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Sample records for oocyte meiotic arrest

  1. 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

  2. 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

  3. Role for endocytosis of a constitutively active GPCR (GPR185) in releasing vertebrate oocyte meiotic arrest.

    PubMed

    Nader, Nancy; Dib, Maya; Daalis, Arwa; Kulkarni, Rashmi P; Machaca, Khaled

    2014-11-15

    Vertebrate oocytes are naturally arrested at prophase of meiosis I for sustained periods of time before resuming meiosis in a process called oocyte maturation that prepares the egg for fertilization. Members of the constitutively active GPR3/6/12 family of G-protein coupled receptors represent important mediators of meiotic arrest. In the frog oocyte the GPR3/12 homolog GPRx (renamed GPR185) has been shown to sustain meiotic arrest by increasing intracellular cAMP levels through GαSβγ. Here we show that GPRx is enriched at the cell membrane (~80%), recycles through an endosomal compartment at steady state, and loses its ability to signal once trapped intracellularly. Progesterone-mediated oocyte maturation is associated with significant internalization of both endogenous and overexpressed GPRx. Furthermore, a GPRx mutant that does not internalize in response to progesterone is significantly more efficient than wild-type GPRx at blocking oocyte maturation. Collectively our results argue that internalization of the constitutively active GPRx is important to release oocyte meiotic arrest. PMID:25220151

  4. Time course of the meiotic arrest in sheep cumulus-oocyte complexes treated with roscovitine.

    PubMed

    Crocomo, Letícia Ferrari; Marques Filho, Wolff Camargo; Ackermann, Camila Louise; Paschoal, Daniela Martins; Guastali, Midyan Daroz; Dias Maziero, Rosiára Rosária; Sudano, Mateus José; Landim-Alvarenga, Fernanda da Cruz; Bicudo, Sony Dimas

    2016-04-01

    Temporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus-oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent. PMID:26170094

  5. The C.elegans MAPK phosphatase LIP-1 is required for the G2/M meiotic arrest of developing oocytes

    PubMed Central

    Hajnal, Alex; Berset, Thomas

    2002-01-01

    In the Caenorhabditis elegans hermaphrodite germline, spatially restricted mitogen-activated protein kinase (MAPK) signalling controls the meiotic cell cycle. First, the MAPK signal is necessary for the germ cells to progress through pachytene of meiotic prophase I. As the germ cells exit pachytene and enter diplotene/diakinesis, MAPK is inactivated and the developing oocytes arrest in diakinesis (G2/M arrest). During oocyte maturation, a signal from the sperm reactivates MAPK to promote M phase entry. Here, we show that the MAPK phosphatase LIP-1 dephosphorylates MAPK as germ cells exit pachytene in order to maintain MAPK in an inactive state during oocyte development. Germ cells lacking LIP-1 fail to arrest the cell cycle at the G2/M boundary, and they enter a mitotic cell cycle without fertilization. LIP-1 thus coordinates oocyte cell cycle progression and maturation with ovulation and fertilization. PMID:12169634

  6. 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

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

    PubMed

    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

  8. Release from Xenopus oocyte prophase I meiotic arrest is independent of a decrease in cAMP levels or PKA activity.

    PubMed

    Nader, Nancy; Courjaret, Raphael; Dib, Maya; Kulkarni, Rashmi P; Machaca, Khaled

    2016-06-01

    Vertebrate oocytes arrest at prophase of meiosis I as a result of high levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity. In Xenopus, progesterone is believed to release meiotic arrest by inhibiting adenylate cyclase, lowering cAMP levels and repressing PKA. However, the exact timing and extent of the cAMP decrease is unclear, with conflicting reports in the literature. Using various in vivo reporters for cAMP and PKA at the single-cell level in real time, we fail to detect any significant changes in cAMP or PKA in response to progesterone. More interestingly, there was no correlation between the levels of PKA inhibition and the release of meiotic arrest. Furthermore, we devised conditions whereby meiotic arrest could be released in the presence of sustained high levels of cAMP. Consistently, lowering endogenous cAMP levels by >65% for prolonged time periods failed to induce spontaneous maturation. These results argue that the release of oocyte meiotic arrest in Xenopus is independent of a reduction in either cAMP levels or PKA activity, but rather proceeds through a parallel cAMP/PKA-independent pathway. PMID:27122173

  9. 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

  10. 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

  11. Mature cystic teratomas arise from meiotic oocytes, but not from pre-meiotic oogonia.

    PubMed

    Kaku, Hiroshi; Usui, Hirokazu; Qu, Jia; Shozu, Makio

    2016-04-01

    Mature cystic teratomas (MCTs) in the ovaries have been thought to originate from germ cells from all developmental stages, i.e., from pre-meiotic oogonia through meiotic oocytes to mature post-meiotic ova. This view was based on research on MCTs by classical methods, including those involving centromeric heteromorphisms in karyotypes, enzyme polymorphisms, and DNA polymorphisms. However, insufficient genomic information was obtained in those studies. The current study aimed to confirm the cytogenetic origin of ovarian MCTs by using short tandem repeat (STR) polymorphism analysis to obtain sufficient genomic information, especially in connection with centromeric loci. Tissue samples of MCTs (57 ovaries from 51 patients, 91 MCTs, 156 specimens in total) obtained from cystectomies or oophorectomies were used. We categorized the specimens into two groups: i) solid components of MCTs and ii) cyst walls. The numbers of solid components of MCTs from pre-meiotic oogonia, primary oocytes, secondary oocytes, and ova were 0, 33, 16, and 15, respectively. There were no pre-meiotic oogonia in this series of solid-component specimens. We propose a hypothesis for the tumorigenesis of ovarian MCTs: the precursors of ovarian MCTs are not functional oocytes or ova, but are primary oocytes that have escaped from meiotic arrest. This hypothesis could satisfactorily explain the lack of pre-meiotic teratomas observed in this study and the nearly equal distribution of teratomas originating from primary oocytes, secondary oocytes, and ova in previous studies. Furthermore, this hypothesis could provide a starting point for determining the mechanism underlying tumorigenesis of ovarian MCTs. © 2016 Wiley Periodicals, Inc. PMID:26791142

  12. 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

  13. Nek11 regulates asymmetric cell division during mouse oocyte meiotic maturation.

    PubMed

    Guo, Lei; Wang, Zhen-Bo; Wang, Hong-Hui; Zhang, Teng; Qi, Shu-Tao; Ouyang, Ying-Chun; Hou, Yi; Sun, Qing-Yuan

    2016-06-10

    Nek11, a member of the never in mitosis gene A (NIMA) family, is activated in somatic cells associated with G1/S or G2/M arrest. However, its function in meiosis is unknown. In this research, the expression, localization and functions of NEK11 in the mouse oocyte meiotic maturation were examined. Western blotting indicated that NEK11S was the major NEK11 protein in mouse oocyte. MYC-tagged Nek11 mRNA microinjection and immunofluorescent staining showed that NEK11 was localized to the meiotic spindles at MI and MII stage. Knockdown of Nek11 by microinjection of siRNA did not affect germinal vesicle breakdown (GVBD) and the first polar body extrusion, but caused formation of 2-cell-like eggs. These results demonstrate that Nek11 regulates asymmetric cell division during oocyte meiotic maturation. PMID:27150633

  14. Rab6a is a novel regulator of meiotic apparatus and maturational progression in mouse oocytes

    PubMed Central

    Hou, Xiaojing; Zhang, Jiaqi; Li, Ling; Ma, Rujun; Ge, Juan; Han, Longsen; Wang, Qiang

    2016-01-01

    Rab family GTPases have been well known to regulate intracellular vesicle transport, however their function in mammalian oocytes has not been addressed. In this study, we report that when Rab6a is specifically knockdown, mouse oocytes are unable to progress normally through meiosis, arresting at metaphase I. Moreover, in these oocytes, the defects of chromosome alignment and spindle organization are readily observed during maturation, and resultantly increasing the aneuploidy incidence. We further reveal that kinetochore-microtubule attachments are severely compromised in Rab6a-depleted oocytes, which may in part mediate the meiotic phenotypes described above. In addition, when Rab6a function is altered, BubR1 levels on the kinetochores are markedly increased in metaphase oocytes, indicating the activation of spindle assembly checkpoint. In sum, we identify Rab6a as an important player in modulating oocyte meiosis, specifically the chromosome/spindle organization and metaphase-anaphase transition. PMID:26915694

  15. 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

  16. Mos is not required for the initiation of meiotic maturation in Xenopus oocytes

    PubMed Central

    Dupré, Aude; Jessus, Catherine; Ozon, René; Haccard, Olivier

    2002-01-01

    In Xenopus oocytes, the c-mos proto-oncogene product has been proposed to act downstream of progesterone to control the entry into meiosis I, the transition from meiosis I to meiosis II, which is characterized by the absence of S phase, and the metaphase II arrest seen prior to fertilization. Here, we report that inhibition of Mos synthesis by morpholino antisense oligonucleotides does not prevent the progesterone-induced initiation of Xenopus oocyte meiotic maturation, as previously thought. Mos-depleted oocytes complete meiosis I but fail to arrest at metaphase II, entering a series of embryonic-like cell cycles accompanied by oscillations of Cdc2 activity and DNA replication. We propose that the unique and conserved role of Mos is to prevent mitotic cell cycles of the female gamete until the fertilization in Xenopus, starfish and mouse oocytes. PMID:12145203

  17. Mos is not required for the initiation of meiotic maturation in Xenopus oocytes.

    PubMed

    Dupré, Aude; Jessus, Catherine; Ozon, René; Haccard, Olivier

    2002-08-01

    In Xenopus oocytes, the c-mos proto-oncogene product has been proposed to act downstream of progesterone to control the entry into meiosis I, the transition from meiosis I to meiosis II, which is characterized by the absence of S phase, and the metaphase II arrest seen prior to fertilization. Here, we report that inhibition of Mos synthesis by morpholino antisense oligonucleotides does not prevent the progesterone-induced initiation of Xenopus oocyte meiotic maturation, as previously thought. Mos-depleted oocytes complete meiosis I but fail to arrest at metaphase II, entering a series of embryonic-like cell cycles accompanied by oscillations of Cdc2 activity and DNA replication. We propose that the unique and conserved role of Mos is to prevent mitotic cell cycles of the female gamete until the fertilization in Xenopus, starfish and mouse oocytes. PMID:12145203

  18. 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

  19. 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

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

    PubMed

    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

  1. 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

  2. How does Xenopus oocyte acquire its competence to undergo meiotic maturation?

    PubMed

    Jessus, Catherine; Ozon, René

    2004-04-01

    During Xenopus oogenesis, the follicle-enclosed oocyte, arrested at the diplotene stage of meiotic prophase, accumulates pre-MPF. Pre-MPF is an heterodimer formed of cyclin B2 and Cdc2 protein kinase, which is maintained inactive by inhibitory phosphorylations on Thr14 and Tyr15. When the oocyte reaches its full size, it becomes competent to respond to progesterone and to activate MPF through a positive feedback loop. In this paper, we present experimental data indicating that the molecular network involved in the autoamplification loop of MPF is progressively established during late oogenesis. PMID:15182701

  3. 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

  4. MOUSE VERSUS RAT: PROFOUND DIFFERENCES IN MEIOTIC REGULATION AT THE LEVEL OF THE ISOLATED OOCYTE

    PubMed Central

    Downs, Stephen M.

    2011-01-01

    Cumulus cell-enclosed oocytes (CEO), denuded oocytes (DO) or dissected follicles were obtained 44–48 h after priming immature mice (20–23-days-old) with 5 IU or immature rats (25–27-days-old) with 12.5 IU of equine chorionic gonadotropin, and exposed to a variety of culture conditions. Mouse oocytes were more effectively maintained in meiotic arrest by hypoxanthine, dbcAMP, IBMX, milrinone, and 8-Br-cGMP. The guanylate cyclase activator, atrial natriuretic peptide, suppressed maturation in CEO from both species, but mycophenolic acid reversed IBMX-maintained meiotic arrest in mouse CEO with little activity in rat CEO. IBMX-arrested mouse, but not rat, CEO were induced to undergo germinal vesicle breakdown (GVB) by follicle stimulating hormone (FSH) and amphiregulin, while human chorionic gonadotropin (hCG) was ineffective in both species. Nevertheless, FSH and amphiregulin stimulated cumulus expansion in both species. FSH and hCG were both effective inducers of GVB in cultured mouse and rat follicles while amphiregulin was stimulatory only in mouse follicles. Changing the culture medium or altering macromolecular supplementation had no effect on FSH-induced maturation in rat CEO. The AMP-activated protein kinase (AMPK) activator, AICAR, was a potent stimulator of maturation in mouse CEO and DO, but only marginally stimulatory in rat CEO and ineffective in rat DO. The AMPK inhibitor, compound C, blocked meiotic induction more effectively in hCG-treated mouse follicles and heat-treated mouse CEO. Both agents produced contrasting results on polar body formation in cultured CEO in the two species. Active AMPK was detected in germinal vesicles of immature mouse, but not rat, oocytes prior to hCG-induced maturation in vivo; it colocalized with chromatin after GVB in rat and mouse oocytes, but did not appear at the spindle poles in rat oocytes as it did in mouse oocytes. Finally, cultured mouse and rat CEO displayed disparate maturation responses to energy substrate

  5. 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

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

    PubMed

    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

  7. 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

  8. 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

  9. p90Rsk is not involved in cytostatic factor arrest in mouse oocytes

    PubMed Central

    Dumont, Julien; Umbhauer, Muriel; Rassinier, Pascale; Hanauer, André; Verlhac, Marie-Hélène

    2005-01-01

    Vertebrate oocytes arrest in metaphase of the second meiotic division (MII), where they maintain a high cdc2/cyclin B activity and a stable, bipolar spindle because of cytostatic factor (CSF) activity. The Mos–MAPK pathway is essential for establishing CSF. Indeed, oocytes from the mos−/− strain do not arrest in MII and activate without fertilization, as do Xenopus laevis oocytes injected with morpholino oligonucleotides directed against Mos. In Xenopus oocytes, p90Rsk (ribosomal S6 kinase), a MAPK substrate, is the main mediator of CSF activity. We show here that this is not the case in mouse oocytes. The injection of constitutively active mutant forms of Rsk1 and Rsk2 does not induce a cell cycle arrest in two-cell mouse embryos. Moreover, these two mutant forms do not restore MII arrest after their injection into mos−/− oocytes. Eventually, oocytes from the triple Rsk (1, 2, 3) knockout present a normal CSF arrest. We demonstrate that p90Rsk is not involved in the MII arrest of mouse oocytes. PMID:15837801

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

    PubMed

    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

  11. 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

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

    PubMed

    Ya, Ru; Downs, Stephen M

    2014-02-01

    The oocyte meiotic spindle is comprised of microtubules (MT) that bind chromatin and regulate both metaphase plate formation and karyokinesis during meiotic maturation; however, little information is known about their role in meiosis reinitiation. This study was conducted to determine if microtubule integrity is required for meiotic induction and to ascertain how it affects activation of AMP-activated protein kinase (AMPK), an important participant in the meiotic induction process. Treatment with microtubule-disrupting agents nocodazole and vinblastine suppressed meiotic resumption in a dose-dependent manner in both arrested cumulus cell-enclosed oocytes (CEO) stimulated with follicle-stimulating hormone (FSH) and arrested denuded oocytes (DO) stimulated with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR). This effect coincided with suppression of AMPK activation as determined by western blotting and germinal vesicle immunostaining. Treatment with the MT stabilizer paclitaxel also suppressed meiotic induction. Targeting actin filament polymerization had only a marginal effect on meiotic induction. Immunolocalization experiments revealed that active AMPK colocalized with γ-tubulin during metaphase I and II stages, while it localized at the spindle midzone during anaphase. This discrete localization pattern was dependent on MT integrity. Treatment with nocodazole led to disruption of proper spindle pole localization of active AMPK, while paclitaxel induced excessive polymerization of spindle MT and formation of ectopic asters with accentuated AMPK colocalization. Although stimulation of AMPK increased the rate of germinal vesicle breakdown (GVB), spindle formation and polar body (PB) extrusion, the kinase had no effect on 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

  13. In vitro follicle growth supports human oocyte meiotic maturation

    PubMed Central

    Xiao, Shuo; Zhang, Jiyang; Romero, Megan M.; Smith, Kristin N.; Shea, Lonnie D.; Woodruff, Teresa K.

    2015-01-01

    In vitro follicle growth is a potential approach to preserve fertility for young women who are facing a risk of premature ovarian failure (POF) caused by radiation or chemotherapy. Our two-step follicle culture strategy recapitulated the dynamic human follicle growth environment in vitro. Follicles developed from the preantral to antral stage, and, for the first time, produced meiotically competent metaphase II (MII) oocytes after in vitro maturation (IVM). PMID:26612176

  14. In vitro follicle growth supports human oocyte meiotic maturation.

    PubMed

    Xiao, Shuo; Zhang, Jiyang; Romero, Megan M; Smith, Kristin N; Shea, Lonnie D; Woodruff, Teresa K

    2015-01-01

    In vitro follicle growth is a potential approach to preserve fertility for young women who are facing a risk of premature ovarian failure (POF) caused by radiation or chemotherapy. Our two-step follicle culture strategy recapitulated the dynamic human follicle growth environment in vitro. Follicles developed from the preantral to antral stage, and, for the first time, produced meiotically competent metaphase II (MII) oocytes after in vitro maturation (IVM). PMID:26612176

  15. Ras family proteins: new players involved in the diplotene arrest of Xenopus oocytes.

    PubMed

    Jessus, C; Rime, H; Ozon, R

    1998-11-01

    Oogonia undergo numerous mitotic cell cycles before completing the last DNA replication and entering the meiotic prophase I. After chromosome pairing and chromatid exchanges between paired chromosomes, the oocyte I remains arrested at the diplotene stage of the first meiotic prophase. Oocyte growth then occurs independently of cell division; indeed, during this growth period, oocytes (4n DNA) are prevented from completing the meiotic divisions. How is the prophase arrest regulated? One of the players of the prophase block is the high level of intracellular cAMP, maintained by an active adenylate cyclase. By using lethal toxin from Clostridium sordellii (LT), a glucosyltransferase that glucosylates and inactivates small G proteins of the Ras subfamily, we have shown that inhibition of either Ras or Rap or both proteins is sufficient to release the prophase block of Xenopus oocytes in a cAMP-dependent manner. The implications of Ras family proteins as new players involved in the prophase arrest of Xenopus oocytes will be discussed here. PMID:10069002

  16. Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes.

    PubMed

    Franciosi, Federica; Coticchio, Giovanni; Lodde, Valentina; Tessaro, Irene; Modina, Silvia C; Fadini, Rubens; Dal Canto, Mariabeatrice; Renzini, Mario Mignini; Albertini, David F; Luciano, Alberto M

    2014-09-01

    Oocyte in vitro maturation (IVM) has become a valuable technological tool for animal breeding and cloning and the treatment of human infertility because it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either prematuration culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a nonpharmacologic meiosis-arresting agent during IVM in a monoovulatory species. NPPC has been shown to maintain meiotic arrest in vivo and in vitro in mice and pigs; however, the use of this molecule for PMC has yet to have been explored. Toward this end, meiotic cell cycle reentry, gap-junction functionality, and chromatin configuration changes were investigated in bovine cumulus-enclosed oocytes cultured in the presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, in vitro fertilization, and embryo culture and compared to standard IVM-in vitro fertilization protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols. PMID:25078681

  17. 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

  18. 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

  19. The functional role of oxytocin in the induction of oocyte meiotic resumption in cattle.

    PubMed

    De Cesaro, M P; Trois, R L; Gutierrez, K; Siqueira, L; Rigo, M L; Glanzner, W G; Oliveira, J F; Gonçalves, P B

    2013-10-01

    The aim of the present study was to examine the role of oxytocin (OT) in the progesterone (P4) and prostaglandins (PGs) pathway to induce oocyte meiotic resumption. Cumulus-oocyte complexes were co-cultured with follicular hemisections for 15 h to determine the effects of different doses of OT or atosiban (ATO; oxytocin receptor antagonist) on oocyte meiotic resumption. In another experiment, we examined the effect of the interaction between P4, OT and PGs on the regulatory cascade of the oocyte meiotic resumption. Oxytocin at 1 μm was effective in inducing meiotic resumption in oocytes co-cultured with follicular cells (84.0%), not differing from the positive control group (74.4%). Atosiban inhibited in a dose-dependent manner the positive effect of OT on the meiotic resumption (27.6% metaphase I with 10 μm of ATO, which did not differ from the 25.5% of the negative control group). Furthermore, a third experiment showed that P4 was able to induce oocyte meiotic resumption, which was inhibited by ATO. However, the OT positive effect was not blocked by mifepristone (P4 antagonist), but was inhibited by indomethacin (a non-selective PTGS2 inhibitor). Collectively, these data suggest a sequential role of P4, OT and PGs in the induction of oocyte meiotic resumption. PMID:23691948

  20. Aurora B inhibitor barasertib prevents meiotic maturation and subsequent embryo development in pig oocytes.

    PubMed

    Ju, Shiqiang; Peng, Xu; Yang, Xiaoliu; Sozar, Sparksi; Muneri, Caroline W; Xu, Yaping; Chen, Changchao; Cui, Panpan; Xu, Weichao; Rui, Rong

    2016-07-15

    Barasertib, a highly selective Aurora B inhibitor, has been widely used in a variety of cells to investigate the role of Aurora B kinase, which has been implicated in various functions in the mitotic process. However, effects of barasertib on the meiotic maturation process are not fully understood, particularly in porcine oocyte meiotic maturation. In the present study, the effects of barasertib on the meiotic maturation and developmental competence of pig oocytes were investigated, and the possible roles of Aurora B were also evaluated in porcine oocytes undergoing meiosis. Initially, we examined the expression and subcellular localization of Aurora B using Western blot analysis and immunofluorescent staining. Aurora B was found to express and exhibit specific dynamic intracellular localization during porcine oocyte meiotic maturation. Aurora B was observed around the chromosomes after germinal vesicle breakdown. Then it was transferred to the spindle region after metaphase I stage, and was particularly concentrated at the central spindles at telophase I stage. barasertib treatment resulted in the failure of polar body extrusion in pig oocytes, with a larger percentage of barasertib-treated oocytes remaining at the pro-metaphase I stage. Additional results reported that barasertib treatment had no effect on chromosome condensation but resulted in a significantly higher percentage of the treated oocytes with aberrant spindles and misaligned chromosomes during the first meiotic division. In addition, inhibition of Aurora B with lower concentrations of barasertib during pig oocyte meiotic maturation decreased the subsequent embryo developmental competence. Thus, these results illustrate that barasertib has significant effects on porcine oocyte meiotic maturation and subsequent development through Aurora B inhibition, and this regulation is related to its effects on spindle formation and chromosome alignment during the first meiotic division in porcine oocytes. PMID

  1. The GTPase SPAG-1 orchestrates meiotic program by dictating meiotic resumption and cytoskeleton architecture in mouse oocytes.

    PubMed

    Huang, Chunjie; Wu, Di; Khan, Faheem Ahmed; Jiao, Xiaofei; Guan, Kaifeng; Huo, Lijun

    2016-06-01

    In mammals, a finite population of oocytes is generated during embryogenesis, and proper oocyte meiotic divisions are crucial for fertility. Sperm-associated antigen 1 (SPAG-1) has been implicated in infertility and tumorigenesis; however, its relevance in cell cycle programs remains rudimentary. Here we explore a novel role of SPAG-1 during oocyte meiotic progression. SPAG-1 associated with meiotic spindles and its depletion severely compromised M-phase entry (germinal vesicle breakdown [GVBD]) and polar body extrusion. The GVBD defect observed was due to an increase in intraoocyte cAMP abundance and decrease in ATP production, as confirmed by the activation of AMP-dependent kinase (AMPK). SPAG-1 RNA interference (RNAi)-elicited defective spindle morphogenesis was evidenced by the dysfunction of γ-tubulin, which resulted from substantially reduced phosphorylation of MAPK and irregularly dispersed distribution of phospho-MAPK around spindles instead of concentration at spindle poles. Significantly, actin expression abruptly decreased and formation of cortical granule-free domains, actin caps, and contractile ring disrupted by SPAG-1 RNAi. In addition, the spindle assembly checkpoint remained functional upon SPAG-1 depletion. The findings broaden our knowledge of SPAG-1, showing that it exerts a role in oocyte meiotic execution via its involvement in AMPK and MAPK signaling pathways. PMID:27053660

  2. Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing.

    PubMed

    Baumann, Claudia; Viveiros, Maria M

    2015-01-01

    Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down's syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization. PMID:26485537

  3. Disrupting Cyclin Dependent Kinase 1 in Spermatocytes Causes Late Meiotic Arrest and Infertility in Mice.

    PubMed

    Clement, Tracy M; Inselman, Amy L; Goulding, Eugenia H; Willis, William D; Eddy, Edward M

    2015-12-01

    While cyclin dependent kinase 1 (CDK1) has a critical role in controlling resumption of meiosis in oocytes, its role has not been investigated directly in spermatocytes. Unique aspects of male meiosis led us to hypothesize that its role is different in male meiosis than in female meiosis. We generated a conditional knockout (cKO) of the Cdk1 gene in mouse spermatocytes to test this hypothesis. We found that CDK1-null spermatocytes undergo synapsis, chiasmata formation, and desynapsis as is seen in oocytes. Additionally, CDK1-null spermatocytes relocalize SYCP3 to centromeric foci, express H3pSer10, and initiate chromosome condensation. However, CDK1-null spermatocytes fail to form condensed bivalent chromosomes in prophase of meiosis I and instead are arrested at prometaphase. Thus, CDK1 has an essential role in male meiosis that is consistent with what is known about the role of CDK1 in female meiosis, where it is required for formation of condensed bivalent metaphase chromosomes and progression to the first meiotic division. We found that cKO spermatocytes formed fully condensed bivalent chromosomes in the presence of okadaic acid, suggesting that cKO chromosomes are competent to condense, although they do not do so in vivo. Additionally, arrested cKO spermatocytes exhibited irregular cell shape, irregular large nuclei, and large distinctive nucleoli. These cells persist in the seminiferous epithelium through the next seminiferous epithelial cycle with a lack of stage XII checkpoint-associated cell death. This indicates that CDK1 is required upstream of a checkpoint-associated cell death as well as meiotic metaphase progression in mouse spermatocytes. PMID:26490841

  4. Cilostazol Improves Developmental Competence of Pig Oocytes by Increasing Intraoocyte Cyclic Adenosine Monophosphate Level and Delaying Meiotic Resumption.

    PubMed

    Elahi, F; Lee, H; Lee, Y; Park, B; Lee, J; Hyun, S-H; Lee, E

    2016-04-01

    Cilostazol (CLZ) is a cyclic adenosine monophosphate (cAMP) modulator that influences the steady state of the meiotic stage. This study was conducted to determine the effects of CLZ treatment during in vitro maturation (IVM) on developmental competence of pig oocytes. Immature oocytes were exposed to 0 (control), 0.5, 2 and 4 μm CLZ during the first 22 h of IVM. Nuclear maturation, intraoocyte glutathione content and embryo cleavage after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were not influenced by CLZ at any concentrations. However, 4 μm CLZ significantly (p < 0.05) improved blastocyst formation after PA (52.1% vs 38.7-46.0%) and SCNT relative to other concentrations (40.8% vs 25.0-30.7%). The mean cell numbers of SCNT blastocysts were significantly increased by 4 μm CLZ compared to the control (42.6 cells vs 35.3 cells/blastocyst). CLZ treatment significantly increased the intraoocyte cAMP level and effectively arrested oocytes at the germinal vesicle (GV) and GV break down stages compared to the control (74.5% vs 45.4%). Our results demonstrated that improved developmental competence of PA and SCNT pig embryos occurred via better synchronization of nuclear and cytoplasmic maturation induced by increased cAMP and delayed meiotic resumption after CLZ treatment. PMID:26834044

  5. Meiotic maturation of incompetent prepubertal sheep oocytes is induced by paracrine factor(s) released by gonadotropin-stimulated oocyte-cumulus cell complexes and involves mitogen-activated protein kinase activation.

    PubMed

    Cecconi, Sandra; Mauro, Annunziata; Capacchietti, Giulia; Berardinelli, Paolo; Bernabò, Nicola; Di Vincenzo, Anna Rita; Mattioli, Mauro; Barboni, Barbara

    2008-01-01

    In this study, sheep oocyte-cumulus cell complexes (OCC) derived from medium (M) antral follicles (M-OCC) were in vitro matured alone or in coculture with OCC derived from small (S) antral follicles (S-OCC) to investigate the contribution of cumulus cells (CC) and oocytes to the process of oocyte meiotic maturation and cumulus expansion (CE). Experiments were conducted with or without gonadotropins (FSH/LH). Regardless of culture conditions, about 12% of S-oocytes reached the metaphase II stage, and S-CC showed a low degree of CE. In contrast, both maturational processes were significantly stimulated by gonadotropins in M-OCC. However, about 48% of S-oocytes progressed to metaphase II, and S-CC expanded after coculture with gonadotropin-stimulated M-OCC and M-CC but not with mural granulosa cells. Both maturational processes were inhibited when S-OCC were cocultured with M-denuded oocytes, or when S-denuded oocytes were cocultured with M-CC. The capacity of these paracrine factor(s) to activate the MAPK pathway in somatic and germ cells of S-complexes was investigated. It was found that MAPK kinase/MAPK phosphorylation levels in M-OCC but not in S-OCC were significantly increased by gonadotropins, first in CC and later in the oocytes. Kinase phosphorylations were activated only in S-oocytes cocultured with M-OCC or M-CC. These results demonstrate that soluble factors specifically produced by M-CC are capable to induce meiotic maturation and CE in S-complexes by acting via CC. These factors can induce MAPK activation only in S-oocytes, whose meiotic arrest could be due to the inability of surrounding CC to respond to gonadotropin stimulation. PMID:17884935

  6. 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

  7. Nuf2 is required for chromosome segregation during mouse oocyte meiotic maturation

    PubMed Central

    Zhang, Teng; Zhou, Yang; Qi, Shu-Tao; Wang, Zhen-Bo; Qian, Wei-Ping; Ouyang, Ying-Chun; Shen, Wei; Schatten, Heide; Sun, Qing-Yuan

    2015-01-01

    Nuf2 plays an important role in kinetochore-microtubule attachment and thus is involved in regulation of the spindle assembly checkpoint in mitosis. In this study, we examined the localization and function of Nuf2 during mouse oocyte meiotic maturation. Myc6-Nuf2 mRNA injection and immunofluorescent staining showed that Nuf2 localized to kinetochores from germinal vesicle breakdown to metaphase I stages, while it disappeared from the kinetochores at the anaphase I stage, but relocated to kinetochores at the MII stage. Overexpression of Nuf2 caused defective spindles, misaligned chromosomes, and activated spindle assembly checkpoint, and thus inhibited chromosome segregation and metaphase-anaphase transition in oocyte meiosis. Conversely, precocious polar body extrusion was observed in the presence of misaligned chromosomes and abnormal spindle formation in Nuf2 knock-down oocytes, causing aneuploidy. Our data suggest that Nuf2 is a critical regulator of meiotic cell cycle progression in mammalian oocytes. PMID:26054848

  8. Sirt3 prevents maternal obesity-associated oxidative stress and meiotic defects in mouse oocytes

    PubMed Central

    Zhang, Liang; Han, Longsen; Ma, Rujun; Hou, Xiaojing; Yu, Yang; Sun, Shaochen; Xu, Yinxue; Schedl, Tim; Moley, Kelle H; Wang, Qiang

    2015-01-01

    Maternal obese environment has been reported to induce oxidative stress and meiotic defects in oocytes, however the underlying molecular mechanism remains unclear. Here, using mice fed a high fat diet (HFD) as an obesity model, we first detected enhanced reactive oxygen species (ROS) content and reduced Sirt3 expression in HFD oocytes. We further observed that specific depletion of Sirt3 in control oocytes elevates ROS levels while Sirt3 overexpression attenuates ROS production in HFD oocytes, with significant suppression of spindle disorganization and chromosome misalignment phenotypes that have been reported in the obesity model. Candidate screening revealed that the acetylation status of lysine 68 on superoxide dismutase (SOD2K68) is dependent on Sirt3 deacetylase activity in oocytes, and acetylation-mimetic mutant SOD2K68Q results in almost threefold increase in intracellular ROS. Moreover, we found that acetylation levels of SOD2K68 are increased by ∼80% in HFD oocytes and importantly, that the non-acetylatable-mimetic mutant SOD2K68R is capable of partially rescuing their deficient phenotypes. Together, our data identify Sirt3 as an important player in modulating ROS homeostasis during oocyte development, and indicate that Sirt3-dependent deacetylation of SOD2 plays a protective role against oxidative stress and meiotic defects in oocytes under maternal obese conditions. PMID:25790176

  9. 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

  10. Zearalenone exposure affects mouse oocyte meiotic maturation and granulosa cell proliferation.

    PubMed

    Hou, Yan-Jun; Zhu, Cheng-Cheng; Xu, Yin-Xue; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2015-09-01

    Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN-treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule-free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose-dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse. PMID:24733567

  11. 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

  12. 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

  13. Naturally occurring steroids in Xenopus oocyte during meiotic maturation. Unexpected presence and role of steroid sulfates.

    PubMed

    Haccard, Olivier; Dupré, Aude; Liere, Philippe; Pianos, Antoine; Eychenne, Bernard; Jessus, Catherine; Ozon, René

    2012-10-15

    In the ovary, oocytes are surrounded by follicle cells and arrested in prophase of meiosis I. Although steroidogenic activity of follicle cells is involved in oogenesis regulation, clear qualitative and quantitative data about the steroid content of follicles are missing. We measured steroid levels of Xenopus oocytes and follicles by gas chromatography-mass spectrometry. We show that dehydroepiandrosterone sulfate is the main steroid present in oocytes. Lower levels of free steroids are also detected, e.g., androgens, whereas progesterone is almost undetectable. We propose that sulfatation is a protective mechanism against local variations of active steroids that could be deleterious for follicle-enclosed oocytes. Steroid levels were measured after LH stimulation, responsible for the release by follicle cells of a steroid signal triggering oocyte meiosis resumption. Oocyte levels of androgens rise slowly during meiosis re-entry whereas progesterone increases abruptly to micromolar concentration, therefore representing the main physiological mediator of meiosis resumption in Xenopus oocyte. PMID:22687883

  14. 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

  15. 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

  16. Guanine Nucleotides in the Meiotic Maturation of Starfish Oocytes: Regulation of the Actin Cytoskeleton and of Ca2+ Signaling

    PubMed Central

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

    2009-01-01

    Background Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca2+ and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca2+ signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation. Methodology/Principal Findings By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPγS) or inactive (GDPβS) forms of G-proteins, we have demonstrated that: i) GTPγS induces Ca2+ release that mimics the effect of 1-MA; ii) GDPβS completely blocks 1-MA-induced Ca2+; iii) GDPβS has little effect on the amplitude of the Ca2+ peak, but significantly expedites the initial Ca2+ waves induced by InsP3 photoactivation, iv) GDPβS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca2+ release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPβS, or actinase E, all led to significant changes of 1-MA-induced Ca2+ signaling. Conclusions/Significance Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca2+ release. PMID:19617909

  17. 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

  18. A moderate increase of hydrogen peroxide level is beneficial for spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro.

    PubMed

    Pandey, Ashutosh N; Chaube, Shail K

    2014-08-01

    Hydrogen peroxide (H2O2) acts as a signaling molecule and modulates various aspects of cell functions in a wide variety of cells including mammalian germ cells. We examined whether a decreased level of intra-oocyte cyclic 3',5'-adenosine monophosphate (cAMP) leads to accumulation of H2O2, and if so, whether a moderate increase of H2O2 inactivates maturation promoting factor (MPF) during spontaneous resumption of meiosis in rat oocytes cultured in vitro. Removal of cumulus cells and culture of denuded oocytes in vitro significantly decreased oocyte cAMP level and led to spontaneous meiotic resumption from diplotene arrest. The reduced oocyte cAMP level was associated with an increased oocyte H2O2 level and reduced catalase activity. Exogenous supplementation of H2O2 induced meiotic resumption from diplotene arrest in a concentration- and time-dependent manner in oocytes treated with 0.1 mM of 3-isobutyl-1-methylxanthine, while dibutyryl-cAMP and 3-t-butyl-4-hydroxyanisole inhibited the stimulatory effect of exogenous H2O2. The increased intra-oocyte H2O2 level induced Thr-14/Tyr-15 phosphorylation of CDK1, while Thr-161 phosphorylated CDK1 and cyclin B1 levels were reduced significantly. These results suggest that a decreased level of intra-oocyte cAMP is associated with an increased level of H2O2. The increased level of H2O2 was associated with high phosphorylation of Thr-14/Tyr-15 and dephosphorylation of the Thr-161 residue of CDK1 and reduced the cyclin B1 level, which eventually inactivated MPF. The MPF inactivation triggered spontaneous resumption of meiosis from diplotene arrest in rat oocytes cultured in vitro. PMID:25126482

  19. 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.

