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Sample records for oocyte meiotic arrest

  1. Cytoplasm replacement following germinal vesicle transfer restores meiotic maturation and spindle assembly in meiotically arrested oocytes.

    PubMed

    Zhang, John; Liu, Hui

    2015-07-01

    Both the cytoplasmic and nuclear compartments are essential for the acquisition of meiotic competence. This study assessed the role of the cytoplasm in meiosis resumption in meiotically arrested oocytes at the germinal vesicle (GV) stage. Mouse oocytes at GV stage were meiotically arrested with 3-isobutyl-1-methylxanthine (IBMX). GV transfer was performed between IBMX-treated and non-treated (control) mouse oocytes, and between control mouse and human GV oocytes. Extrusion of first polar body (PB) was examined as an indication of nuclear maturation. Meiotic spindle assembly and chromosome alignment were examined by immunostaining. Results indicated that oocytes arrested with IBMX for 24 and 48 h exhibited reduced ability for meiotic maturation and for extruding the first PB when compared with controls (P < 0.01). IBMX-treated oocytes reconstituted with cytoplasm, but not GV, of control oocytes restored the assembly of meiotic spindle and meiotic maturation. Mouse oocytes reconstituted with GV of human oocytes underwent meiosis similar to that observed in mice, but not humans. Additionally, human oocytes reconstituted by mouse GV underwent meiosis similar to that observed in humans, but not mice. These findings suggest that cytoplasm replacement by GV transfer could represent a potential therapeutic option for women who do not produce mature oocytes during IVF. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  2. Mutations in TUBB8 and Human Oocyte Meiotic Arrest

    DOE PAGES

    Feng, Ruizhi; Sang, Qing; Kuang, Yanping; ...

    2016-01-21

    BACKGROUND: We present that 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, earlymore » 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. RESULTSL: 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: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.« less

  3. Mutations in TUBB8 and Human Oocyte Meiotic Arrest

    SciTech Connect

    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: We present that 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. RESULTSL: 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: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.

  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.

  5. Role of G-protein-coupled estrogen receptor (GPER/GPR30) in maintenance of meiotic arrest in fish oocytes.

    PubMed

    Thomas, Peter

    2017-03-01

    An essential role for GPER (formerly known as GPR30) in regulating mammalian reproduction has not been identified to date, although it has shown to be involved in the regulation a broad range of other estrogen-dependent functions. In contrast, an important reproductive role for GPER in the maintenance of oocyte meiotic arrest has been identified in teleost fishes, which is briefly reviewed here. Recent studies have clearly shown that ovarian follicle production of estradiol-17β (E2) maintains meiotic arrest in several teleost species through activation of GPER coupled to a stimulatory G protein (Gs) on oocyte plasma membranes resulting in stimulation of cAMP production and maintenance of elevated cAMP levels. Studies with denuded zebrafish oocytes and with microinjection of GPER antisense oligonucleotides into oocytes have demonstrated the requirement for both ovarian follicle production of estrogens and expression of GPER on the oocyte surface for maintenance of meiotic arrest. This inhibitory action of E2 on the resumption of meiosis is mimicked by the GPER-selective agonist G-1, by the GPER agonists and nuclear ER antagonists, ICI 182,780 and tamoxifen, and also by the xenoestrogen bisphenol-A (BPA) and related alkylphenols. GPER also maintains meiotic arrest of zebrafish oocytes through estrogen- and BPA-dependent GPER activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (MAPK) signaling. Interestingly, progesterone receptor component 1 (PGRMC1) is also involved in estrogen maintenance of meiotic arrest through regulation of EGFR expression on the oocyte plasma membrane. The preovulatory surge in LH secretion induces the ovarian synthesis of progestin hormones that activate a membrane progestin receptor alpha (mPRα)/inhibitory G protein (Gi) pathway. It also increases ovarian synthesis of the catecholestrogen, 2-hydroxy-estradiol-17β (2-OHE2) which inhibits the GPER/Gs/adenylyl cyclase pathway. Both of these LH actions

  6. Functional heterologous gap junctions in Fundulus ovarian follicles maintain meiotic arrest and permit hydration during oocyte maturation.

    PubMed

    Cerdá, J L; Petrino, T R; Wallace, R A

    1993-11-01

    The physiological significance of heterologous gap junctions between granulosa cells and the oocyte was investigated in late vitellogenic ovarian follicles of the teleost Fundulus heteroclitus. Lucifer Yellow injected into the oocyte readily passed to the overlying granulosa cells, demonstrating effective dye-coupling. Passage of the fluorescent dye, and hence intercellular communication, was inhibited both by the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and by 1-octanol, known uncouplers of gap junctions in a variety of invertebrate and vertebrate cell types. Octanol alone also initiated resumption of meiosis in follicle-enclosed oocytes, indicating that granulosa cells normally maintain meiotic arrest, as apparently occurs in mammalian and amphibian follicles. Both PMA and octanol also consistently inhibited the hydration process that normally accompanies meiotic maturation. These results support a previously suggested hypothesis that K+, which is the primary osmotic effector for oocyte hydration, is translocated via gap junction from granulosa cells to the maturing oocyte.

  7. A Gs-linked receptor maintains meiotic arrest in mouse oocytes, but luteinizing hormone does not cause meiotic resumption by terminating receptor-Gs signaling

    PubMed Central

    Norris, Rachael P.; Freudzon, Leon; Freudzon, Marina; Hand, Arthur R.; Mehlmann, Lisa M.; Jaffe, Laurinda A.

    2008-01-01

    The maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on the activity of a Gs G-protein that activates adenylyl cyclase and elevates cAMP, and in the mouse oocyte, Gs is activated by a constitutively active orphan receptor, GPR3. To determine whether the action of luteinizing hormone (LH) on the mouse ovarian follicle causes meiotic resumption by inhibiting GPR3-Gs signaling, we examined the effect of LH on the localization of Gαs. Gs activation in response to stimulation of an exogenously expressed β2-adrenergic receptor causes Gαs to move from the oocyte plasma membrane into the cytoplasm, whereas Gs inactivation in response to inhibition of the β2-adrenergic receptor causes Gαs to move back to the plasma membrane. However, LH does not cause a change in Gαs localization, indicating that LH does not act by terminating receptor-Gs signaling. PMID:17850783

  8. In vitro and in vivo studies reveal that hamster oocyte meiotic arrest is maintained only transiently by follicular fluid, but persistently by membrana/cumulus granulosa cell contact.

    PubMed

    Racowsky, C; Baldwin, K V

    1989-08-01

    Studies were carried out with the golden Syrian hamster to investigate the capacity of follicular fluid to maintain oocyte meiotic arrest and to determine the importance of cumulus-membrana granulosa cell contact in the regulation of meiotic status. The follicular fluid studies were conducted by cytological assessment of meiotic stage up to 6 hr after transferring cumulus-free oocytes into antra of explanted "host" follicles in vitro or into follicles of anesthetized animals prior to the gonadotropin surge at proestrus in vivo. The cumulus-membrana granulosa contact studies were undertaken with explanted follicles in which the oocyte-cumulus complex was dislodged from the underlying membrana granulosa, released into the antrum, and subsequently allowed to reestablish contact during 6 hr of incubation within the follicle. The extent of recontact of the dislodged complex with the underlying membrana granulosa was assessed visually at the end of incubation and was classified as close, moderate, or none. These various degrees of contact typically involved the following number of cumulus cells, as determined by serial sectioning of a representative sample of follicles after dislodgement and subsequent incubation: close, 32.7 +/- 1.78; moderate, 9.0 +/- 2.1; and no contact, 0. After 6 hr of incubation either in vitro or in vivo, few transferred oocytes remained at the germinal vesicle (GV) stage (18.8 +/- 8.7 and 17.3 +/- 4.0% GV, respectively). However, time course experiments revealed that meiotic resumption was significantly delayed in transferred oocytes compared with either liberated oocytes, spontaneously maturing oocytes, or follicle-enclosed oocytes induced to mature by luteinizing hormone in vitro (after 4 hr, transferred, 31.3 +/- 6.0% GV; liberated, 0% GV; follicle-enclosed, 0% GV; after 6 hr, 0% transferred oocytes exhibited a GV). In the dislodgement studies, after 6 hr of incubation, 26% of complexes reestablished close contact with the underlying membrana

  9. A pre-in vitro maturation medium containing cumulus oocyte complex ligand-receptor signaling molecules maintains meiotic arrest, supports the cumulus oocyte complex and improves oocyte developmental competence.

    PubMed

    Santiquet, Nicolas W; Greene, Alison F; Becker, John; Barfield, Jennifer P; Schoolcraft, William B; Krisher, Rebecca L

    2017-09-01

    Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following

  10. NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation during follicular development in the mouse ovary.

    PubMed

    Kiyosu, Chiyo; Tsuji, Takehito; Yamada, Kaoru; Kajita, Shimpei; Kunieda, Tetsuo

    2012-08-01

    Natriuretic peptide type C (NPPC) and its high affinity receptor, natriuretic peptide receptor 2 (NPR2), have been assumed to be involved in female reproduction and have recently been shown to play an essential role in maintaining meiotic arrest of oocytes. However, the overall role of NPPC/NPR2 signaling in female reproduction and ovarian function is still less clear. Here we report the defects observed in oocytes and follicles of mice homozygous for Nppc(lbab) or Npr2(cn), mutant alleles of Nppc or Npr2 respectively to clarify the exact consequences of lack of NPPC/NPR2 signaling in female reproductive systems. We found that: i) Npr2(cn)/Npr2(cn) female mice ovulated a comparable number of oocytes as normal mice but never produced a litter; ii) all ovulated oocytes of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice exhibited abnormalities, such as fragmented or degenerated ooplasm and never developed to the two-cell stage after fertilization; iii) histological examination of the ovaries of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice showed that oocytes in antral follicles prematurely resumed meiosis and that immediately before ovulation, oocytes showed disorganized chromosomes or fragmented ooplasm; and iv) ovulated oocytes and oocytes in the periovulatory follicles of the mutant mice were devoid of cumulus cells. These findings demonstrate that NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation, which affects female fertility through the production of oocytes with developmental capacity.

  11. Effects of sperm insemination on the final meiotic maturation of mouse oocytes arrested at metaphase I after in vitro maturation.

    PubMed

    Yoon, Jeong; Juhn, Kyoung-Mi; Yoon, San-Hyun; Ko, Yong; Lim, Jin-Ho

    2017-03-01

    The aims of this study were to investigate whether fertilization could induce the resumption of meiosis in mouse oocytes arrested at metaphase I (MI) after in vitro maturation (IVM), and to investigate the effect of Ca(2)(+) chelator treatment at the time of fertilization on the transition from MI to metaphase II (MII). MII-stage and arrested MI-stage mouse oocytes after IVM were fertilized, and then embryonic development was monitored. Blastocysts from each group were transferred into 2.5 days post-coitum pseudo-pregnant ICR mice. MI oocytes after IVM were treated with a Ca(2)(+) chelator to investigate the effect of Ca(2)(+) oscillations on their maturation. As insemination time increased, the number of oocytes in the MI group that reached the MII stage also increased. The blastocyst rates and total cell numbers in the MII group were significantly higher than in the MI group. No pregnancy occurred in the MI group, but 10 pregnancies were achieved (10 of 12) in the MII group. The proportion of MI oocytes that matured to MII oocytes after fertilization was significantly higher in the non-treated group than in the Ca(2)(+) chelator-treated group. The findings that a higher proportion of MI-arrested oocytes progressed to MII after fertilization and that the MI-to-MII transition was blocked by Ca(2)(+) chelator treatments before fertilization indicate that the maturation of MI oocytes to MII oocytes is associated with intracellular Ca(2)(+) oscillations driven by fertilization.

  12. Meiotic arrest with roscovitine and follicular fluid improves cytoplasmic maturation of porcine oocytes by promoting chromatin de-condensation and gene transcription.

    PubMed

    Zhang, Min; Zhang, Chuan-Xin; Pan, Liu-Zhu; Gong, Shuai; Cui, Wei; Yuan, Hong-Jie; Zhang, Wei-Ling; Tan, Jing-He

    2017-09-14

    The developmental capacity of in vitro matured oocytes is inferior to that of the in vivo matured ones due to insufficient cytoplasmic maturation. Although great efforts were made to accomplish better cytoplasmic maturation by meiotic arrest maintenance (MAM) before in vitro maturation (IVM), limited progress has been achieved in various species. This study showed that MAM of porcine oocytes was better achieved with roscovitine than with dibutyryl cyclic adenosine monophosphate (db-cAMP) or 3-isobutyl-1-methylxanthine. Oocyte developmental competence after IVM was significantly improved following MAM in 199 + FF medium (TCM-199 containing 10% porcine follicular fluid and 25 µM roscovitine) to a level even higher than that in control oocytes matured without pre-MAM. Observations on other markers further confirmed the positive effects of MAM in 199 + FF on oocyte cytoplasmic maturation. During MAM culture in 199 + FF, re-decondensation (RDC) of condensed chromatin occurred, and transcription of genes beneficial to cytoplasmic maturation was evident in some of the oocytes with surrounded nucleoli (SN). However, MAM with db-cAMP neither induced RDC nor improved oocyte developmental potential. Together, the results suggest that MAM in the presence of FF and roscovitine improved the developmental competence of porcine oocytes by promoting a pre-GVBD chromatin de-condensation and expression of beneficial genes.

  13. Effects of sperm insemination on the final meiotic maturation of mouse oocytes arrested at metaphase I after in vitro maturation

    PubMed Central

    Yoon, Jeong; Juhn, Kyoung-Mi; Yoon, San-Hyun; Lim, Jin-Ho

    2017-01-01

    Objective The aims of this study were to investigate whether fertilization could induce the resumption of meiosis in mouse oocytes arrested at metaphase I (MI) after in vitro maturation (IVM), and to investigate the effect of Ca2+ chelator treatment at the time of fertilization on the transition from MI to metaphase II (MII). Methods MII-stage and arrested MI-stage mouse oocytes after IVM were fertilized, and then embryonic development was monitored. Blastocysts from each group were transferred into 2.5 days post-coitum pseudo-pregnant ICR mice. MI oocytes after IVM were treated with a Ca2+ chelator to investigate the effect of Ca2+ oscillations on their maturation. Results As insemination time increased, the number of oocytes in the MI group that reached the MII stage also increased. The blastocyst rates and total cell numbers in the MII group were significantly higher than in the MI group. No pregnancy occurred in the MI group, but 10 pregnancies were achieved (10 of 12) in the MII group. The proportion of MI oocytes that matured to MII oocytes after fertilization was significantly higher in the non-treated group than in the Ca2+ chelator-treated group. Conclusion The findings that a higher proportion of MI-arrested oocytes progressed to MII after fertilization and that the MI-to-MII transition was blocked by Ca2+ chelator treatments before fertilization indicate that the maturation of MI oocytes to MII oocytes is associated with intracellular Ca2+ oscillations driven by fertilization. PMID:28428939

  14. 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

  15. 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.

  16. Oocyte nucleus controls progression through meiotic maturation.

    PubMed

    Polanski, Zbigniew; Hoffmann, Steffen; Tsurumi, Chizuko

    2005-05-15

    We analyzed progression through the meiotic maturation in oocytes manipulated to replace the prophase oocyte nucleus with the nucleus from a cumulus cell, a pachytene spermatocyte or the pronucleus from a fertilized egg. Removal of the oocyte nucleus led to a significant reduction in histone H1 kinase activity. Replacement of the oocyte nucleus by a pronucleus followed by culture resulted in premature pseudomeiotic division and occasional abnormal cytokinesis; however, histone H1 kinase activity was rescued, microtubules formed a bipolar spindle, and chromosomes were condensed. In addition to the anomalies observed after pronuclear transfer, those after transfer of the nucleus from a cumulus cell or spermatocyte included a dramatically impaired ability to form the bipolar spindle or to condense chromosomes, and histone H1 kinase activity was not rescued. Expression of a cyclin B-YFP in enucleated oocytes receiving the cumulus cell nucleus rescued histone H1 kinase activity, but spindle formation and chromosome condensation remained impaired, indicating a pleiotropic effect of oocyte nucleus removal. However, when the cumulus cell nucleus was first transformed into pronuclei (transfer into a metaphase II oocyte followed by activation), such pronuclei supported maturation after transfer into the oocyte in a manner similar to that of normal pronuclei. These results show that the oocyte nucleus contains specific components required for the control of progression through the meiotic maturation and that some of these components are also present in pronuclei.

  17. Calcium Signaling During Meiotic Cell Cycle Regulation and Apoptosis in Mammalian Oocytes.

    PubMed

    Tiwari, Meenakshi; Prasad, Shilpa; Shrivastav, Tulsidas G; Chaube, Shail K

    2017-05-01

    Calcium (Ca(++) ) is one of the major signal molecules that regulate various aspects of cell functions including cell cycle progression, arrest, and apoptosis in wide variety of cells. This review summarizes current knowledge on the differential roles of Ca(++) in meiotic cell cycle resumption, arrest, and apoptosis in mammalian oocytes. Release of Ca(++) from internal stores and/or Ca(++) influx from extracellular medium causes moderate increase of intracellular Ca(++) ([Ca(++) ]i) level and reactive oxygen species (ROS). Increase of Ca(++) as well as ROS levels under physiological range trigger maturation promoting factor (MPF) destabilization, thereby meiotic resumption from diplotene as well as metaphase-II (M-II) arrest in oocytes. A sustained increase of [Ca(++) ]i level beyond physiological range induces generation of ROS sufficient enough to cause oxidative stress (OS) in aging oocytes. The increased [Ca(++) ]i triggers Fas ligand-mediated oocyte apoptosis. Further, OS triggers mitochondria-mediated oocyte apoptosis in several mammalian species. Thus, Ca(++) exerts differential roles on oocyte physiology depending upon its intracellular concentration. A moderate increase of [Ca(++) ]i as well as ROS mediate spontaneous resumption of meiosis from diplotene as well as M-II arrest, while their high levels cause meiotic cell cycle arrest and apoptosis by operating both mitochondria- as well as Fas ligand-mediated apoptotic pathways. Indeed, Ca(++) regulates cellular physiology by modulating meiotic cell cycle and apoptosis in mammalian oocytes. J. Cell. Physiol. 232: 976-981, 2017. © 2016 Wiley Periodicals, Inc.

  18. Estrogen receptors in granulosa cells govern meiotic resumption of pre-ovulatory oocytes in mammals.

    PubMed

    Liu, Wei; Xin, Qiliang; Wang, Xiao; Wang, Sheng; Wang, Huarong; Zhang, Wenqiang; Yang, Ye; Zhang, Yanhao; Zhang, Zhiyuan; Wang, Chao; Xu, Yang; Duan, Enkui; Xia, Guoliang

    2017-03-09

    In mammals, oocytes are arrested at the diplotene stage of meiosis I until the pre-ovulatory luteinizing hormone (LH) surge triggers meiotic resumption through the signals in follicular granulosa cells. In this study, we show that the estradiol (E2)-estrogen receptors (ERs) system in follicular granulosa cells has a dominant role in controlling oocyte meiotic resumption in mammals. We found that the expression of ERs was controlled by gonadotropins under physiological conditions. E2-ERs system was functional in maintaining oocyte meiotic arrest by regulating the expression of natriuretic peptide C and natriuretic peptide receptor 2 (NPPC/NPR2), which was achieved through binding to the promoter regions of Nppc and Npr2 genes directly. In ER knockout mice, meiotic arrest was not sustained by E2 in most cumulus-oocyte complexes in vitro and meiosis resumed precociously in pre-ovulatory follicles in vivo. In human granulosa cells, similar conclusions are reached that ER levels were controlled by gonadotropins and E2-ERs regulated the expression of NPPC/NPR2 levels. In addition, our results revealed that the different regulating patterns of follicle-stimulating hormone and LH on ER levels in vivo versus in vitro determined their distinct actions on oocyte maturation. Taken together, these findings suggest a critical role of E2-ERs system during oocyte meiotic progression and may propose a novel approach for oocyte in vitro maturation treatment in clinical practice.

