human chorionic gonadotropin) for optimisation of oocyte developmental competence. The results of the present study could be applied to therapeutic clinical stimulation protocols and help improve IVF success rates.

  7. Recent Progress in Cryopreservation of Bovine Oocytes

    PubMed Central

    Hochi, Shinichi

    2014-01-01

    Principle of oocyte cryoinjury is first overviewed and then research history of cryopreservation using bovine oocytes is summarized for the last two decades with a few special references to recent progresses. Various types of cryodevices have been developed to accelerate the cooling rate and applied to the oocytes from large domestic species enriched with cytoplasmic lipid droplets. Two recent approaches include the qualitative improvement of IVM oocytes prior to the vitrification and the short-term recovery culture of vitrified-warmed oocytes prior to the subsequent IVF. Supplementation of L-carnitine to IVM medium of bovine oocytes has been reported to reduce the amount of cytoplasmic lipid droplets and improve the cryotolerance of the oocytes, but it is still controversial whether the positive effect of L-carnitine is reproducible. Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes, was inhibited by a short-term culture of the postwarm oocytes in the presence of Rho-associated coiled-coil kinase (ROCK) inhibitor. Use of an antioxidant α-tocopherol, instead of the ROCK inhibitor, also supported the revivability of the postwarm bovine oocytes. Further improvements of the vitrification procedure, combined with pre- and postvitrification chemical treatment, would overcome the high sensitivity of bovine oocytes to cryopreservation. PMID:24738063

  8. Cold-induced changes in amphibian oocytes

    SciTech Connect

    Angelier, N.; Moreau, N.A.; N'Da, E.A.; Lautredou, N.F. )

    1989-08-01

    Female Pleurodeles waltl newts (Amphibia, urodele), usually raised at 20 degrees C, were submitted to low temperatures; oocytes responded to this cold stress by drastic changes both in lampbrush chromosome structure and in protein pattern. Preexisting lateral loops of lampbrush chromosomes were reduced in size and number, while cold-induced loops which were tremendously developed, occurred on defined bivalents of the oocyte at constant, reproducible sites. A comparison of protein patterns in control and stressed oocytes showed two main differences: in stressed oocytes, overall protein synthesis was reduced, except for a set of polypeptides, the cold-stress proteins; second, there was a striking inversion of the relative amount of beta- and gamma-actin found in the oocyte nucleus before and after cold stress. Whereas beta-actin was the predominant form in control oocytes, gamma-actin became the major form in stressed oocytes.

  9. Gene expression, oocyte nuclear maturation and developmental competence of bovine oocytes and embryos produced after in vivo and in vitro heat shock.

    PubMed

    Pavani, Krishna C; Baron, Erica; Correia, Pedro; Lourenço, Joana; Bettencourt, Bruno Filipe; Sousa, Madalena; da Silva, Fernando Moreira

    2016-10-01

    Three assays were performed. In assay 1, oocytes harvested during the winter months were subjected to kinetic heat shock by stressing the oocytes at 39.5°C (HS1) or at 40.5°C (HS2) for either 6, 12, 18 or 24 h and then matured at control temperature (38.5°C). The nuclear maturation rates (NMR) of all oocytes were recorded after 24 h. In assay 2, oocytes collected year-round maturated, were implanted via in vitro fertilization (IVF) and developed for 9 days. Gene expression analysis was performed on target genes (Cx43, CDH1, DNMT1, HSPA14) with reference to the two housekeeping genes (GAPDH and SDHA) in embryos. Similarly, in assay 3, genetic analysis was performed on the embryos produced from heat-stressed oocytes (from HS1 and HS2). In assay 1, the duration of heat stress resulted in a significant decline in NMR (P < 0.05) with HS1 for maturated oocytes at 86.4 ± 4.3; 65.5 ± 0.7; 51.3 ± 0.9; 38.1 ± 1.9 and 36.3 ± 0.9, for control, 6 h, 12 h, 18 h and 24 h, respectively. For assays 2 and 3, results demonstrated that DNMT1, Cx43 and HSPA14 were down-regulated in the embryos produced in the warm with respect to the cold months (P < 0.05). A constant up- and down-regulation of DNMT1 and HSPA14 genes were observed in both HS-treated samples. Also, an inconsistent pattern of gene expression was observed in Cx43 and CDH1 genes (P < 0.05). Targeted gene expression was aberrant in embryo development, which can provide evidence on early embryo arrest and slowed embryo development.