  20. Beclin-1 knockdown shows abscission failure but not autophagy defect during oocyte meiotic maturation.

    PubMed

    You, Seung Yeop; Park, Yong Seok; Jeon, Hyuk-Joon; Cho, Dong-Hyung; Jeon, Hong Bae; Kim, Sung Hyun; Chang, Jong Wook; Kim, Jae-Sung; Oh, Jeong Su

    2016-06-17

    Cytokinesis is the final step in cell division that results in the separation of a parent cell into daughter cells. Unlike somatic cells that undergo symmetric division, meiotic division is highly asymmetric, allowing the preservation of maternal resources for embryo development. Beclin-1/BECN1, the mammalian homolog of yeast Atg6, is a key molecule of autophagy. As part of a class III phosphatidylinositol 3-kinase (PI3K-III) complex, BECN1 initiates autophagosome formation by coordinating membrane trafficking. However, emerging evidence suggests that BECN1 regulates chromosome segregation and cytokinesis during mitosis. Thus, we investigated the function of BECN1 during oocyte meiotic maturation. BECN1 was widely distributed during meiotic maturation forming small vesicles. Interestingly, BECN1 is also detected at the midbody ring during cytokinesis. Depletion of BECN1 impaired the cytokinetic abscission, perturbing the recruitment of ZFYVE26 at the midbody. Similar phenotypes were observed when PI3K-III activity was inhibited. However, inhibition of autophagy by depleting Atg14L did not disturb meiotic maturation. Therefore, our results not only demonstrate that BECN1 as a PI3K-III component is essential for cytokinesis, but also suggest that BECN1 is not associated with autophagy pathway in mouse oocytes. PMID:27149384

  1. 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

  2. Metaphase I arrest and spontaneous parthenogenetic activation of strain LTXBO oocytes: chimeric reaggregated ovaries establish primary lesion in oocytes.

    PubMed

    Eppig, J J; Wigglesworth, K; Hirao, Y

    2000-08-01

    Oocytes of strain LT mice, and related strains such as LTXBO, exhibit a high incidence of arrest in the progression of meiosis at metaphase I (MI) and in spontaneous parthenogenetic activation. Activation of these oocytes within the ovary leads to the formation of ovarian teratomas. In this study, the role of the oocyte's companion granulosa cells, the cumulus cells, was investigated using fully grown oocytes matured in vitro after isolation from LTXBO mice. Results showed that the role of cumulus cells in MI arrest is dichotomous. Cumulus cells temporarily helped to sustain MI arrest, but they also promoted a delayed progression to metaphase II. Cumulus cells also promoted parthenogenetic activation that occurred in association with the delayed progression to metaphase II. Next, the question of whether the lesion(s) promoting MI arrest and spontaneous activation is due to defects in the somatic cells or is intrinsic to the oocyte was addressed using chimeric reaggregated ovaries. An improved method for completely exchanging the germ cell and the somatic cell compartments of ovaries from newborn mice is described. These chimeric reaggregated ovaries, grafted beneath the renal capsule of SCID mice, allowed the complete development of LTXBO oocytes to occur in association with somatic cells from control (B6SJLF(1)) ovaries and development of control oocytes in association with LTXBO somatic cells. Oocyte growth and follicular development appeared generally normal in reaggregated ovaries. High incidences of MI arrest and spontaneous activation of LTXBO oocytes occurred regardless of the genotype of the somatic cells. Moreover, there was a low incidence of MI arrest and spontaneous activation of control oocytes, even though they underwent complete development and maturation associated with LTXBO somatic cells. It is concluded that the phenotypes of MI arrest and parthenogenetic activation in LTXBO oocytes are defects caused by lesions intrinsic to the oocyte

  3. Rab3A, Rab27A, and Rab35 regulate different events during mouse oocyte meiotic maturation and activation.

    PubMed

    Wang, H H; Cui, Q; Zhang, T; Wang, Z B; Ouyang, Y C; Shen, W; Ma, J Y; Schatten, H; Sun, Q Y

    2016-06-01

    Rab family members play important roles in membrane trafficking, cell growth, and differentiation. Almost all components of the cell endomembrane system, the nucleus, and the plasma membrane are closely related to RAB proteins. In this study, we investigated the distribution and functions of three members of the Rab family, Rab3A, Rab27A, and Rab35, in mouse oocyte meiotic maturation and activation. The three Rab family members showed different localization patterns in oocytes. Microinjection of siRNA, antibody injection, or inhibitor treatment showed that (1) Rab3A regulates peripheral spindle and cortical granule (CG) migration, polarity establishment, and asymmetric division; (2) Rab27A regulates CG exocytosis following MII-stage oocyte activation; and (3) Rab35 plays an important role in spindle organization and morphology maintenance, and thus meiotic nuclear maturation. These results show that Rab proteins play important roles in mouse oocyte meiotic maturation and activation and that different members exert different distinct functions. PMID:26791531

  4. 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

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

    PubMed

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

    2007-01-01

    We have developed a new technique called Computer-Aided Meiotic Maturation Assay (CAMMA) for imaging large arrays of zebrafish oocytes and automatically collecting image files at regular intervals during meiotic maturation. This novel method uses a transparency scanner interfaced to a computer with macro programming that automatically scans and archives the image files. Images are stacked and analyzed with ImageJ to quantify changes in optical density characteristic of zebrafish oocyte maturation. Major advantages of CAMMA include (1) ability to image very large arrays of oocytes and follow individual cells over time, (2) simultaneously image many treatment groups, (3) digitized images may be stacked, animated, and analyzed in programs such as ImageJ, NIH-Image, or ScionImage, and (4) CAMMA system is inexpensive, costing less than most microscopes used in traditional assays. We have used CAMMA to determine the dose response and time course of oocyte maturation induced by 17alpha-hydroxyprogesterone (HP). Maximal decrease in optical density occurs around 5 hr after 0.1 micro g/ml HP (28.5 degrees C), approximately 3 hr after germinal vesicle migration (GVM) and dissolution (GVD). In addition to changes in optical density, GVD is accompanied by streaming of ooplasm to the animal pole to form a blastodisc. These dynamic changes are readily visualized by animating image stacks from CAMMA; thus, CAMMA provides a valuable source of time-lapse movies for those studying zebrafish oocyte maturation. The oocyte clearing documented by CAMMA is correlated to changes in size distribution of major yolk proteins upon SDS-PAGE, and, this in turn, is related to increased cyclin B(1) protein. PMID:16998847

  6. 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

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

    PubMed

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

    2016-09-01

    The maintenance of genomic integrity and stability is essential for the survival of every organism. Unfortunately, DNA is vulnerable to attack by a variety of damaging agents. Oxidative stress is a major cause of DNA damage because reactive oxygen species (ROS) are produced as by-products of normal cellular metabolism. Cells have developed eloquent antioxidant defense systems to protect themselves from oxidative damage along with aerobic metabolism. Here, we show that catalase (CAT) is present in mouse oocytes to protect the genome from oxidative damage during meiotic maturation. CAT was expressed in the nucleus to form unique vesicular structures. However, after nuclear envelope breakdown, CAT was redistributed in the cytoplasm with particular focus at the chromosomes. Inhibition of CAT activity increased endogenous ROS levels, but did not perturb meiotic maturation. In addition, CAT inhibition produced chromosomal defects, including chromosome misalignment and DNA damage. Therefore, our data suggest that CAT is required not only to scavenge ROS, but also to protect DNA from oxidative damage during meiotic maturation in mouse oocytes. PMID:27160095

  8. Cyclin B synthesis and rapamycin-sensitive regulation of protein synthesis during starfish oocyte meiotic divisions.

    PubMed

    Lapasset, Laure; Pradet-Balade, Bérengère; Vergé, Valérie; Lozano, Jean-Claude; Oulhen, Nathalie; Cormier, Patrick; Peaucellier, Gérard

    2008-11-01

    Translation of cyclin mRNAs represents an important event for proper meiotic maturation and post-fertilization mitoses in many species. Translational control of cyclin B mRNA has been described to be achieved through two separate but related mechanisms: translational repression and polyadenylation. In this paper, we evaluated the contribution of global translational regulation by the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-binding protein) on the cyclin B protein synthesis during meiotic maturation of the starfish oocytes. We used the immunosupressant drug rapamycin, a strong inhibitor of cap-dependent translation, to check for the involvement of this protein synthesis during this physiological process. Rapamycin was found to prevent dissociation of 4E-BP from the initiation factor eIF4E and to suppress correlatively a burst of global protein synthesis occurring at the G2/M transition. The drug had no effect on first meiotic division but defects in meiotic spindle formation prevented second polar body emission, demonstrating that a rapamycin-sensitive pathway is involved in this mechanism. While rapamycin affected the global protein synthesis, the drug altered neither the specific translation of cyclin B mRNA nor the expression of the Mos protein. The expression of these two proteins was correlated with the phosphorylation and the dissociation of the cytoplasmic polyadenylation element-binding protein from eIF4E. PMID:18361417

  9. 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.

  10. 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

  11. Fas expression correlates with human germ cell degeneration in meiotic and post-meiotic arrest of spermatogenesis.

    PubMed

    Francavilla, Sandro; D'Abrizio, Piera; Cordeschi, Giuliana; Pelliccione, Fiore; Necozione, Stefano; Ulisse, Salvatore; Properzi, Giuliana; Francavilla, Felice

    2002-03-01

    Degeneration of human male germ cells was analysed by means of light (LM) and transmission electron (TEM) microscopy. The frequency of degenerating cells was correlated with that of Fas-expressing germ cells in human testes with normal spermatogenesis (n = 10), complete early maturation arrest (EMA) (n = 10) or incomplete late maturation arrest (LMA; n = 10) of spermatogenesis. LM analysis of testis sections with normal spermatogenesis indicated that degenerating germ cells were localized in the adluminal compartment of the seminiferous epithelium. TEM showed that apoptotic cells were mostly primary spermatocytes and, to a lesser extent, round or early elongating spermatids. Apoptotic germ cells appeared to be eliminated either in the seminiferous lumen or by Sertoli cell phagocytosis. An increased number of degenerating cells was observed in testes with LMA as compared with normal testes and testes with EMA of spermatogenesis (P < 0.001, Wilcoxon's rank sum test). Comparison of these results with those obtained from immunohistochemistry experiments demonstrated a tight correlation between the number of apoptotic cells and the number of Fas-expressing germ cells (P = 0.001, Spearman's rank = 0.69). These findings suggest that altered meiotic and post-meiotic germ cell maturation might be associated with an up-regulation of Fas gene expression capable of triggering apoptotic elimination of defective germ cells. PMID:11870228

  12. Meiosis arrest female 1 (MARF1) has nuage-like function in mammalian oocytes

    PubMed Central

    Su, You-Qiang; Sun, Fengyun; Handel, Mary Ann; Schimenti, John C.; Eppig, John J.

    2012-01-01

    Orderly regulation of meiosis and protection of germline genomic integrity from transposable elements are essential for male and female gamete development. In the male germline, these processes are ensured by proteins associated with cytoplasmic nuage, but morphologically similar germ granules or nuage have not been identified in mammalian female germ cells. Indeed, many mutations affecting nuage-associated proteins such as PIWI and tudor domain containing proteins 5 and 7 (TDRD5/7) can result in failure of meiosis, up-regulation of retrotransposons, and infertility only in males and not in females. We recently identified MARF1 (meiosis arrest female 1) as a protein essential for controlling meiosis and retrotransposon surveillance in oocytes; and in contrast to PIWI-pathway mutations, Marf1 mutant females are infertile, whereas mutant males are fertile. Here we put forward the hypothesis that MARF1 in mouse oocytes is a functional counterpart of the nuage-associated components of spermatocytes. We describe the developmental pattern of Marf1 expression and its roles in retrotransposon silencing and protection from DNA double-strand breaks. Analysis of MARF1 protein domains compared with PIWI and TDRD5/7 revealed that these functional similarities are reflected in remarkable structural analogies. Thus, functions that in the male germline require protein interactions and cooperative scaffolding are combined in MARF1, allowing a single molecule to execute crucial activities of meiotic regulation and protection of germline genomic integrity. PMID:23090997

  13. A requirement for fatty acid oxidation in the hormone-induced meiotic maturation of mouse oocytes.

    PubMed

    Valsangkar, Deepa; Downs, Stephen M

    2013-08-01

    We have previously shown that fatty acid oxidation (FAO) is required for AMP-activated protein kinase (PRKA)-induced maturation in vitro. In the present study, we have further investigated the role of this metabolic pathway in hormone-induced meiotic maturation. Incorporating an assay with (3)H-palmitic acid as the substrate, we first examined the effect of PRKA activators on FAO levels. There was a significant stimulation of FAO in cumulus cell-enclosed oocytes (CEO) treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and RSVA405. In denuded oocytes (DO), AICAR stimulated FAO only in the presence of carnitine, the molecule that facilitates fatty acyl CoA entry into the mitochondria. The carnitine palmitoyltransferase 1 activator C75 successfully stimulated FAO in CEO. All three of these activators trigger germinal vesicle breakdown. Meiotic resumption induced by follicle-stimulating hormone (FSH) or amphiregulin was completely inhibited by the FAO inhibitors etomoxir, mercaptoacetate, and malonyl CoA. Importantly, FAO was increased in CEO stimulated by FSH and epidermal growth factor, and this increase was blocked by FAO inhibitors. Moreover, compound C, a PRKA inhibitor, prevented the FSH-induced increase in FAO. Both carnitine and palmitic acid augmented hormonal induction of maturation. In a more physiological setting, etomoxir eliminated human chorionic gonadotropin (hCG)-induced maturation in follicle-enclosed oocytes. In addition, CEO and DO from hCG-treated mice displayed an etomoxir-sensitive increase in FAO, indicating that this pathway was stimulated during in vivo meiotic resumption. Taken together, our data indicate that hormone-induced maturation in mice requires a PRKA-dependent increase in FAO. PMID:23863407

  14. 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

  15. The Inhibition of Polo Kinase by Matrimony Maintains G2 Arrest in the Meiotic Cell Cycle

    PubMed Central

    Xiang, Youbin; Takeo, Satomi; Florens, Laurence; Hughes, Stacie E; Huo, Li-Jun; Gilliland, William D; Swanson, Selene K; Teeter, Kathy; Schwartz, Joel W; Washburn, Michael P; Jaspersen, Sue L; Hawley, R. Scott

    2007-01-01

    Many meiotic systems in female animals include a lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB). However, the mechanisms by which a meiotic cell can arrest for long periods of time (decades in human females) have remained a mystery. The Drosophila Matrimony (Mtrm) protein is expressed from the end of pachytene until the completion of meiosis I. Loss-of-function mtrm mutants result in precocious NEB. Coimmunoprecipitation experiments reveal that Mtrm physically interacts with Polo kinase (Polo) in vivo, and multidimensional protein identification technology mass spectrometry analysis reveals that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Mutation of a Polo-Box Domain (PBD) binding site in Mtrm ablates the function of Mtrm and the physical interaction of Mtrm with Polo. The meiotic defects observed in mtrm/+ heterozygotes are fully suppressed by reducing the dose of polo+, demonstrating that Mtrm acts as an inhibitor of Polo. Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. Our data suggest a model in which the eventual activation of Cdc25 by an excess of Polo at stage 13 triggers NEB and entry into prometaphase. PMID:18052611

  16. Gene expression in bovine oocytes and cumulus cells after meiotic inhibition with the cyclin-dependent kinase inhibitor butyrolactone I.

    PubMed

    Leal, C L V; Mamo, S; Fair, T; Lonergan, P

    2012-08-01

    The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulus-oocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 μm BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation. PMID:22034924

  17. 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

  18. 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

  19. Apoptotic index within cumulus cells is a questionable marker of meiotic competence of bovine oocytes matured in vitro.

    PubMed

    Warzych, Ewelina; Pers-Kamczyc, Emilia; Krzywak, Anna; Dudzińska, Stanisława; Lechniak, Dorota

    2013-03-01

    Information gained from most human studies indicate a negative correlation between the apoptotic index (AI) in cumulus cells (CC) and the quality of the corresponding oocytes. However, results obtained in other species are not so consistent. The rate of apoptosis-free COCs (cumulus oocytes complexes) subjected to IVM (in vitro maturation) also varies among studies. The aim of the present study was to investigate whether the AI in cumulus cells of post-IVM COCs is related to the morphology of pre-IVM COCs and to meiotic competence of bovine oocytes. COCs of known morphology (four grade scale) obtained from individual follicles were matured in a well-in-drop system. After IVM, the external layers of CC of each COC were analyzed by TUNEL. In order to determine the meiotic stage, oocytes were stained with DAPI. It was found that 25.6% of bovine COCs contained apoptosis-free cumulus cells. Moreover, the majority of COCs with apoptotic cells were characterized by apoptotic index lower than 15%. The level of apoptosis in CC was related neither to COC morphology nor to the oocyte meiotic stage. It is suggested that the extent of apoptosis in cumulus cells is not a reliable quality marker of the corresponding oocyte after IVM. PMID:23522075

  20. Spindle Dynamics during Meiosis in Drosophila Oocytes

    PubMed Central

    Endow, Sharyn A.; Komma, Donald J.

    1997-01-01

    Mature oocytes of Drosophila are arrested in metaphase of meiosis I. Upon activation by ovulation or fertilization, oocytes undergo a series of rapid changes that have not been directly visualized previously. We report here the use of the Nonclaret disjunctional (Ncd) microtubule motor protein fused to the green fluorescent protein (GFP) to monitor changes in the meiotic spindle of live oocytes after activation in vitro. Meiotic spindles of metaphase-arrested oocytes are relatively stable, however, meiotic spindles of in vitro–activated oocytes are highly dynamic: the spindles elongate, rotate around their long axis, and undergo an acute pivoting movement to reorient perpendicular to the oocyte surface. Many oocytes spontaneously complete the meiotic divisions, permitting visualization of progression from meiosis I to II. The movements of the spindle after oocyte activation provide new information about the dynamic changes in the spindle that occur upon re-entry into meiosis and completion of the meiotic divisions. Spindles in live oocytes mutant for a lossof-function ncd allele fused to gfp were also imaged. The genesis of spindle defects in the live mutant oocytes provides new insights into the mechanism of Ncd function in the spindle during the meiotic divisions. PMID:9182665

  1. Progesterone-induced changes in the phosphoryl potential during the meiotic divisions in amphibian oocytes: Role of Na/K-ATPase

    PubMed Central

    2011-01-01

    Background Progesterone triggers resumption of the first meiotic division in the Rana pipiens oocyte by binding to the N-terminal external loop of the catalytic subunit of Na/K-ATPase, releasing a cascade of lipid second messengers. This is followed by internalization of specific membrane proteins, plasma membrane depolarization and nuclear membrane breakdown, culminating in arrest at second metaphase. Results Progesterone initiates an increase in phosphoryl potential during the first meiotic division, resulting in the accumulation of high energy protein phosphate by second metaphase arrest. 31P-NMR, with saturation transfer, demonstrates that the phosphocreatine level rises ~2 fold and that the "pseudo" first order rate constant for the creatine kinase reaction falls to ~20% of the control by the onset of nuclear membrane breakdown. 32PO4 pulse-labeling reveals a net increase in phosphorylation of yolk protein phosvitin during this period. The increased yolk protein phosphorylation coincides with internalization of membrane Na/K-ATPase and membrane depolarizatio Conclusions These results indicate that progesterone binding to the catalytic subunit of the Na-pump diverts ATP from cation regulation at the plasma membrane to storage of high energy phosphate in yolk protein. Phosvitin serves as a major energy source during fertilization and early cleavage stages and is also a storage site for cations (e.g. Na+, K+, Ca2+, Fe2+/3+) essential for embryonic development. PMID:22054214

  2. JNK does not regulate meiotic progression in Xenopus oocytes: The strange case of pJNK and pERK.

    PubMed

    Yue, Jicheng; López, José M

    2016-08-01

    Xenopus ERK2, also known as Xp42 MAPK, is activated by progesterone and regulates meiotic progression in the oocytes through activation of the phosphatase Cdc25C and inhibition of the protein kinase Myt1, thus promoting dephosphorylation and activation of cyclinB/Cdc2 (MPF). Indeed, it has been reported that stress protein kinases p38 and JNK are activated during meiotic progression and, more specifically, that p38γ regulates meiosis through activation of Cdc25C. However, the role of JNK in meiotic progression is not so clear, and despite a 42kDa protein is detected with pJNK antibodies (XpJNK-p42), the specific isoform activated by progesterone has not been characterized in detail. The serine/threonine kinase MEKK1, an upstream activator of JNK and p38, is activated during stress conditions and regulates apoptosis in different cell types. Here we show that ectopic expression of a constitutively active MEKK1 in Xenopus oocytes induces phosphorylation of p38, JNK and ERK and accelerates meiotic progression induced by progesterone. Inhibition of each individual pathway reduces the acceleration of meiosis induced by MEKK1. However, constitutively active MEKK1 induces phosphorylation of two JNK isoforms (p40 and p49, corresponding to JNK1-1 and JNK1-2 respectively) distinct to the p42 protein detected with pJNK antibodies during meiotic progression (XpJNK-p42). Moreover, a constitutively active MKK7, which specifically activates the JNK signaling pathway and induces phosphorylation of the p40 and p49 isoforms, does not accelerate meiotic progression. Immunoprecipitation of the p42 protein with pJNK antibodies and subsequent analysis by mass spectrometry shows that XpJNK-p42 is, in fact, pERK2. Ectopic expression of ERK2 in oocytes treated with progesterone or hyperosmotic shock indicates that ERK2 is phosphorylated in both conditions but is only detected with pJNK antibodies in progesterone-treated oocytes. In addition, mature oocytes only present a moderate increase

  3. 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

  4. 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

  5. Lifespan Extension in a Semelparous Chordate Occurs via Developmental Growth Arrest Just Prior to Meiotic Entry

    PubMed Central

    Subramaniam, Gunasekaran; Campsteijn, Coen; Thompson, Eric M.

    2014-01-01

    It is proposed that the ageing process is linked to signaling from the germline such that the rate of ageing can be adjusted to the state of the reproductive system, allowing these two processes to co-evolve. Mechanistic insight into this link has been primarily derived from iteroparous reproductive models, the nematode C. elegans, and the arthropod Drosophila. Here, we examined to what extent these mechanisms are evolutionarily conserved in a semelparous chordate, Oikopleura dioica, where we identify a developmental growth arrest (GA) in response to crowded, diet-restricted conditions, which can extend its lifespan at least three-fold. Under nutritional stress, the iteroparative models sacrifice germ cells that have entered meiosis, while maintaining a reduced pool of active germline stem cells (GSCs). In contrast, O. dioica only entered GA prior to meiotic entry. Stress conditions encountered after this point led to maturation in a normal time frame but with reduced reproductive output. During GA, TOR signaling was inhibited, whereas MAPK, ERK1/2 and p38 pathways were activated, and under such conditions, activation of these pathways was shown to be critical for survival. Direct inhibition of TOR signaling alone was sufficient to prevent meiotic entry and germline differentiation. This inhibition activated the p38 pathway, but did not activate the ERK1/2 pathway. Thus, the link between reproductive status and lifespan extension in response to nutrient-limited conditions is interpreted in a significantly different manner in these iteroparative versus semelparous models. In the latter case, meiotic entry is a definitive signal that lifespan extension can no longer occur, whereas in the former, meiotic entry is not a unique chronological event, and can be largely erased during lifespan extension in response to nutrient stress, and reactivated from a pool of maintained GSCs when conditions improve. PMID:24695788

  6. In vitro oocyte maturation from unstimulated cycles: does it offer a realistic chance to overcome the problem of repeated oocyte maturation arrest in IVF?

    PubMed

    Gulekli, Bulent; Olgan, Safak; Aydiner, Fulya

    2011-03-01

    This report describes the use of in vitro oocyte maturation in two patients with history of repeated oocyte maturation arrest (OMA) from in vitro fertilization. Based on these two cases, in vitro oocyte maturation from unstimulated cycles seems not an alternative treatment option for the patients with history of OMA. PMID:21210135

  7. 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

  8. The meiotic response of cumulus cell-enclosed mouse oocytes to follicle-stimulating hormone in the presence of different macromolecules.

    PubMed

    Downs, S M; Dow, M P; Fagbohun, C F

    1991-06-01

    Experiments were carried out to determine the effect of different macromolecules on the follicle-stimulating hormone (FSH)-induced maturation of mouse oocytes in culture. Cumulus cell-enclosed oocytes (CEO) were isolated from gonadotropin-primed mice and maintained in meiotic arrest for 17-18 h with the cAMP analogue, dibutyryl cAMP (dbcAMP). Germinal vesicle breakdown (GVB) was stimulated by the addition of FSH. Medium was supplemented with either no macromolecule or with varying concentrations of polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), crystallized bovine serum albumin (BSA), or fetal bovine serum (FBS). Oocyte maturation in all FSH-free cultures occurred at a frequency of about 30% or below. High frequencies of maturation were achieved when FSH was added to macromolecule-free medium or to cultures containing PVP, PVA, or BSA. Crystallized BSA was the most effective of these in supporting stimulation of maturation (94% GVB at 3 mg/ml, compared with 72-74% with synthetic polymer-supplemented or macromolecule-free media). The BSA effect was not due to contaminating fatty acids, and a less pure fraction V BSA was not as effective in supporting FSH-induced maturation. FBS suppressed FSH stimulation of maturation in a dose-dependent fashion. Sera from pigs, goats, horses, and rats were also inhibitory, but bovine calf serum (BCS) permitted a high maturation frequency (80% GVB). When added to medium containing either FBS or BCS, crystallized BSA had no effect on FSH-stimulated maturation, but fraction V BSA suppressed maturation in both serum-supplemented media. Under no conditions did FSH stimulate maturation in cumulus cell-free oocytes. These results demonstrate that hormone-induced oocyte maturation is supported in vitro by nonprotein polymers as well as BSA and that the behavior of the oocyte-cumulus cell complex depends on the purity of the BSA sample. In addition, serum contains inhibitory factors that suppress the positive response to FSH. Thus, the

  9. 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.

  10. 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

  11. Chromosomal meiotic segregation, embryonic developmental kinetics and DNA (hydroxy)methylation analysis consolidate the safety of human oocyte vitrification.

    PubMed

    De Munck, N; Petrussa, L; Verheyen, G; Staessen, C; Vandeskelde, Y; Sterckx, J; Bocken, G; Jacobs, K; Stoop, D; De Rycke, M; Van de Velde, H

    2015-06-01

    Oocyte vitrification has been introduced into clinical settings without extensive pre-clinical safety testing. In this study, we analysed major safety aspects of human oocyte vitrification in a high security closed system: (i) chromosomal meiotic segregation, (ii) embryonic developmental kinetics and (iii) DNA (hydroxy)methylation status. Fresh and vitrified sibling oocytes from young donors after intracytoplasmic sperm injection (ICSI) were compared in three different assays. Firstly, the chromosomal constitution of the fertilized zygotes was deduced from array comparative genomic hybridization results obtained from both polar bodies biopsied at Day 1. Secondly, embryo development up to Day 3 was analysed by time-lapse imaging. Ten specific time points, six morphokinetic time intervals and the average cell number on Day 3 were recorded. Thirdly, global DNA methylation and hydroxymethylation patterns were analysed by immunostaining on Day 3 embryos. The nuclear fluorescence intensity was measured by Volocity imaging software. Comprehensive chromosomal screening of the polar bodies demonstrated that at least half of the zygotes obtained after ICSI of fresh and vitrified oocytes were euploid. Time-lapse analysis showed that there was no significant difference in cleavage timings, the predictive morphokinetic time intervals nor the average cell number between embryos developed from fresh and vitrified oocytes. Finally, global DNA (hydroxy)methylation patterns were not significantly different between Day 3 embryos obtained from fresh and from vitrified oocytes. Our data further consolidate the safety of the oocyte vitrification technique. Nevertheless, additional testing in young and older sub-fertile/infertile patients and sound follow-up studies of children born after oocyte cryopreservation remain mandatory. PMID:25833840

  12. [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

  13. 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.

  14. 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

  15. 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

  16. 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

  17. Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3 complex-driven cytoplasmic streaming in mouse oocytes

    PubMed Central

    Yi, Kexi; Unruh, Jay R.; Deng, Manqi; Slaughter, Brian D.; Rubinstein, Boris; Li, Rong

    2012-01-01

    Mature mammalian oocytes are poised for the completion of second polar body extrusion upon fertilization by positioning the metaphase spindle in close proximity to an actomyosin-rich cortical cap. Loss of this spindle position asymmetry is often associated with poor oocyte quality and infertility 1–3. Here, we report a novel role for the Arp2/3 actin nucleation complex in the maintenance of asymmetric spindle position in mature mouse oocytes. The Arp2/3 complex localizes to the cortical cap in a Ran GTPase-dependent manner and accounts for the nucleation of the majority of actin filaments in both the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 complex activity or localization leads to rapid dissociation of the spindle from the cortex. High resolution live imaging and spatiotemporal image correlation spectroscopy (STICS) analysis reveal that in normal oocytes actin filaments flow continuously away from the Arp2/3-rich cortex, generating a cytoplamic streaming that results in a net pushing force on the spindle toward the actomyosin cap. Arp2/3 inhibition not only diminishes this actin flow and cytoplamic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, leading to spindle movement away from the cortex. We conclude that the Arp2/3 complex maintains asymmetric meiotic spindle position by generating an actin polymerization-driven cytoplamic streaming and by suppressing a counteracting force from myosin-II-based contractility. PMID:21874009

  18. Ultrastructural evaluation of recurrent and in-vitro maturation resistant metaphase I arrested oocytes.

    PubMed

    Windt, M L; Coetzee, K; Kruger, T F; Marino, H; Kitshoff, M S; Sousa, M

    2001-11-01

    An infertile couple whose female partner showed recurrent retrieval of immature metaphase I (MI) oocytes that were resistant to in-vitro maturation, was studied. Four spermiograms revealed teratozoospermia. Consistent non-fertilization and negative pregnancy outcomes were obtained after intrauterine insemination, gamete intra-Fallopian transfer and IVF. Two intracytoplasmic sperm injection (ICSI) cycles were finally performed. All oocytes (n = 17) in both cycles were arrested at MI and failed to mature after 48 h culture. ICSI also resulted in total non-fertilization. In the last cycle, two oocytes were analysed by transmission electron microscopy and showed almost identical results. All organelles showed normal characteristics of an MI oocyte. The main abnormality found was related to the MI spindle, with absence of microtubules and dispersion of the female chromosomes. Minor abnormalities were observed (immature fibrous appearance of the zona pellucida; the presence of small vesicle aggregates which formed a foam-like body). The injected sperm nucleus was arrested in the middle of the chromatin decondensation process, with no visible nuclear envelope reformation. Normal disruption of sperm acrosomal and flagellar components were observed. Only a partial cortical reaction was observed. This represents the first documented case of developmental arrest due to complete absence of spindle formation in association with an otherwise mature ooplasm. PMID:11679527

  19. 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

    2016-01-01

    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

  20. 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

  1. 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

  2. Accelerated follicle growth during the culture of isolated caprine preantral follicles is detrimental to follicular survival and oocyte meiotic resumption.

    PubMed

    Apolloni, Livia Brunetti; Bruno, Jamily Bezerra; Alves, Benner Geraldo; Ferreira, Anna Clara Accioly; Paes, Victor Macêdo; Moreno, Jesus de Los Reyes Cadenas; de Aguiar, Francisco Léo Nascimento; Brandão, Felipe Zandonadi; Smitz, Johan; Apgar, Gary; de Figueiredo, José Ricardo

    2016-10-01

    This study investigated the effect of androstenedione (A4) alone or in association with different concentrations of bovine recombinant FSH on the IVC of isolated goat preantral follicles. Follicles were mechanically isolated from ovarian tissue and cultured for 18 days in α-minimum essential medium supplemented or not with A4 (10 ng/mL) alone or in association with fixed (A4 + FixFSH: 100 ng/mL) or sequential (A4 + SeqFSH: Day 0, 100 ng/mL; Day 6, 500 ng/mL; Day 12, 1000 ng/mL) concentrations of FSH. After 18 days, the oocytes were recovered for IVM and fluorescence analysis. At Day 18 of culture, only A4 + SeqFSH treatment showed a lower (P < 0.05) rate of intact follicles, survival probability, and meiotic resumption, as well as higher (P < 0.05) percentage of degeneration and/or extrusion after antrum formation. Taken together, these results reported a positive correlation between fast-growing follicles and follicles that degenerated and/or extruded after antrum formation. When compared with control, the addition of A4 alone or in association of FSH did not increase (P > 0.05) the estradiol production or androstenedione levels on Day 6. However, on Day 18, the androstenedione levels were significantly lower in A4 + SeqFSH treatment when compared with A4 alone or to A4 + FixFSH treatments, whereas the estradiol production did not differ (P > 0.05). In summary, this study found that accelerated follicle growth negatively impacted the morphology of caprine preantral follicle cultured in vitro. In addition, the association of androstenedione with increasing concentration of FSH was detrimental to follicular survival and oocyte meiotic resumption. PMID:27371972

  3. 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

  4. Exposing cultured mouse ovarian follicles under increased gonadotropin tonus to aromatizable androgens influences the steroid balance and reduces oocyte meiotic capacity.

    PubMed

    Romero, Sergio; Smitz, Johan

    2010-10-01

    Acquisition of oocyte developmental competence relies on the well-controlled events accompanying antral follicular development. Elevated basal androgen levels, as in PCOS, potentially affect oocyte quality. Current experiments in an in vitro follicle bioassay studied dose-effects of androstenedione and testosterone on FSH and hCG stimulated antral follicle growth and meiotic maturation. The addition of either androgens altered follicle's endogenous production of androstenedione, testosterone, estradiol, and progesterone and affected the oocyte's capacity to resume meiosis. Exposure to 200 nM androstenedione induced an increased production of testosterone and estradiol. Exposure to a concentration of ≥200 nM testosterone induced elevated levels of estradiol and progesterone. Significant dose-dependent negative effects on polar body extrusion were seen at concentrations of ≥200 nM of either androgen. In addition, chromosome displacement on the metaphase plate was observed in oocytes obtained from androstenedione-treated follicles. Follicles exposed to a combination of 25 mIU/ml FSH and 3 mIU/ml hCG and elevated aromatizable androgens altered the steroid production profile, affected the follicular development and impaired oocyte meiotic competence. PMID:21046485

  5. Unfertilized frog eggs die by apoptosis following meiotic exit

    PubMed Central

    2011-01-01

    Background A characteristic feature of frog reproduction is external fertilization accomplished outside the female's body. Mature fertilization-competent frog eggs are arrested at the meiotic metaphase II with high activity of the key meiotic regulators, maturation promoting factor (MPF) and cytostatic factor (CSF), awaiting fertilization. If the eggs are not fertilized within several hours of ovulation, they deteriorate and ultimately die by as yet unknown mechanism. Results Here, we report that the vast majority of naturally laid unfertilized eggs of the African clawed frog Xenopus laevis spontaneously exit metaphase arrest under various environmental conditions and degrade by a well-defined apoptotic process within 48 hours after ovulation. The main features of this process include cytochrome c release, caspase activation, ATP depletion, increase of ADP/ATP ratio, apoptotic nuclear morphology, progressive intracellular acidification, and egg swelling. Meiotic exit seems to be a prerequisite for execution of the apoptotic program, since (i) it precedes apoptosis, (ii) apoptotic events cannot be observed in the eggs maintaining high activity of MPF and CSF, and (iii) apoptosis in unfertilized frog eggs is accelerated upon early meiotic exit. The apoptotic features cannot be observed in the immature prophase-arrested oocytes, however, the maturation-inducing hormone progesterone renders oocytes susceptible to apoptosis. Conclusions The study reveals that naturally laid intact frog eggs die by apoptosis if they are not fertilized. A maternal apoptotic program is evoked in frog oocytes upon maturation and executed after meiotic exit in unfertilized eggs. The meiotic exit is required for execution of the apoptotic program in eggs. The emerging anti-apoptotic role of meiotic metaphase arrest needs further investigation. PMID:22195698

  6. 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

  7. N-Acetyl-Cysteine and l-Carnitine Prevent Meiotic Oocyte Damage Induced by Follicular Fluid From Infertile Women With Mild Endometriosis.

    PubMed

    Giorgi, Vanessa S I; Da Broi, Michele G; Paz, Claudia C P; Ferriani, Rui A; Navarro, Paula A

    2016-03-01

    This study evaluated the potential protective effect of the antioxidants, l-carnitine (LC) and N-acetyl-cysteine (NAC), in preventing meiotic oocyte damage induced by follicular fluid (FF) from infertile women with mild endometriosis (ME). We performed an experimental study. The FF samples were obtained from 22 infertile women undergoing stimulated cycles for intracytoplasmic sperm injection (11 with ME and 11 without endometriosis). Immature bovine oocytes were submitted to in vitro maturation (IVM) divided into 9 groups: no-FF (No-FF); with FF from control (CFF) or ME (EFF) groups; and with LC (C + LC and E + LC), NAC (C + NAC and E + NAC), or both antioxidants (C + 2Ao and E + 2Ao). After IVM, oocytes were immunostained for visualization of microtubules and chromatin by confocal microscopy. The percentage of meiotically normal metaphase II (MII) oocytes was significantly lower in the EFF group (51.35%) compared to No-FF (86.36%) and CFF (83.52%) groups. The E + NAC (62.22%), E + LC (80.61%), and E + 2Ao (61.40%) groups showed higher percentage of normal MII than EFF group. The E + LC group showed higher percentage of normal MII than E + NAC and E + 2Ao groups and a similar percentage to No-FF and CFF groups. Therefore, FF from infertile women with ME causes meiotic abnormalities in bovine oocytes, and, for the first time, we demonstrated that the use of NAC and LC prevents these damages. Our findings elucidate part of the pathogenic mechanisms involved in infertility associated with ME and open perspectives for further studies investigating whether the use of LC could improve the natural fertility and/or the results of in vitro fertilization of women with ME. PMID:26342050

  8. 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

  9. ROLE OF AMPK THOUGHOUT MEIOTIC MATURATION IN THE MOUSE OOCYTE: EVIDENCE FOR PROMOTION OF POLAR BODY FORMATION AND SUPPRESSION OF PREMATURE ACTIVATION

    PubMed Central

    Downs, Stephen M.; Ya, Ru; Davis, Christopher

    2014-01-01

    This study was conducted to assess the role of AMPK in regulating meiosis in mouse oocytes from the germinal vesicle stage to metaphase II. Exposure of mouse cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) during spontaneous maturation in vitro to AMPK-activating agents resulted in augmentation of the rate and frequency of polar body formation. Inhibitors of AMPK had an opposite, inhibitory effect. In addition, the AMPK inhibitor, compound C (Cmpd C) increased the frequency of oocyte activation. The stimulatory action of the AMPK-activating agent, AICAR, and the inhibitory action of Cmpd C were diminished if exposure was delayed, indicating an early action of AMPK on polar body formation. The frequency of spontaneous and Cmpd C-induced activation in CEO was reduced as the period of hormonal priming was increased, and AMPK stimulation eliminated the activation response. Immunostaining of oocytes with antibody to active AMPK revealed an association of active kinase with chromatin, spindle poles and midbody during maturation. Immunolocalization of the α1 catalytic subunit of AMPK showed an association with condensed chromatin and the meiotic spindle, but not in the spindle poles or midbody; α2 stained only diffusely throughout the oocyte. These data suggest that AMPK is involved in a regulatory capacity throughout maturation and helps promote the completion of meiosis while suppressing premature activation. PMID:20830737

  10. 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

  11. DNA damage responses in mammalian oocytes.

    PubMed

    Collins, Josie K; Jones, Keith T

    2016-07-01

    DNA damage acquired during meiosis can lead to infertility and miscarriage. Hence, it should be important for an oocyte to be able to detect and respond to such events in order to make a healthy egg. Here, the strategies taken by oocytes during their stages of growth to respond to DNA damaging events are reviewed. In particular, recent evidence of a novel pathway in fully grown oocytes helps prevent the formation of mature eggs with DNA damage. It has been found that fully grown germinal vesicle stage oocytes that have been DNA damaged do not arrest at this point in meiosis, but instead undergo meiotic resumption and stall during the first meiotic division. The Spindle Assembly Checkpoint, which is a well-known mitotic pathway employed by somatic cells to monitor chromosome attachment to spindle microtubules, appears to be utilised by oocytes also to respond to DNA damage. As such maturing oocytes are arrested at metaphase I due to an active Spindle Assembly Checkpoint. This is surprising given this checkpoint has been previously studied in oocytes and considered to be weak and ineffectual because of its poor ability to be activated in response to microtubule attachment errors. Therefore, the involvement of the Spindle Assembly Checkpoint in DNA damage responses of mature oocytes during meiosis I uncovers a novel second function for this ubiquitous cellular checkpoint. PMID:27069010

  12. 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

  13. Oocyte Maturation: The Coming of Age of a Germ Cell

    PubMed Central

    Jamnongjit, Michelle; Hammes, Stephen R.