  19. 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

  20. Cyclic AMP in oocytes controls meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary.

    PubMed

    Wang, Yijing; Teng, Zhen; Li, Ge; Mu, Xinyi; Wang, Zhengpin; Feng, Lizhao; Niu, Wanbao; Huang, Kun; Xiang, Xi; Wang, Chao; Zhang, Hua; Xia, Guoliang

    2015-01-15

    In mammalian ovaries, a fixed population of primordial follicles forms during the perinatal stage and the oocytes contained within are arrested at the dictyate stage of meiotic prophase I. In the current study, we provide evidence that the level of cyclic AMP (cAMP) in oocytes regulates oocyte meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary. Our results show that the early meiotic development of oocytes is closely correlated with increased levels of intra-oocyte cAMP. Inhibiting cAMP synthesis in fetal ovaries delayed oocyte meiotic progression and inhibited the disassembly and degradation of synaptonemal complex protein 1. In addition, inhibiting cAMP synthesis in in vitro cultured fetal ovaries prevented primordial follicle formation. Finally, using an in situ oocyte chromosome analysis approach, we found that the dictyate arrest of oocytes is essential for primordial follicle formation under physiological conditions. Taken together, these results suggest a role for cAMP in early meiotic development and primordial follicle formation in the mouse ovary. © 2015. Published by The Company of Biologists Ltd.

  1. Natriuretic peptides stimulate oocyte meiotic resumption in bovine.

    PubMed

    De Cesaro, Matheus P; Macedo, Mariana P; Santos, Joabel T; Rosa, Paulo R A; Ludke, Charles A; Rissi, Vitor B; Gasperin, Bernardo G; Gonçalves, Paulo B D

    2015-08-01

    The aim of the present study was to evaluate the expression of mRNA encoding natriuretic peptides (NPs) and their receptors in the cumulus-oocyte complex in cattle, a monovular mammalian species, and also to investigate the role of NPs in oocyte meiotic resumption in vitro. mRNA was observed for the NP precursor type-A (NPPA), type-C (NPPC), NP receptor-1 (NPR-1), receptor-2 (NPR-2) and receptor-3 (NPR-3) in bovine cumulus cells, and NPR-2 mRNA was observed in oocytes. These results are different from those obtained in mouse and pig models. The effects of NPPA, NP precursor type-B (NPPB) and NPPC on the resumption of arrested meiosis maintained by forskolin were studied at three different doses (10, 100 and 1000nM) with a 12h culture system. The germinal vesicle breakdown rates were greater (P≤0.05) in oocytes that were cultured in the presence of one or a combination of NPs (from 44% to 73%) than the negative control (from 24% to 27%). Additionally, it was demonstrated that the concentration of cyclic guanosine 3',5'-monophosphate (cGMP) is increased by NPPA and NPPC in oocytes and cumulus cells after 3h of in vitro maturation. However, in both groups, the concentration of cyclic adenosine 3',5'-monophosphate (cAMP) in the oocyte did not increase between 3 and 6h of culture, even when forskolin was used. In summary, we observed the presence of mRNA for NPs and their receptors in the bovine cumulus-oocyte complex and demonstrated that, in vitro, NPPA, NPPB and NPPC stimulate oocyte meiotic resumption in a monovular species.

  2. Acetyl CoA carboxylase inactivation and meiotic maturation in mouse oocytes.

    PubMed

    Valsangkar, Deepa S; Downs, Stephen M

    2015-09-01

    In mouse oocytes, meiotic induction by pharmacological activation of PRKA (adenosine monophosphate-activated protein kinase; formerly known as AMPK) or by hormones depends on stimulation of fatty acid oxidation (FAO). PRKA stimulates FAO by phosphorylating and inactivating acetyl CoA carboxylase (ACAC; formerly ACC), leading to decreased malonyl CoA levels and augmenting fatty-acid transport into mitochondria. We investigated a role for ACAC inactivation in meiotic resumption by testing the effect of two ACAC inhibitors, CP-640186 and Soraphen A, on mouse oocytes maintained in meiotic arrest in vitro. These inhibitors significantly stimulated the resumption of meiosis in arrested cumulus cell-enclosed oocytes, denuded oocytes, and follicle-enclosed oocytes. This stimulation was accompanied by an increase in FAO. Etomoxir, a malonyl CoA analogue, prevented meiotic resumption as well as the increase in FAO induced by ACAC inhibition. Citrate, an ACAC activator, and CBM-301106, an inhibitor of malonyl CoA decarboxylase, which converts malonyl CoA to acetyl CoA, suppressed both meiotic induction and FAO induced by follicle-stimulating hormone, presumably by maintaining elevated malonyl CoA levels. Mouse oocyte-cumulus cell complexes contain both isoforms of ACAC (ACACA and ACACB); when wild-type and Acacb(-/-) oocytes characteristics were compared, we found that these single-knockout oocytes showed a significantly higher FAO level and a reduced ability to maintain meiotic arrest, resulting in higher rates of germinal vesicle breakdown. Collectively, these data support the model that ACAC inactivation contributes to the maturation-promoting activity of PRKA through stimulation of FAO.

  3. G beta gamma signaling reduces intracellular cAMP to promote meiotic progression in mouse oocytes.

    PubMed

    Gill, Arvind; Hammes, Stephen R

    2007-02-01

    In nearly every vertebrate species, elevated intracellular cAMP maintains oocytes in prophase I of meiosis. Prior to ovulation, gonadotropins trigger various intra-ovarian processes, including the breakdown of gap junctions, the activation of EGF receptors, and the secretion of steroids. These events in turn decrease intracellular cAMP levels in select oocytes to allow meiotic progression, or maturation, to resume. Studies suggest that cAMP levels are kept elevated in resting oocytes by constitutive G protein signaling, and that the drop in intracellular cAMP that accompanies maturation may be due in part to attenuation of this inhibitory G protein-mediated signaling. Interestingly, one of these G protein regulators of meiotic arrest is the Galpha(s) protein, which stimulates adenylyl cyclase to raise intracellular cAMP in two important animal models of oocyte development: Xenopus leavis frogs and mice. In addition to G(alpha)(s), constitutive Gbetagamma activity similarly stimulates adenylyl cyclase to raise cAMP and prevent maturation in Xenopus oocytes; however, the role of Gbetagamma in regulating meiosis in mouse oocytes has not been examined. Here we show that Gbetagamma does not contribute to the maintenance of murine oocyte meiotic arrest. In fact, contrary to observations in frog oocytes, Gbetagamma signaling in mouse oocytes reduces cAMP and promotes oocyte maturation, suggesting that Gbetagamma might in fact play a positive role in promoting oocyte maturation. These observations emphasize that, while many general concepts and components of meiotic regulation are conserved from frogs to mice, specific differences exist that may lead to important insights regarding ovarian development in vertebrates.

  4. The Role of RING Box Protein 1 in Mouse Oocyte Meiotic Maturation

    PubMed Central

    Zhou, Lin; Yang, Ye; Zhang, Juanjuan; Guo, Xuejiang; Bi, Ye; Li, Xin; Zhang, Ping; Zhang, Junqiang; Lin, Min; Zhou, Zuomin; Shen, Rong; Guo, Xirong; Huo, Ran; Ling, Xiufeng; Sha, Jiahao

    2013-01-01

    RING box protein-1 (RBX1) is an essential component of Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase and participates in diverse cellular processes by targeting various substrates for degradation. However, the physiological function of RBX1 in mouse oocyte maturation remains unknown. Here, we examined the expression, localization and function of RBX1 during mouse oocyte meiotic maturation. Immunofluorescence analysis showed that RBX1 displayed dynamic distribution during the maturation process: it localized around and migrated along with the spindle and condensed chromosomes. Rbx1 knockdown with the appropriate siRNAs led to a decreased rate of first polar body extrusion and most oocytes were arrested at metaphase I. Moreover, downregulation of Rbx1 caused accumulation of Emi1, an inhibitor of the anaphase-promoting complex/cyclosome (APC/C), which is required for mouse meiotic maturation. In addition, we found apparently increased expression of the homologue disjunction-associated protein securin and cyclin B1, which are substrates of APC/C E3 ligase and need to be degraded for meiotic progression. These results indicate the essential role of the SCFβTrCP-EMI1-APC/C axis in mouse oocyte meiotic maturation. In conclusion, we provide evidence for the indispensable role of RBX1 in mouse oocyte meiotic maturation. PMID:23874827

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

    SciTech Connect

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

    1987-04-01

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

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

    PubMed

    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.

  7. 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

  8. 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

  9. 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.

  10. Effect of the meiotic inhibitor cilostamide on resumption of meiosis and cytoskeletal distribution in buffalo oocytes.

    PubMed

    Li, Qing-Yang; Lou, Juan; Yang, Xiao-Gan; Lu, Yang-Qing; Lu, Sheng-Sheng; Lu, Ke-Huan

    2016-11-01

    Improving the quality of in vitro maturated buffalo oocytes is essential for embryo production. We report here the effects on microtubules and microfilaments in oocytes and embryo development that result from treating buffalo oocytes with the phosphodiesterase 3 (PDE3) inhibitor cilostamide. Addition of 20μM or 50μM cilostamide for 24h during in vitro maturation showed no differences in the percentage of oocytes arrested at the germinal vesicle (GV) stage. When 20μM cilostamide was added to the pre-maturation culture for 6h, 12h or 24h and continued for another 24h without cilostamide, oocytes resumed meiosis, but with significantly lower (P<0.01) maturation rates in the 24h group than that in the other two groups. During oocyte maturation in vitro, no microtubules were detected before GV breakdown (GVBD). After GVBD, microtubules combined with condensed chromatin to form the meiotic metaphase spindle. Microfilaments covered a thick area around the cellular cortex and overlying chromosomes. Cilostamide had no effects on microtubules and microfilaments in metaphase II oocytes, and there were no significant differences in the rates of cleavage, blastocyst formation and number of blastocyst cells between oocytes treated pre-maturation with inhibitor for 6h and those of the control group (P>0.05). In summary, cilostamide reversibly arrested the resumption of meiosis without any adverse impact on the dynamic changes in microtubules and microfilaments in buffalo oocytes and their in vitro developmental capacity.

  11. 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

  12. 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

    2015-12-02

    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.

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

    PubMed

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

    2011-10-01

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

  14. 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.

  15. Zinc deficiency during in vitro maturation of porcine oocytes causes meiotic block and developmental failure.

    PubMed

    Jeon, Yubyeol; Yoon, Junchul David; Cai, Lian; Hwang, Seon-Ung; Kim, Eunhye; Zheng, Zhong; Jeung, Euibae; Lee, Eunsong; Hyun, Sang-Hwan

    2015-10-01

    The present study investigated the effects of zinc deficiency during in vitro maturation (IVM) of porcine oocytes. Zinc deficiency was induced by administering the membrane‑permeable zinc chelator N,N,N',N'‑tetrakis‑(2‑pyridylmethyl)‑ethylendiamine (TPEN). First, the effects of zinc deficiency during IVM on a TPEN‑treated group and a TPEN+zinc-treated group compared with a control group were assessed. The oocyte maturation rates and subsequent embryonic developmental competence of the TPEN+zinc‑treated oocytes were similar to those of the control oocytes (metaphase II [MII] rate, 93.0 and 92.7%, respectively, and blastocyst [BL] formation rate, 42.0 and 40.0%, respectively). These results were significantly different from those obtained for the TPEN‑treated oocytes (MII rate, 0.61%; BL formation rate, 0%). Although the TPEN‑treated oocytes were arrested at metaphase I (MI), the distribution of microtubules was normal. However, microfilament formation was abnormal in the TPEN‑treated oocytes. Furthermore, the effect of a temporary zinc deficiency during IVM on oocyte maturation and subsequent embryonic development was assessed. TPEN (10 µM) was added to the IVM medium for 0, 7, 15 or 22 h. The 0 h‑treated oocytes showed an 83.9% MII rate, while the 7 h‑treated oocytes had a significantly lower MII rate (44.8%). Most of the 15- and 22 h‑treated oocytes were arrested at MI (MI rate: 98.0 and 97.2%, respectively; MII rate, 0% in both groups). Reductions in the BL formation were dependent on the TPEN treatment duration (29.3, 9.2, 0, and 0% after 0, 7, 15 and 22 h, respectively). In conclusion, zinc is an essential element for successful oocyte maturation and embryonic development in pigs. Zinc deficiency caused a meiotic block and had lasting effects on early embryonic development.

  16. Effects of meiotic inhibitors and gonadotrophins on porcine oocytes in vitro maturation, fertilization and development.

    PubMed

    Gil, M A; Nohalez, A; Martinez, C A; Ake-Villanueva, J R; Centurion-Castro, F; Maside, C; Cuello, C; Roca, J; Parrilla, I; Martinez, E A

    2017-10-01

    This study evaluated the effect of three reversible meiotic inhibitors (MINs) and their interaction with gonadotrophins (Gns) on the meiotic maturation and developmental competence of porcine oocytes. In experiment 1, the oocytes were matured for 22 hr in the presence or absence of dbcAMP (1 mM), cycloheximide (7 μM) or cilostamide (20 μM) with or without Gns, and for an additional 22 hr in the absence of MINs and Gns. At 22 hr of maturation, regardless of the presence of Gns, a higher proportion (p < .001) of oocytes cultured in the presence of MINs were effectively arrested at the germinal vesicle stage compared with the oocytes cultured without MINs. At 44 hr of maturation, the proportion of oocytes that reached MII was higher (p < .05) in groups with Gns compared with groups without Gns. In experiment 2, oocytes that were matured as in experiment 1 were inseminated and cultured for 7 days to evaluate fertilization parameters and blastocyst formation. Only oocytes from the dbcAMP + Gns group had higher (p < .05) efficiency of fertilization compared with the other treatment groups. The presence of dbcAMP during maturation also increased (p < .05) blastocyst formation and efficiency of blastocyst formation in both the presence and absence of Gns. These results indicate that the interaction of Gns with the tested MINs improved meiotic progression. In addition, regardless of supplementation with Gns, the presence of dbcAMP during the first maturation period increased and even doubled the capacity of oocytes to develop to the blastocyst stage. © 2017 Blackwell Verlag GmbH.

  17. 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

  18. Cytoskeletal abnormalities in relation with meiotic competence and ageing in porcine and bovine oocytes during in vitro maturation.

    PubMed

    Somfai, T; Kikuchi, K; Kaneda, M; Akagi, S; Watanabe, S; Mizutani, E; Haraguchi, S; Dang-Nguyen, T Q; Inaba, Y; Geshi, M; Nagai, T

    2011-10-01

    We investigated the frequencies of cytoskeletal anomalies in metaphase-II (M-II) and incompetent [arrested at an immature metaphase (IM) stage] porcine and bovine oocytes during in vitro maturation (IVM) in relation with ageing by immunostaining and confocal microscopy. In porcine oocytes, meiotic arrest at the IM stage was associated with abnormalities of cortical actin but not with abnormal spindles. Prolongation of IVM culture to 52 h did not affect microfilament and spindle abnormalities, but reduced the microfilament-rich area overlaying the spindle. Meiotic arrest of bovine oocytes at the IM stage was associated with degenerations of microfilaments, and the frequencies of abnormal spindles were also higher than those of M-II oocytes. Ageing of bovine oocytes (IVM for 30 h) did not affect cortical microfilaments but increased the frequency of spindle alterations in both M-II and IM bovine oocytes. These results suggest that, in both species, altered ability of oocytes to polymerize F-actin might be a possible reason for the failure of polar body extrusion during IVM. Also, there seem to be differences between the two species in the sensitivity of oocytes to suffer ageing-related spindle damages.

  19. SACY-1 DEAD-Box Helicase Links the Somatic Control of Oocyte Meiotic Maturation to the Sperm-to-Oocyte Switch and Gamete Maintenance in Caenorhabditis elegans

    PubMed Central

    Kim, Seongseop; Govindan, J. Amaranath; Tu, Zheng Jin; Greenstein, David

    2012-01-01

    In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. In Caenorhabditis elegans, major sperm protein triggers meiotic resumption through a mechanism involving somatic Gαs–adenylate cyclase signaling and soma-to-germline gap-junctional communication. Using genetic mosaic analysis, we show that the major effector of Gαs–adenylate cyclase signaling, protein kinase A (PKA), is required in gonadal sheath cells for oocyte meiotic maturation and dispensable in the germ line. This result rules out a model in which cyclic nucleotides must transit through sheath-oocyte gap junctions to activate PKA in the germ line, as proposed in vertebrate systems. We conducted a genetic screen to identify regulators of oocyte meiotic maturation functioning downstream of Gαs–adenylate cyclase–PKA signaling. We molecularly identified 10 regulatory loci, which include essential and nonessential factors. sacy-1, which encodes a highly conserved DEAD-box helicase, is an essential germline factor that negatively regulates meiotic maturation. SACY-1 is a multifunctional protein that establishes a mechanistic link connecting the somatic control of meiotic maturation to germline sex determination and gamete maintenance. Modulatory factors include multiple subunits of a CoREST-like complex and the TWK-1 two-pore potassium channel. These factors are not absolutely required for meiotic maturation or its negative regulation in the absence of sperm, but function cumulatively to enable somatic control of meiotic maturation. This work provides insights into the genetic control of meiotic maturation signaling in C. elegans, and the conserved factors identified here might inform analysis in other systems through either homology or analogy. PMID:22887816

  20. 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

  1. 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.

  2. Effects of oocyte culture density on meiotic competence of canine oocytes.

    PubMed

    Otoi, T; Willingham, L; Shin, T; Kraemer, D C; Westhusin, M

    2002-12-01

    This study was conducted to determine a suitable ratio of oocytes to medium for in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) collected from bitches at anoestrus and dioestrus and to examine the meiotic competence of COCs cultured singly or in different group sizes. In the first experiment, different numbers of COCs (5, 10, 15 and 20 per drop) were cultured for 72 h in 100 microl drops of maturation medium. The meiotic competence of oocytes from ovaries at anoestrus was affected by the number of COCs incubated, whereas at dioestrus, the incubation number of COCs had no effect. In the second experiment, COCs were cultured singly or in different group sizes for 72 h by suitable oocyte density according to the reproductive cycle of the donor. In the anoestrous group, 1, 5 and 10 COCs were cultured in 10, 50 and 100 microl drops of the medium (10 microl per COC), respectively. In the dioestrous group, 1, 5 and 15 COCs were cultured in 7, 35 and 105 microl drops of the medium (7 microl per COC), respectively. There were no differences in the proportions of oocytes reaching metaphase II among the different group sizes in each stage of the reproductive cycle of the donor. The results indicate that the influence of oocyte density on the meiotic competence of oocytes differs according to the stage of the reproductive cycle of the donor. Moreover, the group sizes have no effect on the meiotic competence of oocytes cultured at suitable oocyte density according to the reproductive cycle of the donor.

  3. 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. © 2014 by the Society for the Study of Reproduction, Inc.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  6. Non-meiotic chromosome instability in human immature oocytes.

    PubMed

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

    2014-02-01

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

  7. Non-meiotic chromosome instability in human immature oocytes

    PubMed Central

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

    2014-01-01

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

  8. Unifying theory of adult resting follicle recruitment and fetal oocyte arrest.

    PubMed

    Silber, Sherman

    2015-10-01

    One of the biggest mysteries of ovarian physiology is what controls the emergence of adult primordial follicles from the resting stage, and their steady depletion over the woman's lifetime. A related mystery is why do early oogonia begin meiosis in the fetus and then suddenly arrest for most of fetal and adult life. If fetal oocyte arrest did not occur after meiotic activation, there would be no oocytes left in the female baby by the time she is born. Similarly, without a steady controlled release in the adult ovary of resting follicles, the adult woman would run out of her eggs prematurely and have an early menopause. Could there be a similarity between what causes fetal oocyte arrest and what causes adult oocyte recruitment? The answer begins with the observation of a sudden massive recruitment of primordial follicles after human ovarian transplantation, and the embryologic discoveries about oocyte activation and the time of differentiation of cortex and medulla. The unifying theory is that ovarian cortical tissue pressure controls both fetal oocyte arrest and adult oocyte recruitment. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  9. Energy Status Characteristics of Porcine Oocytes During In Vitro Maturation is Influenced by Their Meiotic Competence.