  10. Extrachromosomal amplification of rDNA in oocytes of Hemerobius spp. (Insecta, Neuroptera).

    PubMed

    Kubrakiewicz, J; Biliński, S M

    1995-05-01

    In previtellogenic oocytes of the neuropteran, Hemerobius spp., two distinct, DNA-positive intranuclear structures have been observed. Chromosomes of meiotic prophase assemble in the center of the oocyte nucleus forming a highly polymorphic karyosphere, which persists in this position until the very late stages of vitellogenesis. The extrachromosomal DNA body, containing amplified ribosomal genes, undergoes fragmentation and dispersion in the nucleoplasm. At the onset of previtellogenic growth, transcription of extra rDNA starts, which is accompanied by the appearance of dense, granular material (multiple nucleoli). Arising nucleoli gradually fill the nucleoplasm. At the electron microscopic (EM) level two electron dense structural forms of the granular material have been described. Together with general histological and ultrastructural analysis the amplification of rDNA genes in Hemerobius spp. oocytes has been demonstrated by means of the spreading technique, which has shown that extra rDNA is organized in rings containing various numbers of active ribosomal genes. The transcription activity of amplified genes is manifested in the form of typical "Christmas tree" structures.

  11. Coevolutionary dynamics of polyandry and sex-linked meiotic drive.

    PubMed

    Holman, Luke; Price, Thomas A R; Wedell, Nina; Kokko, Hanna

    2015-03-01

    Segregation distorters located on sex chromosomes are predicted to sweep to fixation and cause extinction via a shortage of one sex, but in nature they are often found at low, stable frequencies. One potential resolution to this longstanding puzzle involves female multiple mating (polyandry). Because many meiotic drivers severely reduce the sperm competitive ability of their male carriers, females are predicted to evolve more frequent polyandry and thereby promote sperm competition when a meiotic driver invades. Consequently, the driving chromosome's relative fitness should decline, halting or reversing its spread. We used formal modeling to show that this initially appealing hypothesis cannot resolve the puzzle alone: other selective pressures (e.g., low fitness of drive homozygotes) are required to establish a stable meiotic drive polymorphism. However, polyandry and meiotic drive can strongly affect one another's frequency, and polyandrous populations may be resistant to the invasion of rare drive mutants.

  12. Heterochromatin-Associated Proteins HP1a and Piwi Collaborate to Maintain the Association of Achiasmate Homologs in Drosophila Oocytes.

    PubMed

    Giauque, Christopher C; Bickel, Sharon E

    2016-05-01

    Accurate segregation of homologous chromosomes during meiosis depends on their ability to remain physically connected throughout prophase I. For homologs that achieve a crossover, sister chromatid cohesion distal to the chiasma keeps them attached until anaphase I. However, in Drosophila melanogaster wild-type oocytes, chromosome 4 never recombines, and the X chromosome fails to cross over in 6-10% of oocytes. Proper segregation of these achiasmate homologs relies on their pericentric heterochromatin-mediated association, but the mechanism(s) underlying this attachment remains poorly understood. Using an inducible RNA interference (RNAi) strategy combined with fluorescence in situ hybridization (FISH) to monitor centromere proximal association of the achiasmate FM7a/X homolog pair, we asked whether specific heterochromatin-associated proteins are required for the association and proper segregation of achiasmate homologs in Drosophila oocytes. When we knock down HP1a, H3K9 methytransferases, or the HP1a binding partner Piwi during mid-prophase, we observe significant disruption of pericentric heterochromatin-mediated association of FM7a/X homologs. Furthermore, for both HP1a and Piwi knockdown oocytes, transgenic coexpression of the corresponding wild-type protein is able to rescue RNAi-induced defects, but expression of a mutant protein with a single amino acid change that disrupts the HP1a-Piwi interaction is unable to do so. We show that Piwi is stably bound to numerous sites along the meiotic chromosomes, including centromere proximal regions. In addition, reduction of HP1a or Piwi during meiotic prophase induces a significant increase in FM7a/X segregation errors. We present a speculative model outlining how HP1a and Piwi could collaborate to keep achiasmate chromosomes associated in a homology-dependent manner. PMID:26984058

  13. Two new competing pathways establish the threshold for cyclin-B–Cdk1 activation at the meiotic G2/M transition

    PubMed Central

    Aono, Ryota; Hanada, Shin-ichiro; Okumura, Eiichi; Kishimoto, Takeo

    2016-01-01

    ABSTRACT Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K–Akt pathway through the Gβγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B–Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B–Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gβγ-dependent pathway enhances PI3K–Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gβγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries. PMID:27390173