    2006-01-01

    Normal female fertility relies on proper development of the oocyte. This growth culminates just prior to ovulation, when oocyte maturation occurs. Oocyte maturation refers to a release of meiotic arrest that allows oocytes to advance from prophase I to metaphase II of meiosis. This precisely regulated meiotic progression is essential for normal ovulation and subsequent fertilization, and involves changes in the delicate balance between factors promoting meiotic arrest and others that are stimulating maturation. Most of the inhibitory mechanisms appear to involve the upregulation of intracellular cyclic adenosine monophosphate levels. These processes may include direct transport of the nucleotide into oocytes via gap junctions, G protein-mediated stimulation of adenylyl cyclase, and inhibition of intracellular phosphodiesterases. In contrast, potential factors that play roles in triggering oocyte maturation include gonadotropins (e.g., follicle-stimulating factor and luteinizing hormone), growth factors (e.g., amphiregulin and epiregulin), sterols (e.g., follicular fluid-derived meiosis-activating sterol), and steroids (e.g., testosterone progesterone, and estradiol). Delineating the complex interactions between these positive and negative components is critical for determining the role that oocyte maturation plays in regulating follicle development and ovulation, and may lead to novel methods that can be used to modulate these processes in women with both normal and aberrant fertility. PMID:16059829

  14. 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

  15. 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

  16. A nuclear factor required for specific translation of cyclin B may control the timing of first meiotic cleavage in starfish oocytes.

    PubMed Central

    Galas, S; Barakat, H; Dorée, M; Picard, A

    1993-01-01

    In most animals, the rate of cyclin B synthesis increases after nuclear envelope breakdown during the first meiotic cell cycle. We have found that cyclin B-cdc2 kinase activity drops earlier in emetine-treated than in control starfish oocytes, although the protein synthesis inhibitor does not activate the cyclin degradation pathway prematurely. Moreover, protein synthesis is required to prevent meiotic cleavage to occur prematurely, sometimes before chromosomes have segregated on the metaphase plate. In normal conditions, increased synthesis of cyclin B after germinal vesicle breakdown (GVBD) balances cyclin degradation and increases the time required for cyclin B-cdc2 kinase to drop below the level that inhibits cleavage. Taken together, these results point to cyclin B as a possible candidate that could explain the need for increased protein synthesis during meiosis I. Although direct experimental evidence was not provided in the present work, cyclin B synthesis after GVBD may be important for correct segregation of homologous chromosomes at the end of first meiotic metaphase, as shown by a variety of cytological disorders that accompany premature cleavage. Although the overall stimulation of protein synthesis because of cdc2 kinase activation is still observed in oocytes from which the germinal vesicle has been removed before hormonal stimulation, the main increase of cyclin B synthesis normally observed after germinal vesicle breakdown is suppressed. The nuclear factor required for specific translation of cyclin B after GVBD is not cyclin B mRNA, as shown by using a highly sensitive reverse transcription followed by polymerase chain reaction procedure that failed to detect any cyclin B mRNA in isolated germinal vesicles. Images PMID:7513215

  17. Redundant pathways for Cdc2 activation in Xenopus oocyte: either cyclin B or Mos synthesis

    PubMed Central

    Haccard, Olivier; Jessus, Catherine

    2006-01-01

    Xenopus oocytes are arrested in meiotic prophase I. Progesterone induces the resumption of meiotic maturation, which requires continuous protein synthesis to bring about Cdc2 activation. The identification of the newly synthesized proteins has long been a goal. Two plausible candidates have received extensive study. The synthesis of cyclin B and of c-Mos, a kinase that activates the mitogen-activated protein kinase pathway in oocytes, is clearly upregulated by translational control in response to progesterone. Recent studies suggest that ablation of either c-Mos or cyclin B synthesis by antisense oligonucleotides does not block meiotic maturation. Here, however, we show that when both pathways are simultaneously inhibited, progesterone no longer triggers maturation; adding back either c-Mos or cyclin B restores meiotic maturation. We conclude that the specific synthesis of either B-type cyclins or c-Mos, induced by progesterone, is required to induce meiotic maturation. The two pathways seem to be functionally redundant. PMID:16374506

  18. Calcium ion currents mediating oocyte maturation events

    PubMed Central

    Tosti, Elisabetta

    2006-01-01

    During maturation, the last phase of oogenesis, the oocyte undergoes several changes which prepare it to be ovulated and fertilized. Immature oocytes are arrested in the first meiotic process prophase, that is morphologically identified by a germinal vesicle. The removal of the first meiotic block marks the initiation of maturation. Although a large number of molecules are involved in complex sequences of events, there is evidence that a calcium increase plays a pivotal role in meiosis re-initiation. It is well established that, during this process, calcium is released from the intracellular stores, whereas less is known on the role of external calcium entering the cell through the plasma membrane ion channels. This review is focused on the functional role of calcium currents during oocyte maturation in all the species, from invertebrates to mammals. The emerging role of specific L-type calcium channels will be discussed. PMID:16684344

  19. 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

  20. Disruption of pairing and synapsis of chromosomes causes stage-specific apoptosis of male meiotic cells.

    PubMed

    Hamer, G; Novak, I; Kouznetsova, A; Höög, C

    2008-02-01

    During meiosis, DNA replication is followed by two successive rounds of chromosome segregation (meiosis I and II), which give rise to genetically diverse haploid gametes. The prophase of the first meiotic division is highly regulated and alignment and synapsis of the homologous chromosomes during this stage are mediated by the synaptonemal complex. Incorrect assembly of the synaptonemal complex results in cell death, impaired meiotic recombination and aneuploidy. Oocytes with meiotic defects often survive the first meiotic prophase and give rise to aneuploid gametes. Similarly affected spermatocytes, on the other hand, almost always undergo apoptosis at a male-specific meiotic checkpoint, located specifically at epithelial stage IV during spermatogenesis. Many examples of this stage IV-specific arrest have been described for several genetic mouse models in which DNA repair or meiotic recombination are abrogated. Interestingly, in C. elegans, meiotic recombination and synapsis are monitored by two separate checkpoint pathways. Therefore we studied spermatogenesis in several knockout mice (Sycp1(-/-), Sycp3(-/-), Smc1beta(-/-) and Sycp3/Sycp1 and Sycp3/Smc1beta double-knockouts) that are specifically defective in meiotic pairing and synapsis. Like for recombination defects, we found that all these genotypes also specifically arrest at epithelial stage IV. It seems that the epithelial stage IV checkpoint eliminates spermatocytes that fail a certain quality check, being either synapsis or DNA damage related. PMID:17997150

  1. 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

  2. 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

  3. 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

  4. In Vitro Acute Exposure to DEHP Affects Oocyte Meiotic Maturation, Energy and Oxidative Stress Parameters in a Large Animal Model

    PubMed Central

    Sardanelli, Anna Maria; Pocar, Paola; Martino, Nicola Antonio; Paternoster, Maria Stefania; Amati, Francesca; Dell'Aquila, Maria Elena

    2011-01-01

    Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 µM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos

  5. Progesterone induces meiotic division in the amphibian oocyte by releasing lipid second messengers from the plasma membrane.

    PubMed

    Morrill, G A; Kostellow, A B

    1999-01-01

    Meiosis in the amphibian oocyte is normally initiated by gonadotropins, which stimulate follicle cells to secret progesterone. The progesterone-induced G2/M transition in the amphibian oocyte was the first well-defined example of a steroid effect at the plasma membrane, since it could be shown that exogenous, but not injected, progesterone induced meiosis and that many of the progesterone-induced changes associated with meiosis occurred in enucleated oocytes. We find that [3H]progesterone binding to isolated plasma membranes of Rana pipiens oocytes is saturable, specific and temperature-dependent. Photoaffinity labeling with the synthetic progestin [3H]R5020 followed by gel electrophoresis demonstrated progestin binding to both 80 and 110 kDa proteins in the oocyte cytosol, whereas only the 110 kDa R5020 binding protein was present in the oocyte plasma membrane. We have shown that progesterone acts at Rana oocyte plasma membrane receptors within seconds to release a cascade of lipid messengers. Membrane-receptor binding causes the successive activation of: 1) N-methyltransferases, which convert phosphatidylethanolamine to phosphatidylcholine (PC); 2) an exchange reaction between PC and ceramide to form sphingomyelin (SM) and 1,2-diacylglycerol (DAG); 3) phospholipase D/phosphatidate phosphohydrolase, releasing a second DAG transient; and 4) phosphatidylinositol-specific phospholipase C, generating inositol trisphosphate and a third DAG transient. Within minutes, diglyceride kinase converts newly formed DAG species to phosphatidic acid, turning off the successive DAG signals. A transient fall (0-30 s) in intracellular ceramide is followed (within 1-2 min) by a sustained rise in intracellular ceramide lasting 3-4 h. This ceramide may be significant in later cyclin-dependent steps. We conclude that the initial action of progesterone at its plasma membrane receptor triggers a series of enzyme activations that modify the membrane and release multiple DAG species. PMID

  6. Suppression of Chemically Induced and Spontaneous Mouse Oocyte Activation by AMP-Activated Protein Kinase1

    PubMed Central

    Ya, Ru; Downs, Stephen M.

    2013-01-01

    ABSTRACT Oocyte activation is an important process triggered by fertilization that initiates embryonic development. However, parthenogenetic activation can occur either spontaneously or with chemical treatments. The LT/Sv mouse strain is genetically predisposed to spontaneous activation. LT oocytes have a cell cycle defect and are ovulated at the metaphase I stage instead of metaphase II. A thorough understanding of the female meiosis defects in this strain remains elusive. We have reported that AMP-activated protein kinase (PRKA) has an important role in stimulating meiotic resumption and promoting completion of meiosis I while suppressing premature parthenogenetic activation. Here we show that early activation of PRKA during the oocyte maturation period blocked chemically induced activation in B6SJL oocytes and spontaneous activation in LT/SvEiJ oocytes. This inhibitory effect was associated with high levels of MAPK1/3 activity. Furthermore, stimulation of PRKA partially rescued the meiotic defects of LT/Sv mouse oocytes in concert with correction of abnormal spindle pole localization of PRKA and loss of prolonged spindle assembly checkpoint activity. Altogether, these results confirm a role for PRKA in helping sustain the MII arrest in mature oocytes and suggest that dysfunctional PRKA contributes to meiotic defects in LT/SvEiJ oocytes. PMID:23390161

  7. Role of cyclic AMP in the maturation of Ciona intestinalis oocytes.

    PubMed

    Silvestre, Francesco; Gallo, Alessandra; Cuomo, Annunziata; Covino, Tiziana; Tosti, Elisabetta

    2011-11-01

    Immature oocytes are arrested at prophase I of the meiotic process and maturation onset is indicated by oocyte nuclear disassembly (germinal vesicle breakdown or GVBD). Signaling pathways that elevate intracellular cyclic AMP (cAMP) may either prevent or induce oocyte maturation depending on the species. In some marine invertebrates and, in particular, in ascidian oocytes, cAMP triggers GVBD rather than blocking it. In this paper, we tested different cAMP elevators in fully grown oocytes at the germinal vesicle stage (GV) of the ascidian Ciona intestinalis. We demonstrated that through the activation of adenylate cyclase or the inhibition and phosphodiesterases the oocyte remained at the GV stage. This effect was reversible as the GV-arrested oocytes, rinsed and incubated in sea water, are able to undergo spontaneous maturation and extrusion of follicle cells. In addition, oocytes acquire the ability to be fertilized and start early development. However, morphology of follicle cells, embryos and larvae from in vitro matured oocytes showed different morphology from those derived from in vivo mature oocytes. The role and the transduction mechanism of cAMP in the regulation of oocyte maturation were discussed. Finally, we indicated a variation of biological mechanisms present in the ascidian species; moreover, we sustain evidence proving that tunicates share some biological mechanisms with vertebrates. This information provided new hints on the importance of ascidians in the evolution of chordates. PMID:20810008

  8. Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes.

    PubMed Central

    Abrieu, A; Dorée, M; Picard, A

    1997-01-01

    The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation. Images PMID:9190205

  9. 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

  10. 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

  11. New Insights into the Genetic Regulation of Homologue Disjunction in Mammalian Oocytes

    PubMed Central

    Homer, H.

    2011-01-01

    Mammalian oocytes execute a unique meiotic programme involving 2 arrest stages and an unusually protracted preamble to chromosome segregation during the first meiotic division (meiosis I). How mammalian oocytes successfully navigate their exceptional meiotic journey has long been a question of immense interest. Understanding the minutiae of female mammalian meiosis I is not merely of academic interest as 80–90% of human aneuploidy is the consequence of errors arising at this particular stage of oocyte maturation, a stage with a peculiar vulnerability to aging. Recent evidence indicates that oocytes employ many of the same cast of proteins during meiosis I as somatic cells do during mitosis, often to execute similar tasks, but intriguingly, occasionally delegate them to unexpected and unprecedented roles. This is epitomised by the master cell-cycle regulon, the anaphase-promoting complex or cyclosome (APC/C), acting in concert with a critical APC/C-targeted surveillance mechanism, the spindle assembly checkpoint (SAC). Together, the APC/C and the SAC are among the most influential entities overseeing the fidelity of cell-cycle progression and the precision of chromosome segregation. Here I review the current status of pivotal elements underpinning homologue disjunction in mammalian oocytes including spindle assembly, critical biochemical anaphase-initiating events, APC/C activity and SAC signalling along with contemporary findings relevant to progressive oocyte SAC dysfunction as a model for age-related human aneuploidy. PMID:21335952

  12. 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.

  13. 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

  14. Newly assembled cyclin B-cdc2 kinase is required to suppress DNA replication between meiosis I and meiosis II in starfish oocytes.

    PubMed Central

    Picard, A; Galas, S; Peaucellier, G; Dorée, M

    1996-01-01

    Micro-injection of catalytically inactive GST-cdc2-K33R or GST-cdk2-K33R fusion proteins, each of which efficiently titrates cyclin B in oocytes and prevents assembly of cyclin B-cdc2 complexes, readily induces premature DNA replication in starfish oocytes after emission of the first polar body. Moreover, partial ablation of cyclin B mRNA by micro-injection of antisense oligonucleotides facilitates premature DNA replication induced by the dominant-negative cdc2 and cdk2 mutant proteins. We thus propose that enhanced translation of cyclin B after GVBD, a universal feature of oocyte maturation in the animal kingdom, and subsequent assembly of cyclin B-cdc2 complexes, are part of the checkpoint that prevents DNA replication in the oocyte after emission of the first polar body. MAPK inactivation is neither required for premature DNA replication after the first meiotic cell cycle nor for DNA replication after completion of meiotic maturation. However, micro-injection of a N-terminally truncated form of the budding yeast STE11 protein, that constitutively maintains MAPK active after the second meiotic cleavage, prevents fertilized eggs from proceeding into embryogenesis, and arrests them at G2, as is the case in unfertilized eggs that cannot inactivate MAPK after the second meiotic cleavage. We thus propose that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development. Images PMID:8670862

  15. Dynamic of expression and localization of cannabinoid-degrading enzymes FAAH and MGLL in relation to CB1 during meiotic maturation of human oocytes.

    PubMed

    Agirregoitia, Ekaitz; Totorikaguena, Lide; Expósito, Antonia; Mendoza, Rosario; Matorras, Roberto; Agirregoitia, Naiara

    2016-08-01

    The endogenous cannabinoid system has been characterized in some female reproductive organs but little is known about the expression and localization pattern of cannabinoid-degrading enzymes in relation to the CB1 cannabinoid receptor in human oocytes. In this study, we focus on the investigation of the presence and differential distribution of fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGLL) in relation to CB1 during the maturation of human oocytes. We used a total of 290 human oocytes not suitable for in vitro fertilization/intracytoplasmic sperm injection (ICSI): germinal-vesicle (GV) and metaphase-I (MI) stages and metaphase-II (MII) oocytes that had not developed into an embryo after ICSI. Cannabinoid-degrading enzymes and the cannabinoid CB1 receptor were present in human oocytes. Specifically, FAAH was detected in the periphery of the oocyte from the GV to MI stage and co-localized with CB1. Later, by the MII stage, FAAH was spread within the oocyte, whereas MGLL immunostaining was homogeneous across the oocyte at all stages of maturation and only overlapped with CB1 at the GV stage. This coordinated redistribution of cannabinoid system proteins suggests a role for this system in the maturation of the female gamete. PMID:26948343

  16. Germinal vesicle material drives meiotic cell cycle of mouse oocyte through the 3'UTR-dependent control of cyclin B1 synthesis.

    PubMed

    Hoffmann, Steffen; Tsurumi, Chizuko; Kubiak, Jacek Z; Polanski, Zbigniew

    2006-04-01

    We compared the profile of histone H1 kinase activity, reflecting Maturation Promoting Factor (MPF) activity in oocytes bisected at the germinal vesicle (GV) stage and allowed to mature as separate oocyte halves in vitro. Whereas the oocyte halves containing the nucleus exhibited the same profile of increased kinase activity as that typical for intact oocytes, the anuclear halves revealed strong inhibition of the increase in this activity soon after germinal vesicle breakdown (GVBD). In contrast, the profile of MAP kinase activity did not differ significantly between anuclear and nucleus-containing oocyte halves throughout maturation. Of the two MPF components, CDK1 and cyclin B1, the amount of the latter was significantly reduced in anuclear halves, a reduction due to low-level synthesis and not to enhanced degradation. Expression of three reporter luciferase RNAs constructed, respectively, to contain cyclin B1-specific 3'UTR, the globin-specific 3'UTR, or no 3'UTR sequence was enhanced in nuclear halves, with significantly greater enhancement for the construct containing cyclin B1-specific 3'UTR as compared to the two other RNAs. We conclude that the profile of activity of MPF during mouse oocyte maturation is controlled by an unknown GV-associated factor(s) acting via 3'UTR-dependent control of cyclin B1 synthesis. These results require the revision of the hitherto prevailing view that the control of MPF activity during mouse oocyte maturation is independent of GV-derived material. PMID:16490186

  17. Bisphenol-A and human oocyte maturation in vitro

    PubMed Central

    Machtinger, Ronit; Combelles, Catherine M.H.; Missmer, Stacey A.; Correia, Katharine F.; Williams, Paige; Hauser, Russ; Racowsky, Catherine

    2013-01-01

    STUDY QUESTION Does exposure to bisphenol-A (BPA) affect the maturation of human oocytes in vitro? SUMMARY ANSWER There was a dose–response association of BPA exposure with altered human oocyte maturation in vitro. WHAT IS KNOWN ALREADY There is widespread exposure of the general population to BPA. BPA has been detected in the human follicular fluid. Animal studies have shown that BPA exposure is associated with maturation arrest and spindle abnormalities in maturing oocytes. STUDY DESIGN, SIZE, DURATION A randomized trial, using 352 clinically discarded oocytes from 121 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS The study population was drawn from patients undergoing IVF/ICSI cycles in our program at Brigham and Women's Hospital from March 2011 to April 2012. Oocytes from only one cycle for each patient were included in the study. Cycles with at least two germinal vesicle stage oocytes were included with random allocation of one oocyte to culture for 30 h without BPA and remaining sibling oocytes to medium-containing BPA (20, 200 ng/ml or 20 µg/ml). Oocytes were fixed and labeled for tubulin, actin and chromatin and examined with immunofluorescence and confocal microscopy. Oocytes were assessed for meiotic stage (n = 292), and those at metaphase II (MII, n = 175) were further classified according to their spindle configurations and patterns of chromosome alignment. McNemar's test was used to compare dichotomized maturation status. Generalized estimating equations were used to account for the correlation between oocytes from the same woman and for the spindle analysis. MAIN RESULTS AND THE ROLE OF CHANCE As the BPA dose increased, there was a decrease in the percentage of oocytes that progressed to MII (P = 0.002) and increases in the percentage of oocytes that were degenerated (P = 0.01) or that had undergone spontaneous activation (P = 0.007). Among MII oocytes, as the BPA dose increased, there was a significant trend (by test for trend) for a

  18. 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

  19. CYTOPLASMIC MICROTUBULAR DYNAMIC AND CHROMATIN ORGANIZATION DURING MAMMALIAN OOCYTE MATURATION

    EPA Science Inventory

    Coordinated alterations in oocyte chromosome and microtubule disposition occur during oogenesis and oocyte maturation in the mammal. imely transitions in meiotic spindle and cytoplasmic microtubules, due to modifications in both the assembly competence of the tubulin pool and nuc...

  20. The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

    PubMed Central

    Choi, T; Fukasawa, K; Zhou, R; Tessarollo, L; Borror, K; Resau, J; Vande Woude, G F

    1996-01-01

    Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692939

  1. 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

  2. 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...

  3. Effect of Temporary Meiotic Attenuation of Oocytes with Butyrolactone I and Roscovitine in Resistance to Bovine Embryos on Vitrification.

    PubMed

    Maziero, Rrd; Guaitolini, Crf; Paschoal, D M; Kievitsbosch, T; Guastali, M D; Moraes, C N; Landim-Alvarenga, F C

    2016-04-01

    This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL-I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO2 atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 μm), BL-I (50 μm) and association of drugs (ROS 6.25 μm and BL-I 25 μm). Oocytes were cultured for 18 h in an agent-free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO2 atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL-I and its association with the ROS increased the rates of cleavage and blastocyst formation (p < 0.05). The ROS alone was inefficient, impairing embryonic development, with low rates of blastocyst formation when compared to the control group and other treatments (p < 0.05). The embryos from BL-I and ROS+BL-I groups presented higher number of cells and lower rates of cellular apoptosis compared to other groups, either for the fresh or for post-thawing embryos. Embryos from ROS+BL-I group showed to be more resistant to the vitrification process, presenting a higher rate of embryonic re-expansion (p < 0.05). In conclusion, block of meiosis using BL-I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL-I resulted in a better resistance to the embryo cryopreservation process. PMID:26812982

  4. Vg1RBP phosphorylation by Erk2 MAP kinase correlates with the cortical release of Vg1 mRNA during meiotic maturation of Xenopus oocytes

    PubMed Central

    Git, Anna; Allison, Rachel; Perdiguero, Eusebio; Nebreda, Angel R.; Houliston, Evelyn; Standart, Nancy

    2009-01-01

    Xenopus Vg1RBP is a member of the highly conserved IMP family of four KH-domain RNA binding proteins, with roles in RNA localization, translational control, RNA stability, and cell motility. Vg1RBP has been implicated in localizing Vg1 mRNAs to the vegetal cortex during oogenesis, in a process mediated by microtubules and microfilaments, and in migration of neural crest cells in embryos. Using c-mos morpholino, kinase inhibitors, and constitutely active recombinant kinases we show that Vg1RBP undergoes regulated phosphorylation by Erk2 MAPK during meiotic maturation, on a single residue, S402, located between the KH2 and KH3 domains. Phosphorylation temporally correlates with the release of Vg1 mRNA from its tight cortical association, assayed in lysates in physiological salt buffers, but does not affect RNA binding, nor self-association of Vg1RBP. U0126, a MAP kinase inhibitor, prevents Vg1RBP cortical release and Vg1 mRNA solubilization in meiotically maturing eggs, while injection of MKK6-DD, a constitutively activated MAP kinase kinase, promotes the release of both Vg1RBP and Vg1 mRNA from insoluble cortical structures. We propose that Erk2 MAP kinase phosphorylation of Vg1RBP regulates the protein:protein-mediated association of Vg1 mRNP with the cytoskeleton and/or ER. Since the MAP kinase site in Vg1RBP is conserved in several IMP homologs, this modification also has important implications for the regulation of IMP proteins in somatic cells. PMID:19376927

  5. 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

  6. 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.

  7. Mammalian meiotic silencing exhibits sexually dimorphic features.

    PubMed

    Cloutier, J M; Mahadevaiah, S K; ElInati, E; Tóth, A; Turner, James

    2016-06-01

    During mammalian meiotic prophase I, surveillance mechanisms exist to ensure that germ cells with defective synapsis or recombination are eliminated, thereby preventing the generation of aneuploid gametes and embryos. Meiosis in females is more error-prone than in males, and this is in part because the prophase I surveillance mechanisms are less efficient in females. A mechanistic understanding of this sexual dimorphism is currently lacking. In both sexes, asynapsed chromosomes are transcriptionally inactivated by ATR-dependent phosphorylation of histone H2AFX. This process, termed meiotic silencing, has been proposed to perform an important prophase I surveillance role. While the transcriptional effects of meiotic silencing at individual genes are well described in the male germ line, analogous studies in the female germ line have not been performed. Here we apply single- and multigene RNA fluorescence in situ hybridization (RNA FISH) to oocytes from chromosomally abnormal mouse models to uncover potential sex differences in the silencing response. Notably, we find that meiotic silencing in females is less efficient than in males. Within individual oocytes, genes located on the same asynapsed chromosome are silenced to differing extents, thereby generating mosaicism in gene expression profiles across oocyte populations. Analysis of sex-reversed XY female mice reveals that the sexual dimorphism in silencing is determined by gonadal sex rather than sex chromosome constitution. We propose that sex differences in meiotic silencing impact on the sexually dimorphic prophase I response to asynapsis. PMID:26712235

  8. 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

  9. Selective Disruption of Aurora C Kinase Reveals Distinct Functions from Aurora B Kinase during Meiosis in Mouse Oocytes

    PubMed Central

    Balboula, Ahmed Z.; Schindler, Karen

    2014-01-01

    Aurora B kinase (AURKB) is the catalytic subunit of the chromosomal passenger complex (CPC), an essential regulator of chromosome segregation. In mitosis, the CPC is required to regulate kinetochore microtubule (K-MT) attachments, the spindle assembly checkpoint, and cytokinesis. Germ cells express an AURKB homolog, AURKC, which can also function in the CPC. Separation of AURKB and AURKC function during meiosis in oocytes by conventional approaches has not been successful. Therefore, the meiotic function of AURKC is still not fully understood. Here, we describe an ATP-binding-pocket-AURKC mutant, that when expressed in mouse oocytes specifically perturbs AURKC-CPC and not AURKB-CPC function. Using this mutant we show for the first time that AURKC has functions that do not overlap with AURKB. These functions include regulating localized CPC activity and regulating chromosome alignment and K-MT attachments at metaphase of meiosis I (Met I). We find that AURKC-CPC is not the sole CPC complex that regulates the spindle assembly checkpoint in meiosis, and as a result most AURKC-perturbed oocytes arrest at Met I. A small subset of oocytes do proceed through cytokinesis normally, suggesting that AURKC-CPC is not the sole CPC complex during telophase I. But, the resulting eggs are aneuploid, indicating that AURKC is a critical regulator of meiotic chromosome segregation in female gametes. Taken together, these data suggest that mammalian oocytes contain AURKC to efficiently execute meiosis I and ensure high-quality eggs necessary for sexual reproduction. PMID:24586209

  10. KIF20A regulates porcine oocyte maturation and early embryo development.

    PubMed

    Zhang, Yu; Liu, Jun; Peng, Xu; Zhu, Cheng-Cheng; Han, Jun; Luo, Jia; Rui, Rong

    2014-01-01

    KIF20A (Kinesin-like family member 20A), also called mitotic kinesin-like proteins 2 (MKLP2), is a mammalian mitotic kinesin-like motor protein of the Kinesin superfamily proteins (KIFs), which was originally involved in Golgi apparatus dynamics and thought to essential for cell cycle regulation during successful cytokinesis. In the present study, we investigated whether KIF20A has roles on porcine oocyte meiotic maturation and subsequent early embryo development. By immunofluorescence staining, KIF20A was found to exhibit a dynamic localization pattern during meiosis. KIF20A was restricted to centromeres after germinal vesicle breakdown (GVBD), transferred to the midbody at telophase I (TI), and again associated with centromeres at metaphase II (MII). Inhibition of endogenous KIF20A via a specific inhibitor, Paprotrain, resulted in failure of polar body extrusion. Further cell cycle analysis showed that the percentage of oocytes that arrested at early metaphase I (MI) stage increased after KIF20A activity inhibition; however, the proportion of oocytes at anaphase/telophase I (ATI) and MII stages decreased significantly. Our results also showed that KIF20A inhibition did not affect spindle morphology. In addition, KIF20A was localized at the nucleus of early embryos, and KIF20A inhibition resulted in failure of early parthenogenetic embryo development. These results demonstrated that KIF20A is critical for porcine oocyte meiotic maturation and subsequent early embryo development. PMID:25036038

  11. KIF20A Regulates Porcine Oocyte Maturation and Early Embryo Development

    PubMed Central

    Zhang, Yu; Liu, Jun; Peng, Xu; Zhu, Cheng-Cheng; Han, Jun; Luo, Jia; Rui, Rong

    2014-01-01

    KIF20A (Kinesin-like family member 20A), also called mitotic kinesin-like proteins 2 (MKLP2), is a mammalian mitotic kinesin-like motor protein of the Kinesin superfamily proteins (KIFs), which was originally involved in Golgi apparatus dynamics and thought to essential for cell cycle regulation during successful cytokinesis. In the present study, we investigated whether KIF20A has roles on porcine oocyte meiotic maturation and subsequent early embryo development. By immunofluorescence staining, KIF20A was found to exhibit a dynamic localization pattern during meiosis. KIF20A was restricted to centromeres after germinal vesicle breakdown (GVBD), transferred to the midbody at telophase I (TI), and again associated with centromeres at metaphase II (MII). Inhibition of endogenous KIF20A via a specific inhibitor, Paprotrain, resulted in failure of polar body extrusion. Further cell cycle analysis showed that the percentage of oocytes that arrested at early metaphase I (MI) stage increased after KIF20A activity inhibition; however, the proportion of oocytes at anaphase/telophase I (ATI) and MII stages decreased significantly. Our results also showed that KIF20A inhibition did not affect spindle morphology. In addition, KIF20A was localized at the nucleus of early embryos, and KIF20A inhibition resulted in failure of early parthenogenetic embryo development. These results demonstrated that KIF20A is critical for porcine oocyte meiotic maturation and subsequent early embryo development. PMID:25036038

  12. 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

  13. 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

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

    PubMed

    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

    2016-06-17

    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

  15. Phosphorylation of ARPP19 by protein kinase A prevents meiosis resumption in Xenopus oocytes

    PubMed Central

    Dupré, Aude; Daldello, Enrico M.; Nairn, Angus C.; Jessus, Catherine; Haccard, Olivier

    2014-01-01

    During oogenesis, oocytes are arrested in prophase and resume meiosis by activating the kinase Cdk1 upon hormonal stimulation. In all vertebrates, release from prophase arrest relies on protein kinase A (PKA) downregulation and on the dephosphorylation of a long-sought but still unidentified substrate. Here we show that ARPP19 is the PKA substrate whose phosphorylation at serine 109 is necessary and sufficient for maintaining Xenopus oocytes arrested in prophase. By downregulating PKA, progesterone, the meiotic inducer in Xenopus, promotes partial dephosphorylation of ARPP19 that is required for the formation of a threshold level of active Cdk1. Active Cdk1 then initiates MPF autoamplification loop that occurs independently of both PKA and ARPP19 phosphorylation at serine 109 but requires the Greatwall-dependent phosphorylation of ARPP19 at serine 67. Therefore, ARPP19 stands at a crossroads in the meiotic M-phase control network by integrating differential effects of PKA and Greatwall, two essential kinases for meiosis resumption. PMID:24525567

  16. Mouse Emi2 as a distinctive regulatory hub in second meiotic metaphase.

    PubMed

    Suzuki, Toru; Suzuki, Emi; Yoshida, Naoko; Kubo, Atsuko; Li, Hongmei; Okuda, Erina; Amanai, Manami; Perry, Anthony C F

    2010-10-01

    The oocytes of vertebrates are typically arrested at metaphase II (mII) by the cytostatic factor Emi2 until fertilization. Regulatory mechanisms in Xenopus Emi2 (xEmi2) are understood in detail but contrastingly little is known about the corresponding mechanisms in mammals. Here, we analyze Emi2 and its regulatory neighbours at the molecular level in intact mouse oocytes. Emi2, but not xEmi2, exhibited nuclear targeting. Unlike xEmi2, separable N- and C-terminal domains of mouse Emi2 modulated metaphase establishment and maintenance, respectively, through indirect and direct mechanisms. The C-terminal activity was mapped to the potential phosphorylation target Tx(5)SxS, a destruction box (D-box), a lattice of Zn(2+)-coordinating residues and an RL domain. The minimal region of Emi2 required for its cytostatic activity was mapped to a region containing these motifs, from residue 491 to the C terminus. The cytostatic factor Mos-MAPK promoted Emi2-dependent metaphase establishment, but Mos autonomously disappeared from meiotically competent mII oocytes. The N-terminal Plx1-interacting phosphodegron of xEmi2 was apparently shifted to within a minimal fragment (residues 51-300) of mouse Emi2 that also contained a calmodulin kinase II (CaMKII) phosphorylation motif and which was efficiently degraded during mII exit. Two equimolar CaMKII gamma isoform variants were present in mII oocytes, neither of which phosphorylated Emi2 in vitro, consistent with the involvement of additional factors. No evidence was found that calcineurin is required for mouse mII exit. These data support a model in which mammalian meiotic establishment, maintenance and exit converge upon a modular Emi2 hub via evolutionarily conserved and divergent mechanisms. PMID:20724447

  17. 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)

  18. 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

  19. 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

  20. 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.

  1. 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

  2. Fertilization causes a single Ca2+ increase that fully depends on Ca2+ influx in oocytes of limpets (Phylum Mollusca, Class Gastropoda).

    PubMed

    Deguchi, Ryusaku

    2007-04-15

    Mature limpet oocytes arrested at the first metaphase (MI) of meiosis are activated by the stimulation of fertilizing sperm. The aim of the present study was to clarify the spatiotemporal property and mechanism of intracellular Ca2+ increase in limpet oocytes, which is a prerequisite signal for initiation of development at fertilization. In all of the five limpet species tested, the initial Ca2+ rising phase just after fertilization took the form of a centripetal Ca2+ wave spreading from the whole cortex to the center (cortical flash), yielding a homogeneous Ca2+ elevation throughout the oocyte. The Ca2+ level remained high during the subsequent plateau phase lasting for several minutes and then returned nearly to the original value. No additional Ca2+ increase followed the plateau phase at least by the time of first cleavage. Both rising and plateau phases of Ca2+ increase at fertilization were inhibited by removal of external Ca2+, suggesting that continuous Ca2+ entry occurs throughout the Ca2+ increase. Injection of inositol 1,4,5-trisphosphate (IP3) was effective in generating a Ca2+ increase in mature limpet oocytes arrested at MI; however, their ability to show an IP3-induced Ca2+ increase was extremely low, as compared with other animals. Responsiveness to IP3 injection in immature oocytes arrested at the first prophase (PI) was similar to that in the mature oocytes, suggesting that the IP3-induced Ca2+ release system does not develop during the process of meiotic maturation in limpet oocytes. Caffeine, cyclic adenosine diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), the agents known to stimulate internal Ca2+ release mechanisms distinct from an IP3-dependent pathway, had no effect on intracellular Ca2+ changes in mature limpet oocytes. Labeling of the endoplasmic reticulum (ER) with DiI revealed that cortical ER clusters are only present in the localized region around meiotic chromosomes in mature oocytes. These data

  3. Could oxidative stress influence the in-vitro maturation of oocytes?

    PubMed Central

    Combelles, Catherine MH; Gupta, Sajal; Agarwal, Ashok

    2011-01-01

    In the efforts aimed at improving the quality of in-vitro-matured human oocytes, the dynamic balance and roles of pro-/antioxidants merit further consideration. In-vitro maturation (IVM) is emerging as a popular technology at the forefront of fertility treatment and preservation. However, standard in-vitro culture conditions exert oxidative stress or an imbalance between oxidants and antioxidants. Reactive oxygen species (ROS) are oxygen-derived molecules formed as intermediary products of cellular metabolism. By acting as powerful oxidants, ROS can oxidatively modify any molecule, resulting in structural and functional alterations. ROS are neutralized by an elaborate defence system consisting of enzymatic and non-enzymatic antioxidants. This review captures the inherent and external factors that may modulate the oxidative stress status of oocytes. It discusses the suspected impacts of oxidative stress on the gamut of events associated with IVM, including prematuration arrest, meiotic progression, chromosomal segregation, cytoskeletal architecture and gene expression. In-vivo and in-vitro strategies that may overcome the potential influences of oxidative stress on oocyte IVM are presented. Future studies profiling the oxidative stress status of the oocyte may permit not only the formulation of a superior IVM medium that maintains an adequate pro-/antioxidant balance, but also the identification of predictors of oocyte quality. PMID:19490793

  4. Activation of protein kinase C induces mitogen-activated protein kinase dephosphorylation and pronucleus formation in rat oocytes.