    PubMed

    Milakovic, I; Jeseta, M; Hanulakova, S; Knitlova, D; Hanzalova, K; Hulinska, P; Machal, L; Kempisty, B; Antosik, P; Machatkova, M

    2015-10-01

    The characteristics of energy status in porcine oocytes as related to their meiotic competence and in vitro maturation were studied. Cycling pubertal gilts in the early luteal to early follicular phases of the ovarian cycle were used as oocyte donors. The oocytes recovered from medium (MF) or small follicles (SF) were considered meiotically more or less competent, respectively. A half of oocytes from each category was matured by the standard protocol. The oocytes were examined before or after maturation by confocal microscopy, a bioluminescent cell assay and Western blotting. Four experiments, each in triplicate, were performed to assess both SF and MF oocytes in terms of metabolic units formed by mitochondria and lipids, ATP and lipid consumption and lipid droplets with adipose differentiation-related protein (ADRP) expression. The proportion of oocytes with metabolic units, the mean ATP content and the number of lipid droplets per oocyte, and the relative number of lipid droplets with ADRP expression were significantly higher in the MF compared to SF oocytes before maturation. On the other hand, after maturation, there was an increase in the proportion of oocytes with metabolic units and the relative number of lipid droplets with ADRP expression in the SF compared to MF oocytes. In conclusion, specific differences in energy characteristics between porcine oocytes with different meiotic competence were found. Meiotically more competent oocytes are more advanced in terms of energy reserves before maturation, while meiotically less competent oocytes are more active in replenishing energy stores during maturation.

  10. Functional role of the bovine oocyte-specific protein JY-1 in meiotic maturation, cumulus expansion, and subsequent embryonic development.

    PubMed

    Lee, Kyung-Bon; Wee, Gabbine; Zhang, Kun; Folger, Joseph K; Knott, Jason G; Smith, George W

    2014-03-01

    Oocyte-expressed genes regulate key aspects of ovarian follicular development and early embryogenesis. We previously demonstrated a requirement of the oocyte-specific protein JY-1 for bovine early embryogenesis. Given that JY-1 is present in oocytes throughout folliculogenesis, and oocyte-derived JY-1 mRNA is temporally regulated postfertilization, we hypothesized that JY-1 levels in oocytes impact nuclear maturation and subsequent early embryogenesis. A novel model system, whereby JY-1 small interfering RNA was microinjected into cumulus-enclosed germinal vesicle-stage oocytes and meiotic arrest maintained for 48 h prior to in vitro maturation (IVM), was validated and used to determine the effect of reduced oocyte JY-1 expression on nuclear maturation, cumulus expansion, and embryonic development after in vitro fertilization. Depletion of JY-1 protein during IVM effectively reduced cumulus expansion, percentage of oocytes progressing to metaphase II, proportion of embryos that cleaved early, total cleavage rates and development to 8- to 16-cell stage, and totally blocked development to the blastocyst stage relative to controls. Supplementation with JY-1 protein during oocyte culture rescued effects of JY-1 depletion on meiotic maturation, cumulus expansion, and early cleavage, but did not rescue development to 8- to 16-cell and blastocyst stages. However, effects of JY-1 depletion postfertilization on development to 8- to 16-cell and blastocyst stages were rescued by JY-1 supplementation during embryo culture. In conclusion, these results support an important functional role for oocyte-derived JY-1 protein during meiotic maturation in promoting progression to metaphase II, cumulus expansion, and subsequent embryonic development.

  11. Microinjected progesterone reinitiates meiotic maturation of Xenopus laevis oocytes.

    PubMed Central

    Tso, J; Thibier, C; Mulner, O; Ozon, R

    1982-01-01

    Microinjection of progesterone dissolved in paraffin oil induces the reinitiation of meiotic maturation in the Xenopus oocyte; 50% maturation is obtained when 50 nl of a 50 microM solution is microinjected into the oocyte. The kinetics of the response to microinjected progesterone are similar to the kinetics of response to externally applied hormone. When an aqueous solution of progesterone is microinjected instead of an oil solution, maturation is never observed, a result which confirms previous work. Leakage of the steroid into the external medium was estimated to range from 1.6 pmol/hr when microinjection was performed in oil to 3.6 pmol/hr when it was performed in aqueous solution. Metabolism of the hormone microinjected in oil is weak (less than 20%) as compared to that after aqueous microinjection (greater than 80%). Progesterone microinjected in oil decreases the cAMP content as does externally applied hormone. We therefore conclude that progesterone acts initially on an intracellular site in order to trigger meiotic maturation of the Xenopus oocyte. PMID:6291050

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

    PubMed

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

    2016-03-07

    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 [2-6]. 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.

  13. Epidermal growth factor receptor signaling-dependent calcium elevation in cumulus cells is required for NPR2 inhibition and meiotic resumption in mouse oocytes.

    PubMed

    Wang, Yakun; Kong, Nana; Li, Na; Hao, Xiaoqiong; Wei, Kaiwen; Xiang, Xi; Xia, Guoliang; Zhang, Meijia

    2013-09-01

    In preovulatory ovarian follicles, the oocyte is maintained in meiotic prophase arrest by natriuretic peptide precursor C (NPPC) and its receptor natriuretic peptide receptor 2 (NPR2). LH treatment results in the decrease of NPR2 guanylyl cyclase activity that promotes resumption of meiosis. We investigated the regulatory mechanism of LH-activated epidermal growth factor (EGF) receptor signaling on NPR2 function. Cumulus cell-oocyte complex is cultured in the medium with 30 nM NPPC to prevent oocyte spontaneous maturation. In this system, EGF could stimulate oocyte meiotic resumption after 4 hours of incubation. Further study showed that EGF elevated intracellular calcium concentrations of cumulus cells and decreased cGMP levels in cumulus cells and oocytes, and calcium-elevating reagents ionomycin and sphingosine-1-phosphate mimicked the effects of EGF on oocyte maturation and cGMP levels. EGF-mediated cGMP levels and meiotic resumption could be reversed by EGF receptor inhibitor AG1478 and the calcium chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester. EGF also decreased the expression of Npr2 mRNA in cumulus cells, which may not be involved in meiotic resumption, because the block of NPR2 protein de novo synthesis by cycloheximide had no effect on NPPC and EGF-mediated oocyte maturation. However, EGF had no effect on oocyte maturation when meiotic arrest was maintained in the present of cGMP analog 8-bromoadenosine-cGMP. These results suggest that EGF receptor signaling induces meiotic resumption by elevating calcium concentrations of cumulus cells to decrease NPR2 guanylyl cyclase activity.

  14. Cumulus Cells Block Oocyte Meiotic Resumption via Gap Junctions in Cumulus Oocyte Complexes Subjected to DNA Double-Strand Breaks.

    PubMed

    Sun, Ming-Hong; Zheng, Jie; Xie, Feng-Yun; Shen, Wei; Yin, Shen; Ma, Jun-Yu

    2015-01-01

    During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.

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

    PubMed

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

    2003-04-28

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

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

    PubMed Central

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

    2016-01-01

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

  17. 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.

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

    PubMed

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

    2017-02-24

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

  19. Prophase I arrest of mouse oocytes mediated by natriuretic peptide precursor C requires GJA1 (connexin-43) and GJA4 (connexin-37) gap junctions in the antral follicle and cumulus-oocyte complex.

    PubMed

    Richard, Samantha; Baltz, Jay M

    2014-06-01

    Fully grown germinal vesicle stage mouse oocytes remain arrested in meiotic prophase I until ovulation. This arrest is maintained by cGMP produced in cumulus granulosa cells surrounding the oocyte. Recently, it was found that cGMP production in cumulus cells depends on NPR2 guanylate cyclase activated by its ligand natriuretic peptide precursor C (NPPC). It is assumed that cGMP reaches the oocyte through gap junctions that couple cumulus granulosa cells to each other and to the oocyte. Previous work identified two main types of gap junctions in the follicle, connexin-43 gap junctions (GJA1 protein) between granulosa cells and connexin-37 gap junctions (GJA4) between cumulus cells and the oocyte. However, it had not been established that both types are required for meiotic arrest mediated by NPPC/NPR2 signaling. To investigate this, we used connexin mimetic peptides (CMPs) that specifically disrupt gap junction isoforms within cumulus-oocyte complexes (COCs) and isolated antral follicles in culture. We furthermore developed a punctured antral follicle preparation to permit CMP access to the antral cavity in an otherwise intact follicle. CMP directed against connexin-43 (Cx43 CMP) overcame NPPC-mediated meiotic arrest in both isolated COCs and antral follicles. Cx37 CMP, in contrast, had no effect when present in the medium, but released oocyte arrest in the presence of NPPC when microinjected into the perivitelline space near the oocyte surface in COCs. This is consistent with both connexin isoforms being required for meiotic arrest and with the reported localization of connexin-43 throughout the cumulus cells and connexin-37 at the oocyte surface. © 2014 by the Society for the Study of Reproduction, Inc.

  20. Protein transport from endoplasmic reticulum to the Golgi complex can occur during meiotic metaphase in Xenopus oocytes

    PubMed Central

    1989-01-01

    We have previously shown that Xenopus oocytes arrested at second meiotic metaphase lost their characteristic multicisternal Golgi apparati and cannot secrete proteins into the surrounding medium. In this paper, we extend these studies to ask whether intracellular transport events affecting the movement of secretory proteins from the endoplasmic reticulum to the Golgi apparatus are also similarly inhibited in such oocytes. Using the acquisition of resistance to endoglycosidase H (endo H) as an assay for movement to the Golgi, we find that within 6 h, up to 66% of the influenza virus membrane protein, hemagglutinin (HA), synthesized from injected synthetic RNA, can move to the Golgi apparati in nonmatured oocytes; indeed after longer periods some correctly folded HA can be detected at the cell surface where it distributes in a nonpolarized fashion. In matured oocytes, up to 49% of the HA becomes endo H resistant in the same 6-h period. We conclude that movement from the endoplasmic reticulum to the Golgi can occur in matured oocytes despite the dramatic fragmentation of the Golgi apparati that we observe to occur on maturation. This observation of residual protein movement during meiotic metaphase contrasts with the situation at mitotic metabphase in cultured mammalian cells where all movement ceases, but resembles that in the budding yeast Saccharomyces cerevisiae where transport is unaffected. PMID:2793929

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

    PubMed

    Wang, Lu; Lu, Angeleem; Zhou, Hong-Xia; Sun, Ran; Zhao, Jie; Zhou, Cheng-Jie; Shen, Jiang-Peng; Wu, Sha-Na; Liang, Cheng-Guang

    2013-01-01

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

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

    PubMed Central

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

    2016-01-01

    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

  3. Effect of holding equine oocytes in meiosis inhibitor-free medium before in vitro maturation and of holding temperature on meiotic suppression and mitochondrial energy/redox potential.

    PubMed

    Martino, Nicola A; Dell'Aquila, Maria E; Filioli Uranio, Manuel; Rutigliano, Lucia; Nicassio, Michele; Lacalandra, Giovanni M; Hinrichs, Katrin

    2014-10-11

    of mitochondrial distribution pattern and there were no significant differences in oocyte energy/redox parameters between these oocytes and controls. Mitochondrial distribution in equine GV-stage oocytes is correlated with chromatin configuration within the GV. Progression of chromatin configuration and mitochondrial status during holding are dependent on temperature. EH holding at 25°C maintains meiotic arrest, viability and mitochondrial potential of equine oocytes. This is the first report on the effects of EH treatment on oocyte mitochondrial energy/redox potential.

  4. Protein Phosphatase 6 Protects Prophase I-Arrested Oocytes by Safeguarding Genomic Integrity

    PubMed Central

    Jiang, Zong-Zhe; Dong, Ming-Zhe; Schatten, Heide; Xu, Xingzhi; Wang, Zhen-Bo; Sun, Qing-Yuan

    2016-01-01

    Mammalian oocytes are arrested at prophase of the first meiotic division in the primordial follicle pool for months, even years, after birth depending on species, and only a limited number of oocytes resume meiosis, complete maturation, and ovulate with each reproductive cycle. We recently reported that protein phosphatase 6 (PP6), a member of the PP2A-like subfamily, which accounts for cellular serine/threonine phosphatase activity, functions in completing the second meiosis. Here, we generated mutant mice with a specific deletion of Ppp6c in oocytes from the primordial follicle stage by crossing Ppp6cF/F mice with Gdf9-Cre mice and found that Ppp6cF/F; GCre+ mice are infertile. Depletion of PP6c caused folliculogenesis defects and germ cell loss independent of the traditional AKT/mTOR pathway, but due to persistent phosphorylation of H2AX (a marker of double strand breaks), increased susceptibility to DNA damage and defective DNA repair, which led to massive oocyte elimination and eventually premature ovarian failure (POF). Our findings uncover an important role for PP6 as an indispensable guardian of genomic integrity of the lengthy prophase I oocyte arrest, maintenance of primordial follicle pool, and thus female fertility. PMID:27930667

  5. Effects of cryopreservation on the meiotic spindle, cortical granule distribution and development of rabbit oocytes.

    PubMed

    Jiménez-Trigos, E; Naturil-Alfonso, C; Vicente, J S; Marco-Jiménez, F

    2012-06-01

    Although much progress has been made in oocyte cryopreservation since 1971, live offspring have only been obtained in a few species and in rabbits. The aim of our study was to evaluate the effect of vitrification and slow freezing on the meiotic spindle, cortical granule (CG) distribution and their developmental competence. Oocytes were vitrified in 16.84% ethylene glycol, 12.86% formamide, 22.3% dimethyl sulphoxide, 7% PVP and 1% of synthetic ice blockers using Cryotop as device or slow freezing in 1.5 m PROH and 0.2 m sucrose in 0.25 ml sterile French mini straws. Meiotic spindle and CG distribution were assessed using a confocal laser-scanning microscope. To determine oocyte competence, in vitro development of oocytes from each cryopreservation procedure was assessed using parthenogenesis activation. Our data showed that oocytes were significantly affected by both cryopreservation procedures. In particular, meiotic spindle organization was dramatically altered after cryopreservation. Oocytes with peripheral CG distribution have a better chance of survival in cryopreservation after slow-freezing procedures compared to vitrification. In addition, slow freezing of oocytes led to higher cleavage and blastocyst rates compared to vitrification. Our data showed that, in rabbits, structural alterations are more evident in vitrified oocytes than in slow-frozen oocytes, probably as a consequence of sensitivity to high levels of cryoprotectants. Slow-freezing method is currently the recommended option for rabbit oocyte cryopreservation. © 2011 Blackwell Verlag GmbH.

  6. Epsin2 promotes polarity establishment and meiotic division through activating Cdc42 in mouse oocyte

    PubMed Central

    Zhang, Jiaqi; Liu, Xiaohui; Ma, Rujun; Hou, Xiaojing; Ge, Juan; Wang, Qiang

    2016-01-01

    Epsins are a conserved family of endocytic adaptors essential for diverse biological events. However, its role in oocytes remains completely unknown. Here, we report that specific depletion of Epsin2 in mouse oocytes significantly disrupts meiotic progression. Confocal microscopy reveals that Epsin2 knockdown results in the failure of actin cap formation and polar body extrusion during meiosis, indicative of the importance of Epsin2 in polarity establishment and cytokinesis. In addition, spindle defects and chromosome misalignment are readily observed in oocytes depleted of Epsin2. Moreover, we find that Epsin2 knockdown markedly decreases the activity of Cdc42 in oocytes and importantly, that the dominant-positive mutant of Cdc42 (Cdc42Q61L) is capable of partially rescuing the deficient phenotypes of Epsin2-knockdown oocytes. Together, our data identify Epsin2 as a novel player in regulating oocyte maturation, and demonstrate that Epsin2 promotes polarity establishment and meiotic division via activating Cdc42. PMID:27463009

  7. Reversible phosphorylation and regulation of mammalian oocyte meiotic chromatin remodeling and segregation.

    PubMed

    Swain, J E; Smith, G D

    2007-01-01

    The mammalian oocyte is notorious for high rates of chromosomal abnormalities. This results in subsequent embryonic aneuploidy, resulting in infertility and congenital defects. Therefore, understanding regulatory mechanisms involved in chromatin remodeling and chromosome segregation during oocyte meiotic maturation is imperative to fully understand the complex process and establish potential therapies. This review will focus on major events occurring during oocyte meiosis, critical to ensure proper cellular ploidy. Mechanistic and cellular events such as chromosome condensation, meiotic spindle formation, as well as cohesion of homologues and sister chromatids will be discussed, focusing on the role of reversible phosphorylation in control of these processes.

  8. A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic induction in isolated mouse oocytes.

    PubMed

    Downs, S M

    2001-02-01

    This study was carried out to compare the possible role of a secreted paracrine factor versus that of a gap-junction-transmitted signal in mediating meiotic induction in isolated mouse oocytes from PMSG-primed, immature mice. In the first set of experiments, oocyte-cumulus cell complexes (OCC) were pretreated for 3 h with 2 mM dbcAMP or FSH, washed, and the oocytes then cultured for 17-18 h in 40 microl drops containing either 300 microM dbcAMP or 4 mM hypoxanthine (HX). Each set of pretreated oocytes was cultured under three different conditions: (1) intact cumulus-cell-enclosed oocytes (CEO); (2) denuded oocytes (DO), cultured alone after removal of cumulus cells; and (3) co-cultured cumulus cells and oocytes (CC/DO), where the cumulus cells were removed in the same drop with a mouth-operated pipette and cultured alongside the oocytes. When pretreated with high dbcAMP or FSH, maturation was stimulated in CEO when cultured in either inhibitor (by 41.4-53.7%). Pretreatment failed to affect the maturation rate in DO. DO maturation was not altered appreciably by co-cultured cumulus cells when arrest was maintained with dbcAMP. However, an increase in maturation of 21-23% was observed in CC/DO in the HX-containing cultures that was not dependent on prior treatment with a meiosis-inducing stimulus. When DO were co-cultured with intact, FSH-treated OCC, there was no evidence of a positive factor secreted by the stimulated complexes, despite the fact that oocytes within the OCC were induced to resume maturation. In a second series of experiments the gap junction inhibitor, 18alpha-glycyrrhetinic acid (GA), was utilised. An initial experiment determined that GA dose-dependently blocked OCC metabolic coupling (0.2% coupling at 10 microM compared with 13.6% in controls). When HX-arrested CEO and DO were cultured for 17-18 h in medium containing increasing concentrations of GA, meiotic maturation was induced in CEO but not DO, suggesting that the cumulus cells provided a

  9. Spindle assembly checkpoint-related meiotic defect in oocytes from LT/Sv mice has cytoplasmic origin and diminishes in older females.

    PubMed

    Hoffmann, Steffen; Król, Marzena; Polanski, Zbigniew

    2012-09-01

    The spindle assembly checkpoint (SAC) ensures proper segregation of chromosomes by delaying anaphase onset until all kinetochores are properly attached to the spindle microtubules. Oocytes from the mouse strain LT/Sv arrest at the first meiotic metaphase (MI) due to, as reported recently, enormously prolonged activity of the SAC. We compared the dynamics of cyclin B1-GFP degradation, the process which is a measure of the SAC activity, in chromosomal and achromosomal halves of LT/Sv oocytes. In chromosome-containing oocyte halves arrested at MI, cyclin B1-GFP was not degraded indicating active SAC. However, in the halves lacking chromosomes, which is a condition precluding the SAC function, degradation always occurred confirming that MI arrest in LT/Sv oocytes is SAC dependent. Transferring the germinal vesicle (GV) from LT/Sv oocytes into the enucleated oocytes from wild-type mice resulted in the progression through meiosis one, indicating that a SAC-activating defect in LT/Sv oocytes is cytoplasmic, yet can be rescued by foreign cytoplasm. These results may help to define the etiology of the human infertility related to the oocyte MI arrest, indicating the involvement of the SAC as likely candidate, and point to GV transfer as the possible therapy. Finally, we found that majority of oocytes isolated from old LT/Sv mice complete the first meiosis. Reciprocal transfers of the GV between the oocytes from young and old LT/Sv females suggest that the factor(s) responsible for the reversal of the phenotype in oocytes from old mice is located both in the GV and in the cytoplasm.