  14. Two new competing pathways establish the threshold for cyclin-B-Cdk1 activation at the meiotic G2/M transition.

    PubMed

    Hiraoka, Daisaku; Aono, Ryota; Hanada, Shin-Ichiro; Okumura, Eiichi; Kishimoto, Takeo

    2016-08-15

    Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K-Akt pathway through the Gβγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B-Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B-Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gβγ-dependent pathway enhances PI3K-Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gβγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries. PMID:27390173

  15. Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice.

    PubMed

    de Boer, Esther; Jasin, Maria; Keeney, Scott

    2015-08-15

    Meiotic recombination initiated by programmed double-strand breaks (DSBs) yields two types of interhomolog recombination products, crossovers and noncrossovers, but what determines whether a DSB will yield a crossover or noncrossover is not understood. In this study, we analyzed the influence of sex and chromosomal location on mammalian recombination outcomes by constructing fine-scale recombination maps in both males and females at two mouse hot spots located in different regions of the same chromosome. These include the most comprehensive maps of recombination hot spots in oocytes to date. One hot spot, located centrally on chromosome 1, behaved similarly in male and female meiosis: Crossovers and noncrossovers formed at comparable levels and ratios in both sexes. In contrast, at a distal hot spot, crossovers were recovered only in males even though noncrossovers were obtained at similar frequencies in both sexes. These findings reveal an example of extreme sex-specific bias in recombination outcome. We further found that estimates of relative DSB levels are surprisingly poor predictors of relative crossover frequencies between hot spots in males. Our results demonstrate that the outcome of mammalian meiotic recombination can be biased, that this bias can vary depending on location and cellular context, and that DSB frequency is not the only determinant of crossover frequency.

  16. Microtubule-Depolymerizing Kinesin KLP10A Restricts the Length of the Acentrosomal Meiotic Spindle in Drosophila Females

    PubMed Central

    Radford, Sarah J.; Harrison, Andrew M.; McKim, Kim S.

    2012-01-01

    During cell division, a bipolar array of microtubules forms the spindle through which the forces required for chromosome segregation are transmitted. Interestingly, the spindle as a whole is stable enough to support these forces even though it is composed of dynamic microtubules, which are constantly undergoing periods of growth and shrinkage. Indeed, the regulation of microtubule dynamics is essential to the integrity and function of the spindle. We show here that a member of an important class of microtubule-depolymerizing kinesins, KLP10A, is required for the proper organization of the acentrosomal meiotic spindle in Drosophila melanogaster oocytes. In the absence of KLP10A, microtubule length is not controlled, resulting in extraordinarily long and disorganized spindles. In addition, the interactions between chromosomes and spindle microtubules are disturbed and can result in the loss of contact. These results indicate that the regulation of microtubule dynamics through KLP10A plays a critical role in restricting the length and maintaining bipolarity of the acentrosomal meiotic spindle and in promoting the contacts that the chromosomes make with microtubules required for meiosis I segregation. PMID:22865737

  17. Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice

    PubMed Central

    de Boer, Esther; Jasin, Maria; Keeney, Scott

    2015-01-01

    Meiotic recombination initiated by programmed double-strand breaks (DSBs) yields two types of interhomolog recombination products, crossovers and noncrossovers, but what determines whether a DSB will yield a crossover or noncrossover is not understood. In this study, we analyzed the influence of sex and chromosomal location on mammalian recombination outcomes by constructing fine-scale recombination maps in both males and females at two mouse hot spots located in different regions of the same chromosome. These include the most comprehensive maps of recombination hot spots in oocytes to date. One hot spot, located centrally on chromosome 1, behaved similarly in male and female meiosis: Crossovers and noncrossovers formed at comparable levels and ratios in both sexes. In contrast, at a distal hot spot, crossovers were recovered only in males even though noncrossovers were obtained at similar frequencies in both sexes. These findings reveal an example of extreme sex-specific bias in recombination outcome. We further found that estimates of relative DSB levels are surprisingly poor predictors of relative crossover frequencies between hot spots in males. Our results demonstrate that the outcome of mammalian meiotic recombination can be biased, that this bias can vary depending on location and cellular context, and that DSB frequency is not the only determinant of crossover frequency. PMID:26251527