    PubMed

    Lu, Qing; Smith, Gary D; Chen, Da-Yuan; Han, Zhi-Ming; Sun, Qing-Yuan

    2002-07-01

    Mammalian oocytes are arrested at metaphase of the second meiotic division (MII) before fertilization. When oocytes are stimulated by spermatozoa, they exit MII stage and complete meiosis. It has been suggested that an immediate increase in intracellular free calcium concentration and inactivation of maturation promoting factor (MPF) are required for oocyte activation. However, the underlying mechanism is still unclear. In the present study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase, and their interplay in rat oocyte activation. We found that MAP kinase became dephosphorylated in correlation with pronucleus formation after fertilization. Protein kinase C activators, phorbol 12-myriatate 13-acetate (PMA) and 1,2-dioctanoyl-rac-glycerol (diC8), triggered dephosphorylation of MAP kinase and pronucleus formation in a dose-dependent and time-dependent manner. Dephosphorylation of MAP kinase was also correlated with pronucleus formation when oocytes were treated with PKC activators. Effects of PKC activators were abolished by the PKC inhibitors, calphostin C and staurosporine, as well as a protein phosphatase blocker, okadaic acid (OA). These results suggest that PKC activation may cause rat oocyte pronucleus formation via MAP kinase dephosphorylation, which is probably mediated by OA-sensitive protein phosphatases. We also provide evidence supporting the involvement of such a process in fertilization. PMID:12080000

  5. 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.

  6. 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

  7. Temporary developmental arrest after storage of fertilized mouse oocytes at 4 degrees C: effects on embryonic development, maternal mRNA processing and cell cycle.

    PubMed

    Sakurai, Takayuki; Kimura, Minoru; Sato, Masahiro

    2005-05-01

    The aim of this study was to examine whether fertilized mouse oocytes can survive after short-term incubation (for 6-48 h) at 4 degrees C. When fertilized oocytes of ICR and C57BL/6 (B6) strain were incubated at 4 degrees C and returned to normal culture conditions (37 degrees C), development of these 4 degrees C-treated embryos for up to 12 h (for ICR) to blastocyst stage did not differ from that of untreated oocytes. Even 4 degrees C-treated embryos for 48 h developed to blastocysts at relatively good rates (33.3% for ICR and 50.8% for B6). The in vivo development of 4 degrees C-treated embryos for 12, 24 and 36 h to fetal stage was similar to that of untreated ones. BrdU labelling assay revealed temporary cessation of DNA replication in 4 degrees C-treated fertilized oocytes. Post-fertilization events including cytoplasmic polyadenylation of maternal mRNAs, mRNA degradation of a cell cycle-related gene and elevated mRNA expression of zygotic gene activation-related genes were temporarily suppressed in 4 degrees C-treated embryos. These findings indicate that 4 degrees C-treatment of fertilized murine oocytes results in temporary cessation of molecular events. We also show that 4 degrees C-treated fertilized oocytes for 12 h can be used for preparation of transgenic mice. PMID:15805144

  8. 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

  9. 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

  10. Involvement of Mos-MEK-MAPK pathway in cytostatic factor (CSF) arrest in eggs of the parthenogenetic insect, Athalia rosae.

    PubMed

    Yamamoto, Daisuke S; Tachibana, Kazunori; Sumitani, Megumi; Lee, Jae Min; Hatakeyama, Masatsugu

    2008-01-01

    Extensive survey of meiotic metaphase II arrest during oocyte maturation in vertebrates revealed that the mitogen-activated protein kinase (MAPK) pathway regulated by the c-mos proto-oncogene product, Mos, has an essential role in cytostatic activity, termed cytostatic factor (CSF). In contrast, little is known in invertebrates in which meiotic arrest occurs in most cases at metaphase I (MI arrest). A parthenogenetic insect, the sawfly Athalia rosae, in which artificial egg activation is practicable, has advantages to investigate the mechanisms of MI arrest. Both the MAPK/extracellular signal-regulated protein kinase kinase (MEK) and MAPK were phosphorylated and maintained active in MI-arrested sawfly eggs, whereas they were dephosphorylated soon after egg activation. Treatment of MI-arrested eggs with U0126, an inhibitor of MEK, resulted in dephosphorylation of MAPK and MI arrest was resumed. The sawfly c-mos gene orthologue encoding a serine/threonine kinase was cloned and analyzed. It was expressed in nurse cells in the ovaries. To examine CSF activity of the sawfly Mos, synthesized glutathione S-transferase (GST)-fusion sawfly Mos protein was injected into MI-resumed eggs in which MEK and MAPK were dephosphorylated. Both MEK and MAPK were phosphorylated again upon injection. In these GST-fusion sawfly Mos-injected eggs subsequent mitotic (syncytial) divisions were blocked and embryonic development was ceased. These results demonstrated that the MEK-MAPK pathway was involved in maintaining CSF arrest in sawfly eggs and Mos functioned as its upstream regulatory molecule. PMID:18793721

  11. High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis.

    PubMed

    Shim, Hee Jin; Lee, Eun-Mi; Nguyen, Long Duy; Shim, Jaekyung; Song, Young-Han

    2014-01-01

    Ionizing radiation (IR) treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs) generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis. PMID:24551207

  12. [The activation of bovine oocytes to parthenogenetic development by ethanol].

    PubMed

    Kuznietsova, I B; Kuznietsov, V Ie; Lukashenko, O O

    2000-01-01

    Dependence of parthenogenetic development ability of in vitro matured cow oocytes from their "aging" had been studied. It had been showed the possibility of parthenogenetic development of physiologically matured cow oocytes after inhibition of the first meiotic division and treatment with ethanol at least to 8-16-cell stage embryos. PMID:10808543

  13. Cytoplasmic Determination of Meiotic Spindle Size Revealed by a Unique Inter-Species Germinal Vesicle Transfer Model

    PubMed Central

    Wang, Zhong-Wei; Zhang, Guang-Li; Schatten, Heide; Carroll, John; Sun, Qing-Yuan

    2016-01-01

    Spindle sizes are different in diverse species and cell types. In frogs, the meiotic spindle size is positively correlated with the egg cell volume. Across species, relatively small mouse oocytes (70–80 μm) have a relatively large spindle while larger pig oocytes (about 120 μm) have a considerably smaller spindle. In this study we investigated whether species-specific oocyte spindle size was determined by cytoplasmic or nuclear factors. By exchanging the germinal vesicle between mouse and pig oocytes, we obtained two kinds of reconstructed oocytes: one with mouse ooplasm and pig GV (mCy-pGV oocyte), and the other with pig ooplasm and mouse GV (pCy-mGV oocyte). We show that the MII spindle size of the mCy-pGV oocyte is similar to that of the mouse meiotic spindle and significantly larger than that of the pig meiotic spindle. The timing of oocyte maturation also followed that of the species from which the oocyte cytoplasm arose, although some impact of the origin of the GV was observed. These data suggest that spindle size and the timing of meiotic progression are governed by cytoplasmic components rather than cytoplasmic volume and GV materials. PMID:26813698

  14. Apoptosis in mammalian oocytes: a review.

    PubMed

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

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals. PMID:25958165

  15. 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

  16. 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

  17. 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

  18. 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

  19. Effect of different types of sesquiterpene lactones on the maturation of Rhinella arenarum oocytes.

    PubMed

    Sánchez-Toranzo, G; Zapata-Martínez, J; Catalán, C; Bühler, M I

    2015-06-01

    The sesquiterpene lactones (STLs) are a large class of plant secondary metabolites that are generally found in the Asteraceae family and that have high diversity with respect to chemical structure as well as biological activity. STLs have been classified into different groups, such as guaianolides, germacranolides, and melampolides etc., based on their carboxylic skeleton. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under the stimulus of progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. The purpose of this work was to determine whether sesquiterpene lactones from the germacranolide and melampolide groups act as inhibitor agents on the meiosis of amphibian oocytes in vitro. Results for germacranolides indicated that the addition of deoxyelephantopins caused a high degree of inhibition and that minimolide showed a moderate inhibitory effect, whereas glaucolide A was inactive. Furthermore, the addition of melampolides (uvedalin, enhydrin, polymatin A and polymatin B) showed inhibitory effects. For enhydrin and uvedalin, inhibitory effects were observed at the higher concentrations assayed. The results of this study suggest that the inhibitory activity of the tested sesquiterpene lactones on the meiosis of Rhinella arenarum oocytes is not dependent on the group to which they belong, i.e. not on the carboxylic skeleton, but probably due to the arrangement and type of function groups present in the molecules. All assayed lactones in the germacranolide group showed low toxicity. In contrast, important differences in toxicity were observed for lactones from the melampolide group: enhydrin and uvedalin showed low toxicity, but polymatin A and B were highly toxic. PMID:24522008

  20. 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

  1. 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.

  2. What criteria for the definition of oocyte quality?

    PubMed

    Coticchio, Giovanni; Sereni, Elena; Serrao, Lucia; Mazzone, Silvia; Iadarola, Immacolata; Borini, Andrea

    2004-12-01

    Although the spermatozoon provides an essential contribution to the generation of a new individual, the developmental fate of the embryo is principally dictated by the oocyte. Oocyte competencies are acquired throughout oogenesis, via the interaction with somatic cells. The ability to reinitiate the meiotic process and undergo preimplantation development is progressively determined during the antral phase. It is known that these changes involve the nuclear and cytoplasmic compartments, respectively, but the underlying cellular and molecular mechanisms are still poorly understood. Analysis of various aspects of oocyte morphology (cytoplasm, zona pellucida, and polar body) via conventional phase-contrast microscopy has generated contrasting evidence on the possibility of establishing reliable criteria for the prediction of developmental potential. The introduction of a newly developed microscopy technique based on the detection of polarized light generated by birefringent cell structures has offered the possibility of visualizing noninvasively the meiotic spindle, whose presence is critical for fertilization and later developmental stages. However, further studies are needed to standardize and interpret the information accessible through such a technique. Although unable to preserve cell viability and therefore provide a method by which to select oocytes with superior developmental competence, invasive techniques can make a fundamental contribution to defining objective criteria of oocyte quality. In particular, immunofluorescence analysis, which is able to identify critical anomalies of the meiotic spindle and cytoskeleton organization that can account for oocyte quality, is an important method for assessing the efficiency of in vitro maturation systems. PMID:15731306

  3. Interplay between Cdc2 kinase and the c-Mos/MAPK pathway between metaphase I and metaphase II in Xenopus oocytes.

    PubMed

    Frank-Vaillant, M; Haccard, O; Ozon, R; Jessus, C

    2001-03-01

    Xenopus oocytes arrested in prophase I resume meiotic division in response to progesterone and arrest at metaphase II. Entry into meiosis I depends on the activation of Cdc2 kinase [M-phase promoting factor (MPF)]. To better understand the role of Cdc2, MPF activity was specifically inhibited by injection of the CDK inhibitor, Cip1. When Cip1 is injected at germinal vesicle breakdown (GVBD) time, Cdc25 and Plx1 are both dephosphorylated and Cdc2 is rephosphorylated on tyrosine. The autoamplification loop characterizing MPF is therefore not only required for MPF generation before GVBD, but also for its stability during the GVBD period. The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), responsible for cyclin degradation, is also under the control of Cdc2; therefore, Cdc2 activity itself induces its own inactivation through cyclin degradation, allowing the exit from the first meiotic division. In contrast, cyclin accumulation, responsible for Cdc2 activity increase allowing entry into metaphase II, is independent of Cdc2. The c-Mos/mitogen-activated protein kinase (MAPK) pathway remains active when Cdc2 activity is inhibited at GVBD time. This pathway could be responsible for the sustained cyclin neosynthesis. In contrast, during the metaphase II block, the c-Mos/MAPK pathway depends on Cdc2. Therefore, the metaphase II block depends on a dynamic interplay between MPF and CSF, the c-Mos/MAPK pathway stabilizing cyclin B, whereas in turn, MPF prevents c-Mos degradation. PMID:11180968

  4. 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

  5. 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. PMID:26364763

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. Hermes RNA-binding protein targets RNAs-encoding proteins involved in meiotic maturation, early cleavage, and germline development.

    PubMed

    Song, Hye-Won; Cauffman, Karen; Chan, Agnes P; Zhou, Yi; King, Mary Lou; Etkin, Laurence D; Kloc, Malgorzata

    2007-07-01

    The early development of metazoans is mainly regulated by differential translation and localization of maternal mRNAs in the embryo. In general, these processes are orchestrated by RNA-binding proteins interacting with specific sequence motifs in the 3'-untranslated region (UTR) of their target RNAs. Hermes is an RNA-binding protein, which contains a single RNA recognition motif (RRM) and is found in various vertebrate species from fish to human. In Xenopus laevis, Hermes mRNA and protein are localized in the vegetal region of oocytes. A subpopulation of Hermes protein is concentrated in a specific structure in the vegetal cortex, called the germ plasm (believed to contain determinants of the germ cell fate) where Hermes protein co-localizes with Xcat2 and RINGO/Spy mRNAs. The level of total Hermes protein decreases during maturation. The precocious depletion of Hermes protein by injection of Hermes antisense morpholino oligonucleotide (HE-MO) accelerates the process of maturation and results in cleavage defects in vegetal blastomeres of the embryo. It is known that several maternal mRNAs including RINGO/Spy and Mos are regulated at the translational level during meiotic maturation and early cleavage in Xenopus. The ectopic expression of RINGO/Spy or Mos causes resumption of meiotic maturation and cleavage arrests, which resemble the loss of Hermes phenotypes. We found that the injection of HE-MO enhances the acceleration of maturation caused by the injection of RINGO/Spy mRNA, and that Hermes protein is present as mRNP complex containing RINGO/Spy, Mos, and Xcat2 mRNAs in vivo. We propose that as an RNA-binding protein, Hermes may be involved in maturation, cleavage events at the vegetal pole and germ cell development by negatively regulating the expression of RINGO/Spy, Mos, and Xcat2 mRNAs. PMID:17309605

  13. 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

  14. Spermatogenesis-Specific Features of the Meiotic Program in Caenorhabditis elegans

    PubMed Central

    Shakes, Diane C.; Wu, Jui-ching; Sadler, Penny L.; LaPrade, Kristen; Moore, Landon L.; Noritake, Alana; Chu, Diana S.

    2009-01-01

    In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex. PMID:19696886

  15. Yeast meiotic mutants proficient for the induction of ectopic recombination.

    PubMed Central

    Engebrecht, J; Masse, S; Davis, L; Rose, K; Kessel, T

    1998-01-01

    A screen was designed to identify Saccharomyces cerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independently of recombination (SPO14, GSG1, SPOT8/MUM2, 3, 4). Similar to the spoT8-1 mutant, mum2 deletion strains do not undergo premeiotic DNA synthesis, arrest prior to the first meiotic division and fail to sporulate. Surprisingly, although DNA replication does not occur, mum2 mutants are induced for high levels of ectopic recombination. gsg1 diploids are reduced in their ability to complete premeiotic DNA synthesis and the meiotic divisions, and a small percentage of cells produce spores. mum3 mutants sporulate poorly and the spores produced are inviable. Finally, mum4-1 mutants produce inviable spores. The meiotic/sporulation defects of gsg1, mum2, and mum3 are not relieved by spo11 or spo13 mutations, indicating that the mutant defects are not dependent on the initiation of recombination or completion of both meiotic divisions. In contrast, the spore inviability of the mum4-1 mutant is rescued by the spo13 mutation. The mum4-1 spo13 mutant undergoes a single, predominantly equational division, suggesting that MUM4 functions at or prior to the first meiotic division. Although recombination is variably affected in the gsg1 and mum mutants, we hypothesize that these mutants define genes important for aspects of meiosis not directly related to recombination. PMID:9504908

  16. 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

  17. 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

  18. 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.

  19. ATM controls meiotic double-strand-break formation.

    PubMed

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

    2011-11-10

    In many organisms, developmentally programmed double-strand breaks (DSBs) formed by the SPO11 transesterase initiate meiotic recombination, which promotes pairing and segregation of homologous chromosomes. Because every chromosome must receive a minimum number of DSBs, attention has focused on factors that support DSB formation. However, improperly repaired DSBs can cause meiotic arrest or mutation; thus, having too many DSBs is probably as deleterious as having too few. Only a small fraction of SPO11 protein ever makes a DSB in yeast or mouse and SPO11 and its accessory factors remain abundant long after most DSB formation ceases, 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 signalling and promote DSB repair. Levels of SPO11-oligonucleotide complexes, by-products of meiotic DSB formation, are elevated at least tenfold 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. Confocal fluorescence assessment of bioenergy/redox status of dromedary camel (Camelus dromedarius) oocytes before and after in vitro maturation

    PubMed Central

    2014-01-01

    Background Reproductive biotechnologies in dromedary camel (Camelus dromedarius) are less developed than in other livestock species. The in vitro maturation (IVM) technology is a fundamental step for in vitro embryo production (IVP), and its optimization could represent a way to increase the success rate of IVP. The aim of the present study was to investigate the bioenergy/oxidative status of dromedary camel oocytes before and after IVM by confocal microscopy 3D imaging. Methods Oocytes were retrieved by slicing ovaries collected at local slaughterhouses. Recovered oocytes were examined before and after IVM culture for nuclear chromatin configuration and bioenergy/oxidative status, expressed as mitochondria (mt) distribution and activity, intracellular Reactive Oxygen Species (ROS) levels and distribution and mt/ROS colocalization. Results The mean recovery rate was 6 oocytes/ovary. After IVM, 61% of oocytes resumed meiosis and 36% reached the Metaphase II stage (MII). Oocyte bioenergy/redox confocal characterization revealed changes upon meiosis progression. Immature oocytes at the germinal vesicle (GV) stage were characterised by prevailing homogeneous mt distribution in small aggregates while MI and MII oocytes showed significantly higher rates of pericortical mt distribution organized in tubular networks (P < 0.05). Increased mt activity in MI (P < 0.001) and MII (P < 0.01) oocytes compared to GV stage oocytes was also observed. At any meiotic stage, homogeneous distribution of intracellular ROS was observed. Intracellular ROS levels also increased in MI (P < 0.01) and MII (P < 0.05) oocytes compared to GV stage oocytes. The mt/ROS colocalization signal increased in MI oocytes (P < 0.05). Conclusions This study provides indications that qualitative and quantitative indicators of bioenergy and oxidative status in dromedary camel oocytes are modified in relation with oocyte meiotic stage. These data may increase the knowledge of camel

  5. Calcium Signaling and Meiotic Exit at Fertilization in Xenopus Egg

    PubMed Central

    Tokmakov, Alexander A.; Stefanov, Vasily E.; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

    2014-01-01

    Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation. PMID:25322156

  6. The Tension at the Top of the Animal Pole Decreases during Meiotic Cell Division

    PubMed Central

    Satoh, Setsuko K.; Tsuchi, Akifumi; Satoh, Ryohei; Miyoshi, Hiromi; Hamaguchi, Miyako S.; Hamaguchi, Yukihisa

    2013-01-01

    Meiotic maturation is essential for the reproduction procedure of many animals. During this process an oocyte produces a large egg cell and tiny polar bodies by highly asymmetric division. In this study, to fully understand the sophisticated spatiotemporal regulation of accurate oocyte meiotic division, we focused on the global and local changes in the tension at the surface of the starfish (Asterina pectinifera) oocyte in relation to the surface actin remodeling. Before the onset of the bulge formation, the tension at the animal pole globally decreased, and started to increase after the onset of the bulge formation. Locally, at the onset of the bulge formation, tension at the top of the animal pole began to decrease, whereas that at the base of the bulge remarkably increased. As the bulge grew, the tension at the base of the bulge additionally increased. Such a change in the tension at the surface was similar to the changing pattern of actin distribution. Therefore, meiotic cell division was initiated by the bulging of the cortex, which had been weakened by actin reduction, and was followed by contraction at the base of the bulge, which had been reinforced by actin accumulation. The force generation system is assumed to allow the meiotic apparatus to move just under the membrane in the small polar body. Furthermore, a detailed comparison of the tension at the surface and the cortical actin distribution indicated another sophisticated feature, namely that the contraction at the base of the bulge was more vigorous than was presumed based on the actin distribution. These features of the force generation system will ensure the precise chromosome segregation necessary to produce a normal ovum with high accuracy in the meiotic maturation. PMID:24260212

  7. 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

  8. Merotelic attachments allow alignment and stabilization of chromatids in meiosis II oocytes.

    PubMed

    Kouznetsova, Anna; Hernández-Hernández, Abrahan; Höög, Christer

    2014-01-01

    The chromosome segregation process in human oocytes is highly error-prone, generating meiosis II (MII) oocytes with unbalanced chromatids that contribute to aneuploidy in offspring. This raises questions regarding the mechanism for transmission of chromatids and how chromatids evade the error correction mechanisms in MII oocytes. Here, we analyse the behaviour of chromatids in mouse MII oocytes. We find that chromatids align at the spindle equator at the metaphase stage of MII and that their presence does not obstruct entry into the anaphase stage. The alignment process is mediated by merotelic (bi-directional) microtubule-kinetochore attachments, revealing a multi-domain organization of the kinetochore of mammalian meiotic chromosomes. Our results suggest that biorientation of chromatids stabilize microtubule attachments at the kinetochores in a tension-dependent manner. Our results also suggest that merotelic attachments contribute to chromosome mis-segregation in wild-type MII oocytes. Thus, merotely is an important promoter of aneuploidy in mammalian oocytes. PMID:25007239

  9. Analysis of oocyte physiology to improve cryopreservation procedures.

    PubMed

    Gardner, David K; Sheehan, Courtney B; Rienzi, Laura; Katz-Jaffe, Mandy; Larman, Mark G

    2007-01-01

    In contrast to the preimplantation mammalian embryo, it has been notoriously difficult to cryopreserve the metaphase II oocyte. The ability to store oocytes successfully at -196 degrees C has numerous practical and financial advantages, together with ethical considerations, and will positively impact animal breeding programs and assisted conception in the human. Differences in membrane permeability and in physiology are two main reasons why successful oocyte cryopreservation has remained elusive. It is proposed, therefore, that rather than relying on technologies already established for the preimplantation embryo, the development of cryopreservation techniques suitable for the mammalian oocyte needs to take into account the idiosyncratic physiology of this cell. Analysis of intracellular calcium, for example, has revealed that exposure to conventional permeating cryoprotectants, such as propanediol, ethylene glycol and DMSO, all independently result in an increase in calcium, which in turn has the potential to initiate oocyte activation, culminating in zona hardening. Quantification of the metabolome and proteome of the oocyte has revealed that whereas slow freezing has a dramatic effect on cell physiology, vitrification appears to have limited effect. This is plausibly achieved by the limited exposure to cryoprotectants. Analysis of meiotic spindle dynamics and embryo development following IVF, also indicate that vitrification is less traumatic than slow freezing, and therefore has the greatest potential for successful oocyte cryopreservation. PMID:17049589

  10. BLEOMYCIN EFFECTS ON MOUSE MEIOTIC CHROMOSOMES

    EPA Science Inventory

    The effects of a radiomimetic chemical, bleomycin (BLM), on meiotic chromosomes was evaluated in mice. hromosome aberrations were analyzed at meiotic metaphase I, and damage to the synaptonemal complex was analyzed in meiotic prophase cells. n the metaphase aberration studies, an...

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

    PubMed

    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. PMID:26781547

  12. 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.

  13. Effect of estradiol-17β during in vitro growth culture on the growth, maturation, cumulus expansion and development of porcine oocytes from early antral follicles.

    PubMed

    Kubo, Naoko; Cayo-Colca, Ilse Silvia; Miyano, Takashi

    2015-03-01

    Growing porcine oocytes from early antral follicles can acquire meiotic and developmental competence under suitable culture conditions, but at lower rates compared to full-grown oocytes. We postulated that estradiol-17β (E2 ) supported the acquisition of meiotic and developmental competence as well as cumulus-expansion ability during growth culture. Growing oocytes from early antral follicles (1.2 to 1.5 mm in diameter) were grown in vitro for 5 days in a medium containing 0, 10(-7) , 10(-6) , 10(-5) or 10(-4)  mol/L E2 ; after in vitro maturation, 35, 58, 47, 74 and 49% of oocytes matured to metaphase II, 25, 79, 77, 90 and 97% acquired cumulus-expansion ability, and 23, 54, 63, 89 and 64% were fully surrounded by cumulus cells, respectively. Following maturation, electro-stimulation was applied to the oocytes grown with 10(-5)  mol/L E2 . After 6 days of culture, in vitro-grown oocytes developed to the blastocyst stage at a rate similar to that for full-grown oocytes (31% and 40%, respectively). Therefore, we suggest that the use of E2 during growth culture improves the meiotic and developmental competence of oocytes, cumulus-expansion ability, and cumulus cell attachment to the oocytes. PMID:25410821

  14. 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

  15. Peroxynitrite deteriorates oocyte quality through disassembly of microtubule organizing centers.

    PubMed

    Khan, Sana N; Shaeib, Faten; Thakur, Mili; Jeelani, Roohi; Awonuga, Awoniyi O; Goud, Pravin T; Abu-Soud, Husam M

    2016-02-01

    Previous theoretical studies have suggested that utilization of 3-D imaging to acquire morphologic parameters of meiotic spindles may be useful in infertility related procedures as an assessment of oocyte quality. However, our results show that treatment of oocytes with increasing concentrations of peroxynitrite (ONOO(-)) caused a dramatic alteration in spindle shape in which morphologic parameters are not measurable or are uninformative in terms of oocyte quality. Metaphase II mouse oocytes (n=520) were treated with increasing concentrations of ONOO(-), after which all oocytes were fixed and subjected to indirect immunofluorescence. Oocyte quality was assessed by alterations in the microtubule-organizing center (MTOC), pericentrin location, microtubule morphology, and chromosomal alignment. In untreated oocytes, pericentrin is primarily assembled utilizing the acentrosomal MTOC, which appears as a condensation at both spindle poles. The spindle has a symmetrical pointed barrel shape, assembled around the chromosomal plate at the spindle equator. Oocytes treated with low concentrations of ONOO(-) (<2.5 μM) showed shortening of the spindle apparatus, while pericentrin scatters from a tight condensation to a dispersed cluster around each spindle pole. At higher ONOO(-) concentrations (>2.5μM) the central attachments between microtubules are strained and bend or unevenly break, and the MTOC proteins are further dispersed or undetectable. Peroxynitrite mediated MTOC damage, which deranges the chromosomal scaffold at the time of assembly and separation, caused the deterioration in oocyte quality. These results provide a link between reactive oxygen species and poor reproductive outcomes and elucidate the underlying etiology, which could be used as a superior biomarker for oocyte quality compared to existing assessment tools. PMID:26746586

  16. Proteomics reveals a switch in CDK1-associated proteins upon M-phase exit during the Xenopus laevis oocyte to embryo transition.

    PubMed

    Marteil, Gaëlle; Gagné, Jean-Philippe; Borsuk, Ewa; Richard-Parpaillon, Laurent; Poirier, Guy G; Kubiak, Jacek Z

    2012-01-01

    Cyclin-dependent kinase 1 (CDK1) is a major M-phase kinase which requires the binding to a regulatory protein, Cyclin B, to be active. CDK1/Cyclin B complex is called M-phase promoting factor (MPF) for its key role in controlling both meiotic and mitotic M-phase of the cell cycle. CDK1 inactivation is necessary for oocyte activation and initiation of embryo development. This complex process requires both Cyclin B polyubiquitination and proteosomal degradation via the ubiquitin-conjugation pathway, followed by the dephosphorylation of the monomeric CDK1 on Thr161. Previous proteomic analyses revealed a number of CDK1-associated proteins in human HeLa cells. It is, however, unknown whether specific partners are involved in CDK1 inactivation upon M-phase exit. To better understand CDK1 regulation during MII-arrest and oocyte activation, we immunoprecipitated (IPed) CDK1 together with its associated proteins from M-phase-arrested and M-phase-exiting Xenopus laevis oocytes. A mass spectrometry (MS) analysis revealed a number of new putative CDK1 partners. Most importantly, the composition of the CDK1-associated complex changed rapidly during M-phase exit. Additionally, an analysis of CDK1 complexes precipitated with beads covered with p9 protein, a fission yeast suc1 homologue well known for its high affinity for CDKs, was performed to identify the most abundant proteins associated with CDK1. The screen was auto-validated by identification of: (i) two forms of CDK1: Cdc2A and B, (ii) a set of Cyclins B with clearly diminishing number of peptides identified upon M-phase exit, (iii) a number of known CDK1 substrates (e.g. peroxiredoxine) and partners (e.g. HSPA8, a member of the HSP70 family) both in IP and in p9 precipitated pellets. In IP samples we also identified chaperones, which can modulate CDK1 three-dimensional structure, as well as calcineurin, a protein necessary for successful oocyte activation. These results shed a new light on CDK1 regulation via a dynamic

  17. 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

  18. 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

  19. Growth of Mouse Oocytes to Maturity from Premeiotic Germ Cells In Vitro

    PubMed Central

    Zhang, Zhi-Peng; Liang, Gui-Jin; Zhang, Xi-Feng; Zhang, Guo-Liang; Chao, Hu-He; Li, Lan; Sun, Xiao-Feng; Min, Ling-Jiang; Pan, Qing-Jie; Shi, Qing-Hua; Sun, Qing-Yuan; De Felici, Massimo; Shen, Wei

    2012-01-01

    In the present study, we established an in vitro culture system suitable for generating fertilizable oocytes from premeiotic mouse female germ cells. These results were achieved after first establishing an in vitro culture system allowing immature oocytes from 12–14 day- old mice to reach meiotic maturation through culture onto preantral granulosa cell (PAGC) monolayers in the presence of Activin A (ActA). To generate mature oocytes from premeiotic germ cells, pieces of ovaries from 12.5 days post coitum (dpc) embryos were cultured in medium supplemented with ActA for 28 days and the oocytes formed within the explants were isolated and cocultured onto PAGC monolayers in the presence of ActA for 6–7 days. The oocytes were then subjected to a final meiotic maturation assay to evaluate their capability to undergo germinal vesicle break down (GVBD) and reach the metaphase II (MII) stage. We found that during the first 28 days of culture, a significant number of oocytes within the ovarian explants reached nearly full growth and formed preantral follicle-like structures with the surrounding somatic cells. GSH level and Cx37 expression in the oocytes within the explants were indicative of proper developmental conditions. Moreover, the imprinting of Igf2r and Peg3 genes in these oocytes was correctly established. Further culture onto PAGCs in the presence of ActA allowed about 16% of the oocytes to undergo GVBD, among which 17% reached the MII stage during the final 16–18 hr maturation culture. These MII oocytes showed normal spindle and chromosome assembly and a correct ERK1/2 activity. About 35% of the in vitro matured oocytes were fertilized and 53.44% of them were able to reach the 2-cell stage. Finally, around 7% of the 2-cell embryos developed to the morula/blastocyst stage. PMID:22848595

  20. Cardiac arrest

    MedlinePlus

    ... treatment for cardiac arrest. It is a medical device that gives an electrical shock to the heart. The shock can get the heart beating normally again. Small, portable defibrillators are often available in public areas for ...

  1. Protein deubiquitination during oocyte maturation influences sperm function during fertilisation, antipolyspermy defense and embryo development.

    PubMed

    Yi, Young-Joo; Sutovsky, Miriam; Song, Won-Hee; Sutovsky, Peter

    2015-11-01

    Ubiquitination is a covalent post-translational modification of proteins by the chaperone protein ubiquitin. Upon docking to the 26S proteasome, ubiquitin is released from the substrate protein by deubiquitinating enzymes (DUBs). We hypothesised that specific inhibitors of two closely related oocyte DUBs, namely inhibitors of the ubiquitin C-terminal hydrolases (UCH) UCHL1 (L1 inhibitor) and UCHL3 (L3 inhibitor), would alter porcine oocyte maturation and influence sperm function and embryo development. Aberrant cortical granule (CG) migration and meiotic spindle defects were observed in oocytes matured with the L1 or L3 inhibitor. Embryo development was delayed or blocked in oocytes matured with the general DUB inhibitor PR-619. Aggresomes, the cellular stress-inducible aggregates of ubiquitinated proteins, formed in oocytes matured with L1 inhibitor or PR-619, a likely consequence of impaired protein turnover. Proteomic analysis identified the major vault protein (MVP) as the most prominent protein accumulated in oocytes matured with PR-619, suggesting that the inhibition of deubiquitination altered the turnover of MVP. The mitophagy/autophagy of sperm-contributed mitochondria inside the fertilised oocytes was hindered by DUB inhibitors. It is concluded that DUB inhibitors alter porcine oocyte maturation, fertilisation and preimplantation embryo development. By regulating the turnover of oocyte proteins and mono-ubiquitin regeneration, the DUBs may promote the acquisition of developmental competence during oocyte maturation. PMID:24848520

  2. 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

  3. Effect of hyaluronan on developmental competence and quality of oocytes and obtained blastocysts from in vitro maturation of bovine oocytes.

    PubMed

    Opiela, Jolanta; Romanek, Joanna; Lipiński, Daniel; Smorąg, Zdzisław

    2014-01-01

    The objective of the present study was to evaluate the effect of hyaluronan (HA) during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC), and obtained blastocysts. COCs were matured in vitro in control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P < 0.001) was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P < 0.01). Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higher Bax mRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure. PMID:24689043

  4. The activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro

    PubMed Central

    Cotterill, Matthew; Harris, Sarah E.; Collado Fernandez, Esther; Lu, Jianping; Huntriss, John D.; Campbell, Bruce K.; Picton, Helen M.

    2013-01-01

    Mitochondria are responsible for the production of ATP, which drives cellular metabolic and biosynthetic processes. This is the first study to quantify the mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in germinal vesicle (GV) and metaphase II (MII) oocytes through JC1 staining. Primordial to early antral follicles (n = 249) were isolated from the sheep ovarian cortex following digestion at 37°C for 1 h and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n = 133) were aspirated from 3- to 5-mm diameter antral follicles, and mature MII oocytes (n = 71) were generated following in vitro maturation (IVM). The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605 ± 205, n = 8) to mature MII oocytes (744 633 ± 115 799, n = 13; P < 0.05). Mitochondrial activity (P > 0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in the mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganization during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the pre-ovulatory gonadotrophin surge. PMID:23468533

  5. 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.

  6. Bisphenol A Exposure during Oocyte Maturation in vitro Results in Spindle Abnormalities and Chromosome Misalignment in Bos taurus.

    PubMed

    Ferris, Jacqueline; Favetta, Laura A; King, W Allan

    2015-01-01

    Bisphenol A (BPA) exposure in humans is widespread, and BPA has been detected in a variety of samples including follicular fluid. BPA levels have been found to negatively correlate with the developmental potential of oocytes in women undergoing in vitro fertilization and to induce meiotic abnormalities experimentally in human and mouse models. BPA may detrimentally affect oocyte maturation, and different concentrations of exposure can cause various outcomes. Because of the importance of oocyte maturation on developmental potential, disturbances during this time can significantly impact oocyte viability. Here, bovine oocytes were matured in vitro with and without BPA treatment of the media. The levels of BPA taken up by the oocytes were much lower than the initial exposure. Medium treatment with 30 ng/ml resulted in an average of 2.48 ng/ml BPA measured in mature oocytes. These oocytes exhibited decreased maturation and increased incidence of spindle abnormalities. Only 57.4% of oocytes exposed to 30 ng/ml BPA reached maturity compared to 72.4% of controls (p < 0.05). Mature oocytes following BPA exposure displayed increased abnormal spindle morphology (67.9%) and chromosome dispersal (60%) compared to all other groups analyzed (p < 0.05). Thus, exposure to BPA during in vitro oocyte maturation has the potential to decrease oocyte quality. PMID:25871885

  7. Genetic control of recombination partner preference in yeast meiosis. Isolation and characterization of mutants elevated for meiotic unequal sister-chromatid recombination.