  10. Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes.

    PubMed

    Rossi, Gianna; Palmerini, Maria Grazia; Macchiarelli, Guido; Buccione, Roberto; Cecconi, Sandra

    2006-07-01

    In this study the effects of mancozeb, a widely used ethylenebisdithiocarbamate fungicide, on mouse oocyte meiotic maturation and fertilization were analyzed. Oocyte cumulus cell-complexes were matured in vitro with or without increasing concentrations of the fungicide (from 0.001 to 1 microg/ml) that, due to its different stability in organic solvents and in water, was resuspended either in dimethyl sulfoxide or in culture medium. Although, about 95% of oocytes reached the metaphase II stage; mancozeb-exposed oocytes showed a dose-dependent increase of alterations in spindle morphology, and this negative effect was more evident when the fungicide was resuspended in culture medium. Under the latter culture condition, oocytes matured in the presence of 0.1 and 1 microg/ml mancozeb showed a significant reduction also in the formation of male and female pronuclei. These results indicate that mancozeb can adversely affect mammalian reproductive performance, likely by perturbing microtubular organization during meiotic maturation.

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

    PubMed

    Zenzes, Maria Teresa; Bielecki, Ryszard

    2004-09-15

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

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

    PubMed Central

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

    2016-01-01

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

  13. Arrest at the diplotene stage of meiotic prophase I is delayed by progesterone but is not required for primordial follicle formation in mice.

    PubMed

    Dutta, Sudipta; Burks, Deion M; Pepling, Melissa E

    2016-12-05

    In mammalian females, reproductive capacity is determined by the size of the primordial follicle pool. During embryogenesis, oogonia divide mitotically but cytokinesis is incomplete so oogonia remain connected in germ cell cysts. Oogonia begin to enter meiosis at 13.5 days postcoitum in the mouse and over several days, oocytes progress through the stages of meiotic prophase I arresting in the diplotene stage. Concurrently, germ cell cysts break apart and individual oocytes become surrounded by granulosa cells forming primordial follicles. In rats, inhibition of a synaptonemal complex protein caused premature arrival at the diplotene stage and premature primordial follicle assembly suggesting diplotene arrest might trigger primordial follicle formation. Cyst breakdown and primordial follicle formation are blocked by exposure to steroid hormones but hormone effects on the timing of diplotene arrest are unclear. Here, we asked: (1) if oocytes were required to arrest in diplotene before follicles formed, (2) if all oocytes within a germ cell cyst arrested at diplotene synchronously, and (3) if steroid hormones affected progression through prophase I. Meiotic stage and follicle formation were assessed in histological sections. Statistical differences over time were determined using one-way ANOVA followed by Newman-Keuls multiple comparisons test. To determine if steroid hormones affect the rate of progression to the diplotene stage, 17.5 dpc ovaries were placed in organ culture with media containing estradiol, progesterone or both hormones. In this case, differences were determined using one-way ANOVA followed by Dunnett's multiple comparisons test. We found primordial follicles containing oocytes at the diplotene stage as well as follicles containing oocytes at pre-diplotene stages. We also found individual germ cell cysts containing oocytes at both diplotene and pre-diplotene stages. Progesterone but not estradiol reduced the number of diplotene oocytes in ovary

  14. Phosphorylation of ribosomal proteins during meiotic maturation and following activation in starfish oocytes: its relationship with changes of intracellular pH.

    PubMed

    Peaucellier, G; Picard, A; Robert, J J; Capony, J P; Labbe, J C; Doree, M

    1988-01-01

    An increased phosphorylation of ribosomal protein S6 has been shown to be correlated with an increase of intracellular pH (pHi) and with stimulation of protein synthesis in many systems. In this research changes in ribosome phosphorylation following hormone-induced meiotic maturation and fertilization or activation by ionophore A23187 were investigated in starfish oocytes. The hormone was found to stimulate, even in the absence of external Na+, the phosphorylation on serine residues of an Mr 31,000 protein identified as S6, as well as that of an acidic Mr 47,000 protein, presumably S1, on threonine residues. Phosphorylation of ribosomes was an early consequence of hormonal stimulation and did not decrease after completion of meiotic maturation. Fertilization or activation by ionophore of prophase-arrested oocytes also stimulated ribosome phosphorylation. Only S6 was labeled in this case, but to a lesser extent than upon hormone-induced meiotic maturation. Changes in pHi were monitored with ion-specific microelectrodes throughout meiotic maturation and following either fertilization or activation. The pHi did not change before germinal vesicle breakdown (GVBD) following hormone addition, but it increased before first polar body emission. It also increased following fertilization or activation by ionophore or the microinjection of Ca-EGTA. In all cases, alkalinization did not depend on activation of an amiloride-sensitive Na+/H+ exchanger. Microinjection of an alkaline Hepes buffer or external application of ammonia, both of which increased pHi, prevented unfertilized oocytes from arresting after formation of the female pronucleus and induced chromosome cycling. Phosphorylation of S6 was still observed following fertilization or induction of maturation when pHi was decreased by external application of acetate, a treatment which suppressed the emission of polar bodies. Protein synthesis increased in prophase-arrested oocytes after fertilization or activation. It also

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    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.

  17. 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

  18. Efficient mutagenesis by CRISPR/Cas system during meiotic maturation of porcine oocytes.

    PubMed

    Onuma, Asuka; Fujii, Wataru; Sugiura, Koji; Naito, Kunihiko

    2017-02-16

    Genome editing using the CRISPR/Cas system can induce mutations with high efficiency, and allows easier production of genome-modified animals than that offered by the conventional method where embryonic stem cells are used. However, studies using CRISPR/Cas systems have been mostly limited to proliferating somatic cells and pronuclear-stage fertilized eggs. In contrast, the efficiency of a CRISPR/Cas system in immature and maturing oocytes progressing through meiosis has not yet been assessed. In the present study, we evaluated the genome-modification efficiency of the CRISPR/Cas system during meiotic maturation of porcine oocytes. Additionally, the localization of the Cas9 protein in immature oocytes was analyzed in relation to nuclear transport and mutation induction. The results showed that CRISPR/Cas induced mutation with high efficiency even in maturing oocytes with condensed chromosomes, whereas mutations were not induced in GV-stage oocytes. The localization analysis of enhanced green fluorescent protein (EGFP)-tagged Cas9 (Cas9-EGFP) revealed that the nuclei contained lesser Cas9 than the cytoplasm in immature oocytes. Treatment with leptomycin B, a nuclear export inhibitor, increased the amount of nuclear Cas9 and enabled mutation induction in GV oocytes. Our results suggest that CRISPR/Cas systems can be applied to oocytes during meiotic maturation and be implemented in novel applications targeting female genomes.

  19. Efficient mutagenesis by CRISPR/Cas system during meiotic maturation of porcine oocytes

    PubMed Central

    ONUMA, Asuka; FUJII, Wataru; SUGIURA, Koji; NAITO, Kunihiko

    2016-01-01

    Genome editing using the CRISPR/Cas system can induce mutations with high efficiency, and allows easier production of genome-modified animals than that offered by the conventional method where embryonic stem cells are used. However, studies using CRISPR/Cas systems have been mostly limited to proliferating somatic cells and pronuclear-stage fertilized eggs. In contrast, the efficiency of a CRISPR/Cas system in immature and maturing oocytes progressing through meiosis has not yet been assessed. In the present study, we evaluated the genome-modification efficiency of the CRISPR/Cas system during meiotic maturation of porcine oocytes. Additionally, the localization of the Cas9 protein in immature oocytes was analyzed in relation to nuclear transport and mutation induction. The results showed that CRISPR/Cas induced mutation with high efficiency even in maturing oocytes with condensed chromosomes, whereas mutations were not induced in GV-stage oocytes. The localization analysis of enhanced green fluorescent protein (EGFP)-tagged Cas9 (Cas9-EGFP) revealed that the nuclei contained lesser Cas9 than the cytoplasm in immature oocytes. Treatment with leptomycin B, a nuclear export inhibitor, increased the amount of nuclear Cas9 and enabled mutation induction in GV oocytes. Our results suggest that CRISPR/Cas systems can be applied to oocytes during meiotic maturation and be implemented in novel applications targeting female genomes. PMID:27773884

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

    PubMed

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

    2013-06-01

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

  1. In vivo regulation of cytostatic activity in Xenopus metaphase II-arrested oocytes.

    PubMed

    Thibier, C; De Smedt, V; Poulhe, R; Huchon, D; Jessus, C; Ozon, R

    1997-05-01

    Metaphase II arrest of Xenopus oocyte is characterized by the presence of M-phase-promoting factor (MPF) and of a microtubular spindle, both of which are stable in the presence of protein synthesis inhibitors. We studied in vivo this equilibrium state that is settled during meiotic maturation. At time of germinal vesicle breakdown (GVBD), cdc2 kinase and MAP kinase activities are stimulated. A component of the cyclin ubiquitin ligase, CDC27, is phosphorylated at the same time and remains phosphorylated until fertilization, indicating that an important component of the ligase complex is modified as early as GVBD. During a first period extending from GVBD until the cortical anchorage of the metaphase II spindle, homogeneous pools of cdc2 kinase and mitogen-activated protein (MAP) kinase activities are present in oocyte and are strictly dependent on protein turnover, since protein synthesis inhibition induces their total inactivation and drives oocytes into interphase. The metaphase II spindle, once anchored into the cortex, is no more sensitive to protein synthesis inhibition, likewise MAP kinase activity. During this cellular arrest, cdc2 kinase is divided into two distinctly regulated pools. The first one contains cyclin B that actively turns over and is subjected to a microtubular checkpoint. The second one is stable. Alteration of intracellular compartmentation of metaphase II oocytes either by gentle centrifugation or by cold shock inactivates MAP kinase and targets all cyclin B molecules for full destruction. We therefore suggest that MAP kinase participates to the cytostatic activity by preventing part of cyclin B molecules from entering the ubiquitination/degradation machinery which is still turned on in metaphase II oocytes.

  2. Zona pellucida birefringence and meiotic spindle visualisation of human oocytes are not influenced by IVM technology.

    PubMed

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

    2014-03-01

    The aim of the present study was to investigate the relationship between the presence of the meiotic spindle and zona pellucida (ZP) birefringence with morphology of in vivo- and in vitro-matured human oocytes. Germinal vesicles (n=47) and MI (n=38) oocytes obtained from stimulated ovaries of patients undergoing intracytoplasmic sperm injection (ICSI) underwent IVM. Using a PolScope (OCTAX PolarAID; Octax, Herbon, Germany), the presence of spindles and ZP birefringence was assessed in both in vivo-matured (n=56) and IVM (n=56) oocytes. In addition, the morphology of each matured oocyte was evaluated microscopically. There were insignificant differences for ZP birefringence and meiotic spindle between the in vivo-matured and IVM MII oocytes. Subanalysis revealed that the rates of morphologically abnormal oocytes did not differ significantly between the two groups, except in the case of irregular shape (P=0.001), refractile body (P=0.001) and fragmented polar body (P=0.03), which were higher in IVM oocytes. In the case of in vivo-matured oocytes, a significantly higher percentage of oocytes with intracytoplasmic and both intra- and extracytoplasmic abnormalities have a low birefringent ZP (P=0.007 and P=0.02, respectively). There was no relationship between morphological abnormalities and spindle detection. The findings suggest that clinical IVM is a safe technology that maintains the high maturation rate and integrity of oocytes. In addition, the use of the non-invasive PolScope is recommended for the detection of oocytes most suitable for ICSI.

  3. Stag3 regulates microtubule stability to maintain euploidy during mouse oocyte meiotic maturation

    PubMed Central

    Zhang, Mianqun; Dai, Xiaoxin; Sun, Yalu; Lu, Yajuan; Zhou, Changyin; Miao, Yilong; Wang, Ying; Xiong, Bo

    2017-01-01

    Stag3, a meiosis-specific subunit of cohesin complex, has been demonstrated to function in both male and female reproductive systems in mammals. However, its roles during oocyte meiotic maturation have not been fully defined. In the present study, we report that Stag3 uniquely accumulates on the spindle apparatus and colocalizes with microtubule fibers during mouse oocyte meiotic maturation. Depletion of Stag3 by gene-targeting morpholino disrupts normal spindle assembly and chromosome alignment in oocytes. We also find that depletion of Stag3 reduces the acetylated level of tubulin and microtubule resistance to microtubule depolymerizing drug, suggesting that Stag3 is required for microtubule stability. Consistent with these observations, kinetochore-microtubule attachment, an important mechanism controlling chromosome alignment, is severely impaired in Stag3-depleted oocytes, resultantly causing the significantly increased incidence of aneuploid eggs. Collectively, our data reveal that Stag3 is a novel regulator of microtubule dynamics to ensure euploidy during moue oocyte meiotic maturation. PMID:27906670

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

    PubMed Central

    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

  5. Inhibition of denuded mouse oocyte meiotic maturation by forskolin, an activator of adenylate cyclase.

    PubMed

    Urner, F; Herrmann, W L; Baulieu, E E; Schorderet-Slatkine, S

    1983-09-01

    Forskolin, a diterpene that activates rapidly adenylate cyclase activity in several somatic cell systems, prevents spontaneous meiotic maturation of denuded mouse oocytes (ED50 of inhibition approximately 2.5 microM), unlike cholera toxin. The oocyte is sensitive to the action of forskolin during the period preceding germinal vesicle breakdown (GVBD). Washing of the cells abolishes the effect. The diterpene potentiates the inhibitory effect of iso-butyl-methyl-xanthine (IBMX), a phosphodiesterase inhibitor, and it increases cAMP concentration in the oocytes. These findings not only confirm the antagonistic effect of cAMP on the first step of meiosis reinitiation (GVBD) in mammalian oocytes, but also provide the first demonstration of a functional adenylate cyclase system in mammalian oocytes upon which regulatory signals may act.

  6. Prophase I arrest and progression to metaphase I in mouse oocytes: comparison of resumption of meiosis and recovery from G2-arrest in somatic cells.

    PubMed

    Solc, Petr; Schultz, Richard M; Motlik, Jan

    2010-09-01

    Mammalian oocytes are arrested at prophase I until puberty when luteinizing hormone (LH) induces resumption of meiosis of follicle-enclosed oocytes. Resumption of meiosis is tightly coupled with regulating cyclin-dependent kinase 1 (CDK1) activity. Prophase I arrest depends on inhibitory phosphorylation of CDK1 and anaphase-promoting complex-(APC-CDH1)-mediated regulation of cyclin B levels. Prophase I arrest is maintained by endogenously produced cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) that in turn phosphorylates (and activates) the nuclear kinase WEE2. In addition, PKA-mediated phosphorylation of the phosphatase CDC25B results in its cytoplasmic retention. The combined effect maintains low levels of CDK1 activity that are not sufficient to initiate resumption of meiosis. LH triggers synthesis of epidermal growth factor-like factors in mural granulosa cells and leads to reduced cGMP transfer from cumulus cells to oocytes via gap junctions that couple the two cell types. cGMP inhibits oocyte phosphodiesterase 3A (PDE3A) and a decline in oocyte cGMP results in increased PDE3A activity. The ensuing decrease in oocyte cAMP triggers maturation by alleviating the aforementioned phosphorylations of WEE2 and CDC25B. As a direct consequence CDC25B translocates into the nucleus. The resulting activation of CDK1 also promotes extrusion of WEE2 from the nucleus thereby providing a positive amplification mechanism for CDK1 activation. Other kinases, e.g. protein kinase B, Aurora kinase A and polo-like kinase 1, also participate in resumption of meiosis. Mechanisms governing meiotic prophase I arrest and resumption of meiosis share common features with DNA damage-induced mitotic G2-checkpoint arrest and checkpoint recovery, respectively. These common features include CDC14B-dependent activation of APC-CDH1 in prophase I arrested oocytes or G2-arrested somatic cells, and CDC25B-dependent cell cycle resumption in both oocytes and somatic

  7. Mechanisms by which a lack of germinal vesicle (GV) material causes oocyte meiotic defects: a study using oocytes manipulated to replace GV with primary spermatocyte nuclei.

    PubMed

    Zhang, Jie; Cui, Wei; Li, Qing; Wang, Tian-Yang; Sui, Hong-Shu; Wang, Jun-Zuo; Luo, Ming-Jiu; Tan, Jing-He

    2013-10-01

    Oocytes with germinal vesicles (GVs) replaced with somatic nuclei exhibit meiotic abnormalities. Although this suggests an exclusive role for GV material in meiosis, mechanisms by which a lack of GV material causes meiotic defects are unknown. Knowledge of these mechanisms will help us to understand meiotic control, nuclear-cytoplasmic interactions, and cellular reprogramming. This study showed that although oocytes with prometaphase I chromosomes replaced with primary spermatocyte nuclei (PSN) did not, oocytes with GV replaced with PSN (PSG oocytes) did display meiotic defects. Among the defects, insufficient chromosome condensation with chromosome bridges was associated with spindle abnormalities. Abnormal spindle migration, cortical nonpolarization, and the aberrant spindle caused randomly positioning of cleavage furrows, leading to large first polar bodies (PB1) and unequal allocation of chromosomes and mitogen-activated protein kinases (MAPK) between oocyte and PB1. Spindle assembly checkpoint was activated but did not stop the incorrect division. The unequal MAPK allocation resulted in differences in pronuclear formation and PB1 degeneration; oocytes receiving more MAPK were more capable of forming pronuclear rudiments, whereas PB1 receiving more MAPK degenerated sooner than those that received less. Because none of the PSG oocytes or the enucleated GV oocytes injected with sperm heads showed cortical polarization in spite of chromosome localization close to the oolemma and because the PSG oocytes receiving more MAPK could form only pronuclear rudiments and not normal pronuclei, we suggest that the GV material plays essential roles in polarization and pronuclear formation on top of those played by chromosomes or MAPK. In conclusion, using PSG oocytes as models, this study has revealed the primary pathways by which a lack of GV material cause meiotic defects, laying a foundation for future research on the role of GV material in oocyte meiotic control.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-01

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

  10. Four-dimensional visualization and quantitative analysis of meiotic spindle movements in live mouse oocytes.

    PubMed

    Tian, N; Zhang, L; Liu, B; Wang, P; Li, Y; Ma, W

    2012-09-01

    This paper made a different attempt of real-time observation of the meiotic spindle movements in living mouse oocyte using a convenient method. This method was based on an experimental phenomenon discovered in our work. In living mouse oocytes, a high concentration of calcium ions (Ca(2+)) was observed throughout the region occupied by the initial meiotic spindle. After Ca(2+) labelling with Fura-2, a weakly fluorescent area (WFA) appeared on each side of the chromosomes. The activities of the WFAs changed during spindle development. By real-time tracking of WFAs, we were able to indirectly observe the meiotic spindle movements. Occasionally, it was observed that the first meiotic spindle rotated from an orientation parallel to the cortex to become perpendicular, instead of migrating from the oocyte centre to the cortex along its axis. Moreover, we analysed this uncommon rotation of the first meiotic spindle and found that the whole rotation process can be divided into two phases: the early slow-speed rotation and the subsequent rapid-speed rotation. We further characterized this rotation with respect to rotational speed and acceleration at all the stages of development. By using a two-photon laser-scanning microscope in combination with Fura-2 dye that is nondamaging to oocytes, we provide a convenient method for indirect visualization and quantitative analysis of spindle movements by real-time tracking of WFAs. This method is easy to operate and master, and economical with time and effort. © 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.