  18. Complex elaboration: making sense of meiotic cohesin dynamics

    PubMed Central

    Rankin, Susannah

    2015-01-01

    In mitotically dividing cells, the cohesin complex tethers sister chromatids, the products of DNA replication, together from the time they are generated during S phase until anaphase. Cohesion between sister chromatids ensures accurate chromosome segregation, and promotes normal gene regulation and certain kinds of DNA repair. In somatic cells, the core cohesin complex is composed of four subunits: Smc1, Smc3, Rad21 and an SA subunit. During meiotic cell divisions meiosis-specific isoforms of several of the cohesin subunits are also expressed and incorporated into distinct meiotic cohesin complexes. The relative contributions of these meiosis-specific forms of cohesin to chromosome dynamics during meiotic progression have not been fully worked out. However, the localization of these proteins during chromosome pairing and synapsis, and their unique loss-of-function phenotypes, suggest non-overlapping roles in controlling meiotic chromosome behavior. Many of the proteins that regulate cohesin function during mitosis also appear to regulate cohesin during meiosis. Here we review how cohesin contributes to meiotic chromosome dynamics, and explore similarities and differences between cohesin regulation during the mitotic cell cycle and meiotic progression. A deeper understanding of the regulation and function of cohesin in meiosis will provide important new insights into how the cohesin complex is able to promote distinct kinds of chromosome interactions under diverse conditions. PMID:25895170

  19. A histone code in meiosis: the histone kinase, NHK-1, is required for proper chromosomal architecture in Drosophila oocytes

    PubMed Central

    Ivanovska, Irena; Khandan, Tulasi; Ito, Takashi; Orr-Weaver, Terry L.

    2005-01-01

    To promote faithful propagation of the genetic material during sexual reproduction, meiotic chromosomes undergo specialized morphological changes that ensure accurate segregation of homologous chromosomes. The molecular mechanisms that establish the meiotic chromosomal structures are largely unknown. We describe a mutation in a recently identified Histone H2A kinase, nhk-1, in Drosophila that leads to female sterility due to defects in the formation of the meiotic chromosomal structures. The metaphase I arrest and the karyosome, a critical prophase I chromosomal structure, require nucleosomal histone kinase-1 (NHK-1) function. The defects are a result of failure to disassemble the synaptonemal complex and to load condensin onto the mutant chromosomes. Embryos laid by nhk-1-/- mutant females arrest with aberrant polar bodies and mitotic spindles, revealing that mitosis is affected as well. We analyzed the role of Histone H2A phosphorylation with respect to the histone code hypothesis and found that it is required for acetylation of Histone H3 and Histone H4 in meiosis. These studies reveal a critical role for histone modifications in chromosome dynamics in meiosis and mitosis. PMID:16230526

  20. RNA FISH, DNA FISH and Chromosome Painting of Chicken Oocytes.

    PubMed

    Guioli, Silvana; Lovell-Badge, Robin

    2016-01-01

    Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique. It identifies the location of DNA loci and RNAs, including nascent RNAs in the process of being transcribed, within individual cells. Great advances in fluorescent dye technology and technique sensitivity, combined with developments in light microscopy and imaging software have made it widely accessible and have expanded the range of applications in basic research as well as in diagnostics. Being able to perform RNA hybridization, DNA hybridization, and protein immunofluorescence consecutively on the same sample is an invaluable tool to study RNA expression in relation to their gene loci and to map RNA and DNA in relation to nuclear or cellular structures. This has contributed to enormous progress in understanding basal mechanisms of male and female meiosis in different animal model systems. In this chapter we describe in detail the protocols for FISH based techniques applied to study gene expression dynamics and nuclear architecture of chicken oocytes during meiotic prophase I. These techniques can be easily performed in any molecular and cell biology laboratory and be adapted to different systems and to different phases of gametogenesis. PMID:27557582

  1. Changes in homologous and heterologous gap junction contacts during maturation-inducing hormone-dependent meiotic resumption in ovarian follicles of Atlantic croaker