    PubMed Central

    Thompson, D A; Stahl, F W

    1999-01-01

    Meiotic exchange occurs preferentially between homologous chromatids, in contrast to mitotic recombination, which occurs primarily between sister chromatids. To identify functions that direct meiotic recombination events to homologues, we screened for mutants exhibiting an increase in meiotic unequal sister-chromatid recombination (SCR). The msc (meiotic sister-chromatid recombination) mutants were quantified in spo13 meiosis with respect to meiotic unequal SCR frequency, disome segregation pattern, sporulation frequency, and spore viability. Analysis of the msc mutants according to these criteria defines three classes. Mutants with a class I phenotype identified new alleles of the meiosis-specific genes RED1 and MEK1, the DNA damage checkpoint genes RAD24 and MEC3, and a previously unknown gene, MSC6. The genes RED1, MEK1, RAD24, RAD17, and MEC1 are required for meiotic prophase arrest induced by a dmc1 mutation, which defines a meiotic recombination checkpoint. Meiotic unequal SCR was also elevated in a rad17 mutant. Our observation that meiotic unequal SCR is elevated in meiotic recombination checkpoint mutants suggests that, in addition to their proposed monitoring function, these checkpoint genes function to direct meiotic recombination events to homologues. The mutants in class II, including a dmc1 mutant, confer a dominant meiotic lethal phenotype in diploid SPO13 meiosis in our strain background, and they identify alleles of UBR1, INP52, BUD3, PET122, ELA1, and MSC1-MSC3. These results suggest that DMC1 functions to bias the repair of meiosis-specific double-strand breaks to homologues. We hypothesize that the genes identified by the class II mutants function in or are regulators of the DMC1-promoted interhomologue recombination pathway. Class III mutants may be elevated for rates of both SCR and homologue exchange. PMID:10511544

  8. Contribution of human oocyte architecture to success of in vitro maturation technology

    PubMed Central

    Khalili, Mohammad Ali; A Nottola, Stefania; Shahedi, Abbas; Macchiarelli, Guido

    2013-01-01

     The use of ovarian stimulation for infertility treatment is associated with side effects of ovarian hyperstimulation syndrome (OHSS) and potential cancer risk. This is also true in high risk women such as those polycystic with ovary (PCO) and polycystic ovarian syndrome (PCOS). In vitro maturation (IVM) of oocytes was primarily developed to make IVF safe for women with PCO and at high risk of OHSS. The application of IVM of oocytes to assist clinical infertility treatment remains poor because of the reduced developmental competence of oocytes after IVM, despite several decades of research. Reduced meiotic maturation and fertilization rates, as well as low blastocyst production reveal short-term developmental insufficiency of oocytes when compared with in vivo-matured counterparts. In this review, the structural role of human oocytes, revealed by different technical approaches, to the success of IVM technology is highlighted. PMID:24639686

  9. 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

  10. 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.

  11. Acute fasting decreases the expression of GLUT1 and glucose utilisation involved in mouse oocyte maturation and cumulus cell expansion.

    PubMed

    Han, Yingying; Yan, Jun; Zhou, Jinlian; Teng, Zhen; Bian, Fenghua; Guo, Meng; Mao, Guankun; Li, Junxia; Wang, Jianwei; Zhang, Meijia; Xia, Guoliang

    2012-01-01

    Acute fasting impairs meiotic resumption and glucose consumption in mouse cumulus cell and oocyte complexes (COCs). This study examines the effects of acute fasting on the regulation of glucose transporter 1 (GLUT1) expression and glucose consumption in oocyte maturation. Our results indicate that the restriction of glucose utilisation by 2-deoxyglucose (2-DG) mimicked the inhibitory effects of acute fasting on oocyte meiotic resumption and cumulus cell expansion, effects that were rescued by high glucose concentrations in the culture medium. GLUT1 protein levels were higher in cumulus cells compared with oocytes, and GLUT1 expression in COCs increased with FSH treatment in vitro. However, under acute fasting conditions, GLUT1 expression in COCs decreased and the response to FSH disappeared. Exposure to high glucose conditions (27.5mM and 55mM), significantly increased both glucose consumption and GLUT1 levels in COCs. Inhibition of GLUT1 function using an anti-GLUT1 antibody significantly inhibited FSH-induced oocyte meiotic resumption. Taken together, these results suggest that acute fasting decreases GLUT1 expression and glucose utilisation, inhibiting the processes of oocyte maturation and cumulus cell expansion. PMID:22697123

  12. MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination

    PubMed Central

    Hervé, Roxane; Finsterbusch, Friederike; Tourpin, Sophie; Le Bouffant, Ronan; Duquenne, Clotilde; Messiaen, Sébastien; Martini, Emmanuelle; Bernardino-Sgherri, Jacqueline; Toth, Attila; Habert, René; Livera, Gabriel

    2013-01-01

    Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB). This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 −/− spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob −/− meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination. PMID:24068956

  13. 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

  14. Ability of in vitro maturing bovine oocytes to transform sperm nuclei to metaphase chromosomes.

    PubMed

    Abeydeera, L R; Niwa, K

    1992-11-01

    Bovine oocytes at the germinal vesicle stage were inseminated in Brackett & Oliphant's medium with bovine serum albumin, caffeine and heparin. Eight hours after insemination, oocytes were transferred into tissue culture medium-199 containing 10% fetal calf serum and cultured for 5-40 h at 39 degrees C in 5% CO2 in air. The proportions of unpenetrated and penetrated oocytes reaching metaphase II increased as the time of examination increased, reaching 70 and 65% 40 h after transfer, respectively. When oocytes were penetrated by more than four spermatozoa, meiotic maturation was greatly retarded. Sperm nuclei were decondensed in most (81%) penetrated oocytes 5 h after transfer. The decondensed sperm nuclei were recondensed and then transformed to metaphase chromosomes which were morphologically compacted at first but became slightly dispersed later. The formation of the metaphase chromosomes was observed in 86% of penetrated oocytes examined 40 h after transfer, and occurred in all metaphase II oocytes at that time. In oocytes penetrated by more than nine spermatozoa, no such transformation of sperm nuclei was observed. Well-developed male and female pro-nuclei were observed in only three (6%) of 51 oocytes penetrated 40 h after transfer. PMID:1339837

  15. Involvement of Rab6a in organelle rearrangement and cytoskeletal organization during mouse oocyte maturation

    PubMed Central

    Ma, Rujun; Zhang, Jiaqi; Liu, Xiaohui; Li, Ling; Liu, Honglin; Rui, Rong; Gu, Ling; Wang, Qiang

    2016-01-01

    Rab GTPases have been reported to define the identity and transport routes of vesicles. Rab6 is one of the most extensively studied Rab proteins involved in regulating organelle trafficking and integrity maintenance. However, to date, the function of Rab6 in mammalian oocytes has not been addressed. Here we report severe disorganization of endoplasmic reticulum upon specific knockdown of Rab6a in mouse oocytes. In line with this finding, intracellular Ca2+ stores are accordingly reduced in Rab6a-depleted oocytes. Furthermore, in these oocytes, we observe the absence of cortical granule free domain, which is a kind of special organelle in matured oocytes and its exocytosis is calcium dependent. On the other hand, following Rab6a knockdown, the prominent defects of cytoskeletal structures are detected during oocyte meiosis. In particular, the majority of Rab6a-depleted oocytes fail to form the actin cap, and the frequency of spindle defects and chromosome misalignment is significantly elevated. In summary, our data reveal that Rab6a not only participates in modulating the organization of oocyte organelles, but also is a novel regulator of meiotic apparatus in mammalian oocytes. PMID:27030207

  16. 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.

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

    PubMed

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

    2015-01-15

    GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17β-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20β-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes. PMID:25485460

  18. KL/KIT co-expression in mouse fetal oocytes.

    PubMed

    Doneda, Luisa; Klinger, Francesca-Gioia; Larizza, Lidia; De Felici, Massimo

    2002-12-01

    The tyrosine kinase receptor, KIT, and its ligand, KL are important regulators of germ cell development. The aim of this study was to examine in detail the expression of the genes encoding these proteins (White and Steel, respectively) during the fetal period (14.5-18.5 days post coitum, dpc) and the two weeks after birth in mouse ovaries using the highly sensitive in situ reverse-transcriptase polymerase chain reaction (in situ RT-PCR). KL and KIT mRNAs were not detected in 14.5-15.5 dpc ovaries but, between 16.5 and 17.5 dpc, most of the oocytes in the outer regions of the ovaries positively stained for both mRNAs. The majority of the co-expressing oocytes were identified at the zygotene/pachytene stage of meiotic prophase I. At 18.5 dpc, positive staining for KL mRNA was present only in the somatic cells in the outer regions of the ovaries. At birth, faint KL mRNA-labelled somatic cells were mainly found in the central region of the ovaries and, by P7-14, a higher level of expression was detected in the follicle cells of one- and two-layered growing follicles. Between 17.5 dpc and birth, most of the oocytes expressed KIT mRNA and, from P7 onward, there was a considerable accumulation of transcripts in the growing oocytes. The results of in situ RT-PCR were confirmed by RT-PCR on purified populations of oocytes, and at protein level by means of immunohistochemistry. The co-expression of KL and KIT in a fraction of fetal oocytes suggests that the KL/KIT system, besides the well known paracrine functions on germ cells, may exert a novel autocrine role during the mid-stage of the oocyte meiotic prophase. The possibility that this autocrine loop plays a role in sustaining the survival of fetal oocytes in this stage is supported by the finding that the addition to the culture medium of anti-KL or anti-KIT antibodies led to a significant increase in oocyte apoptosis in the absence of exogenous KL. PMID:12533025

  19. 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

  20. The kinase VRK1 is required for normal meiotic progression in mammalian oogenesis.

    PubMed

    Schober, Carolyn S; Aydiner, Fulya; Booth, Carmen J; Seli, Emre; Reinke, Valerie

    2011-01-01

    The kinase VRK1 has been implicated in mitotic and meiotic progression in invertebrate species, but whether it mediates these events during mammalian gametogenesis is not completely understood. Previous work has demonstrated a role for mammalian VRK1 in proliferation of male spermatogonia, yet whether VRK1 plays a role in meiotic progression, as seen in Drosophila, has not been determined. Here, we have established a mouse strain bearing a gene trap insertion in the VRK1 locus that disrupts Vrk1 expression. In addition to the male proliferation defects, we find that reduction of VRK1 activity causes a delay in meiotic progression during oogenesis, results in the presence of lagging chromosomes during formation of the metaphase plate, and ultimately leads to the failure of oocytes to be fertilized. The activity of at least one phosphorylation substrate of VRK1, p53, is not required for these defects. These results are consistent with previously defined functions of VRK1 in meiotic progression in Drosophila oogenesis, and indicate a conserved role for VRK1 in coordinating proper chromosomal configuration in female meiosis. PMID:21277975

  1. Optimal concentration of hyaluronan and plant protein in different culture systems for in vitro maturation of bovine oocytes.

    PubMed

    Opiela, Jolanta; Latasiewicz, Ewa; Smorag, Zdzisław

    2012-12-01

    With a view to search for optimal concentration of hyaluronan (HA) and plant protein (PP) in different culture systems for in vitro maturation of bovine oocytes, cumulus-oocyte complexes (COCs) were matured in vitro in 2 culture systems (first co-cultured with granulose cells and estrus calf serum (ECS) in 2 mL volume, second without co-culture where ECS was replaced by exogenous hormones and BSA or PP in 100 microL dose under mineral oil). Seven types of media were used; 3 in first system and 4 in second system. To evaluate HA and PP effect on oocytes after in vitro culture an estimation of meiosis stage and a level of DNA fragmentation was performed by TUNEL staining. The highest meiotic maturation (84%) was observed in oocytes cultured in medium enriched with ECS in co-culture with granulose cells (1st system). The lowest meiotic maturation was noted in medium with addition of BSA (43%). The addition of HA in the medium enriched with BSA significantly increased the rate of matured oocytes (67%) and also didn't affect the chromatin quality of individual oocytes. The addition of HA to the culture medium supplemented with a PP decreased the rate of matured oocytes to 54% but no statistical differences were noted. The results of the present study showed that HA supplementation didn't have a detrimental impact on oocyte chromatin integrity and improved bovine oocytes' meiotic maturation in medium supplemented only with BSA without co-culture of granulose cells. PMID:23986966

  2. 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

  3. 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

  4. Morphogenetic investigation of metaphase-specific cell death in meiotic spermatocytes in mice.

    PubMed

    Kon, Yasuhiro

    2005-09-01

    An MRL/MpJ strain of mice, including Ipr mutants, reveals the complex pathological manifestations of collagen disease, such as systemic vasculitis, glomerulonephritis, arthritis and sialoadenitis, in association with several autoimmune factors. Studies involving this mouse strain have shown that it exhibits a much-enhanced healing response compared with other mouse strains, together with reduced scarring in the periphery. Recently, unique characteristics were found in the testis of the MRL/MpJ mouse: metaphase-specific apoptosis (MSA) of meiotic spermatocytes, heat stress resistance in spermatocytes and the appearance of oocyte-like cells. The present review describes the morphological and genetic analysis of MSA, culminating in the conclusion that inherent mutation of exonuclease 1 induces checkpoint activity during meiotic division in MRL mice. PMID:16158977

  5. History of oocyte cryopreservation.

    PubMed

    Gook, Debra A

    2011-09-01

    The potential advantages of being able to cryopreserve oocytes have been apparent for many decades. Technical difficulties associated with the unique properties of the mammalian oocyte initially retarded rapid development in this area but recent advances have overcome many of the problems. A stage has now been reached where oocyte cryopreservation can be considered an important component of human assisted reproductive technology. The potential advantages of being able to cryopreserve oocytes have been apparent for many decades. Technical difficulties associated with the unique properties of the mammalian oocyte initially retarded rapid development in this area but recent advances have overcome many of the problems. A stage has now been reached where oocyte cryopreservation can be considered an important component of human assisted reproductive technology. PMID:21549640

  6. Isolation of Xenopus oocytes.

    PubMed

    Sive, Hazel L; Grainger, Robert M; Harland, Richard M

    2010-12-01

    Xenopus oocytes are obtained from sexually mature females by surgically removing parts of the ovary. The operation is not fatal and can be performed on an anesthetized frog several times during its lifetime. However, a recovery period of 2 wk is recommended between operations. A careful record of all operations performed, including details of oocyte quality, should be kept. A frog that produces one good batch of oocytes; e.g., those that translate injected messenger RNAs (mRNAs) efficiently, should be recorded and used again, because oocyte quality is generally frog-dependent. PMID:21123421

  7. 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

  8. Maturation, Fertilization, and the Structure and Function of the Endoplasmic Reticulum in Cryopreserved Mouse Oocytes1

    PubMed Central

    Lowther, Katie M.; Weitzman, Vanessa N.; Maier, Donald; Mehlmann, Lisa M.

    2009-01-01

    Oocyte cryopreservation is a promising technology that could benefit women undergoing assisted reproduction. Most studies examining the effects of cryopreservation on fertilization and developmental competence have been done using metaphase II-stage oocytes, while fewer studies have focused on freezing oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation. Herein, we examined the effects of vitrifying GV-stage mouse oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes necessary for proper fertilization and early embryonic development. We examined the endoplasmic reticulum (ER) as one indicator of cytoplasmic structure, as well as the ability of oocytes to develop Ca2+ release mechanisms following vitrification and in vitro maturation. Vitrified GV-stage oocytes matured in culture to metaphase II at a rate comparable to that of controls. These oocytes had the capacity to release Ca2+ following injection of inositol 1,4,5-trisphosphate, demonstrating that Ca2+ release mechanisms developed during meiotic maturation. The ER remained intact during the vitrification procedure as assessed using the lipophilic fluorescent dye DiI. However, the reorganization of the ER that occurs during in vivo maturation was impaired in oocytes that were vitrified before oocyte maturation. These results show that vitrification of GV-stage oocytes does not affect nuclear maturation or the continuity of the ER, but normal cytoplasmic maturation as assessed by the reorganization of the ER is disrupted. Deficiencies in factors that are responsible for proper ER reorganization during oocyte maturation could contribute to the low developmental potential previously reported in vitrified in vitro-matured oocytes. PMID:19299317

  9. Effects of 5α-Dihydrotestosterone and 17β-Estradiol on the Mouse Ovarian Follicle Development and Oocyte Maturation

    PubMed Central

    Tarumi, Wataru; Itoh, Masanori T.; Suzuki, Nao

    2014-01-01

    We have previously reported that androstenedione induces abnormalities of follicle development and oocyte maturation in the mouse ovary. In granulosa cells of the ovarian follicle, androstenedione is aromatized to 17β-estradiol (E2). To determine whether the androgen or estrogen acts directly on the follicle to induce the above-mentioned abnormalities, we compared the effects of a non-aromatizable androgen, 5α-dihydrotestosterone (DHT), with those of E2 on murine follicular development and oocyte maturation in a single follicle culture system. The high dose (10−6 M) of DHT prompted normal follicular development, and there was no effect on oocyte meiotic maturation after stimulation with human chorionic gonadotropin (hCG) and epidermal growth factor (EGF). In contrast, culture with the high dose (10−6 M) of E2 delayed follicular growth and also suppressed proliferation of granulosa cells and antrum formation. Furthermore, culture with E2 delayed or inhibited oocyte meiotic maturation, such as chromosome alignment on the metaphase plate and extrusion of the first polar body, after addition of hCG and EGF. In conclusion, these findings demonstrate that E2, but not DHT, induces abnormalities of follicular development, which leads to delay or inhibition of oocyte meiotic maturation. PMID:24911314

  10. 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

  11. Essential Role for Endogenous siRNAs during Meiosis in Mouse Oocytes

    PubMed Central

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

    2015-01-01

    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. PMID:25695507

  12. 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

  13. 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

  14. In vivo and in vitro environmental effects on mammalian oocyte quality.

    PubMed

    Krisher, Rebecca L

    2013-01-01

    The oocyte is at the center of the equation that results in female fertility. Many factors influence oocyte quality, including external factors such as maternal nutrition, stress, and environmental exposures, as well as ovarian factors such as steroids, intercellular communication, antral follicle count, and follicular fluid composition. These influences are interconnected; changes in the external environment of the female translate into ovarian changes that affect the oocyte. The lengthy period during which the oocyte remains arrested in the ovary provides ample time and opportunity for environmental factors to take their toll. An appropriate environment for growth and maturation of the oocyte, in vivo and in vitro, is critical to ensure optimal oocyte quality, which determines the success of fertilization and preimplantation embryo development, and has long-term implications for implantation, fetal growth, and offspring health. PMID:25387025

  15. 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

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

    PubMed Central

    Winters, Tristan; McNicoll, Francois; Jessberger, Rolf

    2014-01-01

    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

  17. Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

    PubMed Central

    2011-01-01

    concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology. PMID:21410957

  18. The Mouse INO80 Chromatin-Remodeling Complex Is an Essential Meiotic Factor for Spermatogenesis.

    PubMed

    Serber, Daniel W; Runge, John S; Menon, Debashish U; Magnuson, Terry

    2016-01-01

    The ability to faithfully transmit genetic information across generations via the germ cells is a critical aspect of mammalian reproduction. The process of germ cell development requires a number of large-scale modulations of chromatin within the nucleus. One such occasion arises during meiotic recombination, when hundreds of DNA double-strand breaks are induced and subsequently repaired, enabling the transfer of genetic information between homologous chromosomes. The inability to properly repair DNA damage is known to lead to an arrest in the developing germ cells and sterility within the animal. Chromatin-remodeling activity, and in particular the BRG1 subunit of the SWI/SNF complex, has been shown to be required for successful completion of meiosis. In contrast, remodeling complexes of the ISWI and CHD families are required for postmeiotic processes. Little is known regarding the contribution of the INO80 family of chromatin-remodeling complexes, which is a particularly interesting candidate due to its well described functions during DNA double-strand break repair. Here we show that INO80 is expressed in developing spermatocytes during the early stages of meiotic prophase I. Based on this information, we used a conditional allele to delete the INO80 core ATPase subunit, thereby eliminating INO80 chromatin-remodeling activity in this lineage. The loss of INO80 resulted in an arrest during meiosis associated with a failure to repair DNA damage during meiotic recombination. PMID:26607718

  19. 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. PMID:27096707

  20. 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

  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. 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

  3. 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

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

    PubMed

    Nevoral, Jan; Žalmanová, Tereza; Zámostná, Kateřina; Kott, Tomáš; Kučerová-Chrpová, Veronika; Bodart, Jean-Francois; Gelaude, Armance; Procházka, Radek; Orsák, Matyáš; Šulc, Miloslav; Klein, Pavel; Dvořáková, Markéta; Weingartová, Ivona; Víghová, Aurélia; Hošková, Kristýna; Krejčová, Tereza; Jílek, František; Petr, Jaroslav

    2015-12-01

    Hydrogen sulfide, one of three known gasotransmitters, is involved in physiological processes, including reproductive functions. Oocyte maturation and surrounding cumulus cell expansion play an essential role in female reproduction and subsequent embryonic development. Although the positive effects of exogenous hydrogen sulfide on maturing oocytes are well known, the role of endogenous hydrogen sulfide, which is physiologically released by enzymes, has not yet been described in oocytes. In this study, we observed the presence of Cystathionine β-Synthase (CBS), Cystathionine γ-Lyase (CTH) and 3-Mercaptopyruvate Sulfurtransferase (3-MPST), hydrogen sulfide-releasing enzymes, in porcine oocytes. Endogenous hydrogen sulfide production was detected in immature and matured oocytes as well as its requirement for meiotic maturation. Individual hydrogen sulfide-releasing enzymes seem to be capable of substituting for each other in hydrogen sulfide production. However, meiosis suppression by inhibition of all hydrogen sulfide-releasing enzymes is not irreversible and this effect is a result of M-Phase/Maturation Promoting Factor (MPF) and Mitogen-Activated Protein Kinase (MAPK) activity inhibition. Futhermore, cumulus expansion expressed by hyaluronic acid (HA) production is affected by the inhibition of hydrogen sulfide production. Moreover, quality changes of the expanded cumuli are indicated. These results demonstrate hydrogen sulfide involvement in oocyte maturation as well as cumulus expansion. As such, hydrogen sulfide appears to be an important cell messenger during mammalian oocyte meiosis and adequate cumulus expansion. PMID:26456342

  5. 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

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

    PubMed

    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

    2015-10-01

    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

  7. The Src Homology 2 Domain-Containing Adapter Protein B (SHB) Regulates Mouse Oocyte Maturation

    PubMed Central

    Calounova, Gabriela; Livera, Gabriel; Zhang, Xiao-Qun; Liu, Kui; Gosden, Roger G.; Welsh, Michael

    2010-01-01

    SHB (Src homology 2 domain-containing adapter protein B) is involved in receptor tyrosine kinase signaling. Mice deficient in the Shb gene have been found to exhibit a transmission ratio distortion with respect to inheritance of the Shb null allele among offspring and this phenomenon was linked to female gamete production. Consequently, we postulated that Shb plays a role for oocyte biology and thus decided to investigate oocyte formation, meiotic maturation, and early embryo development in relation to absence of the Shb gene. Oogenesis was apparently accelerated judging from the stages of oocyte development on fetal day 18.5 and one week postnatally in Shb −/− mice; but in adulthood ovarian follicle maturation was impaired in these mice. Completion of meiosis I (first polar body extrusion) was less synchronized, with a fraction of oocytes showing premature polar body extrusion in the absence of Shb. In vitro fertilization of mature oocytes isolated from Shb +/+, +/− and −/− mice revealed impaired early embryo development in the −/− embryos. Moreover, the absence of Shb enhanced ERK (extracellular-signal regulated kinase) and RSK (ribosomal S6 kinase) signaling in oocytes and these effects were paralleled by an increased ribosomal protein S6 phosphorylation and activation. It is concluded that SHB regulates normal oocyte and follicle development and that perturbation of SHB signaling causes defective meiosis I and early embryo development. PMID:20585392

  8. 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

  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. Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin.

    PubMed

    Inoué, S; Borisy, G G; Kiehart, D P

    1974-07-01

    Purified tubulin solutions stabilized and augmented the birefringence (BR) of isolated Chaetopterus spindles. Tubulin was extracted from pig brain tissue in cold PEG buffer (0.1 M piperazine-N-N'-bis[2-ethane sulfonic acid], 1 mM ethylene bis-[oxyethylenenitrilo]tetraacetate, [EGTA], 2.5 mM guanosine triphosphate, [GTP], pH 6.94, at 25 degrees C), and purified by two cycles of a reversible, temperature-dependent assembly-disassembly procedure. The spindle BR of the meiotic metaphase-arrested oocytes of Chaetopterus decreased linearly at a rate of 1.5 nm/min when perfused with PEG buffer without tubulin. In this hypotonic, calcium-chelating solution, the cell lysed within 1.5 min, and after a brief, transient rise, the BR disappeared in ca. 4 min from the time of buffer application. Cells perfused with tubulin in PEG buffer also showed BR decay at the same rate until cell lysis. Immediately upon cell lysis the spindle BR increased, initially at ca. 2.3 nm/min and then more slowly until the BR attained or exceeded intact cell values. Spindle and asters grew considerably larger than those in intact cells. From the kinetics of the transient BR increase after lysis, we infer that, initially, Chaetopterus cytoplasmic tubulin contributes to increased BR; further augmentation required added pig brain tubulin and most probably reflects the addition and incorporation of heterologous porcine tubulin into the spindle and asters. Isolated, augmented spindles depolymerized rapidly at 6 degrees C. Upon return to 23 degrees C, spindle BR returned slowly in tubulin-PEG. The BR of the isolates also decayed in solutions containing calcium ions 2.5 mM in excess of the EGTA. However, the isolates did not respond, or responded very slowly, to 1 mM colchicine or Colcemid and to dilution of tubulin with PEG solution. Microinjection into Chaetopterus oocytes of tubulin-PEG, but not PEG alone, enhanced spindle and aster BR which reversibly disappeared upon chilling the cell. PMID:4407048

  11. 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

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

    PubMed

    Zhaunova, Liudmila; Ohkura, Hiroyuki; Breuer, Manuel

    2016-08-01

    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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  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. Oocyte quality and estradiol supplementation affect in vitro maturation success in the white-tailed deer (Odocoileus virginianus).

    PubMed

    Siriaroonrat, B; Comizzoli, P; Songsasen, N; Monfort, S L; Wildt, D E; Pukazhenthi, B S

    2010-01-01

    White-tailed deer oocyte biology is not well documented. The objective of this study was to determine (1) the influence of estradiol (E(2)) supplementation on meiotic resumption and the ability to "rescue" poorer quality (lower grade) oocytes and (2) the kinetics of oocyte nuclear maturation in vitro in the white-tailed deer. In Experiment 1, immature oocytes harvested during hunting-culling operations were cultured for 24h in the presence or absence of E(2). Incubation in 1mug/mL E(2) promoted nuclear maturation (to telophase I, TI; or to metaphase II, MII) in a higher proportion of Grade 1 oocytes ( approximately 77%; P<0.05) compared with that in Grade 2 or Grade 3 counterparts ( approximately 51%). For Grades 2 and 3 oocytes, there was no advantage (P>0.05) for E(2) supplementation in reaching TI/MII. In Experiment 2, Grade 1 oocytes were cultured in the presence of E(2) and nuclear status evaluated at 0, 3, 6, 12, and 24h of in vitro incubation. At 0h,>70% of oocytes already had undergone germinal vesicle breakdown. After 12h, approximately 70% of oocytes had reached metaphase I of nuclear maturation, with approximately 75% achieving TI/MII by 24h in vitro. In summary, adding E(2) to an in vitro maturation (IVM) culture system for white-tailed deer was advantageous, but only for the highest quality oocytes, with approximately 75% achieving nuclear maturation. In contrast, E(2) supplement did not benefit lower-grade oocytes, half of which will reach MII, with the other half failing. Under the described culture conditions, good-quality white-tailed deer oocytes achieve nuclear maturation over a time duration comparable with that reported in other ungulates. PMID:19853902

  1. Oocyte ageing and epigenetics

    PubMed Central

    Ge, Zhao-Jia; Schatten, Heide; Zhang, Cui-Lian; Sun, Qing-Yuan

    2015-01-01

    It has become a current social trend for women to delay childbearing. However, the quality of oocytes from older females is compromised and the pregnancy rate of older women is lower. With the increased rate of delayed childbearing, it is becoming more and more crucial to understand the mechanisms underlying the compromised quality of oocytes from older women, including mitochondrial dysfunctions, aneuploidy and epigenetic changes. Establishing proper epigenetic modifications during oogenesis and early embryo development is an important aspect in reproduction. The reprogramming process may be influenced by external and internal factors that result in improper epigenetic changes in germ cells. Furthermore, germ cell epigenetic changes might be inherited by the next generations. In this review, we briefly summarise the effects of ageing on oocyte quality. We focus on discussing the relationship between ageing and epigenetic modifications, highlighting the epigenetic changes in oocytes from advanced-age females and in post-ovulatory aged oocytes as well as the possible underlying mechanisms. PMID:25391845

  2. From fresh heterologous oocyte donation to autologous oocyte banking

    PubMed Central

    Stoop, D.

    2012-01-01

    Introduction: Today, oocyte donation has become well established, giving rise to thousands of children born worldwide annually. The introduction of oocyte cryopreservation through vitrification allows the introduction of egg banking, improving the efficiency and comfort of oocyte donation. Moreover, the vitrification technique can now enable autologous donation of oocytes to prevent future infertility. Methods: We evaluated fresh heterologous oocyte donation in terms of obstetrical and perinatal outcome as well as of the reproductive outcome of past donors. We then evaluated the efficiency of a closed vitrification device and its clinical applications within ART. Thirdly, we evaluated the opinion of women with regard to preventive egg freezing and the efficiency of a human oocyte in relation to age. Results: Oocyte donation is associated with an increased risk of first trimester bleeding and pregnancy induced hypertension. Donating oocytes does not seem to increase the likelihood for a later need of fertility treatment. The chance of an oocyte to result in live birth (utilization rate) in women <37 years old remains constant with a mean of 4.47%. A significant proportion of young women would consider safeguarding their reproductive potential through egg freezing or are at least open to the idea. Discussion and Conclusion: The introduction of efficient oocyte cryopreservation has revolutionized oocyte donation through the establishment of eggbank donation. The technique also enables women to perform autologous donation after preventive oocyte storage in order to circumvent their biological clock. PMID:24753920

  3. 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. PMID:12700411

  4. 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. PMID:24905140

  5. Regulation of recombinant human insulin-induced maturational events in Clarias batrachus (L.) oocytes in vitro.

    PubMed

    Hajra, Sudip; Das, Debabrata; Ghosh, Pritha; Pal, Soumojit; Nath, Poulomi; Maitra, Sudipta

    2016-04-01

    Regulation of insulin-mediated resumption of meiotic maturation in catfish oocytes was investigated. Insulin stimulation of post-vitellogenic oocytes promotes the synthesis of cyclin B, histone H1 kinase activation and a germinal vesicle breakdown (GVBD) response in a dose-dependent and duration-dependent manner. The PI3K inhibitor wortmannin abrogates recombinant human (rh)-insulin action on histone H1 kinase activation and meiotic G2-M1 transition in denuded and follicle-enclosed oocytes in vitro. While the translational inhibitor cycloheximide attenuates rh-insulin action, priming with transcriptional blocker actinomycin D prevents insulin-stimulated maturational response appreciably, albeit in low amounts. Compared with rh-insulin, human chorionic gonadotrophin (hCG) stimulation of follicle-enclosed oocytes in vitro triggers a sharp increase in 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DHP) secreted in the incubation medium at 12 h. Interestingly, the insulin, but not the hCG-induced, maturational response shows less susceptibility to steroidogenesis inhibitors, trilostane or dl-aminoglutethimide. In addition, priming with phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) or cell-permeable dbcAMP or adenylyl cyclase activator forskolin reverses the action of insulin on meiotic G2-M1 transition. Conversely, the adenylyl cyclase inhibitor, SQ 22536, or PKA inhibitor H89 promotes the resumption of meiosis alone and further potentiates the GVBD response in the presence of rh-insulin. Furthermore, insulin-mediated meiotic maturation involves the down-regulation of endogenous protein kinase A (PKA) activity in a manner sensitive to PI3K activation, suggesting potential involvement of a cross-talk between cAMP/PKA and insulin-mediated signalling cascade in catfish oocytes in vitro. Taken together, these results suggest that rh-insulin regulation of the maturational response in C. batrachus oocytes involves down-regulation of PKA, synthesis of cyclin

  6. 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

  7. 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

  8. 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

  9. 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

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

    PubMed

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

    2016-06-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications. PMID:26988607

  11. 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

  12. 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

  13. 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

  14. Oocyte pre-IVM with caffeine improves bovine embryo survival after vitrification.

    PubMed

    Bernal-Ulloa, Sandra Milena; Lucas-Hahn, Andrea; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Baulain, Ulrich; Niemann, Heiner

    2016-09-15

    Cryopreservation of in vitro produced bovine embryos is associated with significantly reduced survival rates, mainly due to insufficient quality of the embryos. Caffeine supplementation during IVM has been used to delay meiotic resumption and concomitantly also increased embryo quality. Here, we investigated the influence of pre-IVM with caffeine on oocyte maturation, intraoocyte cAMP concentration, developmental competence after IVF, and blastocyst cryotolerance. Oocytes were obtained by slicing of ovaries and were submitted to either 2 hours culture before IVM with or without caffeine (0, 1, 5, 10, 20, 30 mM), or standard IVM (no pre-IVM). Oocytes were in vitro matured and fertilized and zygotes were cultured under standard in vitro conditions until Day 8. Expanded blastocysts derived from either standard control or the 10-mM caffeine treatments were submitted to vitrification. Caffeine delayed meiotic resumption after 9-hour IVM in a concentration-dependent manner. The cAMP levels were similar before and after IVM. Matured oocytes, cleavage, and blastocyst rates were reduced in the 30-mM caffeine concentration and were similar among the other treatment groups. Number and proportion of inner cell mass and trophectoderm cells in blastocysts did not differ among treatments. Forty-eight hours after thawing, hatching rates were higher in the 10-mM caffeine group (73.8%) compared with the standard control (59.7%). Reexpansion rates and total number of cells after 48 hours were similar in both treatments. The ratio of live/total cells was higher in the caffeine treatment. These results suggest that caffeine supplementation before IVM delayed meiotic resumption and improved blastocyst quality shown in higher cryotolerance. PMID:27180328

  15. Effect of 5-aminoimidazole-4-carboxamide ribonucleoside on the mitochondrial function and developmental ability of bovine oocytes.

    PubMed

    Takeo, Shun; Abe, Takahito; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

    2015-09-01

    Oocyte nuclear maturation depends on sufficient energy supply through oxidative phosphorylation and β-oxidation. AMP-activated protein kinase (AMPK) is an energy sensor controlling the oocyte energy metabolism. The main aim of this study was to examine the effect of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a potent activator of AMPK, on the ATP content and mitochondrial DNA copy number (Mt-number) of bovine oocytes and on their developmental ability. Oocytes were collected from slaughterhouse-derived bovine ovaries. When these oocytes were cultured in a maturation medium containing 0-, 50-, 250-, and 500-μM AICAR, higher AICAR concentrations reduced the rate of meiotic maturation and the ATP content in oocytes, whereas lower AICAR increased the ATP content in oocytes without affecting the maturation rate. Supplementation of the maturation medium with a low concentration of AICAR (50 and 250 μM) increased phospho-AMPK expression level, as determined by immunostaining. In addition, AICAR treatment increased the ATP content in oocytes, which remained elevated for as long as 2 days after fertilization. On culturing the oocytes with AICAR (250 μM), the fertilization outcome, rate of blastulation, and total cell number of the blastocysts significantly improved. When the proteosomal mitochondrial degradation was inhibited by supplementing the maturation medium with MG132, the Mt-number, as determined by real-time polymerase chain reaction, significantly increased. However, the treatment of oocytes with AICAR did not affect the Mt-number in the presence or absence of MG132. From these data, we conclude that low concentrations of AICAR improved the embryonic developmental ability, presumably via the upregulation of the ATP content in oocytes, but the increase in the ATP content was not due to the upregulation of mitochondrial biogeneration. PMID:26001600

  16. 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

  17. 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

  18. SISTER CHROMATID EXCHANGES IN MAMMALIAN MEIOTIC CHROMOSOMES

    EPA Science Inventory

    Very little is known about sister chromatid exchanges (SCEs) in meiotic cells--only that they occur (1) and reveal frequency and distribution patterns apparently unaffected by cross-over (CO) exchange conditions in those cells; (2) unfortunately, the number of studies from which ...

  19. Two distinct mechanisms control the accumulation of cyclin B1 and Mos in Xenopus oocytes in response to progesterone.

    PubMed

    Frank-Vaillant, M; Jessus, C; Ozon, R; Maller, J L; Haccard, O

    1999-10-01

    Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34(cdc2) could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34(cdc2), and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor-induced feedback. We report here that the cdk inhibitor p21(cip1), when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21(cip1), progesterone fails to induce the activation of MAPK or p34(cdc2), and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA. PMID:10512866

  20. Two Distinct Mechanisms Control the Accumulation of Cyclin B1 and Mos in Xenopus Oocytes in Response to Progesterone

    PubMed Central

    Frank-Vaillant, Marie; Jessus, Catherine; Ozon, René; Maller, James L.; Haccard, Olivier

    1999-01-01

    Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34cdc2 could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34cdc2, and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor–induced feedback. We report here that the cdk inhibitor p21cip1, when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21cip1, progesterone fails to induce the activation of MAPK or p34cdc2, and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA. PMID:10512866

  1. Maturation-promoting factor (MPF) is responsible for the transformation of sperm nuclei to metaphase chromosomes in maturing bovine oocytes in vitro.