  11. 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

  12. Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes.

    PubMed

    Shen, Yu-Ting; Song, Yue-Qiang; He, Xiao-Qin; Zhang, Fei; Huang, Xin; Liu, Yu; Ding, Lu; Xu, Lin; Zhu, Mao-Bi; Hu, Wen-Feng; Qi, Zhong-Quan; Wang, Hai-Long; Yang, Xiang-Jun

    2014-10-01

    Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10μgmL(-1) TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P<0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10mgkg(-1) TPTCL (P<0.05). GVBD decreased in a non-significant, dose-dependent manner (P>0.05). PBE was inhibited with 10mgkg(-1) TPTCL (P<0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10mgkg(-1) TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

  13. 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.

  14. 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.

  15. Phosphorylation of ribosomal proteins during meiotic saturation and following activation in starfish oocytes: Its relationship with changes of intracellular pH

    SciTech Connect

    Peaucellier, G.; Picard, A.; Robert, J.J. ); Capony, J.P.; Labbe, J.C. CNRS-CRBM, Montpellier ); Doree, M. INSERM, Montpellier CNRS-CRBM, Montpellier )

    1988-01-01

    An increased phosphorylation of ribosomal protein S6 has been shown to be correlated with an increase of intracellular pH (pH{sub i}) and with stimulation of protein synthesis in many systems. In this research changes in ribosome phosphorylation following hormone-induced meiotic maturation and fertilization or activation by ionophore A23187 were investigated in starfish oocytes. The hormone was found to stimulate the phosphorylation on serine residues of an M{sub r} 31,000 protein identified as S6, as well as that of an acidic M{sub r} 47,000 protein, presumably S1, on threonine residues. Phosphorylation of ribosomes was an early consequence of hormonal stimulation and did not decrease after completion of meiotic maturation. Fertilization or activation by ionophore of prophase-arrested oocytes also stimulated ribosome phosphorylation. The pH{sub i} did not change before germinal vesicle breakdown (GVBD) following hormone addition, but it increased before first polar body emission. It also increased following fertilization or activation by ionophore or the microinjection of Ca-EGTA. Phosphorylation of S6 was still observed following fertilization or induction of maturation when pH{sub i} was decreased by external application of acetate, a treatment which suppressed the emission of polar bodies. Protein synthesis increased in prophase-arrested oocytes after fertilization or activation.

  16. Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800

    PubMed Central

    1990-01-01

    Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo. PMID:2211822

  17. Kif2a regulates spindle organization and cell cycle progression in meiotic oocytes

    PubMed Central

    Yi, Zi-Yun; Ma, Xue-Shan; Liang, Qiu-Xia; Zhang, Teng; Xu, Zhao-Yang; Meng, Tie-Gang; Ouyang, Ying-Chun; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan; Quan, Song

    2016-01-01

    Kif2a is a member of the Kinesin-13 microtubule depolymerases. Here, we report the expression, subcellular localization and functions of Kif2a during mouse oocyte meiotic maturation. Immunoblotting analysis showed that Kif2a was gradually increased form GV to the M I stages, and then decreased slightly at the M II stage. Confocal microscopy identified that Kif2a localized to the meiotic spindle, especially concentrated at the spindle poles and inner centromeres in metaphase and translocated to the midbody at telophase. Kif2a depletion by siRNA microinjection generated severely defective spindles and misaligned chromosomes, reduced microtubule depolymerization, which led to significant pro-M I/M Iarrest and failure of first polar body (PB1) extrusion. Kif2a-depleted oocytes were also defective in spindle pole localization of γ-tubulin and showed spindle assembly checkpoint (SAC) protein Bub3 at the kinetochores even after 10 hr extended culture. These results demonstrate that Kif2a may act as a microtubule depolymerase, regulating microtubule dynamics, spindle assembly and chromosome congression, and thus cell cycle progression during mouse oocyte meiotic maturation. PMID:27991495

  18. 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

  19. Rejuvenation of meiotic cohesion in oocytes during prophase I is required for chiasma maintenance and accurate chromosome segregation.

    PubMed

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

    2014-09-01

    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

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

    PubMed

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

    2014-05-01

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

  1. 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.

  2. The Meiotic Recombination Checkpoint Suppresses NHK-1 Kinase to Prevent Reorganisation of the Oocyte Nucleus in Drosophila

    PubMed Central

    Lancaster, Oscar M.; Breuer, Manuel; Cullen, C. Fiona; Ito, Takashi; Ohkura, Hiroyuki

    2010-01-01

    The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired. PMID:21060809

  3. Meiotic maturation of oocytes recovered from the ovaries of Indian big cats at postmortem.

    PubMed

    Rao, Brahmasani Sambasiva; Mahesh, Yelisetti Uma; Suman, Komjeti; Charan, Katari Venu; Nath, Rhisita; Rao, K Ramachander

    2015-01-01

    In order to increase the available sources of genetic material for endangered members of the great cat family, this study was designed to assess the meiotic competence of oocytes recovered from postmortem ovaries of the Indian leopard, tiger and lion. The average number of oocytes that were recovered per ovary was 11.0 ± 5.0, 11.0 ± 3.5 and 21.3 ± 8.8 for tiger, lion and leopard, respectively. The proportion of culture grade oocytes for tiger, lion and leopard were 72.7, 78.8 and 71.9%, respectively. The culture grade oocytes were matured in tissue culture medium 199 modified with sodium bicarbonate supplemented with 0.3% BSA (fatty acid-free) (w/v), 10 μg/ml FSH, 6 IU/ml LH, 1 μg/ml 17β-estradiol, 0.36 mM pyruvate, 2.2 mM calcium lactate, 2.0 mM L-glutamine, 100 IU/ml penicillin and 0.1 mg/ml streptomycin in an incubator with 5% CO2 under humidified air at 38.5°C for 36 h. After in vitro maturation, 56.3, 53.8 and 58.7% of the tiger, lion and leopard oocytes, respectively, were matured. The proportion of oocytes that extruded first polar body was significantly higher when the oocytes were collected from the animals of less than 15 yr of age compared to above 15 yr. These findings suggest that the oocytes recovered from ovaries of tiger, lion and leopard immediately postmortem can be successfully matured to MII stage.

  4. Caenorhabditis elegans oocyte meiotic spindle pole assembly requires microtubule severing and the calponin homology domain protein ASPM-1

    PubMed Central

    Connolly, Amy A.; Osterberg, Valerie; Christensen, Sara; Price, Meredith; Lu, Chenggang; Chicas-Cruz, Kathy; Lockery, Shawn; Mains, Paul E.; Bowerman, Bruce

    2014-01-01

    In many animals, including vertebrates, oocyte meiotic spindles are bipolar but assemble in the absence of centrosomes. Although meiotic spindle positioning in oocytes has been investigated extensively, much less is known about their assembly. In Caenorhabditis elegans, three genes previously shown to contribute to oocyte meiotic spindle assembly are the calponin homology domain protein encoded by aspm-1, the katanin family member mei-1, and the kinesin-12 family member klp-18. We isolated temperature-sensitive alleles of all three and investigated their requirements using live-cell imaging to reveal previously undocumented requirements for aspm-1 and mei-1. Our results indicate that bipolar but abnormal oocyte meiotic spindles assemble in aspm-1(-) embryos, whereas klp-18(-) and mei-1(-) mutants assemble monopolar and apolar spindles, respectively. Furthermore, two MEI-1 functions—ASPM-1 recruitment to the spindle and microtubule severing—both contribute to monopolar spindle assembly in klp-18(-) mutants. We conclude that microtubule severing and ASPM-1 both promote meiotic spindle pole assembly in C. elegans oocytes, whereas the kinesin 12 family member KLP-18 promotes spindle bipolarity. PMID:24554763

  5. 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.

  6. 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

  7. Progesterone Receptor Membrane Component 1 Mediates Progesterone-Induced Suppression of Oocyte Meiotic Prophase I and Primordial Folliculogenesis.

    PubMed

    Guo, Meng; Zhang, Cheng; Wang, Yan; Feng, Lizhao; Wang, Zhengpin; Niu, Wanbo; Du, Xiaoyan; Tang, Wang; Li, Yuna; Wang, Chao; Chen, Zhenwen

    2016-11-16

    Well-timed progression of primordial folliculogenesis is essential for mammalian female fertility. Progesterone (P4) inhibits primordial follicle formation under physiological conditions; however, P4 receptor that mediates this effect and its underlying mechanisms are unclear. In this study, we used an in vitro organ culture system to show that progesterone receptor membrane component 1 (PGRMC1) mediated P4-induced inhibition of oocyte meiotic prophase I and primordial follicle formation. We found that membrane-impermeable BSA-conjugated P4 inhibited primordial follicle formation similar to that by P4. Interestingly, PGRMC1 and its partner serpine1 mRNA-binding protein 1 were highly expressed in oocytes in perinatal ovaries. Inhibition or RNA interference of PGRMC1 abolished the suppressive effect of P4 on follicle formation. Furthermore, P4-PGRMC1 interaction blocked oocyte meiotic progression and decreased intra-oocyte cyclic AMP (cAMP) levels in perinatal ovaries. cAMP analog dibutyryl cAMP reversed P4-PGRMC1 interaction-induced inhibition of meiotic progression and follicle formation. Thus, our results indicated that PGRMC1 mediated P4-induced suppression of oocyte meiotic progression and primordial folliculogenesis by decreasing intra-oocyte cAMP levels.

  8. Progesterone Receptor Membrane Component 1 Mediates Progesterone-Induced Suppression of Oocyte Meiotic Prophase I and Primordial Folliculogenesis

    PubMed Central

    Guo, Meng; Zhang, Cheng; Wang, Yan; Feng, Lizhao; Wang, Zhengpin; Niu, Wanbo; Du, Xiaoyan; Tang, Wang; Li, Yuna; Wang, Chao; Chen, Zhenwen

    2016-01-01

    Well-timed progression of primordial folliculogenesis is essential for mammalian female fertility. Progesterone (P4) inhibits primordial follicle formation under physiological conditions; however, P4 receptor that mediates this effect and its underlying mechanisms are unclear. In this study, we used an in vitro organ culture system to show that progesterone receptor membrane component 1 (PGRMC1) mediated P4-induced inhibition of oocyte meiotic prophase I and primordial follicle formation. We found that membrane-impermeable BSA-conjugated P4 inhibited primordial follicle formation similar to that by P4. Interestingly, PGRMC1 and its partner serpine1 mRNA-binding protein 1 were highly expressed in oocytes in perinatal ovaries. Inhibition or RNA interference of PGRMC1 abolished the suppressive effect of P4 on follicle formation. Furthermore, P4-PGRMC1 interaction blocked oocyte meiotic progression and decreased intra-oocyte cyclic AMP (cAMP) levels in perinatal ovaries. cAMP analog dibutyryl cAMP reversed P4–PGRMC1 interaction-induced inhibition of meiotic progression and follicle formation. Thus, our results indicated that PGRMC1 mediated P4-induced suppression of oocyte meiotic progression and primordial folliculogenesis by decreasing intra-oocyte cAMP levels. PMID:27848973

  9. The 'second-codon rule' and autophosphorylation govern the stability and activity of Mos during the meiotic cell cycle in Xenopus oocytes.

    PubMed Central

    Nishizawa, M; Okazaki, K; Furuno, N; Watanabe, N; Sagata, N

    1992-01-01

    The c-mos proto-oncogene product, Mos, functions in both early (germinal vesicle breakdown) and late (metaphase II arrest) steps during meiotic maturation in Xenopus oocytes. In the early step, Mos is only partially phosphorylated and metabolically unstable, while in the late step it is fully phosphorylated and highly stable. Using a number of Mos mutants expressed in oocytes, we show here that the instability of Mos in the early step is determined primarily by its penultimate N-terminal residue, or by a rule referred to here as the 'second-codon rule'. We demonstrate that unstable Mos is degraded by the ubiquitin-dependent pathway. In the late step, on the other hand, Mos is stabilized by autophosphorylation at Ser3, which probably acts to prevent the N-terminus of Mos from being recognized by a ubiquitin-protein ligase. Moreover, we show that Ser3 phosphorylation is essential for Mos to exert its full cytostatic factor (CSF) activity in fully mature oocytes. Thus, a few N-terminal amino acids are primary determinants of both the metabolic stability and physiological activity of Mos during the meiotic cell cycle. Images PMID:1321032

  10. 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. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  11. 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

  12. 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.

  13. Meiotic response of in vitro matured canine oocytes under different proteins and heterologous hormone supplementation.

    PubMed

    Rodrigues, B A; Rodrigues, J L

    2003-02-01

    The impact of TCM-199 supplemented with different proteins and heterologous hormones on the in vitro maturation (IVM) rate of bitch oocytes was evaluated by nuclear staining under fluorescence microscopy. Oocytes were recovered by slicing of ovaries from bitches presented at various stages of oestrous cycle to ovariohysterectomy. The basic culture medium was TCM-199 supplemented with 25 mM Hepes/l, with 10% heat-inactivated oestrous cow serum (ECS), 50 microg/ml gentamicin, 2.2 mg/ml sodium bicarbonate and 22-microg/ml pyruvic acid, 1.0-microg/ml oestradiol (E 8875; Sigma), 0.5-microg/ml follicle-stimulating hormone (FSH) (Folltropin-V; Vetrepharm Inc., Ontario, Canada) and 0.03 IU/ml human gonadotropin (hCG) (Profasi HP; Serono, Aubonne, Switzerland). Oocytes were distributed randomly between basic culture medium (control) and the corresponding experimental treatment. Hormone treatments were: oocytes cultured in; (1) medium without FSH, (2) control medium supplemented with 20 microg/ml oestradiol, or (3) medium supplemented with 1 microg/ml human somatotropin (hST; Humatrope, Lilly, Saint Cloud, France). The second experiment consisted of oocytes cultured in medium supplemented with 0.4% (w/v) bovine serum albumin (BSA, fraction V; Gibco Grand Island, NY, USA) instead of ECS, or oocytes cultured in medium with 10% inactivated oestrous bitch serum (EBS) instead of ECS. Oocytes were cultured in 100 microl droplets (up to 25 oocytes per drop) under mineral oil at 37 degrees C in a 100% humidified atmosphere containing 5% CO2 in air. After 72 h of IVM, the highest rates (p < 0.05) of meiotic resumption were achieved with the 0.4% BSA supplementation. A positive influence on the metaphase II (MII) acquisition rate was observed with hST supplement. Oocytes cultured with 10% EBS supplementation did not develop to the MII stage. The results in this study show that the protein and hormone supplements to TCM-199 culture medium tested did not promote the final steps of IVM

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

    PubMed Central

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

    2009-01-01

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

  15. A primer on meiotic resumption in starfish oocytes: the proposed signaling pathway triggered by maturation-inducing hormone.

    PubMed

    Kishimoto, Takeo

    2011-01-01

    This short review updates the maturation-inducing hormonal signaling in starfish oocytes. In this system, the activation of cyclin B-Cdc2 kinase (Cdk1) that leads to meiotic resumption does not require new protein synthesis. The key intracellular mediator after hormonal stimulation by 1-methyladenine is the protein kinase Akt/PKB, which in turn directly downregulates Myt1 and upregulates Cdc25 toward the activation of cyclin B-Cdc2. Mitotic kinases including Aurora, Plk1 and Greatwall are activated downstream of cyclin B-Cdc2. The starfish oocyte thus provides a simple model system for the study of meiotic resumption. Copyright © 2011 Wiley Periodicals, Inc.

  16. Effect of initial cumulus morphology on meiotic dynamic and status of mitochondria in horse oocytes during IVM.

    PubMed

    Torner, H; Alm, H; Kanitz, W; Goellnitz, K; Becker, F; Poehland, R; Bruessow, K-P; Tuchscherer, A

    2007-04-01

    The aim of this investigation was to examine the chromatin configuration of the nucleus, pattern of mitochondrial aggregation and mitochondrial activity in parallel studies in the same horse oocytes. Horse oocytes recovered by ultrasound-guided follicle aspiration in vivo were classified according to two main initial cumulus morphologies as having compact or expanded cumulus. The percentage of oocytes with a diplotene meiotic configuration at the time of recovery from the follicles was highest in compact oocytes. Oocytes with expanded cumulus layers at the time of recovery matured more rapidly in vitro and reached a proportion >50% at the metaphase II stage (M 2) sooner during in vitro maturation (IVM), than did compact oocytes. The mitochondrial aggregation pattern changed from finely distributed (Type 1) through crystalline (Type 2) to an aggregated, granulated appearance (Type 3) during IVM. The pattern of mitochondrial aggregation at the time of recovery was associated with the initial cumulus morphology of the oocyte, in that compact oocytes had a higher proportion of Type 1 aggregation, whereas expanded oocytes had a higher proportion of Type 3. The fluorescence intensity of metabolic active mitochondria, measured by fluorescence intensity (Em 570) per oocyte after MitoTracker CMTM Ros orange labelling, increased in the oocytes during IVM and depended on initial cumulus investment. Oocytes with the granulated type of aggregated mitochondria Type 3 had the highest level of metabolic activity and were in more progressed stages of meiosis (A 1-M 2). Oocytes initially having expanded layers of cumulus reached significantly higher levels of mitochondrial activity after IVM than did oocytes initially having compact cumuli. During resumption of meiosis the mitochondrial activity of oocytes with initially expanded cumulus increased continuously up to M 2, whereas in oocytes from compact cumulus-oocyte complex (COC), the activity declined after A 1/T 1 stages of

  17. APC(FZR1) prevents nondisjunction in mouse oocytes by controlling meiotic spindle assembly timing.

    PubMed

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

    2012-10-01

    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 APC(CDC20) 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 APC(CDC20) activity. This study implicates FZR1 as a major regulator of prometaphase whose activity helps to prevent chromosome nondisjunction.

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

    PubMed

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

    2015-08-18

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

  20. Effects of the anti-androgen cyproterone acetate (CPA) on oocyte meiotic maturation in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Rime, Hélène; Nguyen, Thaovi; Ombredane, Kevin; Fostier, Alexis; Bobe, Julien

    2015-07-01

    In the present study, we aimed at characterizing the effect of cyproterone acetate (CPA), an anti-androgenic compound, on oocyte meiotic maturation in a freshwater teleost fish species, the rainbow trout (Oncorhynchus mykiss). Fully-grown post-vitellogenic ovarian follicles were incubated in vitro with CPA, luteinizing hormone (Lh) or a combination of CPA and Lh. Incubations were also performed using a combination of Lh and testosterone (T). The occurrence of oocyte maturation (i.e., resumption of the meiotic process) was assessed by monitoring germinal vesicle breakdown (GVBD) after a 72h in vitro incubation. The effect of CPA on the production of 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), the natural maturation-inducing steroid (MIS), was quantified by radioimmunoassay. Our results show that CPA dramatically inhibits Lh-induced oocyte maturation and MIS synthesis. We also observed a synergistic effect of Lh and T on oocyte maturation in highly competent oocytes (i.e., able to resume meiosis after stimulation by low doses of Lh). Our results also show that a combination of CPA and Lh inhibits phosphorylation of extracellular signal-regulated kinase (Erk), kinases that are associated with oocyte maturation in many species. As a whole, our results indicate that CPA has a potential to alter meiotic maturation in rainbow trout. Further analyses are, however, needed to determine the mechanisms by which this anti-androgen interferes with the meiotic process. Furthermore, the present study provides a framework for better understanding of the ecological consequences of exposure to anti-androgens and resulting meiotic maturation abnormalities observed in trout.