    USGS Publications Warehouse

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

    2003-01-01

    Homologous (granulosa cell-granulosa cell) gap junction (GJ) contacts increase in ovarian follicles of Atlantic croaker (Micropogonias undulatus) during the early (first) stage of maturation, but their profile during the second stage [i.e., during maturation-inducing hormone (MIH)-mediated meiotic resumption] is unknown. The profile of homologous GJ contacts during the second stage of maturation in croaker follicles was examined in this study and compared to that of heterologous (granulosa cell-oocyte) GJ, for which changes have been previously documented. Follicles were incubated with human chorionic gonadotropin to induce maturational competence (first stage), and then with MIH to induce meiotic resumption. The follicles were collected for examination immediately before and after different durations of MIH exposure until the oocyte had reached the stage of germinal vesicle breakdown (GVBD; index of meiotic resumption). Ultrathin sections were observed by transmission electron microscopy, and homologous and heterologous GJ contacts were quantified along a 100-??m segment of granulosa cell-zona radiata complex per follicle (three follicles/time/fish, n=3 fish). Relatively high numbers of both types of GJ were observed before and after the first few hours of MIH exposure (up to the stage of oil droplet coalescence). GJ numbers declined during partial yolk globule coalescence (at or near GVBD) and were just under 50% of starting values after the completion of GVBD (P<0.05). These results confirm earlier observations that GVBD temporally correlates with declining heterologous GJ contacts, and for the first time in teleosts show that there is a parallel decline in homologous GJ. The significance of the changes in homologous and heterologous GJ is uncertain and deserves further study. ?? 2003 Elsevier Science (USA). All rights reserved.

  2. Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte

    NASA Technical Reports Server (NTRS)

    Tombes, R. M.; Simerly, C.; Borisy, G. G.; Schatten, G.

    1992-01-01

    During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.

  3. Drosophila brca2 Is Required for Mitotic and Meiotic DNA Repair and Efficient Activation of the Meiotic Recombination Checkpoint

    PubMed Central

    Klovstad, Martha; Abdu, Uri; Schüpbach, Trudi

    2008-01-01

    Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made null mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal. PMID:18266476

  4. Relocalization of STIM1 in mouse oocytes at fertilization: early involvement of store-operated calcium entry.

    PubMed

    Gómez-Fernández, Carolina; Pozo-Guisado, Eulalia; Gañán-Parra, Miguel; Perianes, Mario J; Alvarez, Ignacio S; Martín-Romero, Francisco Javier

    2009-08-01

    Calcium waves represent one of the most important intracellular signaling events in oocytes at fertilization required for the exit from metaphase arrest and the resumption of the cell cycle. The molecular mechanism ruling this signaling has been described in terms of the contribution of intracellular calcium stores to calcium spikes. In this work, we considered the possible contribution of store-operated calcium entry (SOCE) to this signaling, by studying the localization of the protein STIM1 in oocytes. STIM1 has been suggested to play a key role in the recruitment and activation of plasma membrane calcium channels, and we show here that mature mouse oocytes express this protein distributed in discrete clusters throughout their periphery in resting cells, colocalizing with the endoplasmic reticulum marker calreticulin. However, immunolocalization of the endogenous STIM1 showed considerable redistribution over larger areas or patches covering the entire periphery of the oocyte during Ca(2+) store depletion induced with thapsigargin or ionomycin. Furthermore, pharmacological activation of endogenous phospholipase C induced a similar pattern of redistribution of STIM1 in the oocyte. Finally, fertilization of mouse oocytes revealed a significant and rapid relocalization of STIM1, similar to that found after pharmacological Ca(2+) store depletion. This particular relocalization supports a role for STIM1 and SOCE in the calcium signaling during early stages of fertilization.

  5. Relocalization of STIM1 in mouse oocytes at fertilization: early involvement of store-operated calcium entry.

    PubMed

    Gómez-Fernández, Carolina; Pozo-Guisado, Eulalia; Gañán-Parra, Miguel; Perianes, Mario J; Alvarez, Ignacio S; Martín-Romero, Francisco Javier

    2009-08-01

    Calcium waves represent one of the most important intracellular signaling events in oocytes at fertilization required for the exit from metaphase arrest and the resumption of the cell cycle. The molecular mechanism ruling this signaling has been described in terms of the contribution of intracellular calcium stores to calcium spikes. In this work, we considered the possible contribution of store-operated calcium entry (SOCE) to this signaling, by studying the localization of the protein STIM1 in oocytes. STIM1 has been suggested to play a key role in the recruitment and activation of plasma membrane calcium channels, and we show here that mature mouse oocytes express this protein distributed in discrete clusters throughout their periphery in resting cells, colocalizing with the endoplasmic reticulum marker calreticulin. However, immunolocalization of the endogenous STIM1 showed considerable redistribution over larger areas or patches covering the entire periphery of the oocyte during Ca(2+) store depletion induced with thapsigargin or ionomycin. Furthermore, pharmacological activation of endogenous phospholipase C induced a similar pattern of redistribution of STIM1 in the oocyte. Finally, fertilization of mouse oocytes revealed a significant and rapid relocalization of STIM1, similar to that found after pharmacological Ca(2+) store depletion. This particular relocalization supports a role for STIM1 and SOCE in the calcium signaling during early stages of fertilization. PMID:19470709