    PubMed

    Abeydeera, L R; Niwa, K; Okuda, K

    1993-07-01

    Bovine oocytes at the germinal vesicle stage were inseminated in Brackett and Oliphant's medium in the presence of BSA (10 mg ml-1), caffeine (5 mmol l-1) and heparin (10 micrograms ml-1). When oocytes were transferred into tissue culture medium (TCM)-199 containing 10% fetal calf serum (FCS) with or without 6-dimethylaminopurine (6-DMAP; 2 mmol l-1) 8 h after insemination and cultured for 15-40 h at 39 degrees C in 5% CO2 in air, 74-83% of oocytes were penetrated and polyspermy (67-80%) was common. At 40 h after culture in 6-DMAP-free medium, 65% and 63% of unpenetrated and penetrated oocytes, respectively, reached metaphase II or beyond. A few (6%) oocytes were activated and contained both male and female pronuclei. Sperm metaphase chromosomes were observed in 90% of the penetrated oocytes. Penetration by more than four spermatozoa greatly retarded the meiotic maturation of the oocyte. However, sperm chromosomes were never observed in oocytes cultured in 6-DMAP supplemented medium and oocyte maturation did not proceed beyond the stage of prometaphase I. These results demonstrate the possible participation of maturation-promoting factor in metaphase chromosome formation in spermatozoa. PMID:8410805

  2. 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

  3. 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

  4. 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

  5. Phosphorylation of the RNA polymerase II largest subunit during Xenopus laevis oocyte maturation.

    PubMed Central

    Bellier, S; Dubois, M F; Nishida, E; Almouzni, G; Bensaude, O

    1997-01-01

    Xenopus laevis oogenesis is characterized by an active transcription which ceases abruptly upon maturation. To survey changes in the characteristics of the transcriptional machinery which might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. We found that the RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes. The C-terminal domain (CTD) of the RPB1 subunit was essentially hypophosphorylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increased dramatically and abruptly. The hyperphosphorylated RPB1 subunits were dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes. Extracts from metaphase II-arrested oocytes showed a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity was attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, we observed a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1. Both events required protein synthesis, which demonstrated that activation of p34(cdc2)off kinase was insufficient to phosphorylate RPB1 ex vivo and was consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation, as previously proposed during stress or growth factor stimulation of mammalian cells. PMID:9032270

  6. Short-term Preservation of Porcine Oocytes in Ambient Temperature: Novel Approaches

    PubMed Central

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

    2010-01-01

    The objective of this study was to evaluate the feasibility of preserving porcine oocytes without freezing. To optimize preservation conditions, porcine cumulus-oocyte complexes (COCs) were preserved in TCM-199, porcine follicular fluid (pFF) and FCS at different temperatures (4°C, 20°C, 25°C, 27.5°C, 30°C and 38.5°C) for 1 day, 2 days or 3 days. After preservation, oocyte morphology, germinal vesicle (GV) rate, actin cytoskeleton organization, cortical granule distribution, mitochondrial translocation and intracellular glutathione level were evaluated. Oocyte maturation was indicated by first polar body emission and spindle morphology after in vitro culture. Strikingly, when COCs were stored at 27.5°C for 3 days in pFF or FCS, more than 60% oocytes were still arrested at the GV stage and more than 50% oocytes matured into MII stages after culture. Almost 80% oocytes showed normal actin organization and cortical granule relocation to the cortex, and approximately 50% oocytes showed diffused mitochondria distribution patterns and normal spindle configurations. While stored in TCM-199, all these criteria decreased significantly. Glutathione (GSH) level in the pFF or FCS group was higher than in the TCM-199 group, but lower than in the non-preserved control group. The preserved oocytes could be fertilized and developed to blastocysts (about 10%) with normal cell number, which is clear evidence for their retaining the developmental potentiality after 3d preservation. Thus, we have developed a simple method for preserving immature pig oocytes at an ambient temperature for several days without evident damage of cytoplasm and keeping oocyte developmental competence. PMID:21151922

  7. 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. PMID:26802898

  8. 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

  9. MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells.

    PubMed

    Pan, Bo; Toms, Derek; Shen, Wei; Li, Julang

    2015-03-15

    We sought to investigate whether miR-378 plays a role in cumulus cells and whether the manipulation of miRNA levels in cumulus cells influences oocyte maturation in vitro. Cumulus-oocyte complexes (COCs) from ovarian follicles had significantly lower levels of precursor and mature miR-378 in cumulus cells surrounding metaphase II (MII) oocytes than cumulus cells surrounding germinal vesicle (GV) oocytes, suggesting a possible role of miR-378 during COC maturation. Overexpression of miR-378 in cumulus cells impaired expansion and decreased expression of genes associated with expansion (HAS2, PTGS2) and oocyte maturation (CX43, ADAMTS1, PGR). Cumulus cell expression of miR-378 also suppressed oocyte progression from the GV to MII stage (from 54 ± 2.7 to 31 ± 5.1%), accompanied by a decrease of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zona pellucida 3 (ZP3), and CX37 in the oocytes. Subsequent in vitro fertilization resulted in fewer oocytes from COCs overexpressing miR-378 reaching the blastocyst stage (7.3 ± 0.7 vs. 16.6 ± 0.5%). miR-378 knockdown led to increased cumulus expansion and oocyte progression to MII, confirming a specific effect of miR-378 in suppressing COC maturation. Aromatase (CYP19A1) expression in cumulus cells was also inhibited by miR-378, leading to a significant decrease in estradiol production. The addition of estradiol to IVM culture medium reversed the effect of miR-378 on cumulus expansion and oocyte meiotic progression, suggesting that decreased estradiol production via suppression of aromatase may be one of the mechanisms by which miR-378 regulates the maturation of COCs. Our data suggest that miR-378 alters gene expression and function in cumulus cells and influences oocyte maturation, possibly via oocyte-cumulus interaction and paracrine regulation. PMID:25628423

  10. 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

  11. 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

  12. 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

  13. 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. PMID:26267363

  14. 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

  15. 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

  16. Oocyte freezing: timely reproductive insurance?

    PubMed

    Molloy, David; Hall, Barbara A; Ilbery, Mariannne; Irving, Jacqui; Harrison, Keith L

    2009-03-01

    Cryopreservation of unfertilised oocytes for later use in initiating pregnancy is now a viable technology, with acceptable pregnancy rates (over 20% per thaw cycle). Oocyte cryopreservation used as a form of insurance against "social" (age-related) infertility can improve the lifetime chance of pregnancy in women who defer pregnancy into their late 30s or early 40s. We report two pregnancies using oocytes that were frozen for social rather than medical reasons, as part of a larger series of nine pregnancies using cryopreserved oocytes. Use of oocytes harvested and frozen from women aged under 35 years may more than double the chance of pregnancy for a 41-year-old woman. The disadvantages of oocyte freezing for social infertility reasons include cost, the usual risks associated with in-vitro fertilisation, and the lack of a guarantee of eventual pregnancy. PMID:19296788

  17. Effects of mono(2-ethylhexyl)phthalate on cytoplasmic maturation of oocytes--The bovine model.

    PubMed

    Kalo, D; Roth, Z

    2015-06-01

    Phthalates are known reproductive toxicants, but their intracellular disruptive effects on oocyte maturation competence are less known. We studied the potential risk associated with acute exposure of oocytes to mono(2-ethylhexyl)phthalate (MEHP). First, bovine oocytes were matured in vitro with or without 50 μM MEHP and examined for mitochondrial features associated with DNA fragmentation. MEHP increased reactive oxygen species levels and reduced the proportion of highly polarized mitochondria along with alterations in genes associated with mitochondrial oxidative phosphorylation (CYC1, MT-CO1 and ATP5B). In a second set of experiments, we associated the effects of MEHP on meiotic progression with those on cytoplasmic maturation. MEHP impaired reorganization of cytoplasmic organelles in matured oocytes reflected by reductions in category I mitochondria, type III cortical granules and class I endoplasmic reticulum. These alterations are associated with the previously reported reduced developmental competence of MEHP-treated bovine oocytes, and reveal the risk associated with acute exposure. PMID:25900598

  18. Maturation and fertilisation of sheep oocytes cultured in serum-free medium containing silk protein sericin.

    PubMed

    Yasmin, Cut; Otoi, Takeshige; Setiadi, Mohamad Agus; Karja, Ni Wayan Kurniani

    2015-03-01

    Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation in a maturation medium on the meiotic competence and fertilisability of sheep oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM199 supplemented with sericin at various concentrations of 0 (control), 0.1, 0.25 and 0.5%, either with or without bovine serum albumin (BSA). When the COCs were matured without BSA, the supplementation of 0.1% sericin significantly increased the rates of maturation to metaphase II and the total fertilisation of oocytes compared with the other concentrations of sericin. When the COCs were matured with BSA, the beneficial effects of 0.1% sericin supplementation on the maturation and fertilisation of oocytes were not observed. Our findings indicate that supplementation with 0.1% sericin during maturation culture may improve the nuclear maturation and fertilisability of sheep oocytes. Moreover, it may be possible to replace BSA with sericin in chemically defined media without the risk of disease transmission. PMID:25655418

  19. Embryos derived from the in vitro fertilization of oocytes of pregnant cows.

    PubMed

    Vajta, G; Macháty, Z; Bárándi, Z; Varga, Z

    1992-04-01

    The aim of our study was to determine if the oocytes of pregnant cattle are capable for undergoing embryonic growth following in vitro fertilization. The ovaries of nine heifers at 4 to 7 months of pregnancy were collected at an abattoir and transferred to the laboratory. A total 191 oocytes (10.6 per ovary) collected by aspiration were matured and fertilized by frozen-thawed semen. Embryos were co-cultured with granulosa cells in modified TCM 199 medium and 20% estrous cow serum. The cleavage rate of embryos was 48%, and 41% of of the cleaved embryos developed to the morula/blastocyst stage 7 days after insemination. Additionally, the ovaries of 10 nonpregnant heifers were also collected, yielding 213 oocytes (10.7 per ovary). The cleavage rate was 51%, and 35% of those which cleaved reached the morula/blastocyst stage. No significant differences were found between the two groups. The average number of transferable-stage embryos obtained from pregnant and nonpregnant animals was 4.1 and 3.7, respectively. Our results indicate that preganancy does not influence the meiotic competence of bovine oocytes, and transferable stage embryos can be obtained by the fertilization of oocytes derived from pregnant animals. PMID:16727081

  20. 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

  1. A light- and electron microscopic analysis of meiotic prophase in female mice.

    PubMed

    Dietrich, A J; Mulder, R J

    1983-01-01

    In the paper we describe meiotic prophase of female mice on successive days of embryonic and early postnatal development. For this purpose we used three different techniques on ovarian material, i.e., Giemsa staining for the light microscopic study of chromatin, silver staining for the light microscopic study of the synaptonemal complex (SC), and agar filtration followed by uranyl acetate staining for the electron microscopic study of the SC. In all types of preparation it was impossible to distinguish leptotene stages, and we conclude that if leptotene really exists, it is of very short duration.--Two types of zygotene stages were found: the "normal" one, resembling zygotene stages in male mice, and a second type that has never been described in males and is characterized by, probably stable, unpaired regions together with totally unpaired axial elements of the SC.--The duration of pachytene was found to be 3-4 days, which is considerably shorter than in males. During early diplotene despiralization of the chromatin and disintegration of the axes of the SC were usually found together with desynapsis.--A considerable variation in distribution of meiotic stages was found between different litters in the same day of gestation. Fetuses in the same litter showed no significant variation. However, the oocytes in an ovary did not pass through meiosis synchronously, with differences up several days. The appearance of chromosomes in a highly contracted state could not be interpreted as a preleptotene condensation stage but probably is a mitotic phenomenon. PMID:6197255

  2. Mouse BRWD1 is critical for spermatid postmeiotic transcription and female meiotic chromosome stability

    PubMed Central

    Pattabiraman, Shrivatsav; Baumann, Claudia; Guisado, Daniela; Eppig, John J.

    2015-01-01

    Postmeiotic gene expression is essential for development and maturation of sperm and eggs. We report that the dual bromodomain-containing protein BRWD1, which is essential for both male and female fertility, promotes haploid spermatid–specific transcription but has distinct roles in oocyte meiotic progression. Brwd1 deficiency caused down-regulation of ∼300 mostly spermatid-specific transcripts in testis, including nearly complete elimination of those encoding the protamines and transition proteins, but was not associated with global epigenetic changes in chromatin, which suggests that BRWD1 acts selectively. In females, Brwd1 ablation caused severe chromosome condensation and structural defects associated with abnormal telomere structure but only minor changes in gene expression at the germinal vesicle stage, including more than twofold overexpression of the histone methyltransferase MLL5 and LINE-1 elements transposons. Thus, loss of BRWD1 function interferes with the completion of oogenesis and spermatogenesis through sexually dimorphic mechanisms: it is essential in females for epigenetic control of meiotic chromosome stability and in males for haploid gene transcription during postmeiotic sperm differentiation. PMID:25547156

  3. 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

  4. Cyclin B in Xenopus oocytes: implications for the mechanism of pre-MPF activation.

    PubMed Central

    Gautier, J; Maller, J L

    1991-01-01

    Using a polyclonal antibody raised against B2 cyclin from Xenopus laevis, we show that prophase-arrested Xenopus oocytes contain a stockpile of cyclin B2 protein. During progesterone-induced maturation, an increase in the synthesis of cyclin B2 is observed, although Western blotting experiments show that this new synthesis does not significantly increase the mass of cyclin over the maternal stockpile. In the oocyte cyclin B2 is already present in two forms which differ in the extent of phosphorylation, but the phosphorylated form becomes predominant as oocytes progress towards germinal vesicle breakdown (GVBD), coincident with cdc2 protein kinase activation. These two events do not depend upon formation of a new complex between cyclin and cdc2 protein kinase, since these two proteins are already found associated in resting oocytes, prior to activation of the kinase. Images PMID:1824935

  5. 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

  6. 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

  7. Meiotic recombination and genome evolution in plants.

    PubMed

    Melamed-Bessudo, Cathy; Shilo, Shay; Levy, Avraham A

    2016-04-01

    Homologous recombination affects genome evolution through crossover, gene conversion and point mutations. Whole genome sequencing together with a detailed epigenome analysis have shed new light on our understanding of how meiotic recombination shapes plant genes and genome structure. Crossover events are associated with DNA sequence motifs, together with an open chromatin signature (hypomethylated CpGs, low nucleosome occupancy or specific histone modifications). The crossover landscape may differ between male and female meiocytes and between species. At the gene level, crossovers occur preferentially in promoter regions in Arabidopsis. In recent years, there is rising support suggesting that biased mismatch repair during meiotic recombination may increase GC content genome-wide and may be responsible for the GC content gradient found in many plant genes. PMID:26939088

  8. The telomere bouquet regulates meiotic centromere assembly.

    PubMed

    Klutstein, Michael; Fennell, Alex; Fernández-Álvarez, Alfonso; Cooper, Julia Promisel

    2015-04-01

    The role of the conserved meiotic telomere bouquet has been enigmatic for over a century. We showed previously that disruption of the fission yeast bouquet impairs spindle formation in approximately half of meiotic cells. Surprisingly, bouquet-deficient meiocytes with functional spindles harbour chromosomes that fail to achieve spindle attachment. Kinetochore proteins and the centromeric histone H3 variant Cnp1 fail to localize to those centromeres that exhibit spindle attachment defects in the bouquet's absence. The HP1 orthologue Swi6 also fails to bind these centromeres, suggesting that compromised pericentromeric heterochromatin underlies the kinetochore defects. We find that centromeres are prone to disassembly during meiosis, but this is reversed by localization of centromeres to the telomere-proximal microenvironment, which is conducive to heterochromatin formation and centromere reassembly. Accordingly, artificially tethering a centromere to a telomere rescues the tethered centromere but not other centromeres. These results reveal an unanticipated level of control of centromeres by telomeres. PMID:25774833

  9. Dynamics of DOT1L localization and H3K79 methylation during meiotic prophase I in mouse spermatocytes

    PubMed Central

    Ontoso, David; Kauppi, Liisa; Keeney, Scott; San-Segundo, Pedro A.

    2013-01-01

    During meiotic prophase I, interactions between maternal and paternal chromosomes, under checkpoint surveillance, establish connections between homologs that promote their accurate distribution to meiotic progeny. In human, faulty meiosis causes aneuploidy resulting in miscarriages and genetic diseases. Meiotic processes occur in the context of chromatin, therefore histone post-translational modifications are expected to play important roles. Here, we report the cytological distribution of the evolutionarily conserved DOT1L methyltransferase and the different H3K79 methylation states resulting from its activity (mono-, di- and tri-methylation; H3K79me1, me2 and me3, respectively) during meiotic prophase I in mouse spermatocytes. In the wild type, whereas low amounts of H3K79me1 are rather uniformly present throughout prophase I, levels of DOT1L, H3K79me2 and H3K79me3 exhibit a notable increase from pachynema onwards, but with differential subnuclear distribution patterns. The heterochromatic centromeric regions and the sex body are enriched for H3K79me3. In contrast, H3K79me2 is present all over the chromatin, but is largely excluded from the sex body despite the accumulation of DOT1L. In meiosis-defective mouse mutants, the increase of DOT1L and H3K79me is blocked at the same stage where meiosis is arrested. H3K79me patterns, combined with the cytological analysis of the H3.3, γH2AX, macroH2A and H2A.Z histone variants, are consistent with a differential role for these epigenetic marks in male mouse meiotic prophase I. We propose that H3K79me2 is related to transcriptional reactivation on autosomes during pachynema, whereas H3K79me3 may contribute to the maintenance of repressive chromatin at centromeric regions and the sex body. PMID:24105599

  10. 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

  11. Toxic effects of HT-2 toxin on mouse oocytes and its possible mechanisms.

    PubMed

    Zhu, Cheng-Cheng; Zhang, Yue; Duan, Xing; Han, Jun; Sun, Shao-Chen

    2016-06-01

    T-2 toxin is one of the type A trichothecene mycotoxins that is considered to be the most toxic of the trichothecenes. T-2 toxin has been shown to exert various toxic effects in farm animals and humans, as it induces lesions in the brain and in lymphoid, hematopoietic, and gastrointestinal tissues. HT-2 toxin is the major metabolite of T-2 toxin. There is little information regarding the effects of HT-2 toxin on the female reproductive system, particularly oocyte maturation. Thus, in this study, we investigated the toxic effects of HT-2 on mouse oocyte maturation and its possible mechanisms of action. HT-2 toxin exposure disrupted oocyte maturation, reduced actin expression in both the oocyte cortex and cytoplasm, and disrupted meiotic spindle morphology by reducing p-MAPK protein level. HT-2 toxin exposure also induced oxidative stress and resulted in oocyte apoptosis, as shown by ROS accumulation, increased SOD mRNA level, and the expression of the early apoptosis marker Annexin V and increased caspase-3 and bax mRNA levels. Additionally, HT-2 toxin exposure increased LC3 and ATG12 protein levels and lc3 and atg14 mRNA levels, which indicated that HT-2 toxin induced autophagy in mouse oocytes. We also examined for possible epigenetic modifications. Fluorescence intensity analysis showed that 5mC level increased after HT-2 toxin exposure, whereas H3K9me2 and H3K27me3 levels decreased after HT-2 toxin exposure, which indicated that DNA and histone methylations were altered. Thus, our results indicated that HT-2 toxin exposure reduced mouse oocyte maturation capability by affecting cytoskeletal dynamics, apoptosis/autophagy, oxidative stress, and epigenetic modifications. PMID:26138683

  12. 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

  13. Deterrence and arrest ratios.

    PubMed

    Carmichael, Stephanie E; Piquero, Alex R

    2006-02-01

    In the limited research on the origins of sanction threat perceptions, researchers have focused on either the effects of actively engaging in crime or the effects of formal sanctioning but rarely on both (i.e., the arrest ratio or the number of arrests relative to the number of crimes committed). This article extends this line of research by using a sample of Colorado inmates and measures arrest ratios and sanction perceptions for eight different crime types. Analyses reveal that the offenders report both significant experiential and arrest ratio effects. Theoretical and policy implications, limitations, and directions for future research are outlined. PMID:16397123

  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. WASH complex regulates Arp2/3 complex for actin-based polar body extrusion in mouse oocytes

    PubMed Central

    Wang, Fei; Zhang, Liang; Zhang, Guang-Li; Wang, Zhen-Bo; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2014-01-01

    Prior to their fertilization, oocytes undergo asymmetric division, which is regulated by actin filaments. Recently, WASH complex were identified as actin nucleation promoting factors (NPF) that activated Arp2/3 complex. However, the roles of WASH complex remain uncertain, particularly for oocyte polarization and asymmetric division. Here, we examined the functions of two important subunits of a WASH complex, WASH1 and Strumpellin, during mouse oocyte meiosis. Depleting WASH1 or disrupting Strumpellin activity by WASH1 morpholino (MO) injection or Strumpellin antibody injection decreased polar body extrusion and caused oocyte symmetric division, and this may have been due to spindle formation and migration defects. Time lapse microscopy showed that actin filaments distribution and relative amount at the membrane and in the cytoplasm of oocytes was significantly decreased after disrupting WASH complex. In addition, Arp2/3 complex expression was reduced after WASH1 depletion. Thus, our data indicated that WASH complex regulated Arp2/3 complex and were required for cytokinesis and following polar body extrusion during mouse oocyte meiotic maturation. PMID:24998208

  16. Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedarius) during maturation in vitro.

    PubMed

    Torner, H; Heleil, B; Alm, H; Ghoneim, I M; Srsen, V; Kanitz, W; Tuchscherer, A; Fattouh, E M

    2003-09-15

    The aim of the present study was to examine the cumulus morphology and the oocyte chromatin quality of camel cumulus-oocyte complexes (COCs) at the time of recovery, and to monitor changes in oocyte chromatin configuration and apoptosis in cumulus cells from camel COCs during in vitro maturation (IVM) (0, 12, 24, 32, 36, 42, and 48 p.IVM) depending on pregnancy of donors. A total of 1023 COCs were isolated from sliced ovaries after slaughtering of 47 pregnant and 43 non-pregnant camels in an abattoir. The mean number of COCs per donor was 10.3 in pregnant and 12.5 in non-pregnant donors. The cumulus morphology of COCs was independent of the type of donor and was divided in COCs with compact (26.9 and 28%), dispersed (39.3 and 46%), corona radiata cumulus investment (27.9 and 21.7%) and without cumulus (6 and 4.2%), respectively for pregnant and non-pregnant donors. The highest proportion of COCs exhibited dispersed cumulus (P<0.05). Oocytes with meiotic stages of diplotene >50% were found only in compact (55 and 56.5%) and in dispersed COCs (58.4 and 60%), respectively for pregnant and non-pregnant donors. During IVM (0-48h) the first significant onset of specific meiotic stages were different in oocytes from pregnant donors: metaphase 1 (24-32h), metaphase 2 (36-42h), versus oocytes from non-pregnant donors: metaphase 1 (24h), metaphase 2 (32-48h) (P<0.05). The level of apoptotic cells in cumuli of matured COCs increased during IVM and was higher in matured COCs from non-pregnant donors for each time point during IVM (P<0.01). Camel oocytes meiosis during IVM is accompanied by a drastic increase of apoptosis in the surrounding cumulus cells 0-32 and 0-24h during IVM, respectively for pregnant and non-pregnant donors. The oocytes of pregnant camels require 36h of maturation to reach levels of >50% metaphase 2 stage in comparison to oocytes from non-pregnant donors where 32h are sufficient. The earlier onset of apoptosis in the COCs derived from non-pregnant donors

  17. The adverse effect of obesity/high fat diet on oocyte quality and metabolism is not reversible with resumption of regular diet in mice

    PubMed Central

    Reynolds, Kasey A; Boudoures, Anna L; Chi, Maggie M-Y; Wang, Qiang; Moley, Kelle H

    2016-01-01

    Obesity, which affects over one-third of reproductive-age women, has negative effects on reproduction and results in oocyte defects in both mice and humans. In this study, we used a mouse model to examine whether the adverse effects of an obesogenic diet, specifically abnormal oocyte spindle formation, mitochondrial metabolism, and lipid accumulation, can be reversed by return to normal weight and metabolic profile. Female C57BL6/J mice were placed on either a high-fat diet (HFD; 35.8% fat and 20.2% protein by nutritional content) or an isocaloric control diet (CD; 13% fat and 25% protein) for six weeks. All mice were then maintained on CD for eight weeks. We found that whereas metabolic parameters (weight, glucose tolerance, and cholesterol levels) of the HFD mice returned to normal after this “diet reversal” period, several oocyte defects were not reversible. Oocytes from the diet reversal mice demonstrated a significantly higher percentage of abnormal meiotic spindles than those from control mice. The HFD diet reversal GV oocytes also had lower mitochondrial membrane potential, lower levels of ATP and citrate, and higher percentages of abnormal lipid accumulation and distribution and abnormally distributed mitochondria than oocytes from control mice. Thus, despite normalization of weight, glucose utilization, and cholesterol levels eight weeks after switching from a high fat to a regular chow, oocytes from diet reversal mice exhibited significantly higher rates of meiotic spindle, lipid, and mitochondrial defects than found in mice maintained on regular chow. These results suggest that the negative effects of an obesogenic diet on oocyte quality are not as reversible as the overall metabolic parameters. These data may provide better insight when counseling obese women regarding reproductive options and success. PMID:25775080

  18. 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

  19. Apoptosis in male germ cells in response to cyclin A1-deficiency and cell cycle arrest.

    PubMed

    Salazar, Glicella; Liu, Dong; Liao, Ching; Batkiewicz, Leah; Arbing, Rachel; Chung, Sanny S W; Lele, Karen; Wolgemuth, Debra J

    2003-10-15

    Male mice homozygous for a mutated allele of the cyclin A1 gene (Ccna1) are sterile due to a block in cell cycle progression before the first meiotic division. Meiosis arrest in Ccna1(-/-) spermatocytes is associated with desynapsis abnormalities, lowered MPF activity, and apoptosis as evidenced by TUNEL-positive staining. With time, adult testicular tubules exhibit severe degeneration: some tubules in the older animals are almost devoid of germ cells at various stages of spermatogenesis. The mechanisms by which the cells sense the cell cycle arrest and the regulation of the decision to undergo cell death are under investigation. PMID:14555236

  20. 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. PMID:23995739

  1. Calcium and actin in the saga of awakening oocytes.

    PubMed

    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(2+) swings and structural changes. In this article, we review some of the key findings on the rapid dynamic rearrangements of the actin cytoskeleton in the oocyte/egg cortex upon hormonal or sperm stimulation and their roles in the modulation of the Ca(2+) signals and in the control of monospermic fertilization. PMID:25998739

  2. 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.

  3. Cilostamide and forskolin treatment during pre-IVM improves preimplantation development of cloned embryos by influencing meiotic progression and gap junction communication in pigs.

    PubMed

    Park, Bola; Lee, Hanna; Lee, Yongjin; Elahi, Fazle; Lee, Joohyeong; Lee, Seung Tae; Park, Choon-Keun; Hyun, Sang-Hwan; Lee, Eunsong

    2016-08-01

    This study was conducted to evaluate the effects of treatment with the cAMP modulators cilostamide and/or forskolin during pre-IVM culture on meiotic progression, gap junction communication, intraoocyte cAMP level and glutathione content, embryonic development after parthenogenesis, and somatic cell nuclear transfer in pigs. Cumulus-oocyte complexes were cultured for 24 hours in unsupplemented medium or media containing 20 μM cilostamide and/or 50 μM forskolin. After pre-IVM, oocytes were cultured for 41 to 44 hours in a standard IVM medium to induce oocyte maturation. When the nuclear status of oocytes was examined after pre-IVM for 24 hours, a higher (P < 0.01) proportion of oocytes treated with forskolin (85.5%) and cilostamide + forskolin (92.6%) remained at the germinal vesicle stage compared with untreated (20.6%) and cilostamide-treated oocytes (54.7%). cAMP level in pre-IVM oocytes was significantly increased by combined treatment with cilostamide + forskolin (21.38 fmol/oocyte) relative to the no pre-IVM control, no treatment, cilostamide, and forskolin groups (2.85, 1.88, 1.74, and 8.95 fmol/oocyte, respectively). Forskolin with or without cilostamide significantly maintained open-gap junction communication relative to no treatment. Blastocyst formation in parthenogenesis was significantly (P < 0.01) improved by forskolin (65.3%) relative to other treatments (28.3% to 48.1%). Supplementation of pre-IVM with dibutyryl cAMP showed similar blastocyst formation as forskolin treatment (61.1% and 61.0%, respectively). In somatic cell nuclear transfer, simultaneous treatment with cilostamide + forskolin significantly (P < 0.05) increased embryonic development to the blastocyst stage (42.9%) relative to the no pre-IVM, control, and cilostamide groups (32.3, 28.6, and 32.8%, respectively). The glutathione contents in pre-IVM oocytes were increased by no treatment, forskolin, and cilostamide + forskolin (1.38, 1.39, and 1.27 pixels/oocyte

  4. 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.

  5. Leptin enhances maturation and development of calf oocytes in vitro.

    PubMed

    Jia, Z; Zhang, J; Wu, Z; Tian, J

    2012-10-01

    We investigated the effects of leptin on the in vitro maturation (IVM) and development of calf oocytes. Cumulus-oocyte complexes were matured in IVM medium containing 0-100 ng/ml leptin. Experiment 1 showed that exposure of calf oocytes to IVM medium containing 1 or 10 ng/ml leptin significantly increased rates of development to the metaphase II stage compared with the control (81.7 ± 3.0% and 83.3 ± 2.1% for 1 and 10 ng/ml leptin, respectively, vs 64.1 ± 5.1% for control; p < 0.05). Experiment 2 showed that 1 or 10 ng/ml leptin significantly improved cleavage rates after in vitro fertilization when compared to control (58.6 ± 3.3% and 59.3 ± 2.9% for 1 and 10 ng/ml leptin, respectively, vs 48.5 ± 2.6% for control; p < 0.05); in addition, when compared to control medium, the addition of 10 ng/ml leptin to the IVM medium resulted in more presumptive zygotes reaching the 4- to 8-cell stage after 48 h of in vitro culture (30.3 ± 2.3% vs 20.1 ± 2.3%; p < 0.05) and developing into blastocysts after 8 days of culture (20.4 ± 1.6% vs 11.7 ± 1.7%; p < 0.05). Experiment 3 showed that the addition of 1 or 10 ng/ml leptin significantly increased the total number of blastocyst cells on day 8 of culture (114.6 ± 7.8 and 117.4 ± 5.9 for 1 and 10 ng/ml leptin, respectively, vs 92.7 ± 8.3 for control; p < 0.05) and trophectoderm (TE) cells (88.5 ± 5.5 and 90.6 ± 3.7 for 1 and 10 ng/ml leptin, respectively, vs 70.1 ± 5.9 for control; p < 0.05). In summary, these results indicate that the addition of leptin to IVM medium enhances meiotic maturation and embryo development from calf oocytes and improves the quality of embryos derived from these oocytes. PMID:22136278

  6. 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

  7. Ran GTPase promotes oocyte polarization by regulating ERM (Ezrin/Radixin/Moesin) inactivation

    PubMed Central

    Dehapiot, Benoit; Halet, Guillaume

    2013-01-01

    Asymmetric meiotic divisions in mammalian oocytes are driven by the eccentric positioning of the spindle, along with a dramatic reorganization of the overlying cortex, including a loss of microvilli and formation of a thick actin cap. Actin polarization relies on a Ran-GTP gradient centered on metaphase chromosomes; however, the downstream signaling cascade is not completely understood. In a recent study, we have shown that Ran promotes actin cap formation via the polarized activation of Cdc42. The related GTPase Rac is also activated in a polarized fashion in the oocyte cortex and co-localizes with active Cdc42. In other cells, microvilli collapse can be triggered by inactivation of the ERM (Ezrin/Radixin/Moesin) family of actin-membrane crosslinkers under the control of Rac. Accordingly, we show here that Ran-GTP promotes a substantial loss of phosphorylated ERMs in the cortex overlying the spindle in mouse oocytes. However, this polarized phospho-ERM exclusion zone was unaffected by Rac or Cdc42 inhibition. Therefore, we suggest that Ran activates two distinct pathways to regulate actin cap formation and microvilli disassembly in the polarized cortex of mouse oocytes. The possibility of a crosstalk between Rho GTPase and ERM signaling and a role for ERM inactivation in promoting cortical actin dynamics are also discussed. PMID:23656777

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

    PubMed

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

    2013-10-01

    Clathrin heavy chain 1 (CLTC) has been considered a “moonlighting protein” which acts in membrane trafficking during interphase and in stabilizing spindle fibers during mitosis. However, its roles in meiosis, especially in mammalian oocyte maturation, remain unclear. This study investigated CLTC expression and function in spindle formation and chromosome congression during mouse oocyte meiotic maturation. Our results showed that the expression level of CLTC increased after germinal vesicle breakdown (GVBD) and peaked in the M phase. Immunostaining results showed CLTC distribution throughout the cytoplasm in a cell cycle-dependent manner. Appearance and disappearance of CLTC along with β-tubulin (TUBB) could be observed during spindle dynamic changes. To explore the relationship between CLTC and microtubule dynamics, oocytes at metaphase were treated with taxol or nocodazole. CLTC colocalized with TUBB at the enlarged spindle and with cytoplasmic asters after taxol treatment; it disassembled and distributed into the cytoplasm along with TUBB after nocodazole treatment. Disruption of CLTC function using stealth siRNA caused a decreased first polar body extrusion rate and extensive spindle formation and chromosome congression defects. Taken together, these results show that CLTC plays an important role in spindle assembly and chromosome congression through a microtubule correlation mechanism during mouse oocyte maturation. PMID:23816345

  9. CUL-2 and ZYG-11 promote meiotic anaphase II and the proper placement of the anterior-posterior axis in C. elegans.

    PubMed

    Liu, Ji; Vasudevan, Srividya; Kipreos, Edward T

    2004-08-01

    The faithful segregation of chromosomes during meiosis is vital for sexual reproduction. Currently, little is known about the molecular mechanisms regulating the initiation and completion of meiotic anaphase. We show that inactivation of CUL-2, a member of the cullin family of ubiquitin ligases, delays or abolishes meiotic anaphase II with no effect on anaphase I, indicating differential regulation during the two meiotic stages. In cul-2 mutants, the cohesin REC-8 is removed from chromosomes normally during meiosis II and sister chromatids separate, suggesting that the failure to complete anaphase results from a defect in chromosome movement rather than from a failure to sever chromosome attachments. CUL-2 is required for the degradation of cyclin B1 in meiosis and inactivation of cyclin B1 partially rescued the meiotic delay in cul-2 mutants. In cul-2 mutants, the failure to degrade cyclin B1 precedes the metaphase II arrest. CUL-2 is also required for at least two aspects of embryonic polarity. The extended meiosis II in cul-2 mutants induces polarity reversals that include reversed orientation of polarity proteins, P granules, pronuclei migration and asymmetric cell division. Independently of its role in meiotic progression, CUL-2 is required to limit the initiation/spread of the polarity protein PAR-2 in regions distant from microtubule organizing centers. Finally, we show that inactivation of the leucine-rich repeat protein ZYG-11 produces meiotic and polarity reversal defects similar to those observed in cul-2 mutants, suggesting that the two proteins function in the same pathways. PMID:15215209

  10. Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation

    PubMed Central

    de Vries, Femke A.T.; de Boer, Esther; van den Bosch, Mike; Baarends, Willy M.; Ooms, Marja; Yuan, Li; Liu, Jian-Guo; van Zeeland, Albert A.; Heyting, Christa; Pastink, Albert

    2005-01-01

    In meiotic prophase, synaptonemal complexes (SCs) closely appose homologous chromosomes (homologs) along their length. SCs are assembled from two axial elements (AEs), one along each homolog, which are connected by numerous transverse filaments (TFs). We disrupted the mouse gene encoding TF protein Sycp1 to analyze the role of TFs in meiotic chromosome behavior and recombination. Sycp1-/- mice are infertile, but otherwise healthy. Sycp1-/- spermatocytes form normal AEs, which align homologously, but do not synapse. Most Sycp1-/- spermatocytes arrest in pachynema, whereas a small proportion reaches diplonema, or, exceptionally, metaphase I. In leptotene Sycp1-/- spermatocytes, γH2AX (indicative of DNA damage, including double-strand breaks) appears normal. In pachynema, Sycp1-/- spermatocytes display a number of discrete γH2AX domains along each chromosome, whereas γH2AX disappears from autosomes in wild-type spermatocytes. RAD51/DMC1, RPA, and MSH4 foci (which mark early and intermediate steps in pairing/recombination) appear in similar numbers as in wild type, but do not all disappear, and MLH1 and MLH3 foci (which mark late steps in crossing over) are not formed. Crossovers were rare in metaphase I of Sycp1-/- mice. We propose that SYCP1 has a coordinating role, and ensures formation of crossovers. Unexpectedly, Sycp1-/- spermatocytes did not form XY bodies. PMID:15937223

  11. 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

  12. Effects of canine serum collected from dogs at different estrous cycle stages on in vitro nuclear maturation of canine oocytes.

    PubMed

    Oh, Hyun Ju; Fibrianto, Yuda Heru; Kim, Min Kyu; Jang, Goo; Hossein, M Shamim; Kim, Hye Jin; Kang, Sung Keun; Lee, Byeong Chun; Hwang, Woo Suk

    2005-08-01

    Canine oocytes are ovulated at prophase of the first meiotic division and undergo maturation in the distal part of the oviduct for at least 48-72 h. Because of these differences from other domestic mammals, the efficiency of in vitro maturation (IVM) of canine oocyte is very low. The present study was conducted to evaluate the effects of canine serum on IVM of canine oocytes recovered from ovaries in various reproductive states (follicular, luteal or anestrous stages). Oocytes were recovered by mincing ovaries from bitches presented for ovariohysterectomy at various stages of the estrous cycle. Heat-inactivated canine serum was prepared with blood taken from dogs at the anestrous, estrous or diestrous stage of the estrous cycle as determined by progesterone concentration and vaginal cytology. Oocytes were cultured for 72 h in tissue culture medium (TCM)-199 supplemented with 10% canine anestrous, estrous or diestrous serum or fetal bovine serum (FBS) (experiment 1), or supplemented with 0 (control), 5%, 10% or 20% canine estrous serum (experiment 2). In experiment 1, IVM of oocytes collected at the follicular stage of the estrous cycle to metaphase II (MII) stage was higher (p < 0.05) with canine estrous serum (14.2%) than with canine anestrous (5.2%) or diestrous serum (6.3%), FBS (2.2%) or in the control (2.2%). In experiment 2, oocytes collected at the follicular stage of the estrous cycle cultured in TCM-199 with 10% canine estrous serum showed a higher maturation rate to MII stage (13.5%, p < 0.05) compared with those cultured with 5% (1.3% MII) or 20% canine estrous serum (5.1% MII) or the control (2.7% MII). In conclusion, our results demonstrate that supplementing culture medium with 10% canine estrous serum improves IVM of canine follicular stage oocytes. PMID:16261767

  13. 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. PMID:26154026

  14. 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

  15. 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. PMID:25925194

  16. ``sex Ratio'' Meiotic Drive in Drosophila Testacea

    PubMed Central

    James, A. C.; Jaenike, J.