  1. Impairment of pachytene spermatogenesis in Dmrt7 deficient mice, possibly causing meiotic arrest.

    PubMed

    Date, Shiori; Nozawa, Osamu; Inoue, Hiroaki; Hidema, Shizu; Nishimori, Katsuhiko

    2012-01-01

    Although Dmrt7 has been reported to be essential for male spermatogenesis, the molecular mechanism underlying pachytene spermatogenesis by Dmrt7 is not known. In the present study, by detailed analysis of Dmrt7 protein distribution in spermatocytes in the first wave of spermatogenesis, we clarified the profile of Dmrt7 expression and localization in pachytene spermatogenesis. Dmrt7-deficient spermatocytes were arrested in the pachytene stage, followed by apoptosis. We analyzed to determine whether every event in the spermatogenesis at the Dmrt7-deficient mice progressed normally, because in several gene knockout mice with spermatogenic arrest described in the previous reports impairments of these events often appeared. Mutant mice showed normal synapsis and XY body formation, while impairment of meiotic sex chromosome inactivation (MSCI), decreased expression of backup genes, and increased expression of retrotransposons indicated incomplete meiotic recombination.

  2. DYNLT3 is required for chromosome alignment during mouse oocyte meiotic maturation.

    PubMed

    Huang, Xin; Wang, Hai-Long; Qi, Shu-Tao; Wang, Zhen-Bo; Tong, Jing-Shan; Zhang, Qing-Hua; Ouyang, Ying-Chun; Hou, Yi; Schatten, Heide; Qi, Zhong-Quan; Sun, Qing-Yuan

    2011-10-01

    Dynein light chain, Tctex-type 3 (DYNLT3), is a member of the cytoplasmic dynein DYNLT light chain family and has been reported to have a potential role in chromosome congression in human mitosis. However, its role in mammalian meiosis is unclear. In this study, we examined its localization, expression, and functions in mouse oocyte meiosis. Immunofluorescent staining showed that DYNLT3 was restricted to the germinal vesicle and associated with kinetochores at the germinal vesicle breakdown stage, metaphase I and metaphase II. The expression level of DYNLT3 was similar at all meiotic stages. Depletion of DYNLT3 by antibody injection resulted in chromosome misalignment and decrease of the polar body extrusion rate. We further found that DYNLT3-depleted oocytes displayed kinetochore-microtubule detachments. Chromosome-spread experiments showed that depletion of DYNLT3 inhibited the metaphase-anaphase transition by preventing homologous chromosome segregation in meiosis I. Our data suggest that DYNLT3 is required for chromosome alignment and homologous chromosome segregation during mouse oocyte meiosis.

  3. Cortical granule translocation is microfilament mediated and linked to meiotic maturation in the sea urchin oocyte.

    PubMed

    Wessel, Gary M; Conner, Sean D; Berg, Linnea

    2002-09-01

    Cortical granules exocytose after the fusion of egg and sperm in most animals, and their contents function in the block to polyspermy by creating an impenetrable extracellular matrix. Cortical granules are synthesized throughout oogenesis and translocate en masse to the cell surface during meiosis where they remain until fertilization. As the mature oocyte is approximately 125 micro m in diameter (Lytechinus variegatus), many of the cortical granules translocate upwards of 60 micro m to reach the cortex within a 4 hour time window. We have investigated the mechanism of this coordinated vesicular translocation event. Although the stimulus to reinitiate meiosis in sea urchin oocytes is not known, we found many different ways to reversibly inhibit germinal vesicle breakdown, and used these findings to discover that meiotic maturation and cortical granule translocation are inseparable. We also learned that cortical granule translocation requires association with microfilaments but not microtubules. It is clear from endocytosis assays that microfilament motors are functional prior to meiosis, even though cortical granules do not use them. However, just after GVBD, cortical granules attach to microfilaments and translocate to the cell surface. This latter conclusion is based on organelle stratification within the oocyte followed by positional quantitation of the cortical granules. We conclude from these studies that maturation promoting factor (MPF) activation stimulates vesicle association with microfilaments, and is a key regulatory step in the coordinated translocation of cortical granules to the egg cortex.

  4. The incorporation of myo-inositol into phosphatidylinositol derivatives is stimulated during hormone-induced meiotic maturation of amphibian oocytes

    SciTech Connect

    Carrasco, D.; Allende, C.C.; Allende, J.E. )

    1990-12-01

    The incorporation of myo-({sup 3}H)inositol into phosphatidylinositol and its phosphorylated derivatives was studied by microinjection of the radioactive precursor into Xenopus laevis oocytes. Induction of meiotic maturation of the oocytes by treatment with either progesterone one or insulin resulted in a significant increase in the incorporation of myo-({sup 3}H)inositol into the phospholipid fraction. This increase occurred 3-6 h after hormonal treatment, a time coincident with the start of the breakdown of the nuclear envelope, and requires protein synthesis. The effect of progesterone and insulin contrasts with the effect of acetylcholine, which acts through a muscarinic receptor causing the activation of phospholipase C, since the latter effector causes an increase in myo-({sup 3}H)inositol incorporation, which is more rapid and does not require protein synthesis. These results suggest that the meiotic maturation process is connected with changes in inositol metabolism in the amphibian oocyte.

  5. Gene expression profiles of Spo11-/- mouse testes with spermatocytes arrested in meiotic prophase I.

    PubMed

    Smirnova, Natalya A; Romanienko, Peter J; Khil, Pavel P; Camerini-Otero, R Daniel

    2006-07-01

    Spo11, a meiosis-specific protein, introduces double-strand breaks on chromosomal DNA and initiates meiotic recombination in a wide variety of organisms. Mouse null Spo11 spermatocytes fail to synapse chromosomes and progress beyond the zygotene stage of meiosis. We analyzed gene expression profiles in Spo11(-/ -)adult and juvenile wild-type testis to describe genes expressed before and after the meiotic arrest resulting from the knocking out of Spo11. These genes were characterized using the Gene Ontology data base. To focus on genes involved in meiosis, we performed comparative gene expression analysis of Spo11(-/ -)and wild-type testes from 15-day mice, when spermatocytes have just entered pachytene. We found that the knockout of Spo11 causes dramatic changes in the level of expression of genes that participate in meiotic recombination (Hop2, Brca2, Mnd1, FancG) and in the meiotic checkpoint (cyclin B2, Cks2), but does not affect genes encoding protein components of the synaptonemal complex. Finally, we discovered unknown genes that are affected by the disruption of the Spo11 gene and therefore may be specifically involved in meiosis and spermatogenesis.

  6. Prevention of maternal aging-associated oocyte aneuploidy and meiotic spindle defects in mice by dietary and genetic strategies.

    PubMed

    Selesniemi, Kaisa; Lee, Ho-Joon; Muhlhauser, Ailene; Tilly, Jonathan L

    2011-07-26

    Increased meiotic spindle abnormalities and aneuploidy in oocytes of women of advanced maternal ages lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Despite the significance of the problem, strategies to sustain oocyte quality with age have remained elusive. Here we report that adult female mice maintained under 40% caloric restriction (CR) did not exhibit aging-related increases in oocyte aneuploidy, chromosomal misalignment on the metaphase plate, meiotic spindle abnormalities, or mitochondrial dysfunction (aggregation, impaired ATP production), all of which occurred in oocytes of age-matched ad libitum-fed controls. The effects of CR on oocyte quality in aging females were reproduced by deletion of the metabolic regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Thus, CR during adulthood or loss of PGC-1α function maintains female germline chromosomal stability and its proper segregation during meiosis, such that ovulated oocytes of aged female mice previously maintained on CR or lacking PGC-1α are comparable to those of young females during prime reproductive life.

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

    PubMed

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

    2015-09-14

    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.

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    1990-01-01

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

  10. Block of CDK1-dependent polyadenosine elongation of Cyclin B mRNA in metaphase-i-arrested starfish oocytes is released by intracellular pH elevation upon spawning.

    PubMed

    Ochi, Hiroe; Aoto, Saki; Tachibana, Kazunori; Hara, Masatoshi; Chiba, Kazuyoshi

    2016-01-01

    Meiotic progression requires the translation of maternal mRNAs in a strict temporal order. In isolated animal oocytes, translation of maternal mRNAs containing a cytoplasmic polyadenylation element (CPE), such as cyclin B, is activated by in vitro stimulation of meiotic resumption which induces phosphorylation of CPEB (CPE-binding protein) and elongation of their polyadenosine (poly(A)) tails; whether or not this model can be applied in vivo to oocytes arrested at metaphase of meiosis I in ovaries is unknown. In this study, we found that active CDK1 (cyclin-dependent kinase 1) phosphorylated CPEB in ovarian oocytes arrested at metphase I in the starfish body cavity, but phosphorylation of CPEB was not sufficient for elongation of cyclin B poly(A) tails. Immediately after spawning, however, mRNA was polyadenylated, suggesting that an increase in intracellular pH (pHi ) upon spawning triggers the elongation of poly(A) tails. Using a cell-free system made from maturing oocytes at metaphase I, we demonstrated that polyadenylation was indeed suppressed at pH below 7.0. These results suggest that a pH-sensitive process, functioning after CPEB phosphorylation, is blocked under physiologically low pHi (<7.0) in metaphase-I-arrested oocytes. The increase in pHi (>7.0) that occurs after spawning triggers polyadenylation of cyclin B mRNA and progression into meiosis II. © 2015 Wiley Periodicals, Inc.

  11. Xp38γ/SAPK3 promotes meiotic G2/M transition in Xenopus oocytes and activates Cdc25C

    PubMed Central

    Perdiguero, Eusebio; Pillaire, Marie-Jeanne; Bodart, Jean-Francois; Hennersdorf, Florian; Frödin, Morten; Duesbery, Nicholas S.; Alonso, Gema; Nebreda, Angel R.

    2003-01-01

    We have studied the role of p38 mitogen-activated protein kinases (MAPKs) in the meiotic maturation of Xenopus oocytes. Overexpression of a constitutively active mutant of the p38 activator MKK6 accelerates progesterone-induced maturation. Immunoprecipit ation experiments indicate that p38γ/SAPK3 is the major p38 activated by MKK6 in the oocytes. We have cloned Xenopus p38γ (Xp38γ) and show that co-expression of active MKK6 with Xp38γ induces oocyte maturation in the absence of progesterone. The maturation induced by Xp38γ requires neither protein synthesis nor activation of the p42 MAPK–p90Rsk pathway, but it is blocked by cAMP-dependent protein kinase. A role for the endogenous Xp38γ in progesterone-induced maturation is supported by the inhibitory effect of kinase-dead mutants of MKK6 and Xp38γ. Furthermore, MKK6 can rescue the inhibition of oocyte maturation by anthrax lethal factor, a protease that inactivates MAPK kinases. We also show that Xp38γ can activate the phosphatase XCdc25C, and we identified Ser205 of XCdc25C as a major phosphorylation site for Xp38γ. Our results indicate that phosphorylation of XCdc25C by Xp38γ/SAPK3 is important for the meiotic G2/M progression of Xenopus oocytes. PMID:14592973

  12. Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1Ser137 involvement in spindle formation and REC8 cleavage

    PubMed Central

    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 (pPlk1Ser137 and pPlk1Thr210) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1Ser137 with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1Thr210 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 pPlk1Ser137 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, pPlk1Thr210 was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1Ser137 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 pPlk1Ser137 and pPlk1Thr210, as well as the subcellular distribution of pPlk1Thr210, were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1Ser137 is the action executor, in mouse oocytes during meiotic division. PMID:26654596

  13. Peritoneal Fluid From Infertile Women With Minimal/Mild Endometriosis Compromises the Meiotic Spindle of Metaphase II Bovine Oocytes.

    PubMed

    Gazeto Melo Jianini, Bruna Talita; Giorgi, Vanessa Silvestre Innocenti; Da Broi, Michele Gomes; de Paz, Cláudia Cristina Paro; Rosa E Silva, Júlio César; Ferriani, Rui Alberto; Navarro, Paula Andrea

    2017-01-01

    Some studies have demonstrated alterations in the composition of peritoneal fluid (PF) from women with endometriosis. Controversial studies have suggested that impaired oocyte quality may be involved in the pathogenesis of endometriosis-related infertility. The aim of this study was to evaluate the spindle and chromosome distribution of in vitro-matured oocytes in the presence of 2 concentrations of PF from infertile women with minimal/mild endometriosis (EI/II) compared to fertile controls. We performed an experimental study using a bovine model. Samples of PF were obtained from 12 women who underwent videolaparoscopy-6 infertile women with EI/II and 6 fertile women without endometriosis (control group). Immature bovine oocytes underwent in vitro maturation (IVM) in the absence of PF and in the presence of 2 concentrations (1% and 10%) of PF from fertile women and from infertile women with EI/II. After 22 to 24 hours of IVM, oocytes were fixed for subsequent immunofluorescence staining for the visualization of microtubules and chromosomes by confocal microscopy. The percentage of meiotically normal oocytes was significantly lower for oocytes that underwent IVM in the presence of 1% (62.50%) and 10% (56.25%) of PF from infertile women with EI/II than in the absence of PF (88.46%) and in the presence of 1% (78.57%) and 10% (84.61%) of PF from fertile women ( P < .01). We demonstrated that PF from infertile women with EI/II promotes meiotic abnormalities in in vitro-matured bovine oocytes. Therefore, our results contribute to the understanding of the etiopathogenic mechanisms of infertility related to EI/II.

  14. 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.

  15. The role of Fyn kinase in the release from metaphase in mammalian oocytes.

    PubMed

    Levi, M; Shalgi, R

    2010-01-27

    Meiosis in mammalian oocytes starts during embryonic life and arrests for the first time before birth, at prophase of the first meiotic division. The second meiotic arrest occurs after spindle formation at metaphase of the second meiotic division (MII) in selected oocytes designated for ovulation. The fertilizing spermatozoon induces the release from MII arrest only after the oocyte's spindle assembly checkpoint (SAC) was deactivated. Src family kinases (SFKs) are nine non-receptor protein tyrosine kinases that regulate many key cellular functions. Fyn is an SFK expressed in many cell types, including oocytes. Recent studies, including ours, imply a role for Fyn in exit from meiotic and mitotic metaphases. Other studies demonstrate that SFKs, particularly Fyn, are required for regulation of microtubules polymerization and spindle stabilization. Altogether, Fyn is suggested to play an essential role in signaling events that implicate SAC pathway and hence in regulating the exit from metaphase in oocytes and zygote. 2009 Elsevier Ireland Ltd. All rights reserved.

  16. Dynamic changes of connexin-43, gap junctional protein, in outer layers of cumulus cells are regulated by PKC and PI 3-kinase during meiotic resumption in porcine oocytes.

    PubMed

    Shimada, M; Maeda, T; Terada, T

    2001-04-01

    Mammalian oocytes are surrounded by numerous layers of cumulus cells, and the loss of gap junctional communication in the outer layers of cumulus cells induces meiotic resumption in oocytes. In this study, we investigated the dynamic changes in the gap junctional protein connexin-43 in cumulus cells during the meiotic resumption of porcine oocytes. The amount of connexin-43 in all layers of cumulus cells recovered from cumulus-oocyte complexes was increased after 4-h cultivation. However, at 12-h cultivation, the positive signal for connexin-43 immunoreactivity was markedly reduced in the outer layers of cumulus cells. When these reductions of connexin-43 were blocked by protein kinase C (PKC) or phosphatidylinositol (PI) 3-kinase inhibitor, networks of filamentous bivalents (i.e., advanced chromosomal status) were undetectable in the germinal vesicle of the oocyte. After 28-h cultivation, when the majority of oocytes were reaching the metaphase I (MI) stage, the connexin-43 in the inner layers of cumulus cells was phosphorylated, regardless of mitogen-activated protein (MAP) kinase activation. These results suggest that the initiation of meiotic resumption, namely, the formation of networks of filamentous bivalents in germinal vesicle, is associated with the reduction of gap junctional protein connexin-43 in the outer layers of cumulus cells via the PKC and/or PI 3-kinase pathway. Moreover, the connexin-43 in the inner layers of cumulus cells is phosphorylated during meiotic progression beyond the MI stage, regardless of MAP kinase activation in cumulus cells surrounding the oocyte.

  17. Di (2-ethylhexyl) phthalate exposure impairs meiotic progression and DNA damage repair in fetal mouse oocytes in vitro

    PubMed Central

    Liu, Jing-Cai; Lai, Fang-Nong; Li, Ling; Sun, Xiao-Feng; Cheng, Shun-Feng; Ge, Wei; Wang, Yu-Feng; Li, Lan; Zhang, Xi-Feng; De Felici, Massimo; Dyce, Paul W; Shen, Wei

    2017-01-01

    Di (2-ethylhexyl) phthalate (DEHP), is the most common member of the class of phthalates that are used as plasticizers and have become common environmental contaminants. A number of studies have shown that DEHP exposure impacts reproductive health in both male and female mammals by acting as an estrogen analog. Here, we investigated the effects of DEHP on meiotic progression of fetal mouse oocytes by using an in vitro model of ovarian tissue culture. The results showed that 10 or 100 μM DEHP exposure inhibited the progression of oocytes throughout meiotic prophase I, specifically from the pachytene to diplotene stages. DEHP possibly impairs the ability to repair DNA double-strand breaks induced by meiotic recombination and as a consequence activates a pachytene check point. At later stages, such defects led to an increased number of oocytes showing apoptotic markers (TUNEL staining, expression of pro-apoptotic genes), resulting in reduced oocyte survival, gap junctions, and follicle assembly in the ovarian tissues. Microarray analysis of ovarian tissues exposed to DEHP showed altered expression of several genes including some involved in apoptosis and gonad development. The expression changes of some genes clustered in cell-cell communication and signal transduction, along with plasma membrane, extracellular matrix and ion channel function classes, were dependent on the DEHP concentration. Together, these results bring new support to the notion that exposure to DEHP during gestation might exert deleterious effects on ovary development, perturbing germ cell meiosis and the expression of genes involved in a wide range of biological processes including ovary development. PMID:28771232

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

    PubMed Central

    Downs, Stephen M.

    2014-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  20. Supplementation with cumulus cell masses improves the in vitro meiotic competence of porcine cumulus-oocytes complexes derived from small follicles.

    PubMed

    Matsunaga, R; Funahashi, H

    2017-03-30

    The present study was conducted to examine the supplemented effect of cumulus cell masses (CCMs) derived from middle follicle (MF; 3-6 mm diameter) on the morphology and the meiotic or developmental competence of oocytes from small follicles (SF; 1-2 mm diameter). The number of cumulus cells surrounding oocytes just after collection was also lower in cumulus-oocyte complexes (COCs) from SF than MF. The ooplasmic diameter of oocytes was significantly smaller in SF-derived oocytes than MF-derived ones before and after in vitro maturation (IVM), whereas the diameter significantly increased during the culture. Co-culture of SF-derived COCs with MF-derived CCMs during IVM significantly improved the meiotic competence of the oocytes to the metaphase-II stage. Furthermore, the ooplasmic diameter of SF-derived COCs during IVM was increased to the similar size of MF-derived those in the presence of MF-derived CCMs. The abilities of oocytes to be penetrated, to form male pronuclear formation and to cleave or develop to the blastocyst stage were not affected by the co-culture with CCMs. Electrophoretic analysis of CCM secretions clearly showed the presence of more protein(s) approximately 27.6 kDa in the conditioned medium when supplemented with MF-derived CCMs. In conclusion, we demonstrate that supplementation with MF-derived CCMs improves the ooplasmic diameter and meiotic competence of SF-derived oocytes.

  1. Oocyte development, meiosis and aneuploidy.

    PubMed

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

    2015-09-01

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

  2. 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

  3. Knockdown of UCHL5IP causes abnormalities in γ-tubulin localisation, spindle organisation and chromosome alignment in mouse oocyte meiotic maturation.

    PubMed

    Wang, Ya-Peng; Qi, Shu-Tao; Wei, Yanchang; Ge, Zhao-Jia; Chen, Lei; Hou, Yi; Ouyang, Ying-Chun; Schatten, Heide; Zhao, Jian-Guo; Sun, Qing-Yuan

    2013-01-01

    UCHL5IP is one of the subunits of the haus complex, which is important for microtubule generation, spindle bipolarity and accurate chromosome segregation in Drosophila and human mitotic cells. In this study, the expression and localisation of UCHL5IP were explored, as well as its functions in mouse oocyte meiotic maturation. The results showed that the UCHL5IP protein level was consistent during oocyte maturation and it was localised to the meiotic spindle in MI and MII stages. Knockdown of UCHL5IP led to spindle defects, chromosome misalignment and disruption of γ-tubulin localisation in the spindle poles. These results suggest that UCHL5IP plays critical roles in spindle formation during mouse oocyte meiotic maturation.