  6. Chromatin and cytoplasmic characteristics of equine oocytes recovered by transvaginal ultrasound-guided follicle aspiration are influenced by the developmental stage of their follicle of origin.

    PubMed

    Vernunft, Andreas; Alm, Hannelore; Tuchscherer, Armin; Kanitz, Wilhelm; Hinrichs, Katrin; Torner, Helmut

    2013-07-01

    were from a more atretic follicle population and exhibited signs of atresia-related acquisition of meiotic and cytoplasmic competence. These findings will help in effective scheduling of oocyte recovery for equine-assisted reproduction techniques.

  7. Two statistical tests for meiotic breakpoint analysis.

    PubMed Central

    Plaetke, R; Schachtel, G A

    1995-01-01

    Meiotic breakpoint analysis (BPA), a statistical method for ordering genetic markers, is increasing in importance as a method for building genetic maps of human chromosomes. Although BPA does not provide estimates of genetic distances between markers, it efficiently locates new markers on already defined dense maps, when likelihood analysis becomes cumbersome or the sample size is small. However, until now no assessments of statistical significance have been available for evaluating the possibility that the results of a BPA were produced by chance. In this paper, we propose two statistical tests to determine whether the size of a sample and its genetic information content are sufficient to distinguish between "no linkage" and "linkage" of a marker mapped by BPA to a certain region. Both tests are exact and should be conducted after a BPA has assigned the marker to an interval on the map. Applications of the new tests are demonstrated by three examples: (1) a synthetic data set, (2) a data set of five markers on human chromosome 8p, and (3) a data set of four markers on human chromosome 17q. PMID:7847387

  8. Vitrification of buffalo oocytes and embryos.

    PubMed

    Parnpai, Rangsun; Liang, Yuanyuan; Ketudat-Cairns, Mariena; Somfai, Tamas; Nagai, Takashi

    2016-07-01

    During the past decade, vitrification has been acknowledged as an efficient alternative to traditional slow-rate freezing in both human and animal embryology. The buffalo is the major milk and meat producing farm animal in many developing countries. Cryopreservation of buffalo oocytes and embryos is very important in preserving this species for future use. This review discusses the recent buffalo oocytes and embryos vitrification procedures, different types of cryoinjuries, and other factors affecting the vitrification of buffalo oocytes and embryos.

  9. Correlations between Synaptic Initiation and Meiotic Recombination: A Study of Humans and Mice

    PubMed Central

    Gruhn, Jennifer R.; Al-Asmar, Nasser; Fasnacht, Rachael; Maylor-Hagen, Heather; Peinado, Vanessa; Rubio, Carmen; Broman, Karl W.; Hunt, Patricia A.; Hassold, Terry

    2016-01-01

    Meiotic recombination is initiated by programmed double strand breaks (DSBs), only a small subset of which are resolved into crossovers (COs). The mechanism determining the location of these COs is not well understood. Studies in plants, fungi, and insects indicate that the same genomic regions are involved in synaptic initiation and COs, suggesting that early homolog alignment is correlated with the eventual resolution of DSBs as COs. It is generally assumed that this relationship extends to mammals, but little effort has been made to test this idea. Accordingly, we conducted an analysis of synaptic initiation sites (SISs) and COs in human and mouse spermatocytes and oocytes. In contrast to our expectation, we observed remarkable sex- and species-specific differences, including pronounced differences between human males and females in both the number and chromosomal location of SISs. Further, the combined data from our studies in mice and humans suggest that the relationship between SISs and COs in mammals is a complex one that is not dictated by the sites of synaptic initiation as reported in other organisms, although it is clearly influenced by them. PMID:26749305