    1990-01-01

    We document the occurrence of ``sex ratio'' meiotic drive in natural populations of Drosophila testacea. ``Sex ratio'' males sire >95% female offspring. Genetic analysis reveals that this effect is due to a meiotically driven X chromosome, as in other species of Drosophila in which ``sex ratio'' has been found. In contrast to other drosophilids, the ``sex ratio'' and standard chromosomes of D. testacea do not differ in gene arrangement, implying that the effect may be due to a single genetic factor in this species. In all likelihood, the ``sex ratio'' condition has evolved independently in D. testacea and in the Drosophila obscura species group, as the loci responsible for the effect occur on different chromosomal elements. An important ecological consequence of ``sex ratio'' is that natural populations of D. testacea exhibit a strong female bias. Because D. testacea mates, oviposits, and feeds as adults and larvae on mushrooms, this species provides an excellent opportunity to study the selective factors in nature that prevent ``sex ratio'' chromosomes from increasing to fixation and causing the extinction of the species. PMID:2249763

  17. Functional Redundancy in the Maize Meiotic Kinetochore

    PubMed Central

    Yu, Hong-Guo; Dawe, R. Kelly

    2000-01-01

    Kinetochores can be thought of as having three major functions in chromosome segregation: (a) moving plateward at prometaphase; (b) participating in spindle checkpoint control; and (c) moving poleward at anaphase. Normally, kinetochores cooperate with opposed sister kinetochores (mitosis, meiosis II) or paired homologous kinetochores (meiosis I) to carry out these functions. Here we exploit three- and four-dimensional light microscopy and the maize meiotic mutant absence of first division 1 (afd1) to investigate the properties of single kinetochores. As an outcome of premature sister kinetochore separation in afd1 meiocytes, all of the chromosomes at meiosis II carry single kinetochores. Approximately 60% of the single kinetochore chromosomes align at the spindle equator during prometaphase/metaphase II, whereas acentric fragments, also generated by afd1, fail to align at the equator. Immunocytochemistry suggests that the plateward movement occurs in part because the single kinetochores separate into half kinetochore units. Single kinetochores stain positive for spindle checkpoint proteins during prometaphase, but lose their staining as tension is applied to the half kinetochores. At anaphase, ∼6% of the kinetochores develop stable interactions with microtubules (kinetochore fibers) from both spindle poles. Our data indicate that maize meiotic kinetochores are plastic, redundant structures that can carry out each of their major functions in duplicate. PMID:11018059

  18. Cardiac arrest in children.

    PubMed

    Tress, Erika E; Kochanek, Patrick M; Saladino, Richard A; Manole, Mioara D

    2010-07-01

    Major advances in the field of pediatric cardiac arrest (CA) were made during the last decade, starting with the publication of pediatric Utstein guidelines, the 2005 recommendations by the International Liaison Committee on Resuscitation, and culminating in multicenter collaborations. The epidemiology and pathophysiology of in-hospital and out-of-hospital CA are now well described. Four phases of CA are described and the term "post-cardiac arrest syndrome" has been proposed, along with treatment goals for each of its four phases: immediate post-arrest, early post-arrest, intermediate and recovery phase. Hypothermia is recommended to be considered as a therapy for post-CA syndrome in comatose patients after CA, and large multicenter prospective studies are underway. We reviewed landmark articles related to pediatric CA published during the last decade. We present the current knowledge of epidemiology, pathophysiology and treatment of CA relevant to pre-hospital and acute care health practitioners. PMID:20930971

  19. The Effects of cAMP-elevating Agents and Alpha Lipoic Acid on In Vitro Maturation of Mouse Germinal Vesicle Oocytes

    PubMed Central

    Rahnama, Ali; Zavareh, Saeed; Ghorbanian, Mohammad Taghi; Karimi, Isaac

    2013-01-01

    Background In spite of extensive efforts to improve in vitro oocyte maturation, the obtained results are not very efficient. This study was conducted to assess impacts of cAMP elevating agents and alpha lipoic acid (ALA) on in vitro oocyte maturation and fertilization. Methods Mouse germinal vesicle (GV) oocytes were categorized into cumulus denuded oocytes (DOs; n=896) and cumulus oocyte complexes (COCs; n=1077) groups. GV oocytes were matured in vitro with or without ALA; (I) without the meiotic inhibitors; (II) supplemented with cilostamide; (III) supplemented with forskolin and (IV) supplemented with Forskolin plus cilostamide. The obtained metaphase II (MII) oocytes were subjected to in vitro fertilization. Independent-samples t-testand ANOVA were used for data analysis. A p-value less than 0.05 was considered to be statistically significant. Results The COCs maturation, fertilization and two cell embryo rates were higher than those of DOs in the control group, while no significant difference was observed between relevant COCs and DOs when they were cultured with cilostamide meiotic inhibitors in two step manner. Combined treatment of cilostamide and forskolin significantly elevated the developmental rates in both COCs and DOs as compared to other groups. The developmental rates of COCs and DOs in the presence of ALA were similar to their respective groups without ALA. Conclusion cAMP elevating agents were more effective on DOs than COCs with regard to rates of maturation and fertilization. However, ALA did not affect the developmental rates of both COCs and DOs in in vitro maturation in one or two step manner. PMID:24551571

  20. 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

  1. 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

  2. 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. PMID:26995551

  3. 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

  4. 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

  5. Dysferlin is essential for endocytosis in the sea star oocyte

    PubMed Central

    Oulhen, Nathalie; Onorato, Thomas M.; Ramos, Isabela; Wessel, Gary M.

    2014-01-01

    Dysferlin is a calcium-binding transmembrane protein involved in membrane fusion and membrane repair. In humans, mutations in the dysferlin gene are associated with muscular dystrophy. In this study, we isolated plasma membrane-enriched fractions from full-grown immature oocytes of the sea star, and identified dysferlin by mass spectrometry analysis. The full-length dysferlin sequence is highly conserved between human and the sea star. We learned that in the sea star Patiria miniata, dysferlin RNA and protein are expressed from oogenesis to gastrulation. Interestingly, the protein is highly enriched in the plasma membrane of oocytes. Injection of a morpholino against dysferlin leads to a decrease of endocytosis in oocytes, and to a developmental arrest during gastrulation. These results suggest that dysferlin is critical for normal endocytosis during oogenesis and for embryogenesis in the sea star and that this animal may be a useful model for studying the relationship of dysferlin structure as it relates to its function. PMID:24368072

  6. c-mos and cdc2 Cooperate in the Translational Activation of Fibroblast Growth Factor Receptor-1 during Xenopus Oocyte Maturation

    PubMed Central

    Culp, Patricia A.; Musci, Thomas J.

    1999-01-01

    During oocyte maturation in Xenopus, previously quiescent maternal mRNAs are translationally activated at specific times. We hypothesized that the translational recruitment of individual messages is triggered by particular cellular events and investigated the potential for known effectors of the meiotic cell cycle to activate the translation of the FGF receptor-1 (XFGFR) maternal mRNA. We found that both c-mos and cdc2 activate the translation of XFGFR. However, although oocytes matured by injection of recombinant cdc2/cyclin B translate normal levels of XFGFR protein, c-mos depletion reduces the level of XFGFR protein induced by cdc2/cyclin B injection. In oocytes blocked for cdc2 activity, injection of mos RNA induced low levels of XFGFR protein, independent of MAPK activity. Through the use of injected reporter RNAs, we show that the XFGFR 3′ untranslated region inhibitory element is completely derepressed by cdc2 alone. In addition, we identified a new inhibitory element through which both mos and cdc2 activate translation. We found that cdc2 derepresses translation in the absence of polyadenylation, whereas mos requires poly(A) extension to activate XFGFR translation. Our results demonstrate that mos and cdc2, in addition to functioning as key regulators of the meiotic cell cycle, cooperate in the translational activation of a specific maternal mRNA during oocyte maturation. PMID:10564256

  7. 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

  8. 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.

  9. 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

  10. Seasonal variations in developmental competence and relative abundance of gene transcripts in buffalo (Bubalus bubalis) oocytes.

    PubMed

    Abdoon, Ahmed S; Gabler, Christoph; Holder, Christoph; Kandil, Omaima M; Einspanier, Ralf

    2014-11-01

    , cleavage rate was lower (P < 0.01) for oocytes collected during HS, and the percentage of oocytes arrested at the two-cell stage was higher (P < 0.01) than oocytes IVF during CS. Oocytes matured during CS showed a higher (P < 0.01) blastocyst rate than those matured during HS. Also, COCs recovered in HS showed significant (P < 0.05) upregulation of HSP70 mRNA expression compared with those recovered in CS. For in vitro matured oocytes, CS down regulated the transcript abundance of ACTB and upregulated GAPDH and HSP70 mRNA levels compared with HS condition. In conclusion, HS could impair buffalo fertility by reducing the number of antral follicles and oocyte quality. In vitro maturation of buffalo oocytes during HS impairs their nuclear and cytoplasmic maturation, fertilization, and subsequent embryo development to the morula and blastocyst stages. This could be in part because of the altered gene expression found in COCs and in vitro matured oocytes. PMID:25156970

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

    PubMed Central

    Tatone, Carla; Carbone, Maria Cristina

    2006-01-01

    Background Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC) and the exocytosis of cortical granules in mouse oocytes. Methods An In-Vitro-Fertilization assay (IVF) was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val), was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val), were evaluated. Results The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P < 0.05, Student-Newman-Keuls Method). When the peptide was applied to the oocytes at these concentrations, a dose-dependent increase of PKC activity was observed, in association with a loss of cortical granules ranging from 38+/-2.5 % to 52+/-5.4 %. Evaluation of meiotic status revealed that cyclic RGD peptide was ineffective in inducing meiosis resumption under conditions used in the

  12. 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. PMID:25565579

  13. 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

  14. Thr-161 phosphorylation of monomeric Cdc2. Regulation by protein phosphatase 2C in Xenopus oocytes.

    PubMed

    De Smedt, Veronique; Poulhe, Robert; Cayla, Xavier; Dessauge, Frederic; Karaiskou, Anthi; Jessus, Catherine; Ozon, Rene

    2002-08-01

    Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B.Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification. PMID:12036957

  15. Proliferating Cell Nuclear Antigen (PCNA) Regulates Primordial Follicle Assembly by Promoting Apoptosis of Oocytes in Fetal and Neonatal Mouse Ovaries

    PubMed Central

    Zhang, Yuanwei; Jiang, Xiaohua; Zhang, Huan; Ma, Tieliang; Zheng, Wei; Sun, Rui; Shen, Wei; Sha, Jiahao; Cooke, Howard J.; Shi, Qinghua

    2011-01-01

    Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer guanulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries. PMID:21253613

  16. Sperm-derived WW domain-binding protein, PAWP, elicits calcium oscillations and oocyte activation in humans and mice.

    PubMed

    Aarabi, Mahmoud; Balakier, Hanna; Bashar, Siamak; Moskovtsev, Sergey I; Sutovsky, Peter; Librach, Clifford L; Oko, Richard

    2014-10-01

    Mammalian zygotic development is initiated by sperm-mediated intracellular calcium oscillations, followed by activation of metaphase II-arrested oocytes. Sperm postacrosomal WW binding protein (PAWP) fulfils the criteria set for an oocyte-activating factor by inducing oocyte activation and being stored in the perinuclear theca, the sperm compartment whose content is first released into oocyte cytoplasm during fertilization. However, proof that PAWP initiates mammalian zygotic development relies on demonstration that it acts upstream of oocyte calcium oscillations. Here, we show that PAWP triggers calcium oscillations and pronuclear formation in human and mouse oocytes similar to what is observed during intracytoplasmic sperm injection (ICSI). Most important, sperm-induced calcium oscillations are blocked by coinjection of a competitive inhibitor, derived from the WWI domain-binding motif of PAWP, implying the requirement of sperm PAWP and an oocyte-derived WWI domain protein substrate of PAWP for successful fertilization. Sperm-delivered PAWP is, therefore, a unique protein with a nonredundant role during human and mouse fertilization, required to trigger zygotic development. Presented data confirm our previous findings in nonmammalian models and suggest potential applications of PAWP in the diagnosis and treatment of infertility.- PMID:24970390

  17. 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

  18. RanGTP is required for meiotic spindle organization and the initiation of embryonic development in Drosophila

    PubMed Central

    Cesario, J.; McKim, K. S.

    2011-01-01

    RanGTP is important for chromosome-dependent spindle assembly in Xenopus extracts. Here we report on experiments to determine the role of the Ran pathway on microtubule dynamics in Drosophila oocytes and embryos. Females expressing a dominant-negative form of Ran have fertility defects, suggesting that RanGTP is required for normal fertility. This is not, however, because of a defect in acentrosomal meiotic spindle assembly. Therefore, RanGTP does not appear to be essential or sufficient for the formation of the acentrosomal spindle. Instead, the most important function of the Ran pathway in spindle assembly appears to be in the tapering of microtubules at the spindle poles, which might be through regulation of proteins such as TACC and the HURP homolog, Mars. One consequence of this spindle organization defect is an increase in the nondisjunction of achiasmate chromosomes. However, the meiotic defects are not severe enough to cause the decreased fertility. Reductions in fertility occur because RanGTP has a role in microtubule assembly that is not directly nucleated by the chromosomes. This includes microtubules nucleated from the sperm aster, which are required for pronuclear fusion. We propose that following nuclear envelope breakdown, RanGTP is released from the nucleus and creates a cytoplasm that is activated for assembling microtubules, which is important for processes such as pronuclear fusion. Around the chromosomes, however, RanGTP might be redundant with other factors such as the chromosome passenger complex. PMID:22100918

  19. 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

  20. 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

  1. 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

  2. Intrinsic and extrinsic mechanisms of oocyte loss.

    PubMed

    Thomson, Travis C; Fitzpatrick, Katherine E; Johnson, Joshua

    2010-12-01

    A great deal of evolutionary conservation has been found in the control of oocyte development, from invertebrates to women. However, little is known of mechanisms that control oocyte loss over time. Oocyte loss is often assumed to be a result of oocyte-intrinsic deficiencies or damage. In fruit flies, starvation results in halted oocyte production by germline stem cells and induces oocyte loss midway through development. When we fed wild-type flies the bacterial compound Rapamycin (RAP) to mimic starvation, production of new oocytes continued, but mid-stage loss sterilized the animals. Surprisingly, follicle cell invasion and phagocytosis of the oocyte preceded any signs of germ cell death. RAP-induced egg chamber loss was prevented when RAP receptor FKBP12 was knocked down specifically in follicle cells. Oogenesis continued past the mid-stages, and these mutants continued to lay embryos that could develop into normal adults. Hence, intact healthy oocytes can be destroyed by somatic cells responding to extrinsic stimuli. We termed this process inducible somatic oocyte destruction. RAP treatment of mouse follicles in vitro resulted in phagocytic uptake of the oocyte by granulosa cells as seen in flies. We hypothesize that extrinsic modes of oocyte loss occur in mammals. PMID:20651035

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

    PubMed

    Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

    2010-02-01

    Recent research has shown that the maternal nucleolus is essential for embryonic development. The morphology of the nucleolus in growing oocytes differs from that in full-grown oocytes. We determined the ability of nucleoli from growing oocytes to substitute for nucleoli of full-grown oocytes in terms of supporting embryonic development in this study. Growing (around 100 microm in diameter) and full-grown porcine oocytes (120 microm) were collected from small (0.6-1.0 mm) and large antral follicles (4-5 mm), respectively. The nucleolus was aspirated from full-grown oocytes by micromanipulation, and the resulting enucleolated oocytes were matured to metaphase II; the nucleoli originating from full-grown and growing oocytes were then injected into the oocytes. The Chromatin of growing oocytes was aspirated with the nucleolus during the enucleolation process. Growing oocytes were thus treated with actinomycin D to release the chromatin from their nucleoli, and the nucleoli were collected and transferred to the enucleolated and matured full-grown oocytes. After activation by electro-stimulation, nucleoli were formed in pronuclei of sham-operated oocytes. Enucleolated oocytes that had been injected with nucleoli from either full-grown or growing, however, did not form any nucleoli in the pronuclei. No enucleolated oocytes developed to blastocysts, whereas enucleolated oocytes injected with nucleoli from full-grown oocytes (15%) or growing oocytes (18%) developed to blastocysts. These results indicate that the nucleoli from growing oocytes can substitute for nucleoli from full-grown oocytes during early embryonic development. PMID:19908251

  4. Progesterone modulation of transmembrane helix-helix interactions between the α-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane

    PubMed Central

    2010-01-01

    Background Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the α-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the α1-subunit. Results Preparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020) successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT) and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME), can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the α1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT. Conclusion We propose that progesterone binding to the first external loop of the α1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions. PMID:20500835

  5. 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.

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

    PubMed

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

    2016-07-15

    The purpose of this study was to investigate the effect of antifreeze glycoprotein 8 (AFGP8) supplementation during vitrification on the survival, fertilization, and embryonic development of bovine oocytes and the underlying molecular mechanism(s). Survival, fertilization, early embryonic development, apoptosis, DNA double-strand breaks, reactive oxygen species levels, meiotic cytoskeleton assembly, chromosome alignment, and energy status of mitochondria were measured in the present experiments. Compared with that in the nonsupplemented group; survival, monospermy, blastocyst formation rates, and blastomere counts were significantly higher in the AFGP8-supplemented animals. Oocytes of the latter group also presented fewer double-strand breaks and lower cathepsin B and caspase activities. Rates of normal spindle organization and chromosome alignment, actin filament impairment, and mitochondrial distribution were significantly higher in the AFGP8-supplemented group. In addition, intracellular reactive oxygen species levels significantly decreased in the AFGP8-supplemented groups, maintaining a higher ΔΨm than that in the nonsupplemented group. Taken together, these results indicated that supplementation with AFGP8 during vitrification has a protective effect on bovine oocytes against chilling injury. PMID:26948296

  7. Expression and functional activity of PACAP and its receptors on cumulus cells: effects on oocyte maturation.

    PubMed

    Barberi, Marzia; Di Paolo, Virginia; Latini, Stefania; Guglielmo, Maria Cristina; Cecconi, Sandra; Canipari, Rita

    2013-08-15

    Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor PAC1-R (PACAP type 1 receptor) are transiently expressed in granulosa cells (GCs) of mouse preovulatory follicles and affect several parameters associated with the ovulatory process. We investigated the expression of PACAP and its receptors in cumulus cells (CCs) after the LH surge and their role on cumulus expansion/apoptosis and oocyte maturation. PACAP and PAC1-R expression increased in CCs isolated at different times after treatment with human chorionic gonadotropin (hCG). Moreover, PACAP was able to reverse the inhibition of oocyte meiotic maturation caused by hypoxantine in cumulus cell-oocyte complexes (COCs) and efficiently promoted male pronuclear formation after fertilisation. PACAP was also able to induce cumulus expansion and prevent CC apoptosis. Our results demonstrated the induction of PACAP and its receptors in CCs by LH and EGF, suggesting that PACAP may play a significant role in the complex interactions of gonadotropin and growth factors during ovulation and fertilisation. PMID:23684890

  8. Oocyte donation programs: strategy for improving results.

    PubMed

    Borini, Andrea; Suriano, Rosanna; Barberi, Marzia; Dal Prato, Luca; Bulletti, Carlo

    2011-03-01

    Oocyte donation is now a useful option for women who cannot start a spontaneous pregnancy for reasons related to advanced age, iatrogenic factors, early depletion of ovarian reserve, or genetic disorders. Embryo implantation rates, pregnancy rates, and pregnancy outcomes among women included in oocyte donation programs were shown to be comparable to those of spontaneous or in vitro fertilization (IVF) pregnancies. With oocyte freezing and cryobanks, recipients may have a successful response to oocyte donation, with no need to be on waiting lists, access to a larger number of oocytes from the same donor, and a lesser risk of infectious disease transmission. PMID:21401626

  9. 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

  10. Driving folliculogenesis by the oocyte-somatic cell dialog: Lessons from genetic models.

    PubMed

    Monniaux, Danielle

    2016-07-01

    This review focuses on the role of the dialog between the oocyte and its companion somatic cells in driving folliculogenesis from the primordial to the preovulatory follicle stage. Mouse and sheep genetic models have brought complementary evidence of these cell interactions and their consequences for ovarian function. In mouse, the deletion of genes encoding connexins has shown that functional gap junction channels between oocytes and granulosa cells and between granulosa cells themselves maintain the follicle in a functionally integrated state. Targeted deletions in oocytes or granulosa cells have revealed the cell- and stage-specific role of ubiquist factors belonging to the phosphatidylinositol 3 kinase signaling pathway in primordial follicle activation, oocyte growth and follicle survival. Various models of transgenic mice and sheep carrying natural loss-of-function mutations associated with sterility have established that the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor 9 orchestrate follicle development, support cumulus metabolism and maturation and participate in oocyte meiosis arrest. Unexpectedly in sheep, mutations resulting in the attenuation of BMP signaling lead to enhanced ovulation rate, likely resulting from a lowered follicular atresia rate and the enhancement of FSH-regulated follicular maturation. Both the activation level of BMP signaling and an adequate equilibrium between BMP15 and growth differentiation factor 9 determine follicle survival, maturation, and development toward ovulation. The physiological approaches which were implemented on genetic animal models during the last 20 years have opened up new perspectives for female fertility by identifying the main signaling pathways of the oocyte-somatic cell dialog. PMID:27155734

  11. Mammalian oocyte growth and development in vitro.

    PubMed

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

    1996-06-01

    This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes form preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk. PMID:9115726

  12. 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

  13. 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.

  14. Relationship of a human oocyte scoring system to oocyte maturity and fertilizing capacity.

    PubMed

    Mahadevan, M M; Fleetham, J

    1990-01-01

    A simple, quick, and semiquantitative human oocyte scoring system is described. Oocyte-corona-cumulus complexes were spread, either by tilting the dish or by aspiration of the fluid, and examined under the dissecting microscope. A maximum of four points was assigned to each of the following: cumulus expansion, cumulus appearance, amount of cumulus, corona expansion, corona appearance, and oocyte appearance. The oocyte score was significantly correlated to two important physiological parameters of oocytes, nuclear maturity (P less than .02) and fertilization rate (P less than .0001). This oocyte scoring system is useful for selecting oocytes for in vitro fertilization and embryo transfer (IVF-ET) or gamete intrafallopian tube transfer (GIFT), for training new laboratory personnel in recognizing the important characteristics of a mature oocyte, and in the standardized reporting of oocyte quality by different IVF-ET or GIFT programs. PMID:1977717

  15. 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.

  16. Analysis of meiotic segregation, using single-sperm typing: meiotic drive at the myotonic dystrophy locus.

    PubMed Central

    Leeflang, E. P.; McPeek, M. S.; Arnheim, N.

    1996-01-01

    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. PMID:8808606

  17. 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.

  18. Linoleic acid stimulates neutral lipid accumulation in lipid droplets of maturing bovine oocytes.

    PubMed

    Carro, M; Buschiazzo, J; Ríos, G L; Oresti, G M; Alberio, R H

    2013-03-01

    Linoleic acid (LA) is a polyunsaturated fatty acid present in high concentrations in bovine follicular fluid; when added to maturation culture media, it affects oocyte competence (depending on the type and concentration of LA used). To date, little is known about the effective level of incorporation of LA and there is apparently no information regarding its esterification into various lipid fractions of the oocyte and its effect on neutral lipid storage. Therefore, the objective was to assess the uptake and subcellular lipid distribution of LA by analyzing incorporation of radiolabeled LA into oocyte polar and neutral lipid classes. The effects of various concentrations of LA on the nuclear status and cytoplasmic lipid content of bovine oocytes matured in vitro was also analyzed, with particular emphasis on intermediate concentrations of LA. Neutral lipids stored in lipid droplets were quantified with a fluorescence approach. Linoleic acid at 9 and 43 μM did not affect the nuclear status of oocytes matured in vitro, and 100 μM LA inhibited germinal vesicle breakdown, resulting in a higher percentage of oocytes arrested at the germinal state (43.5 vs. 3.0 in controls; P < 0.05). Bovine oocytes actively incorporated LA from the maturation medium (83.4 pmol LA per 100 oocytes at 22 hours of incubation; P < 0.05) and metabolized it mainly into major lipid classes, e.g., triacylglycerols and phospholipids (61.1% and 29.3%, respectively). Supplementation of the maturation medium with LA increased triacylglycerol accumulation in cytoplasmic lipid droplets at all concentrations assayed (P < 0.05). In conclusion, LA added to a defined maturation medium at concentrations that did not alter the nuclear status of bovine oocytes matured in vitro (9 and 43 μM) improved their quality by increasing the content of neutral lipids stored in lipid droplets. By directing the free fatty acid (LA) to triacylglycerol synthesis pathways and increasing the degree of unsaturation of

  19. 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

  20. Mouse Pachytene Checkpoint 2 (Trip13) Is Required for Completing Meiotic Recombination but Not Synapsis

    PubMed Central

    Li, Xin; Schimenti, John C

    2007-01-01

    In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice. PMID:17696610

  1. Cardiac arrest and pregnancy

    PubMed Central

    Campbell, Tabitha A; Sanson, Tracy G

    2009-01-01

    Cardiopulmonary arrest in pregnancy is rare occurring in 1 in 30,000 pregnancies. When it does occur, it is important for a clinician to be familiar with the features peculiar to the pregnant state. Knowledge of the anatomic and physiologic changes of pregnancy is helpful in the treatment and diagnosis. Although the main focus should be on the mother, it should not be forgotten that there is another potential life at stake. Resuscitation of the mother is performed in the same manner as in any other patient, except for a few minor adjustments because of the changes of pregnancy. The specialties of obstetrics and neonatology should be involved early in the process to ensure appropriate treatment of both mother and the newborn. This article will explore the changes that occur in pregnancy and their impact on treatment. The common causes of maternal cardiac arrest will be discussed briefly. PMID:19561954

  2. Lateral microtubule bundles promote chromosome alignment during acentrosomal oocyte meiosis

    PubMed Central

    Wignall, Sarah M.; Villeneuve, Anne M.

    2009-01-01

    Although centrosomes serve to organize microtubules in most cell types, oocyte spindles form and mediate meiotic chromosome segregation in their absence. Here, we use high-resolution imaging of both bipolar and experimentally-generated monopolar spindles in C. elegans to reveal a surprising organization of microtubules and chromosomes within acentrosomal structures. We find that homologous chromosome pairs (bivalents) are surrounded by microtubule bundles running along their sides, whereas microtubule density is extremely low at chromosome ends despite a concentration of kinetochore proteins on those regions. Further, we find that the chromokinesin KLP-19 is targeted to a ring around the center of each bivalent and provides a polar ejection force required for congression. Together, these observations create a new picture of chromosome/microtubule association in acentrosomal spindles and reveal a mechanism by which metaphase alignment can be achieved utilizing this organization. Specifically, we propose that: 1) Ensheathment by lateral microtubule bundles places spatial constraints on the chromosomes, thereby promoting biorientation, and 2) Localized motors mediate movement along these bundles, thereby promoting alignment. PMID:19525937

  3. 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

  4. A Link between Meiotic Prophase Progression and CrossoverControl

    SciTech Connect

    Carlton, Peter M.; Farruggio, Alfonso P.; Dernburg, Abby F.

    2005-07-06

    During meiosis, most organisms ensure that homologous chromosomes undergo at least one exchange of DNA, or crossover, to link chromosomes together and accomplish proper segregation. How each chromosome receives a minimum of one crossover is unknown. During early meiosis in Caenorhabditis elegans and many other species, chromosomes adopt a polarized organization within the nucleus, which normally disappears upon completion of homolog synapsis. Mutations that impair synapsis even between a single pair of chromosomes in C. elegans delay this nuclear reorganization. We quantified this delay by developing a classification scheme for discrete stages of meiosis. Immunofluorescence localization of RAD-51 protein revealed that delayed meiotic cells also contained persistent recombination intermediates. Through genetic analysis, we found that this cytological delay in meiotic progression requires double-strand breaks and the function of the crossover-promoting heteroduplex HIM-14 (Msh4) and MSH-5. Failure of X chromosome synapsis also resulted in impaired crossover control on autosomes, which may result from greater numbers and persistence of recombination intermediates in the delayed nuclei. We conclude that maturation of recombination events on chromosomes promotes meiotic progression, and is coupled to the regulation of crossover number and placement. Our results have broad implications for the interpretation of meiotic mutants, as we have shown that asynapsis of a single chromosome pair can exert global effects on meiotic progression and recombination frequency.

  5. A Link between Meiotic Prophase Progression and Crossover Control

    PubMed Central

    Carlton, Peter M; Farruggio, Alfonso P; Dernburg, Abby F

    2006-01-01

    During meiosis, most organisms ensure that homologous chromosomes undergo at least one exchange of DNA, or crossover, to link chromosomes together and accomplish proper segregation. How each chromosome receives a minimum of one crossover is unknown. During early meiosis in Caenorhabditis elegans and many other species, chromosomes adopt a polarized organization within the nucleus, which normally disappears upon completion of homolog synapsis. Mutations that impair synapsis even between a single pair of chromosomes in C. elegans delay this nuclear reorganization. We quantified this delay by developing a classification scheme for discrete stages of meiosis. Immunofluorescence localization of RAD-51 protein revealed that delayed meiotic cells also contained persistent recombination intermediates. Through genetic analysis, we found that this cytological delay in meiotic progression requires double-strand breaks and the function of the crossover-promoting heteroduplex HIM-14 (Msh4) and MSH-5. Failure of X chromosome synapsis also resulted in impaired crossover control on autosomes, which may result from greater numbers and persistence of recombination intermediates in the delayed nuclei. We conclude that maturation of recombination events on chromosomes promotes meiotic progression, and is coupled to the regulation of crossover number and placement. Our results have broad implications for the interpretation of meiotic mutants, as we have shown that asynapsis of a single chromosome pair can exert global effects on meiotic progression and recombination frequency. PMID:16462941

  6. 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

  7. Meiotic recombination and the crossover assurance checkpoint in Caenorhabditis elegans.

    PubMed

    Yu, Zhouliang; Kim, Yumi; Dernburg, Abby F

    2016-06-01

    During meiotic prophase, chromosomes pair and synapse with their homologs and undergo programmed DNA double-strand break (DSB) formation to initiate meiotic recombination. These DSBs are processed to generate a limited number of crossover recombination products on each chromosome, which are essential to ensure faithful segregation of homologous chromosomes. The nematode Caenorhabditis elegans has served as an excellent model organism to investigate the mechanisms that drive and coordinate these chromosome dynamics during meiosis. Here we focus on our current understanding of the regulation of DSB induction in C. elegans. We also review evidence that feedback regulation of crossover formation prolongs the early stages of meiotic prophase, and discuss evidence that this can alter the recombination pattern, most likely by shifting the genome-wide distribution of DSBs. PMID:27013114

  8. MPS3 mediates meiotic bouquet formation in Saccharomyces cerevisiae.

    PubMed

    Conrad, Michael N; Lee, Chih-Ying; Wilkerson, Joseph L; Dresser, Michael E

    2007-05-22

    In meiotic prophase, telomeres associate with the nuclear envelope and accumulate adjacent to the centrosome/spindle pole to form the chromosome bouquet, a well conserved event that in Saccharomyces cerevisiae requires the meiotic telomere protein Ndj1p. Ndj1p interacts with Mps3p, a nuclear envelope SUN domain protein that is required for spindle pole body duplication and for sister chromatid cohesion. Removal of the Ndj1p-interaction domain from MPS3 creates an ndj1 Delta-like separation-of-function allele, and Ndj1p and Mps3p are codependent for stable association with the telomeres. SUN domain proteins are found in the nuclear envelope across phyla and are implicated in mediating interactions between the interior of the nucleus and the cytoskeleton. Our observations indicate a general mechanism for meiotic telomere movements. PMID:17495028

  9. The chromosome passenger complex is required for fidelity of chromosome transmission and cytokinesis in meiosis of mouse oocytes.

    PubMed

    Sharif, Bedra; Na, Jie; Lykke-Hartmann, Karin; McLaughlin, Stephen H; Laue, Ernest; Glover, David M; Zernicka-Goetz, Magdalena

    2010-12-15

    The existence of two forms of the chromosome passenger complex (CPC) in the mammalian oocyte has meant that its role in female meiosis has remained unclear. Here we use loss- and gain-of function approaches to assess the meiotic functions of one of the shared components of these complexes, INCENP, and of the variable kinase subunits, Aurora B or Aurora C. We show that either the depletion of INCENP or the combined inhibition of Aurora kinases B and C activates the anaphase-promoting complex or cyclosome (APC/C) before chromosomes have properly congressed in meiosis I and also prevents cytokinesis and hence extrusion of the first polar body. Overexpression of Aurora C also advances APC/C activation and results in cytokinesis failure in a high proportion of oocytes, indicative of a dominant effect on CPC function. Together, this points to roles for the meiotic CPC in functions similar to the mitotic roles of the complex: correcting chromosome attachment to microtubules, facilitating the spindle-assembly checkpoint (SAC) function and enabling cytokinesis. Surprisingly, overexpression of Aurora B leads to a failure of APC/C activation, stabilization of securin and consequently a failure of chiasmate chromosomes to resolve - a dominant phenotype that is completely suppressed by depletion of INCENP. Taken together with the differential distribution of Aurora proteins B and C on chiasmate chromosomes, this points to differential functions of the two forms of CPC in regulating the separation of homologous chromosomes in meiosis I. PMID:21123620

  10. Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation

    PubMed Central

    Yamagishi, Yuka; Abe, Hiroshi

    2015-01-01

    We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation. PMID:26424802

  11. The CSN/COP9 Signalosome Regulates Synaptonemal Complex Assembly during Meiotic Prophase I of Caenorhabditis elegans

    PubMed Central

    Brockway, Heather; Balukoff, Nathan; Dean, Martha; Alleva, Benjamin; Smolikove, Sarit

    2014-01-01

    The synaptonemal complex (SC) is a conserved protein structure that holds homologous chromosome pairs together throughout much of meiotic prophase I. It is essential for the formation of crossovers, which are required for the proper segregation of chromosomes into gametes. The assembly of the SC is likely to be regulated by post-translational modifications. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via the ubiquitin degradation/proteasome pathway. Here we examine the role of the CSN/COP9 signalosome in SC assembly in the model organism C. elegans. Our work shows that mutants in three subunits of the CSN/COP9 signalosome fail to properly assemble the SC. In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes. The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers. The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes. We also find a marked increase in apoptosis in csn mutants that specifically eliminates nuclei with aggregated SC proteins. csn mutants exhibit defects in germline proliferation, and an almost complete pachytene arrest due to an inability to activate the MAPK pathway. The work described here supports a previously unknown role for the CSN/COP9 signalosome in chromosome behavior during meiotic prophase I. PMID:25375142

  12. 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

  13. Diet-Induced Obesity Model: Abnormal Oocytes and Persistent Growth Abnormalities in the Offspring

    PubMed Central

    Jungheim, Emily S.; Schoeller, Erica L.; Marquard, Kerri L.; Louden, Erica D.; Schaffer, Jean E.; Moley, Kelle H.

    2010-01-01

    Associations between maternal obesity and adverse fetal outcomes are well documented, but the mechanisms involved are largely unknown. Most previous work has focused on postconceptional events, however, our laboratory has shown pre- and periconceptional aberrations in maternal glucose metabolism have adverse effects on oocytes and embryos that carry on to the fetus. To demonstrate effects of maternal obesity in the pre- and periconceptional periods, we compared reproductive tissues from diet-induced obese female mice to those of control mice. Ovaries were either stained for follicular apoptosis or dissected and evaluated for oocyte size and meiotic maturation. Mice were also mated and followed for reproductive outcomes including preimplantation embryonic IGF-I receptor (IGF-IR) immunostaining, midgestation fetal growth, and midgestational placental IGF receptor 2 (Igf2r) mRNA. Delivered pups were followed for growth and development of markers of metabolic syndrome. Compared with controls, obese mice had significantly more apoptotic ovarian follicles, smaller and fewer mature oocytes, decreased embryonic IGF-IR staining, smaller fetuses, increased placental Igf2r mRNA, and smaller pups. All weaned pups were fed a regular diet. At 13 wk pups delivered from obese mice were significantly larger, and these pups demonstrated glucose intolerance and increased cholesterol and body fat suggesting early development of a metabolic-type syndrome. Together, our findings suggest maternal obesity has adverse effects as early as the oocyte and preimplantation embryo stage and that these effects may contribute to lasting morbidity in offspring, underscoring the importance of optimal maternal weight and nutrition before conception. PMID:20573727

  14. [Controversy in ART: should we cryopreserve oocytes or embryos? Do prefer oocytes].

    PubMed

    Boyer, P

    2014-09-01

    Since the beginning of IVF, cryopreservation concern spermatozoa or embryos due to the poor efficiency of oocyte freezing. To date, oocyte vitrification allows changing our practice privileging female gamete vitrification instead of human embryo freezing. PMID:25153440

  15. [New possibilities resulting from oocyte banking].