  4. Loss of function of KIF1B impairs oocyte meiotic maturation and early embryonic development in mice.

    PubMed

    Kong, Xiang-Wei; Wang, Dong-Hui; Zhou, Cheng-Jie; Zhou, Hong-Xia; Liang, Cheng-Guang

    2016-11-01

    Kinesin family member 1B (KIF1B) is an important microtubule-dependent monomeric motor in mammals, although little is known about its role in meiosis. We profiled KIF1B expression and localization during oocyte maturation and early embryonic development in mice, revealing a dynamic pattern throughout meiotic progression. Depletion or inhibition of KIF1B leads to abnormal polar body extrusion, disordered spindle dynamics, defects in chromosome congression, increased aneuploidy, and impaired embryonic development. Further, KIF1B depletion affects the distribution of mitochondria and abundance of ATP. Taken together, our study demonstrates that mouse KIF1B is important for spindle assembly, chromosome congression, and mitochondrial distribution during oocyte maturation and early embryonic development. Mol. Reprod. Dev. 83: 1027-1040, 2016 © 2016 Wiley Periodicals, Inc.

  5. Sensitivity of mouse oocytes to nicotine-induced perturbations during oocyte meiotic maturation and aneuploidy in vivo and in vitro.

    PubMed

    Mailhes, J B; Young, D; Caldito, G; London, S N

    2000-03-01

    Oocyte meiosis is sensitive to endogenous and exogenous perturbations that upset the temporal sequence of biochemical reactions during oocyte maturation (OM) and predispose oocytes to aneuploidy. Nicotine is an alkaloid that has been reported to disrupt the rate of OM, reduce ovulation and fertilization rates, and increase diploidy. The objective of this study was to test the hypothesis that nicotine perturbs the rate of OM and induces aneuploidy in mouse oocytes in vivo and in vitro. Female mice were given 7.5 IU pregnant mare's serum and either 0, 5.0, 7.5, or 10 mg/kg nicotine in vivo at -3, 0, and +3 h relative to a 5 IU injection of HCG. Oocytes were also cultured in vitro in the presence of 0, 1.0, 5.0, or 10.0 mmol/l nicotine. In vivo, significant (P < 0.05) differences in the proportions of oocytes with premature centromere separation and premature anaphase were found at 10.0 mg/kg nicotine suggesting that the rate of OM was advanced. Also, at this dose the proportion of ovulated oocytes was reduced by approximately 50% relative to controls. In vitro, only non-significant differences were found among the parameters measured. Although nicotine reduced the ovulation rate and perturbed the rate of OM in vivo, these data show that the rate of aneuploidy was not significantly elevated.

  6. 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

  7. Cyclopiazonic acid, an inhibitor of calcium-dependent ATPases, induces exit from metaphase I arrest in growing pig oocytes.

    PubMed

    Petr, J; Rozinek, J; Vanourková, Z; Jílek, F

    1999-01-01

    Calcium plays an important role in the regulation of meiotic maturation in mammalian oocytes. In the present study, mycotoxin cyclopiazonic acid (CPA), an inhibitor of calcium-dependent ATPases, was used to mobilize intracellular calcium deposits in growing pig oocytes, which had not attained full meiotic competence and in which maturation is thus spontaneously blocked at the metaphase I stage. CPA treatment significantly increased the ratio of growing oocytes that are able to overcome the spontaneously occurring metaphase I block to complete their maturation at the metaphase II stage. CPA treatment of a least 2 hours' duration is necessary to overcome the metaphase I block in growing oocytes. A similar effect upon release from the spontaneous meiotic block at the metaphase I stage was observed after treatment of growing pig oocytes with thapsigargin, another inhibitor of endogenous calcium-dependent ATPases. Numerous calcium deposits in vacuoles, the mitochondria and on the surface of yolk granules in growing pig oocytes were observed. CPA treatment is able to mobilize calcium from the mitochondria, but deposits in vacuoles and deposits on the surface of yolk granules seem to remain intact after CPA treatment. A microinjection of heparin, which is known to bind with the inositol trisphosphate receptors, significantly decreased the ratio of CPA-treated growing oocytes overcoming the block at the metaphase I stage. This indicates that CPA might mobilize calcium in growing pig oocytes through inositol trisphosphate receptors. On the other hand, a microinjection of procaine or a microinjection of ruthenium red, both inhibitors of ryanodine receptors, did not prevent the overcoming of the metaphase I block, induced by CPA treatment. The calcium channel blocker, verapamil, significantly reduces the proportion of CPA-treated growing oocytes that overcome the metaphase I block. This indicates that the influx of calcium from extracellular sources is necessary to overcome

  8. 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.

  9. Effect of removing cumulus cells from porcine cumulus-oocyte complexes derived from small and medium follicles during IVM on the apoptotic status and meiotic progression of the oocytes.

    PubMed

    Ferré, Pilar; Bui, Tra Mi Thi; Wakai, Takuya; Funahashi, Hiroaki

    2016-10-15

    The present study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small follicle (SF; 0.5-2 mm in diameter) and medium follicle (MF; 3-6 mm in diameter) when the oocytes were denuded before, during, and after IVM. Cumulus-oocyte complexes (COCs) were collected from SF or MF of prepubertal gilt ovaries. Before or 20 hours after the start of IVM culture, some oocytes were denuded and cultured for IVM. At the end of IVM, apoptotic status and meiotic progression of the oocytes were compared with oocytes matured in the presence of cumulus cells (CCs) by Annexin-V/PI assay and 4',6-Diamidino-2-phenylindole staining. Apoptotic status of the oocytes was only affected by time when the oocytes were denuded. In both oocytes from SF and MF, although the incidence of early and late apoptotic oocytes was significantly higher when the CCs were removed before IVM, the rate was significantly lower when CCs were removed 20 and 44 hours after the start of IVM. The incidence of mature oocytes was significantly affected by both the origin of COCs and time when oocytes were denuded from the COCs. Although the percentage of mature oocytes was higher in MF than SF, maturation rates were significantly higher when oocytes were denuded 20 hours, as compared with 0 and 44 hours after the start of IVM. However, the percentage of mature oocytes with a morphologically normal spindle was significantly higher when oocytes were denuded 44 hours, rather than 22 hours of IVM. In conclusion, removing CCs 20 hours after the start of IVM seems to promote meiotic progression of the oocytes to the metaphase-II stage even when the COCs were collected from SF, although factor(s) from or communication with CCs during IVM may need to obtain a morphologically normal spindle in mature oocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Developmental Control of Oocyte Maturation and Egg Activation in Metazoan Models

    PubMed Central

    Von Stetina, Jessica R.; Orr-Weaver, Terry L.

    2011-01-01

    Production of functional eggs requires meiosis to be coordinated with developmental signals. Oocytes arrest in prophase I to permit oocyte differentiation, and in most animals, a second meiotic arrest links completion of meiosis to fertilization. Comparison of oocyte maturation and egg activation between mammals, Caenorhabditis elegans, and Drosophila reveal conserved signaling pathways and regulatory mechanisms as well as unique adaptations for reproductive strategies. Recent studies in mammals and C. elegans show the role of signaling between surrounding somatic cells and the oocyte in maintaining the prophase I arrest and controlling maturation. Proteins that regulate levels of active Cdk1/cyclin B during prophase I arrest have been identified in Drosophila. Protein kinases play crucial roles in the transition from meiosis in the oocyte to mitotic embryonic divisions in C. elegans and Drosophila. Here we will contrast the regulation of key meiotic events in oocytes. PMID:21709181

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

    PubMed

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

    2017-03-01

    The aims of this study were: (1) to evaluate the effect of different insulin concentrations, alone or in combination with either a fixed FSH concentration or increasing FSH concentrations on the in vitro culture of isolated caprine preantral follicles and (2) to analyze the efficiency of two IVM media and maturation culture systems (with or without coculture with in vivo grown oocytes) on the meiosis resumption. Secondary follicles were cultured for 18 days in a basic medium supplemented with low- or high-insulin concentration alone or with a fixed FSH concentration or with increasing FSH concentrations. Oocytes grown in vivo or in vitro were matured alone or cocultured. The high-insulin concentration associated with fixed FSH treatment had higher meiotic resumption rate (P < 0.05) and was the only treatment capable of producing oocytes in metaphase II. The rates of germinal vesicle, germinal vesicle breakdown, metaphase I, metaphase II (MII), meiotic resumption, and oocyte diameter were similar between the maturation media. In conclusion, a basic medium supplemented with 10-μg/mL insulin and 100-μg/mL FSH throughout the culture period improved meiotic resumption rate and produced MII oocytes from caprine preantral follicles cultured in vitro. The MII rate was similar between in vivo and in vitro grown oocytes ≥110 μm.

  12. 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.

  13. 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

  14. 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

  15. Effects of cysteamine during in vitro maturation on viability and meiotic competence of vitrified buffalo oocytes

    PubMed Central

    Mahmoud, K. Gh. M.; El-Sokary, M. M. M.; Kandiel, M. M. M.; Abou El-Roos, M. E. A.; Sosa, G. M. S.

    2016-01-01

    The aims of the present study were to assess the effects of cysteamine as an anti-oxidant on the rate of in vitro maturation (IVM) of buffalo oocytes (experiment 1), and their viability and nuclear status following vitrification (experiment 2). Immature oocytes with compact cumulus cells obtained from the ovaries of slaughtered animals were harvested and then cultured in the maturation medium with no cysteamine (control) or 50 μM cysteamine (treated). Oocytes were vitrified in vitrification solution 1 (VS1): 1.5 M ethylene glycol (EG) + 1.5 M dimethyl sulfoxide (DMSO) for 45 s (step one). After this initial exposure, oocytes were transferred to VS2: 3 M EG + 3 M DMSO in a holding medium for 25 s (step two). After warming, oocytes were evaluated morphologically and then cultured for a further 2 h in the cysteamine-supplemented or control maturation media. The oocytes were evaluated morphologically, stained with trypan blue for viability evaluation. The maturation rate of oocytes was higher (P<0.05) for IVM media with cysteamine compared with controls. There was no significant difference in morphology, survivability and maturation rate between the two vitrification groups (cysteamine-treated and untreated groups) but the morphology, survivability and percentages of metaphase-II oocytes in both groups of vitrified oocytes were lower compared with their respective controls. In conclusion, the addition of cysteamine to the maturation medium improved nuclear maturation of buffalo oocytes but had no positive effect on their cryoresistance during vitrification. PMID:27822245

  16. 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.

  17. Oocyte development, meiosis and aneuploidy

    PubMed Central

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

    2015-01-01

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

  18. Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes.

    PubMed

    Wang, Zhen-Bo; Jiang, Zong-Zhe; Zhang, Qing-Hua; Hu, Meng-Wen; Huang, Lin; Ou, Xiang-Hong; Guo, Lei; Ouyang, Ying-Chun; Hou, Yi; Brakebusch, Cord; Schatten, Heide; Sun, Qing-Yuan

    2013-12-01

    Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.

  19. Follistatin Rescues Blastocyst Development of Poor Quality Porcine Cumulus-Oocyte Complexes by Delaying Meiotic Resumption With Decreased cGMP.

    PubMed

    Lee, Bo Myeong; Chun, Ju Lan; Lee, Ji Hye; Kim, Eun Young; Park, Kang-Sun; Lee, Jin-Hee; Daigneault, Bradford W; Smith, George W; Kim, Keun Jung; Chang, Kyu-Tae; Lee, Sang-Rae; Kim, Sun-Uk; Choi, Seon-A; Lee, Kyung-Bon; Kim, Min Kyu

    2017-01-01

    Mammalian oocytes resume maturation when removed from follicles and cultured in vitro. During folliculogenesis, oocytes are bathed in follicular fluid (FF), which provides an important and specialized microenvironment for oocyte competence. Follistatin (FST) is one component of FF that may play a role in oocyte maturation and embryo development. This study was conducted to examine possible effects of FST on porcine oocyte competence and embryo development. Exogenous FST in oocyte maturation medium for 22 or 44 hours did not improve nuclear maturation and had no effect on good quality cumulus-oocyte complexes (COCs). However, FST improved blastocyst rates in embryos derived from oocytes with less than 2 layers of cumulus. Follistatin treatment of the poor quality COC group increased transcript levels for genes indicative of oocyte quality. Transcript levels were also altered for cumulus expansion-related genes in response to FST when measured during the germinal vesicle breakdown stage. Interestingly, high-quality oocytes remained at germinal vesicle stage much longer than low-quality oocytes, FST treatment induced temporary blockage of spontaneous meiotic resumption when added during culture of both good and poor quality COCs, and levels of cyclic guanosine monophosphate (cGMP) were higher in FST-treated versus untreated groups for both good and poor quality oocytes. In conclusion, FST treatment of porcine oocytes during in vitro maturation can rescue competency of poor quality oocytes to develop to blastocyst stage following in vitro fertilization. Beneficial effects of addition of FST to culture medium may be mediated by inhibiting degradation of cGMP and temporarily delaying nuclear maturation.

  20. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Two zinc finger proteins, OMA-1 and OMA-2, are redundantly required for oocyte maturation in C. elegans.

    PubMed

    Detwiler, M R; Reuben, M; Li, X; Rogers, E; Lin, R

    2001-08-01

    Oocytes are released from meiotic prophase I arrest through a process termed oocyte maturation. We present here a genetic characterization of oocyte maturation, using C. elegans as a model system. We show that two TIS11 zinc finger-containing proteins, OMA-1 and OMA-2, express specifically in maturing oocytes and function redundantly in oocyte maturation. Oocytes in oma-1;oma-2 mutants initiate but do not complete maturation and arrest at a defined point in prophase I. Two maturation signal-induced molecular events, including the maintenance of activated MAP kinase, do not occur in Oma oocytes. The Oma prophase arrest is released by inactivation of a MYT-1-like kinase, suggesting that OMA-1 and OMA-2 function upstream of MYT-1 as positive regulators of prophase progression during meiotic maturation.

  2. Distribution of cortical granules and meiotic maturation of canine oocytes in bi-phasic systems.

    PubMed

    Apparicio, Maricy; Mostachio, Giuliano Q; Motheo, Tathiana F; Alves, Aracelle E; Padilha, Luciana; Pires-Butler, Eliandra A; Savi, Paula A P; Uscategui, Ricardo A R; Luvoni, Gaia C; Vicente, Wilter R R

    2015-09-01

    The aim of this study was to evaluate the influence of different bi-phasic systems with gonadotrophins and steroids on in vitro maturation rates of oocytes obtained from bitches at different reproductive stages (follicular, luteal, anoestrous). In System A (control) oocytes were matured for 72h in base medium (BM) with 10IUmL(-1) human chorionic gonadotrophin (hCG), 1μgmL(-1) progesterone (P4) and 1μgmL(-1) oestradiol (E2); in bi-phasic System B oocytes were matured for 48h in BM with hCG and for 24h in BM with P4; in bi-phasic System C oocytes were matured for 48h in BM with hCG, P4 and E2, and for 24h in BM with P4; in System D, oocytes were cultured in BM without hormonal supplementation. Data were analysed by ANOVA. There was a positive effect of the bi-phasic systems on germinal vesicle breakdown, metaphase I and metaphase II rates, irrespective of reproductive status (P<0.05). Bi-phasic systems were also beneficial for cortical granule distribution (an indication of cytoplasmic maturation) and its relationship to nuclear status: 74.5% of the oocytes cultured in System B and 85.4% of those cultured in System C presented both nuclear and cytoplasmic maturation (P<0.001). The stage of the oestrous cycle did not influence maturation rates.

  3. Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes.

    PubMed Central

    Nielsen, P J; Thomas, G; Maller, J L

    1982-01-01

    A single ribosomal protein (Mr, 32,000) becomes phosphorylated during progesterone-induced in vitro maturation of Xenopus laevis oocytes. The protein is identified as 40S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two-dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in unstimulated oocytes. After progesterone treatment, phosphorylation of S6 precedes germinal vesicle breakdown (GVBD) and is maximal at the time when 50% of the oocytes have undergone GVBD. S6, when maximally phosphorylated, exists in derivatives that correspond to the most highly phosphorylated forms observed in other systems, and the increase in S6 phosphorylation occurs at approximately the same time as the increase in the overall protein synthesis rate reported to occur during oocyte maturation. S6 is also maximally phosphorylated in unfertilized eggs following maturation in vivo. Injection of a partially purified preparation of maturation-promoting factor into immature oocytes induces immediate phosphorylation of S6 and rapidly increases the rate of protein synthesis. Moreover, incubation of ribosomes with this factor and radiolabeled ATP results in labeling of S6. These findings suggest that S6 phosphorylation may be important in the control of protein synthesis during maturation and may also play a role in the mechanism of action of maturation-promoting factor. Images PMID:7045876

  4. Signaling-Mediated Regulation of Meiotic Prophase I and Transition During Oogenesis

    PubMed Central

    2017-01-01

    Generation of healthy oocytes requires coordinated regulation of multiple cellular events and signaling pathways. Oocytes undergo a unique developmental growth and differentiation pattern interspersed with long periods of arrest. Oocytes from almost all species arrest in prophase I of oogenesis that allows for long period of growth and differentiation essential for normal oocyte development. Depending on species, oocytes that transit from prophase I to meiosis I also arrest at meiosis I for fairly long periods of time and then undergo a second arrest at meiosis II that is completed upon fertilization. While there are species-specific differences in C. elegans, D. melanogaster, and mammalian oocytes in stages of prophase I, meiosis I, or meiosis II arrest, in all cases cell signaling pathways coordinate the developmental events controlling oocyte growth and differentiation to regulate these crucial phases of transition. In particular, the ERK MAP kinase signaling pathway, cyclic AMP second messengers, and the cell cycle regulators CDK1/cyclin B are key signaling pathways that seem evolutionarily conserved in their control of oocyte growth and meiotic maturation across species. Here, I identify the common themes and differences in the regulation of key meiotic events during oocyte growth and maturation. PMID:28247047

  5. Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes

    PubMed Central

    1993-01-01

    The role of Raf and MAPK (mitogen-activated protein kinase) during the maturation of Xenopus oocytes was investigated. Treatment of oocytes with progesterone resulted in a shift in the electrophoretic mobility of Raf at the onset of germinal vesicle breakdown (GVBD), which was coincident with the activation of MAPK. Expression of a kinase- defective mutant of the human Raf-1 protein (KD-RAF) inhibited progesterone-mediated MAPK activation. MAPK activation was also inhibited by KD-Raf in oocytes expressing signal transducers of the receptor tyrosine kinase (RTK) pathway, including an activated tyrosine kinase (Tpr-Met), a receptor tyrosine kinase (EGFr), and Ha-RasV12. KD- RAF completely inhibited GVBD induced by the RTK pathway. In contrast, KD-RAF did not inhibit GVBD and the progression to Meiosis II in progesterone-treated oocytes. Injection of Mos-specific antisense oligodeoxyribonucleotides inhibited MAPK activation in response to progesterone and Tpr-Met, but failed to inhibit these events in oocytes expressing an oncogenic deletion mutant of Raf-1 (delta N'Raf). Injection of antisense oligodeoxyribonucleotides to Mos also reduced the progesterone- and Tpr-Met-induced electrophoretic mobility shift of Xenopus Raf. These results demonstrate that RTKs and progesterone participate in distinct yet overlapping signaling pathways resulting in the activation of maturation or M-phase promoting factor (MPF). Maturation induced by the RTK pathway requires activation of Raf and MAPK, while progesterone-induced maturation does not. Furthermore, the activation of MAPK in oocytes appears to require the expression of Mos. PMID:8335690

  6. A casein kinase 1 prevents expulsion of the oocyte meiotic spindle into a polar body by regulating cortical contractility.