  10. Elevated testosterone during meiotic segregation stimulates laying hens to produce more sons than daughters.

    PubMed

    Pinson, Sara E; Wilson, Jeanna L; Navara, Kristen J

    2011-11-01

    Biases in avian sex ratios have been documented in relation to a variety of social and environmental conditions. Previous studies suggest that treatment with hormones can stimulate females to manipulate offspring sex, and that this effect occurs before ovulation. For example, acute and chronic treatments with testosterone stimulated significant skews towards male offspring. Hormones may act by influencing which sex chromosome is donated by the heterogametic female bird into the oocyte. However, it is difficult to pinpoint when effects of testosterone on offspring sex occurred in previous experiments because testosterone treatments were given either chronically over the entire period of follicular development or many hours before the critical period of chromosome segregation. We injected laying hens with testosterone injections 5 h prior to ovulation to target this critical period and quantified the sexes of the subsequently ovulated eggs. We hypothesized that an injection of testosterone coincident with segregation of sex chromosomes would stimulate hens to produce more male than female offspring. As hypothesized, hens injected with testosterone produced a significant bias towards male offspring compared to controls, nearly 70%. These results suggest that acute testosterone elevation during meiotic segregation may mediate skews in avian primary sex ratios.

  11. Making ICSI Safer and More Effective: A Review of the Human Oocyte and ICSI Practice.

    PubMed

    Simopoulou, Mara; Giannelou, Polina; Bakas, Panagiotis; Gkoles, Laertis; Kalampokas, Theodoros; Pantos, Konstantinos; Koutsilieris, Michael

    2016-01-01

    Intracytoplasmic sperm injection (ICSI) has become an indispensable procedure of every assisted reproduction unit. This has created as much controversy as it has awe. As this is a multistep invasive technique, every part of the procedure has become subject to investigation. We contribute this review aspiring to offer the embryologist insight into all available approaches of securing an effective ICSI practice. Herein we present all the different approaches with respect to handling of the human oocyte, taking into consideration the important steps of the technique such as the oocyte positioning, timing of performing ICSI, the option of viewing the meiotic spindle and further individual action such as artificial oocyte activation, rescue ICSI and in vitro maturation. We enrich this by including our view based on our collective experience and current practice. Published studies have led to various options for performing ICSI, resulting in in vitro fertilization units around the world adopting different approaches. The steps that ICSI technique entails discussed from the oocyte perspective are still a long way from being organized into one secure and optimal protocol. Our stressing of the need to secure an efficient ICSI protocol could be the trigger for further well-designed larger scale studies with all the latest technological advantages. We aim to approach this subject in categories and assess them separately. However, ICSI is a multifaceted procedure involving several consecutive steps and when evaluating one we cannot exclude the end effect of the previous, or the overall effect of the different practitioners involved form beginning to end. PMID:27381599

  12. Autoregulation of Musashi1 mRNA translation during Xenopus oocyte maturation.

    PubMed

    Arumugam, Karthik; Macnicol, Melanie C; Macnicol, Angus M

    2012-08-01

    The mRNA translational control protein, Musashi, plays a critical role in cell fate determination through sequence-specific interactions with select target mRNAs. In proliferating stem cells, Musashi exerts repression of target mRNAs to promote cell cycle progression. During stem cell differentiation, Musashi target mRNAs are de-repressed and translated. Recently, we have reported an obligatory requirement for Musashi to direct translational activation of target mRNAs during Xenopus oocyte meiotic cell cycle progression. Despite the importance of Musashi in cell cycle regulation, only a few target mRNAs have been fully characterized. In this study, we report the identification and characterization of a new Musashi target mRNA in Xenopus oocytes. We demonstrate that progesterone-stimulated translational activation of the Xenopus Musashi1 mRNA is regulated through a functional Musashi binding element (MBE) in the Musashi1 mRNA 3' untranslated region (3' UTR). Mutational disruption of the MBE prevented translational activation of Musashi1 mRNA and its interaction with Musashi protein. Further, elimination of Musashi function through microinjection of inhibitory antisense oligonucleotides prevented progesterone-induced polyadenylation and translation of the endogenous Musashi1 mRNA. Thus, Xenopus Musashi proteins regulate translation of the Musashi1 mRNA during oocyte maturation. Our results indicate that the hierarchy of sequential and dependent mRNA translational control programs involved in directing progression through meiosis are reinforced by an intricate series of nested, positive feedback loops, including Musashi mRNA translational autoregulation. These autoregulatory positive feedback loops serve to amplify a weak initiating signal into a robust commitment for the oocyte to progress through the cell cycle and become competent for fertilization.