    PubMed

    Revel, Ariel; Revel, Michel; Laufer, Neri; Kasher, Asa

    2011-06-01

    Oocyte cryopreservation solves the legal and ethical problems associated with the cryopreservation of embryos in patients undergoing in vitro fertilization procedures. Furthermore, it may also offer the possibility of extending the reproductive capability of young women with malignant diseases in cases where the treatment may compromise the ovarian reserve. Moreover, it may also offer alternatives for infertile patients who are subject to ovarian hyper-stimulation syndrome or premature ovarian faiLure or who require oocyte donation. The creation of banks for cryopreserved oocytes avoids the need for cycle synchronization or the formation of an over-supply of embryos destined for cryopreservation. If a Large number of oocytes is obtained it could possibly enable women and couples the opportunity to postpone childbirth according to their wishes. This paper reviews the revolution obtained by oocyte vitrification, reports on ethical issues and discusses the pros and cons of oocyte banking and its potential effects on society. PMID:21800492

  16. Essential role of the Cdk2 activator RingoA in meiotic telomere tethering to the nuclear envelope

    PubMed Central

    Mikolcevic, Petra; Isoda, Michitaka; Shibuya, Hiroki; del Barco Barrantes, Ivan; Igea, Ana; Suja, José A.; Shackleton, Sue; Watanabe, Yoshinori; Nebreda, Angel R.

    2016-01-01

    Cyclin-dependent kinases (CDKs) play key roles in cell cycle regulation. Genetic analysis in mice has revealed an essential role for Cdk2 in meiosis, which renders Cdk2 knockout (KO) mice sterile. Here we show that mice deficient in RingoA, an atypical activator of Cdk1 and Cdk2 that has no amino acid sequence homology to cyclins, are sterile and display meiotic defects virtually identical to those observed in Cdk2 KO mice including non-homologous chromosome pairing, unrepaired double-strand breaks, undetectable sex-body and pachytene arrest. Interestingly, RingoA is required for Cdk2 targeting to telomeres and RingoA KO spermatocytes display severely affected telomere tethering as well as impaired distribution of Sun1, a protein essential for the attachment of telomeres to the nuclear envelope. Our results identify RingoA as an important activator of Cdk2 at meiotic telomeres, and provide genetic evidence for a physiological function of mammalian Cdk2 that is not dependent on cyclins. PMID:27025256

  17. The pam1 gene is required for meiotic bouquet formation and efficient homologous synapsis in maize (Zea mays L.).

    PubMed Central

    Golubovskaya, Inna N; Harper, Lisa C; Pawlowski, Wojciech P; Schichnes, Denise; Cande, W Zacheus

    2002-01-01

    The clustering of telomeres on the nuclear envelope (NE) during meiotic prophase to form the bouquet arrangement of chromosomes may facilitate homologous chromosome synapsis. The pam1 (plural abnormalities of meiosis 1) gene is the first maize gene that appears to be required for telomere clustering, and homologous synapsis is impaired in pam1. Telomere clustering on the NE is arrested or delayed at an intermediate stage in pam1. Telomeres associate with the NE during the leptotene-zygotene transition but cluster slowly if at all as meiosis proceeds. Intermediate stages in telomere clustering including miniclusters are observed in pam1 but not in wild-type meiocytes. The tight bouquet normally seen at zygotene is a rare event. In contrast, the polarization of centromeres vs. telomeres in the nucleus at the leptotene-zygotene transition is the same in mutant and wild-type cells. Defects in homologous chromosome synapsis include incomplete synapsis, nonhomologous synapsis, and unresolved interlocks. However, the number of RAD51 foci on chromosomes in pam1 is similar to that of wild type. We suggest that the defects in homologous synapsis and the retardation of prophase I arise from the irregularity of telomere clustering and propose that pam1 is involved in the control of bouquet formation and downstream meiotic prophase I events. PMID:12524364

  18. Preparation of the cortical reaction: maturation-dependent migration of SNARE proteins, clathrin, and complexin to the porcine oocyte's surface blocks membrane traffic until fertilization.

    PubMed

    Tsai, Pei-Shiue; van Haeften, Theo; Gadella, Bart M

    2011-02-01

    The cortical reaction is a calcium-dependent exocytotic process in which the content of secretory granules is released into the perivitellin space immediately after fertilization, which serves to prevent polyspermic fertilization. In this study, we investigated the involvement and the organization of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins in the docking and fusion of the cortical granule membrane with the oolemma in porcine oocytes. During meiotic maturation, secretory vesicles that were labeled with a granule-specific binding lectin, peanut agglutinin (PNA), migrated toward the oocyte's surface. This surface-orientated redistribution behavior was also observed for the oocyte-specific SNARE proteins SNAP23 and VAMP1 that colocalized with the PNA-labeled structures in the cortex area just under the oolemma and with the exclusive localization area of complexin (a trans-SNARE complex-stabilizing protein). The coming together of these proteins serves to prevent the spontaneous secretion of the docked cortical granules and to prepare the oocyte's surface for the cortical reaction, which should probably be immediately compensated for by a clathrin-mediated endocytosis. In vitro fertilization resulted in the secretion of the cortical granule content and the concomitant release of complexin and clathrin into the oocyte's cytosol, and this is considered to stimulate the observed endocytosis of SNARE-containing membrane vesicles. PMID:20944080

  19. Distinct subtypes of zona pellucida morphology reflect canine oocyte viability and cumulus-oocyte complex quality.

    PubMed

    Lunn, Matthew O; Wright, Shirley J

    2013-09-15

    The aim of this study was to analyze surface morphology of the zona pellucida (ZP) and assess its relationship with oocyte viability, cumulus-oocyte complex (COC) quality, and oocyte donor age in dogs. Canine ovaries were sliced to release COCs for use in three experiments. In Experiment 1, oocytes from high-quality (grade I) COCs were viewed with scanning electron microscopy to visualize the zona surface. Four zonae, classified as types I, II, III, and IV, were detectable on high-quality oocytes. Most (95.5%) dog donors had oocytes with two or three ZP types. The ZP type I had a smooth compact surface with few pores. The ZP type II was less compact with many distinct circular or elliptical pores. The ZP type III had a rough surface with folds and many irregular shaped pores and hollows. The ZP type IV also had a rough surface with folds, but in addition, stringy filaments obscured the pores and hollows. The frequency of ZP type I in the oocyte population was low (2.7%), whereas ZP types II, III, and IV each occurred in approximately one-third of the oocyte population. In Experiment 2, oocytes from high-quality COCs were stained with propidium iodide (PI) before scanning electron microscopy to investigate the relationship of oocyte viability with ZP morphology. In Experiment 3, oocytes were collected from low-quality (grade 2) and high-quality (grade 1) COCs to investigate the role of COC quality on zona structure. Zonae types I and II were characteristic of PI-positive (dead) oocytes and oocytes from low-quality COCs, whereas ZP types III and IV were prevalent on PI-negative (living) oocytes and oocytes from high-quality COCs. We concluded that the heterogeneous ZP surface underwent structural rearrangements related to oocyte viability and COC quality. This warrants further investigation into ZP structure and may be useful for canine-assisted reproduction. PMID:23790239

  20. 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. PMID:27160442

  1. Impact of sperm DNA damage and oocyte-repairing capacity on trout development.

    PubMed

    Fernández-Díez, C; González-Rojo, S; Lombó, M; Herráez, M P

    2016-07-01

    Zygotic repair of paternal DNA is essential during embryo development. In spite of the interest devoted to sperm DNA damage, its combined effect with defect-repairing oocytes has not been analyzed. Modification of the breeding season is a common practice in aquaculture. This practice reduces developmental success and could affect the both factors: sperm DNA integrity and oocyte repair capacity. To evaluate the maternal role, we analyzed the progeny outcome after fertilizing in-season trout oocytes with untreated and with UV-irradiated sperm. We also analyzed the offspring obtained out of season with untreated sperm. The analysis of the number of lesions in 4 sperm nuclear genes revealed an increase of 1.22-11.18 lesions/10 kb in out-of-season sperm, similar to that obtained after sperm UV irradiation (400 µW/cm(2)5 min). Gene expression showed in out-of-season oocytes the overexpression of repair genes (ogg1, ung, lig3, rad1) and downregulation of tp53, indicating an enhanced repairing activity and reduced capacity to arrest development upon damage. The analysis of the progeny in out-of-season embryos revealed a similar profile tolerant to DNA damage, leading to a much lower apoptotic activity at organogenesis, lower hatching rates and increased rate of malformations. The effects were milder in descendants from in-season-irradiated sperm, showing an enhanced repairing activity at epibolia. Results point out the importance of the repairing machinery provided by the oocyte and show how susceptible it is to environmental changes. Transcripts related to DNA damage signalization and repair could be used as markers of oocyte quality. PMID:27071918

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

    PubMed

    Li, Min; Yu, Yang; Yan, Jie; Yan, Li-Ying; Zhao, Yue; Li, Rong; Liu, Ping; Hsueh, Aaron J; Qiao, Jie

    2012-01-01

    It is important to identify effective contraceptive drugs that cause minimal disruption to physiological processes. Phosphodiesterase 3 (PDE3) inhibitors suppress meiosis in oocytes by decreasing the level of cAMP and blocking the extrusion of the first polar body. In this study, we tested the PDE3 inhibitor, cilostazol, as a potential contraceptive agent. The effects of cilostazol treatment in vitro and in vivo on the suppression of oocyte maturation in a mouse model were investigated. The results indicated that treatment with increasing concentrations of cilostazol led to a dose-dependent arrest in meiosis progression. The effective in vitro concentration was 1 µM and was 300 mg/kg in vivo. The effect of cilostazol was reversible. After removal of the drug, meiosis resumed and mouse oocytes matured in vitro, and showed normal chromosome alignment and spindle organization. After fertilization using an ICSI method, the oocytes showed normal morphology, fertilization rate, embryo cleavage, blastocyst formation, and number of viable pups when compared with controls. The offspring showed similar body weight and fertility. In vivo, the mice became infertile if the drug was injected sequentially, and became pregnant following discontinuation of cilostazol. More importantly, no side effects of cilostazol were observed in treated female mice as demonstrated by blood pressure and heart rate monitoring. It is concluded that cilostazol, a drug routinely used for intermittent claudication, can effectively inhibit oocyte maturation in vitro and in vivo, does not affect the developmental potential of oocytes following drug removal and has few side effects in female mice treated with this drug. These findings suggest that cilostazol may be a potential new contraceptive agent that may facilitate an efficacy and safety study of this drug. PMID:22292006

  3. 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.

  4. Maintenance of CSF arrest: A role for Cdc2 and PP2A-mediated regulation of Emi2

    PubMed Central

    Wu, Qiju; Guo, Yanxiang; Yamada, Ayumi; Perry, Jennifer A.; Wang, Michael Z.; Araki, Marito; Freel, Christopher D.; Tung, Jeffrey J.; Tang, Wanli; Margolis, Seth S.; Jackson, Peter K.; Yamano, Hiroyuki; Asano, Maki; Kornbluth, Sally

    2009-01-01

    Summary Background Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell cycle arrest by inhibiting the anaphase promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. Results We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor, Emi2. Emi2 bound to the core APC and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2 mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. Conclusions Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest. PMID:17276914

  5. Registry of Unexplained Cardiac Arrest

    ClinicalTrials.gov

    2016-05-16

    Cardiac Arrest; Long QT Syndrome; Brugada Syndrome; Catecholaminergi Polymorphic Ventricular Tachycardia; Idiopathic VentricularFibrillation; Early Repolarization Syndrome; Arrhythmogenic Right Ventricular Cardiomyopathy

  6. Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone.

    PubMed

    Shuhaibar, Leia C; Egbert, Jeremy R; Edmund, Aaron B; Uliasz, Tracy F; Dickey, Deborah M; Yee, Siu-Pok; Potter, Lincoln R; Jaffe, Laurinda A

    2016-01-01

    The meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (Npr2-7E). Npr2-7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events. PMID:26522847

  7. Modulating Mek1 kinase alters outcomes of meiotic recombination and the stringency of the recombination checkpoint response

    PubMed Central

    Hsin-Yen, Wu; Hsuan-Chung, Ho; Burgess, Sean M.

    2010-01-01

    Summary Background During meiosis, recombination between homologous chromosomes promotes their proper segregation. In budding yeast, programmed double-strand breaks (DSBs) promote recombination between homologs versus sister chromatids by dimerizing and activating Mek1, a chromosome axis-associated kinase. Mek1 is also a proposed effector kinase in the recombination checkpoint that arrests exit from pachytene in response to aberrant DNA/axis structures. Elucidating a role for Mek1 in the recombination checkpoint has been difficult since in mek1 loss-of-function mutants DSBs are rapidly repaired using a sister chromatid thereby bypassing formation of checkpoint-activating lesions. Here we tested the hypothesis that a MEK1 gain-of-function allele would enhance interhomolog bias and the recombination checkpoint response. Results When Mek1 activation was artificially maintained through GST-mediated dimerization, there was an enhanced skew toward interhomolog recombination and reduction of intersister events including multi-chromatid joint molecules. Increased interhomolog events were specifically repaired as noncrossovers rather than crossovers. Ectopic Mek1 dimerization was also sufficient to impose interhomolog bias in the absence of recombination checkpoint functions, thereby uncoupling these two processes. Finally, the stringency of the recombination checkpoint was enhanced in weak meiotic recombination mutants by blocking prophase exit in a subset of cells where arrest is not absolute. Conclusions We propose that Mek1 plays dual roles during meiotic prophase I by phosphorylating targets directly involved in the recombination checkpoint as well as targets involved in sister chromatid recombination. We discuss how regulation of pachytene exit by Mek1 or similar kinases could influence checkpoint stringency, which may differ among species and between sexes. PMID:20888230

  8. Progesterone regulates the accumulation and the activation of Eg2 kinase in Xenopus oocytes.

    PubMed

    Frank-Vaillant, M; Haccard, O; Thibier, C; Ozon, R; Arlot-Bonnemains, Y; Prigent, C; Jessus, C

    2000-04-01

    Xenopus prophase oocytes reenter meiotic division in response to progesterone. The signaling pathway leading to Cdc2 activation depends on neosynthesized proteins and a decrease in PKA activity. We demonstrate that Eg2 protein, a Xenopus member of the Aurora/Ipl1 family of protein kinases, accumulates in response to progesterone and is degraded after parthenogenetic activation. The polyadenylation and cap ribose methylation of Eg2 mRNA are not needed for the protein accumulation. Eg2 protein accumulation is induced by progesterone through a decrease in PKA activity, upstream of Cdc2 activation. Eg2 kinase activity is undetectable in prophase and is raised in parallel with Cdc2 activation. In contrast to Eg2 protein accumulation, Eg2 kinase activation is under Cdc2 control. Furthermore, by using an anti-sense strategy, we show that Eg2 accumulation is not required in the transduction pathway leading to Cdc2 activation. Altogether, our results strongly suggest that Eg2 is not necessary for Cdc2 activation, though it could participate in the organization of the meiotic spindles, in agreement with the well-conserved roles of the members of the Aurora family, from yeast to man. PMID:10704364

  9. Novel Ca2+ increases in the maturing oocytes of starfish during the germinal vesicle breakdown.

    PubMed

    Limatola, Nunzia; Chun, Jong T; Kyozuka, Keiichiro; Santella, Luigia

    2015-11-01

    It has been known that the intracellular Ca(2+) level transiently rises at the specific stages of mitosis such as the moment of nuclear envelope breakdown and at the metaphase-anaphase transition. Comparable intracellular Ca(2+) increases may also take place during meiosis, as was intermittently reported in mouse, Xenopus, and starfish oocytes. In a majority of starfish species, the maturing oocytes display an intracellular Ca(2+) increase within few minutes after the addition of the maturation hormone, 1-methyladenine (1-MA). Although starfish oocytes at meiosis also manifest a Ca(2+) increase at the time of polar body extrusion, a similar Ca(2+) increase has never been observed during the envelope breakdown of the nucleus (germinal vesicle, GV). Here, we report, for the first time, the existence of an additional Ca(2+) response in the maturing oocytes of Asterina pectinifera at the time of GV breakdown. In contrast to the immediate early Ca(2+) response to 1-MA, which is independent of external Ca(2+) and takes a form of intracellular Ca(2+) wave traveling three times as fast as that in the fertilized eggs, this late stage Ca(2+) response comprised a train of numerous spikes representing Ca(2+) influx. These Ca(2+) spikes coinciding with GV breakdown were mostly eliminated when the GV was removed from the oocytes prior to the addition of 1-MA, suggesting that the Ca(2+) spikes are rather a consequence of the GV breakdown. In support of the idea that these Ca(2+) spikes play a physiological role, the oocytes matured in calcium-free seawater had a higher rate of cleavage failure 2h after the fertilization in natural seawater. Specific inhibitors of L-type Ca(2+) channels, verapamil and diltiazem, severely suppressed the amplitude of the individual Ca(2+) spikes, but not their frequencies. On the other hand, latrunculin-A (LAT-A), which promotes net depolymerization of the actin cytoskeleton, had a dual effect on this late Ca(2+) response. When added immediately

  10. Biochemistry of Meiotic Recombination: Formation, Processing, and Resolution of Recombination Intermediates

    PubMed Central

    Ehmsen, Kirk T.

    2009-01-01

    Meiotic recombination ensures accurate chromosome segregation during the first meiotic division and provides a mechanism to increase genetic heterogeneity among the meiotic products. Unlike homologous recombination in somatic (vegetative) cells, where sister chromatid interactions prevail and crossover formation is avoided, meiotic recombination is targeted to involve homologs, resulting in crossovers to connect the homologs before anaphase of the first meiotic division. The mechanisms responsible for homolog choice and crossover control are poorly understood, but likely involve meiosis-specific recombination proteins, as well as meiosis-specific chromosome organization and architecture. Much progress has been made to identify and biochemically characterize many of the proteins acting during meiotic recombination. This review will focus on the proteins that generate and process heteroduplex DNA, as well as those that process DNA junctions during meiotic recombination, with particular attention to how recombination activities promote crossover resolution between homologs. PMID:20098639

  11. Meiotic Segregation and Male Recombination in Interspecific Hybrids of Drosophila

    PubMed Central

    Coyne, Jerry A.

    1986-01-01

    Male hybrids between three pairs of Drosophila species show no substantial distortion of Mendelian segregation and no appreciable male recombination. These results do not support the theories that meiotic drive alleles of large effect are often fixed within species and that transposable genetic elements cause speciation. PMID:3021573

  12. RPA homologs and ssDNA processing during meiotic recombination.

    PubMed

    Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

    2016-06-01

    Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair. PMID:26520106

  13. Soluble sperm extract specifically recapitulates the initial phase of the Ca2+ response in the fertilized oocyte of P. occelata following a G-protein/ PLCβ signaling pathway.

    PubMed

    Nakano, Takeshi; Kyozuka, Keiichiro

    2015-12-01

    Matured oocytes of the annelidan worm Pseudopotamilla occelata are fertilized at the first metaphase of the meiotic division. During the activation by fertilizing spermatozoa, the mature oocyte shows a two-step intracellular Ca2+ increase. Whereas the first Ca2+ increase is localized and appears to utilize the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, the second Ca2+ increase is global and involves Ca2+ influx via voltage-gated Ca2+ channels on the entire surface of the oocyte. To study how sperm trigger the Ca2+ increases during fertilization, we prepared soluble sperm extract (SE) and examined its ability to induce Ca2+ increases in the oocyte. The SE could evoke a Ca2+ increase in the oocyte when it was added to the medium, but not when it was delivered by microinjection. However, the second-step Ca2+ increase leading to the resumption of meiosis did not follow in these eggs. Local application of SE induced a non-propagating Ca2+ increase and formed a cytoplasmic protrusion that was similar to that created by the fertilizing sperm at the first stage of the Ca2+ response, important for sperm incorporation into the oocyte. Our results suggest that the fertilizing spermatozoon may trigger the first-step Ca2+ increase before it fuses with the oocyte in a pathway that involves the G-protein-coupled receptor and phospholipase C. Thus, the first phase of the Ca2+ response in the fertilized egg of this species is independent of the second phase of the Ca2+ increase for egg activation. PMID:25318389

  14. Uptake of betaine into mouse cumulus-oocyte complexes via the SLC7A6 isoform of y+L transporter.

    PubMed

    Corbett, Hannah E; Dubé, Chantal D; Slow, Sandy; Lever, Michael; Trasler, Jacquetta M; Baltz, Jay M

    2014-04-01

    Betaine (N,N,N-trimethylglycine) has previously been shown to function in cell volume homeostasis in early mouse embryos and also to be a key donor to the methyl pool in the blastocyst. A betaine transporter (SLC6A20A or SIT1) has been shown to be activated after fertilization, but there is no saturable betaine uptake in mouse oocytes or eggs. Unexpectedly, the same high level of betaine is present in mature metaphase II (MII) eggs as is found in one-cell embryos despite the lack of transport in oocytes or eggs. Significant saturable betaine transport is, however, present in intact cumulus-oocyte complexes (COCs). This transport system has an affinity for betaine of ∼227 μM. The inhibition profile indicates that betaine transport by COCs could be completely blocked by methionine, proline, leucine, lysine, and arginine, and transport is dependent on Na(+) but not Cl(-). This is consistent with transport by a y+L-type amino acid transport system. Both transcripts and protein of one y+L isoform, SLC7A6 (y+LAT2), are present in COCs, with little or no expression in isolated germinal vesicle (GV)-stage oocytes, MII eggs, or one-cell embryos. Betaine accumulated by COCs is transferred into the enclosed GV oocyte, which requires functional gap junctions. Thus, at least a portion of the endogenous betaine in MII eggs could be derived from transport into cumulus cells and subsequent transfer into the enclosed oocyte before gap junction closure during meiotic maturation. The oocyte-derived betaine then could be regulated and supplemented by the SIT1 transporter that arises in the embryo after fertilization. PMID:24599290

  15. Linolenic acid improves oocyte developmental competence and decreases apoptosis of in vitro-produced blastocysts in goat.

    PubMed

    Veshkini, Arash; Khadem, Ali Akbar; Mohammadi-Sangcheshmeh, Abdollah; Alamouti, Ali Asadi; Soleimani, Masoud; Gastal, Eduardo L

    2016-08-01

    The effects of α-linolenic acid (ALA) on developmental competence of oocytes in goats were evaluated in this study. Initially, the level of ALA in small and large antral follicles was determined to be in a range of 0.018-0.028 mg/ml (64.6-100.6 μM, respectively). In vitro maturation was performed in the presence of various concentrations (10, 50, 100, or 200 μM) of ALA. Cumulus expansion, meiotic maturation, levels of intracellular glutathione (GSH), embryonic cleavage, blastocyst formation following parthenogenetic activation (PA) and in vitro fertilization (IVF), number of total and apoptotic cells in blastocyst, and expression of Bax, Bcl-2, and p53 genes in blastocyst cells were determined. Compared with the control, no improvement was observed in cumulus expansion in ALA-treated groups. At 50 μM concentration, ALA increased meiotic maturation rate but had no effect on GSH level. When oocytes treated with 50 μM ALA were subsequently used for PA or IVF, a higher rate of blastocyst formation was observed, and these embryos had a higher total cell number and a lower apoptotic cell number. Expression analyses of genes in blastocysts revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in 50 μM ALA group. Expression of p53 gene was also less observed in ALA-treated blastocysts. Our results show that ALA treatment at 50 μM during in vitro maturation (IVM) had a beneficial effect on maturation of goat oocytes and this, in turn, stimulated embryonic development and regulated apoptotic gene expression. PMID:26584822

  16. Sex Chromosome Meiotic Drive in Stalk-Eyed Flies

    PubMed Central

    Presgraves, D. C.; Severance, E.; Wilkinson, G. S.

    1997-01-01

    Meiotically driven sex chromosomes can quickly spread to fixation and cause population extinction unless balanced by selection or suppressed by genetic modifiers. We report results of genetic analyses that demonstrate that extreme female-biased sex ratios in two sister species of stalk-eyed flies, Cyrtodiopsis dalmanni and C. whitei, are due to a meiotic drive element on the X chromosome (X(d)). Relatively high frequencies of X(d) in C. dalmanni and C. whitei (13-17% and 29%, respectively) cause female-biased sex ratios in natural populations of both species. Sex ratio distortion is associated with spermatid degeneration in male carriers of X(d). Variation in sex ratios is caused by Y-linked and autosomal factors that decrease the intensity of meiotic drive. Y-linked polymorphism for resistance to drive exists in C. dalmanni in which a resistant Y chromosome reduces the intensity and reverses the direction of meiotic drive. When paired with X(d), modifying Y chromosomes (Y(m)) cause the transmission of predominantly Y-bearing sperm, and on average, production of 63% male progeny. The absence of sex ratio distortion in closely related monomorphic outgroup species suggests that this meiotic drive system may predate the origin of C. whitei and C. dalmanni. We discuss factors likely to be involved in the persistence of these sex-linked polymorphisms and consider the impact of X(d) on the operational sex ratio and the intensity of sexual selection in these extremely sexually dimorphic flies. PMID:9383060

  17. Immature oocyte quality and maturational competence of porcine cumulus-oocyte complexes subpopulations.

    PubMed

    Alvarez, Gabriel Martin; Dalvit, Gabriel Carlos; Achi, María Verónica; Miguez, Marcelo Sergio; Cetica, Pablo Daniel

    2009-12-01

    Porcine immature oocyte quality (i.e., that of live oocytes at the germinal vesicle stage) was evaluated according to features of the surrounding cumulus, aiming to establish maturational competence of different subpopulations of such cumulus-oocyte complexes. Six subpopulations were identified: A1 (with a dense cumulus), A2 (with a translucent cumulus), B1 (with the corona radiata), B2 (partly naked oocytes), C (naked oocytes), D (with a dark cumulus). The percent incidence of live oocyte in these subpopulations changed significantly as related to cumulus features, however the occurrence of oocytes in the germinal vesicle stage was lower in class D only. Similar metaphase II rates achieved in A1, A2, B1 and B2 classes after in vitro maturation suggest that the nucleus may in fact mature in vitro, in spite of the different accompanying cumulus features which are typical of these classes. In contrast, a higher cytoplasmic maturation rate obtained in class A may indicate a stronger dependence of this variable upon cumulus features than that shown by nuclear maturation. When different types of cumulus expansion after in vitro maturation were considered (i.e., fully expanded cumulus, partly expanded cumulus, and partly naked oocyte), no differences were found in the percent of oocytes reaching metaphase II or cytoplasmic maturation. It is concluded that morphological features of the collected porcine cumulus-oocyte complexes (rather than cumulus behavior during culture) may be useful for selection of potentially competent oocytes for in vitro fertilization and embryo production. PMID:20067032

  18. Effect of aspiration pressure during oocyte harvesting on oocyte recovery and in vitro development of ovine oocytes.

    PubMed

    Morton, K M; Maxwell, W M C; Evans, G

    2008-02-01

    Oocytes from abattoir-sourced ovine ovaries were aspirated from 2- to 4-mm follicles using 25, 50 or 100 mmHg pressure and an aspiration pump, or a needle (20-G) and syringe (2.5 ml) and subjected to in vitro maturation, fertilization and culture to determine the effect of aspiration pressure on the number and quality of oocytes recovered, and early embryonic development. Oocyte recovery rate was similar between groups (range: 57.1-73.1%; p > 0.05). The number and percentage of grade I and II oocytes recovered was reduced for 100 mm (24.5 +/- 3.6 and 31.1 +/- 6.1%) compared with 25 mm (51.4 +/- 7.0 and 60.2 +/- 7.8%) and 50 mm pressure (40.8 +/- 5.6 and 50.3 +/- 4.4%) and a syringe (40.3 +/- 12.0 and 45.2 +/- 2.1%; p < 0.05). Oocyte cleavage was similar for all groups at 24 (range: 30.9-49.6%) and 48 h post-insemination (49.7-65.5%), but blastocyst formation (% cleaved oocytes) was lower for oocytes aspirated with 25 mm (37.8%) than 50 (69.2%) or 100 mm (67.2%) pressure, and a syringe (72.0%; p < 0.05). Embryo production efficiency (% of oocytes cultured developing to the blastocyst stage) was higher for oocytes aspirated with 50 mm (45.4%) and 100 mm pressure (43.8%) and a syringe (45.0%) than 25 mm pressure (18.8%; p < 0.05). These results demonstrate that the aspiration of ovine oocytes with an aspiration pressure of 50 mm, or aspiration with a needle and syringe are equally efficacious for the in vitro production of embryos. PMID:18199266

  19. Alteration of energy metabolism gene expression in cumulus cells affects oocyte maturation via MOS-mitogen-activated protein kinase pathway in dairy cows with an unfavorable "Fertil-" haplotype of one female fertility quantitative trait locus.

    PubMed

    Brisard, Daphné; Desmarchais, Alice; Touzé, Jean-Luc; Lardic, Lionel; Freret, Sandrine; Elis, Sebastien; Nuttinck, Fabienne; Camous, Sylvaine; Dupont, Joelle; Uzbekova, Svetlana

    2014-03-01

    Prim'Holstein heifers selected for the "Fertil-" homozygous haplotype of QTL-Female-Fert ility-BTA3 showed a greater rate of early pregnancy failure and slower embryo development after IVM suggesting lower oocyte quality than those selected for "Fertile+". We aimed to ascertain intrafollicular factors related to lower oocyte quality in "Fertil-" cows. Analysis of individual oocytes showed meiotic progression delay in "Fertil-" compared with "Fertil+" dairy cows after in vivo maturation and IVM (P < 0.05). Expression of several genes localized to QTL-F-Fert-BTA3 or related to meiosis and mitogen-activated protein kinase pathway was analyzed in individual metaphase-II oocytes using reverse transcription- real-time polymerase chain reaction. Energy metabolism, apoptosis, extracellular matrix, and QTL-F-Fert-BTA3 genes were analyzed in surrounding cumulus cells (CC). In vivo, a significant decrease in prostaglandin synthase PTGES1 and PTGS2 expression coupled with lower PTGS2 protein abundance in CC and reduced expression of MOS in enclosed metaphase-II oocytes from "Fertil-" cows was observed. IVM strongly deregulated gene expression in CC and in oocytes compared with in vivo; nevertheless, differential expression of several genes including PEX19, NAMPT and MOS was observed between the two haplotypes. During IVM, PTGS2 activity inhibitor NS398 (50 μM) led to lower expression of fatty acid synthase (FASN) in CC and of MOS in treated metaphase-II oocytes. Using immunofluorescence, MOS protein was localized to a midbody-like contractile ring separating the polar body from the ooplasm, suggesting a role in the terminal stage of oocyte maturation. Our results suggest that factors involved in prostaglandin synthesis and lipid metabolism in CC could impair oocyte maturation, and might be involved in the reduced fertility of "Fertil-" cows. PMID:24377862

  20. The effects of season and moderate nutritional restriction on ovarian function and oocyte nuclear maturation in cycling gilts.

    PubMed

    Swinbourne, A M; Kelly, J M; Kind, K L; Kennaway, D J; van Wettere, W H E J

    2014-12-01

    The fertility of female pigs is impaired during summer and in response to restriction of feed intake, resulting in reduced productivity of the breeding herd. This study determined the effect of season and moderate nutritional restriction on ovarian function and oocyte developmental competence of cycling gilts. Eighty prepubescent gilts were used across two seasons-summer (S: January to March) and winter (W: June to August)-and received either a high (2.5× maintenance) or a moderately restricted (1.5× maintenance) feeding level for the first 19 days of their second estrous cycle. On Day 19, ovaries were collected post-slaughter. Diameters of all surface follicles over 1 mm were measured. All follicles ≥4 mm were aspirated and cumulus-oocyte complexes underwent in vitro maturation for ∼44 hours to assess oocyte developmental competence on the basis of metaphase II (MII) attainment. Moderate dietary nutrition reduced daily liveweight gain but did not affect the ovarian follicle population or oocyte developmental competence. The number of large follicles (≥6 mm) was lower during summer (S: 10.7 ± 1.74 vs. W: 15.5 ± 1.15, P < 0.05), as was the proportion of oocytes at the germinal vesicle stage of meiosis (S: 0.06 ± 0.02 vs. W: 0.08 ± 0.02, P < 0.05). However, the proportion of oocytes attaining MII was similar in summer and winter (S: 0.72 ± 0.04 and W: 0.69 ± 0.06, P > 0.05). Intrafollicular concentrations of luteinizing hormone were higher in summer (S: 43.05 ± 6.44 vs. W: 12.05 ± 5.12 ng/mL, P < 0.001), whereas estradiol was lower (S: 1.27 ± 0.36 vs. W: 27.52 ± 5.59 ng/mL, P < 0.001). In conclusion, our data demonstrated that in summer, follicle growth beyond 6 mm is impaired during the periovulatory period, without affecting oocyte meiotic competence. Importantly, these data also demonstrated that ovarian follicle growth and the capacity of oocytes to reach MII in vitro appear unaffected by moderate nutritional restriction during the preceding

  1. Meiosis-activating sterol promotes resumption of meiosis in mouse oocytes cultured in vitro in contrast to related oxysterols.

    PubMed

    Grøndahl, C; Ottesen, J L; Lessl, M; Faarup, P; Murray, A; Grønvald, F C; Hegele-Hartung, C; Ahnfelt-Rønne, I

    1998-05-01

    The sterol 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS [follicular-fluid meiosis-activating sterol]) from human follicular fluid has recently been identified as a compound that induces the resumption of meiosis. FF-MAS and various oxysterols have been reported to transactivate the orphan receptor LXRalpha. The objective was to determine the biological activity of synthetic FF-MAS on the resumption of meiosis and final maturation of mouse oocytes in vitro. In order to evaluate whether LXRalpha might mediate FF-MAS action on the oocyte, we compared the capability of various compounds to activate LXRalpha-dependent transcription and to induce resumption of meiosis in the oocyte assay. Ovaries were isolated from immature mice primed with FSH 48 h before collection. Naked oocytes (NkO) and cumulus enclosed oocytes (CEO) were isolated from follicles. The oocytes were cultured in two groups, NkO and CEO, respectively, in media containing either 3 mM hypoxanthine, 5 microM IBMX, or 0.100 mM dbcAMP to maintain the oocytes in the germinal vesicle stage. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) after 24 h of in vitro culture. FF-MAS overcame the meiotic inhibition by hypoxanthine in both the NkO group and CEO group in a dose-dependent manner within the concentration range 0.07-7 microM. FF-MAS displayed similar potency in all inhibitory agents used. Also, FF-MAS significantly increased the formation of polar bodies in both the CEO and NkO group. The oxysterols 22(R)-hydroxycholesterol (a potent ligand for the LXRalpha receptor), 16-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, as well as cholesterol, were tested without any significant effect on maturation compared to that of controls. Oxysterols and FF-MAS were observed to activate LXRalpha. In conclusion, the results reported here clearly demonstrate that synthetic FF-MAS exclusively is capable of mediating resumption of meiosis in

  2. Increasing age influences uterine integrity, but not ovarian function or oocyte quality, in the cheetah (Acinonyx jubatus).

    PubMed

    Crosier, Adrienne E; Comizzoli, Pierre; Baker, Tom; Davidson, Autumn; Munson, Linda; Howard, JoGayle; Marker, Laurie L; Wildt, David E

    2011-08-01

    Although the cheetah (Acinonyx jubatus) routinely lives for more than 12 yr in ex situ collections, females older than 8 yr reproduce infrequently. We tested the hypothesis that reproduction is compromised in older female cheetahs due to a combination of disrupted gonadal, oocyte, and uterine function/integrity. Specifically, we assessed 1) ovarian response to gonadotropins; 2) oocyte meiotic, fertilization, and developmental competence; and 3) uterine morphology in three age classes of cheetahs (young, 2-5 yr, n = 17; prime, 6-8 yr, n = 8; older, 9-15 yr, n = 9). Ovarian activity was stimulated with a combination of equine chorionic gonadotropin and human chorionic gonadotropin (hCG), and fecal samples were collected for 45 days before gonadotropin treatment and for 30 days after oocyte recovery by laparoscopy. Twenty-six to thirty hours post-hCG, uterine morphology was examined by ultrasound, ovarian follicular size determined by laparoscopy, and aspirated oocytes assessed for nuclear status or inseminated in vitro. Although no influence of age on fecal hormone concentrations or gross uterine morphology was found (P > 0.05), older females produced fewer (P < 0.05) total antral follicles and oocytes compared to younger counterparts. Regardless of donor age, oocytes had equivalent (P > 0.05) nuclear status and ability to reach metaphase II and fertilize in vitro. A histological assessment of voucher specimens revealed an age-related influence on uterine tissue integrity, with more than 87% and more than 56% of older females experiencing endometrial hyperplasia and severe pathologies, respectively. Our collective findings reveal that lower reproductive success in older cheetahs appears to be minimally influenced by ovarian and gamete aging and subsequent dysfunction. Rather, ovaries from older females are responsive to gonadotropins, produce normative estradiol/progestogen concentrations, and develop follicles containing oocytes with the capacity to mature and be

  3. Effects of Escherichia coli- and Staphylococcus aureus-induced mastitis in lactating cows on oocyte developmental competence.

    PubMed

    Asaf, S; Leitner, G; Furman, O; Lavon, Y; Kalo, D; Wolfenson, D; Roth, Z

    2014-01-01

    Mastitis is associated with decreased fertility in dairy cows. In the current study, we created an experimental model to simulate short-term mastitis by a single intramammary administration of Gram-negative endotoxin of Escherichia coli origin (G-), or Gram-positive toxin of Staphylococcus aureus origin (G+), to examine the effect of mastitis on oocyte developmental competence. Healthy Holstein cows were synchronized, and follicular fluid (FF) of cows treated with G+ or G- and of uninfected cows (controls) was aspirated from the preovulatory follicles by transvaginal ultrasound procedure. The aspirated FF was used as maturation medium for in vitro embryo production. The distribution of matured oocytes into different cortical granule classes and meiotic stages was affected by G- administration (P<0.05) but not by G+ administration. The proportion of oocytes that cleaved to two- and four-cell stage embryos (44 h postfertilization) was lower in both G+ and G- groups than in controls (P<0.05). Blastocyst formation rate (7-8 days postfertilization) was lower in the G- group (P<0.05) and numerically lower in the G+ group compared with their uninfected counterparts. The total cell number in blastocysts did not differ among groups; however, the apoptotic index was higher in the G+ group (P<0.05), but not in the G- group, relative to controls. Examining mRNA relative abundance in oo