  13. Analysis of meiotic segregation, using single-sperm typing: Meiotic drive at the myotonic dystrophy locus

    SciTech Connect

    Leeflang, E.P.; Arnheim, N.; McPeek, M.S.

    1996-10-01

    Meiotic drive at the myotonic dystrophy (DM) locus has recently been suggested as being responsible for maintaining the frequency, in the human population, of DM chromosomes capable of expansion to the disease state. In order to test this hypothesis, we have studied samples of single sperm from three individuals heterozygous at the DM locus, each with one allele larger and one allele smaller than 19 CTG repeats. To guard against the possible problem of differential PCR amplification rates based on the lengths of the alleles, the sperm were also typed at another closely linked marker whose allele size was unrelated to the allele size at the DM locus. Using statistical models specifically designed to study single-sperm segregation data, we find no evidence of meiotic segregation distortion. The upper limit of the two-sided 95% confidence interval for the estimate of the common segregation probability for the three donors is at or below .515 for all models considered, and no statistically significant difference from .5 is detected in any of the models. This suggests that any greater amount of segregation distortion at the myotonic dystrophy locus must result from events following sperm ejaculation. The mathematical models developed make it possible to study segregation distortion with high resolution by using sperm-typing data from any locus. 26 refs., 1 fig., 8 tabs.

  14. Fourier analysis of mitochondrial distribution in oocytes

    NASA Astrophysics Data System (ADS)

    Hollmann, Joseph L.; Brooks, Dana H.; Newmark, Judith A.; Warner, Carol M.; DiMarzio, Charles A.

    2011-03-01

    This paper describes a novel approach to quantifying mitochondrial patterns which are typically described using the qualitative terms "diffuse" "aggregated" and are potentially key indicators for an oocyte's health and survival potential post-implantation. An oocyte was isolated in a confocal image and a coarse grid was superimposed upon it. The spatial spectrum was calculated and an aggregation factor was generated. A classifier for healthy cells was developed and verified. The aggregation factor showed a clear distinction between the healthy and unhealthy oocytes. The ultimate goal is to screen oocytes for viability preimplantation, thus improving the outcome of in vitro fertilization (IVF) treatments.

  15. Meiotic wave adds extra asymmetry to the development of female chicken gonads

    PubMed Central

    de Melo Bernardo, Ana; Heeren, A. Marijne; van Iperen, Liesbeth; Fernandes, Maria Gomes; He, Nannan; Anjie, Stafford; Noce, Toshiaki; Ramos, Ester Silveira

    2015-01-01

    SUMMARY Development of female gonads in the chicken is asymmetric. This asymmetry affects gene expression, morphology, and germ cell development; consequently only the left ovary develops into a functional organ, whereas the right ovary remains vestigial. In males, on the other hand, both gonads develop into functional testes. Here, we revisited the development of asymmetric traits in female (and male) chicken gonads between Hamburger Hamilton stage 16 (HH16) and hatching. At HH16, primordial germ cells migrated preferentially to the left gonad, accumulating in the left coelomic hinge between the gut mesentery and developing gonad in both males and females. Using the meiotic markers SYCP3 and phosphorylated H2AFX, we identified a previously undescribed, pronounced asymmetryc meiotic progression in the germ cells located in the central, lateral, and extreme cortical regions of the left female gonad from HH38 until hatching. Moreover, we observed that—in contrast to the current view—medullary germ cells are not apoptotic, but remain arrested in pre‐leptotene until hatching. In addition to the systematic analysis of the asymmetric distribution of germ cells in female chicken gonads, we propose an updated model suggesting that the localization of germ cells—in the left or right gonad; in the cortex or medulla of the left gonad; and in the central part or the extremities of the left cortex—has direct consequences for their development and participation in adult reproduction. Mol. Reprod. Dev. 82: 774–786, 2015. © 2015 The Authors. Molecular Reproduction and Development published by Wiley Periodicals, Inc. PMID:26096940

  16. Cytoplasmic polyadenylation element (CPE)- and CPE-binding protein (CPEB)-independent mechanisms regulate early class maternal mRNA translational activation in Xenopus oocytes.

    PubMed

    Charlesworth, Amanda; Cox, Linda L; MacNicol, Angus M

    2004-04-23

    Meiotic cell cycle progression during vertebrate oocyte maturation requires the correct temporal translation of maternal mRNAs encoding key regulatory proteins. The mechanism by which specific mRNAs are temporally activated is unknown, although both cytoplasmic polyadenylation elemen