    PubMed

    Flynn, Jonathan R; McNally, Francis J

    2017-09-01

    During female meiosis, haploid eggs are generated from diploid oocytes. This reduction in chromosome number occurs through two highly asymmetric cell divisions, resulting in one large egg and two small polar bodies. Unlike mitosis, where an actomyosin contractile ring forms between the sets of segregating chromosomes, the meiotic contractile ring forms on the cortex adjacent to one spindle pole, then ingresses down the length of the spindle to position itself at the exact midpoint between the two sets of segregating chromosomes. Depletion of casein kinase 1 gamma (CSNK-1) in Caenorhabditis elegans led to the formation of large polar bodies that contain all maternal DNA, because the contractile ring ingressed past the spindle midpoint. Depletion of CSNK-1 also resulted in the formation of deep membrane invaginations during meiosis, suggesting an effect on cortical myosin. Both myosin and anillin assemble into dynamic rho-dependent cortical patches that rapidly disassemble in wild-type embryos. CSNK-1 was required for disassembly of both myosin patches and anillin patches. Disassembly of anillin patches was myosin independent, suggesting that CSNK-1 prevents expulsion of the entire meiotic spindle into a polar body by negatively regulating the rho pathway rather than through direct inhibition of myosin. © 2017 Flynn and McNally. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  7. Melatonin protects against maternal obesity-associated oxidative stress and meiotic defects in oocytes via the SIRT3-SOD2-dependent pathway.

    PubMed

    Han, Longsen; Wang, Haichao; Li, Ling; Li, Xiaoyan; Ge, Juan; Reiter, Russel J; Wang, Qiang

    2017-10-01

    Maternal obesity in humans is associated with poor outcomes across the reproductive spectrum. Emerging evidence indicates that these defects are likely attributed to factors within the oocyte. Although various molecules and pathways may contribute to impaired oocyte quality, prevention of fertility issues associated with maternal obesity is a challenge. Using mice fed a high-fat diet (HFD) as an obesity model, we document spindle disorganization, chromosome misalignment, and elevated reactive oxygen species (ROS) levels in oocytes from obese mice. Oral administration of melatonin to HFD mice not only reduces ROS generation, but also prevents spindle/chromosome anomalies in oocytes, consequently promoting the developmental potential of early embryos. Consistent with this finding, we find that melatonin supplement during in vitro maturation also markedly attenuates oxidative stress and meiotic defects in HFD oocytes. Finally, by performing morpholino knockdown and acetylation-mimetic mutant overexpression assays, we reveal that melatonin ameliorates maternal obesity-induced defective phenotypes in oocytes through the SIRT3-SOD2-dependent mechanism. In sum, our data uncover the marked beneficial effects of melatonin on oocyte quality from obese females; this opens a new area for optimizing culture system as well as fertility management. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Characterization of aurora-a in porcine oocytes and early embryos implies its functional roles in the regulation of meiotic maturation, fertilization and cleavage.

    PubMed

    Yao, Li-Juan; Sun, Qing-Yuan

    2005-02-01

    Aurora-A is a serine/threonine protein kinase that plays important regulatory roles during mitotic cell cycle progression. In this study, Aurora-A expression, subcellular localization, and possible functions during porcine oocyte meiotic maturation, fertilization and early embryonic cleavage were studied by using Western blot, confocal microscopy and drug treatments. The quantity of Aurora-A protein remained stable during porcine oocyte meiotic maturation. Confocal microscopy revealed that Aurora-A distributed abundantly in the nucleus at the germinal vesicle stage. After germinal vesicle breakdown, Aurora-A concentrated around the condensed chromosomes and the metaphase I spindle, and finally, Aurora-A was associated with spindle poles during the formation of the metaphase II spindle. Aurora-A concentrated in the pronuclei in fertilized eggs. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. In conclusion, Aurora-A may be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during porcine oocyte meiotic maturation, fertilization and early embryonic mitosis.

  9. Rise and fall of protein phosphorylation during meiotic maturation in oocytes of Sabellaria alveolata (polychaete annelid).

    PubMed

    Peaucellier, G; Dorée, M; Picard, A

    1984-12-01

    Incorporation of [32P]phosphate into proteins was monitored, in preloaded Sabellaria oocytes, during meiosis. After a fourfold increase during the transition from prophase to metaphase I, the incorporated radioactivity decreased steadily by 25% during completion of meiosis, while it slowly increased in metaphase I-blocked oocytes. Measurements of the amount and specific activity of nucleotide pools showed no variation, while total alkali-labile protein-bound phosphate was found to increase and then decrease during meiosis. Autoradiography of sodium dodecyl sulfate slabgels showed that some proteins have peculiar phosphorylation-dephosphorylation kinetics. The changes in the level of phosphorylation of proteins may be related to similar changes in maturation-promoting factor (MPF) activity.

  10. Maternal liver damage delays meiotic resumption in bovine oocytes through impairment of signalling cascades originated from low p38MAPK activity in cumulus cells.

    PubMed

    Tanaka, H; Takeo, S; Monji, Y; Kuwayama, T; Iwata, H

    2014-02-01

    The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p < 0.05). When cumulus cells were removed from cumulus-oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7 h of in vitro maturation (IVM) (p < 0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0 h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p < 0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.

  11. Errors at mitotic segregation early in oogenesis and at first meiotic division in oocytes from donor females: comparative genomic hybridization analyses in metaphase II oocytes and their first polar body.

    PubMed

    Obradors, Albert; Rius, Mariona; Cuzzi, Juliana; Daina, Gemma; Gutiérrez-Mateo, Cristina; Pujol, Aïda; Marina, Fernando; Márquez, Carmen; Benet, Jordi; Navarro, Joaquima

    2010-02-01

    The aim of this work is to analyze, using the comparative genomic hybridization technique, the frequencies and the mechanisms involved in the production of aneuploidy events in donor oocytes. The results showed that 32.1% of them were aneuploid, with 51.7% of those originating from first meiotic division errors and 48.3% from the presence of aneuploid oogonium. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  12. 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.

  13. 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

  14. Localization and Dynamics of Cdc2-Cyclin B during Meiotic Reinitiation in Starfish OocytesV⃞

    PubMed Central

    Terasaki, Mark; Okumura, Ei-ichi; Hinkle, Beth; Kishimoto, Takeo

    2003-01-01

    The Cdc2-cyclin B kinase has a central role in regulating the onset of M phase. In starfish oocytes, Cdc2-cyclin B begins to be activated ∼10 min after application of maturation hormone, followed by accumulation in the nucleus then nuclear envelope breakdown. By immunofluorescence and by expressing a green fluorescent (GFP) chimera of cyclin B, we find that cyclin B is present in aggregates in the cytoplasm of immature oocytes. The aggregates disperse at ∼10 min, suggesting that the dispersal is closely related to the activation of the kinase. Using cyclin B-GFP, the dispersion begins from the region containing the centrosomes. Extractability of Cdc2-cyclin B changes with similar kinetics during maturation. Active Cdc25 phosphatase released Cdc2-cyclin B from the detergent-insoluble fraction independently of its phosphatase activity. Live cell imaging also showed that Cdc2-cyclin B begins to accumulate in the nucleus before changes in nuclear pore permeability, consistent with Cdc2-cyclin B-induced disassembly of the pores. PMID:14551249

  15. Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes

    PubMed Central

    1985-01-01

    A human autoantiserum (5051) directed against pericentriolar material (PCM) was used to study the distribution of microtubule-organizing centers (MTOCs) in the oocyte and during the first cell cycle of mouse development. In oocytes, the PCM was found not only at the poles of the barrel-shaped metaphase II spindle but also at many discrete loci around the cytoplasm near the cell cortex. The spindle poles were also composed of several PCM foci. In metaphase-arrested eggs only the PCM foci located near the chromosomes acted as MTOCs. However, after reduction of the critical concentration for tubulin polymerization by taxol, the cytoplasmic PCM foci were also found to be associated with nucleation of microtubules. After fertilization the cortical PCM foci remained in a peripheral position until the end of the S phase, when they appeared to migrate centrally towards the pronuclei. At prometaphase of the first mitotic division, numerous MTOCs were found around the two sets of chromosomes; these MTOCs then aligned to form two bands on either side of the metaphase plate of the first mitosis. PMID:2865266

  16. Carrying-Over Effects of GVBD Blocking on Post-Blocking Meiotic Progression of Oocytes: Species Difference and the Signaling Pathway Leading to MPF Activation

    PubMed Central

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

    2014-01-01

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

  17. 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

  18. Effects of Mono-(2-Ethylhexyl) Phthalate and Di-(2-Ethylhexyl) Phthalate Administrations on Oocyte Meiotic Maturation, Apoptosis and Gene Quantification in Mouse Model

    PubMed Central

    Absalan, Forouzan; Saremy, Sadegh; Mansori, Esrafil; Taheri Moghadam, Mahin; Eftekhari Moghadam, Ali Reza; Ghanavati, Razie

    2017-01-01

    results indicate that oral administration of MEHP and DEHP could negatively affect mouse oocyte meiotic maturation and development in vivo, suggesting that phthalates could be risk factors for mammalians’ reproductive health. Additionally, phthalate-induced changes in Pou5f1, Asah1 and Ccna1 transcription level could explain in part, the reduced developmental ability of mouse-treated oocytes. PMID:28042535

  19. Plasma membrane events associated with the meiotic divisions in the amphibian oocyte: insights into the evolution of insulin transduction systems and cell signaling

    PubMed Central

    2013-01-01

    Background Insulin and its plasma membrane receptor constitute an ancient response system critical to cell growth and differentiation. Studies using intact Rana pipiens oocytes have shown that insulin can act at receptors on the oocyte surface to initiate resumption of the first meiotic division. We have reexamined the insulin-induced cascade of electrical and ion transport-related plasma membrane events using both oocytes and intact plasma membranes in order to characterize the insulin receptor-steroid response system associated with the meiotic divisions. Results [125I]Insulin binding (Kd = 54 ± 6 nM) at the oocyte plasma membrane activates membrane serine protease(s), followed by the loss of low affinity ouabain binding sites, with a concomitant 3–4 fold increase in high affinity ouabain binding sites. The changes in protease activity and ouabain binding are associated with increased Na+/Ca2+ exchange, increased endocytosis, decreased Na+ conductance resulting in membrane hyperpolarization, increased 2-deoxy-D-glucose uptake and a sustained elevation of intracellular pH (pHi). Hyperpolarization is largely due to Na+-channel inactivation and is the main driving force for glucose uptake by the oocyte via Na+/glucose cotransport. The Na+ sym- and antiporter systems are driven by the Na+ free energy gradient generated by Na+/K+-ATPase. Shifts in α and/or β Na+-pump subunits to caveolar (lipid raft) membrane regions may activate Na/K-ATPase and contribute to the Na+ free energy gradient and the increase in both Na+/glucose co-transport and pHi. Conclusions Under physiological conditions, resumption of meiosis results from the concerted action of insulin and progesterone at the cell membrane. Insulin inactivates Na+ channels and mobilizes fully functional Na+-pumps, generating a Na+ free energy gradient which serves as the energy source for several membrane anti- and symporter systems. PMID:23343451

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

    PubMed

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

    2015-11-01

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

  1. EGG-4 and EGG-5 link events of the oocyte-to-embryo transition with meiotic cell cycle progression in Caenorhabditis elegans

    PubMed Central

    Parry, Jean M.; Velarde, Nathalie V.; Lefkovith, Ariel J.; Zegarek, Matthew H.; Hang, Julie S.; Ohm, Jonathan; Klancer, Richard; Maruyama, Rika; Druzhinina, Marina K.; Grant, Barth D.; Piano, Fabio; Singson, Andrew

    2009-01-01

    Summary The molecular underpinnings of the oocyte-to-embryo transition are poorly understood. Here we show that two protein tyrosine phosphatase-like (PTPL) family proteins, EGG-4 and EGG-5, are required for key events of the oocyte-to-embryo transition in Caenorhabditis elegans. The predicted EGG-4 and EGG-5 amino acid sequences are 99% identical and their functions are redundant. In embryos lacking EGG-4 and EGG-5 we observe defects in meiosis, polar body formation, the block to polyspermy, F-actin dynamics, and eggshell deposition. During oogenesis, EGG-4 and EGG-5 assemble at the oocyte cortex with the previously identified regulators or effectors of the oocyte-to-embryo transition EGG-3, CHS-1 and MBK-2 [1, 2]. All of these molecules share a complex interdependence with regards to their dynamics and subcellular localization. Shortly after fertilization, EGG-4 and EGG-5 are required to properly coordinate a redistribution of CHS-1 and EGG-3 away from the cortex during meiotic anaphase I. Therefore EGG-4 and EGG-5 are not only required for critical events of the oocyte-to-embryo transition but also link the dynamics of the regulatory machinery with the advancing cell cycle. PMID:19879147

  2. Induction of meiotic maturation of follicle-enclosed oocytes of rabbits by a transient increase followed by an abrupt decrease in cyclic AMP concentration.

    PubMed

    Yoshimura, Y; Nakamura, Y; Oda, T; Ando, M; Ubukata, Y; Karube, M; Koyama, N; Yamada, H

    1992-08-01

    The involvement of cyclic adenosine monophosphate (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without forskolin at 10(-4), 10(-5) or 10(-6) mol l-1 for 3-6 h. At 3 or 4 h spontaneous meiotic maturation was significantly (P < 0.05) inhibited by forskolin at 10(-4) mol l-1. With prolonged incubation, spontaneous maturation progressed despite exposure to forskolin. In the second experiment ovaries were perfused for 12 h with forskolin (10(-4), 10(-5) or 10(-6) mol l-1) or medium alone. Neither ovulation nor degeneration of follicular oocytes occurred in any perfused ovary. The percentage of follicular oocytes achieving germinal vesicle breakdown was significantly (P < 0.001) increased in response to forskolin in a dose-related manner. In an additional experiment, ovaries were perfused with forskolin at 10(-4) mol l-1. A significant increase in the cAMP content in the follicle was observed within 30 min, but the ability to produce cAMP in response to forskolin decreased as the duration of perfusion was increased. Intraoocyte cAMP increased significantly within 30 min and reached its maximum 2 h after exposure to forskolin. Thereafter, cAMP levels in the oocytes decreased abruptly. This drop in intraoocyte cAMP concentration was followed by the resumption of meiosis. The alterations of intraoocyte cAMP contents following exposure to hCG in vivo paralleled those observed in the ovaries perfused with forskolin. These data suggest that a transient, but not continuous, increase in cAMP concentration after the gonadotrophin surge may be required to initiate oocyte maturation.

  3. Distribution of prepubertal and adult goat oocyte cortical granules during meiotic maturation and fertilisation: ultrastructural and cytochemical study.

    PubMed

    Velilla, E; Izquierdo, D; Rodríguez-González, E; López-Béjar, M; Vidal, F; Paramio, M T

    2004-08-01

    The aim of this study was evaluate cortical granule (CG) distribution during in vitro maturation (IVM) and fertilisation of prepubertal goat oocytes compared to CG distribution of ovulated and in vitro fertilised oocytes from adult goats. Oocytes from prepubertal goats were recovered from a slaughterhouse and were matured in M199 with hormones and serum for 27 hr. Ovulated oocytes were collected from gonadotrophin treated Murciana goats. Frozen-thawed spermatozoa were selected by centrifugation in percoll gradient and were capacitated in DMH with 20% steer serum for 1 hr. Ovulated and IVM-oocytes were inseminated in DMH medium with steer serum and calcium lactate for 20 hr. Oocytes and presumptive zygotes were stained with FITC-LCA (Lens culinaris agglutinin labelled with fluorescein isothiocyanate) and observed under a confocal laser scanning microscope. Ultrastructure morphology of oocytes and presumptive zygotes were analysed by transmission electron microscopy (TEM). Prepubertal goat oocytes at germinal vesicle stage show a homogeneous CG distribution in the cytoplasm. IVM-oocytes at Metaphase II (MII) and ovulated oocytes presented CGs located in the cortex with the formation of a monolayer beneath to the plasma membrane. At 20 hr postinsemination (hpi), zygotes from IVM-oocytes showed a complete CG exocytosis whereas zygotes from ovulated oocytes presented aggregates of CGs located at the cortical region. Images by TEM detected that CGs were more electrodense and compacts in oocytes from prepubertal than from adult goats.

  4. Microinjection of Cdc25 protein phosphatase into Xenopus prophase oocyte activates MPF and arrests meiosis at metaphase I.

    PubMed

    Rime, H; Huchon, D; De Smedt, V; Thibier, C; Galaktionov, K; Jessus, C; Ozon, R

    1994-01-01

    Microinjection of bacterially expressed human cdc25A protein into Xenopus prophase oocytes provokes the activation of p34cdc2 kinase and the tyrosine dephosphorylation of p34cdc2 in the presence or absence of protein synthesis. The level of p34cdc2 kinase activity then drops in parallel with the degradation of cyclin B2 and finally increases again to stabilize at a high level. Cdc25 microinjection induces the assembly of a metaphase I spindle which is abnormally located in the deep cytoplasm. Moreover, oocytes arrest at the metaphase I stage and do not reach metaphase II even 10 h after cdc25 microinjection. The extended metaphase I period observed in cdc25-injected oocytes results from an equilibrium between degradation of cyclins and synthesis of new cyclins. This is in contrast with progesterone-stimulated oocytes where cyclin degradation is turned off when oocytes enter metaphase II. During metaphase I, the reactivation of MPF activity can be disrupted in two different ways: 1) cycloheximide, an inhibitor of protein synthesis, by preventing the synthesis of new cyclins, provokes the disappearance of MPF kinase activity and the reformation of a nucleus; 2) when the cAMP level is increased during the metaphase I period in cdc25-injected oocytes, MPF kinase activity drops following a rephosphorylation of tyrosine 15 of p34cdc2, while the cyclin turn-over remains unaffected. Moreover, increasing the cAMP level in prophase oocytes totally prevents the action of cdc25. Our results indicate that in Xenopus oocytes, the PKA pathway negatively regulates the activation of MPF and the activity of p34cdc2/cyclin B complex through tyrosine phosphorylation of p34cdc2 during metaphase I.

  5. 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

  6. Two p90 ribosomal S6 kinase isoforms are involved in the regulation of mitotic and meiotic arrest in Artemia.

    PubMed

    Duan, Ru-Bing; Zhang, Li; Chen, Dian-Fu; Yang, Fan; Yang, Jin-Shu; Yang, Wei-Jun

    2014-06-06

    There are multiple isoforms of p90 ribosomal S6 kinase (RSK), which regulate diverse cellular functions such as cell growth, proliferation, maturation, and motility. However, the relationship between the structures and functions of RSK isoforms remains undetermined. Artemia is a useful model in which to study cell cycle arrest because these animals undergo prolonged diapauses, a state of obligate dormancy. A novel RSK isoform was identified in Artemia, which was termed Ar-Rsk2. This isoform was compared with an RSK isoform that we previously identified in Artemia, termed Ar-Rsk1. Ar-Rsk2 has an ERK-docking motif, whereas Ar-Rsk1 does not. Western blot analysis revealed that Ar-Rsk1 was activated by phosphorylation, which blocked meiosis in oocytes. Knockdown of Ar-Rsk1 reduced the level of phosphorylated cdc2 and thereby suppressed cytostatic factor activity. This indicates that Ar-Rsk1 regulates the cytostatic factor in meiosis. Expression of Ar-Rsk2 was down-regulated in Artemia cysts in which mitosis was arrested. Knockdown of Ar-Rsk2 resulted in decreased levels of cyclin D3 and phosphorylated histone H3, and the production of pseudo-diapause cysts. This indicates that Ar-Rsk2 regulates mitotic arrest. PLK and ERK RNAi showed that Ar-Rsk2, but not Ar-Rsk1, could be activated by PLK-ERK in Artemia. This is the first study to report that RSK isoforms with and without an ERK-docking motif regulate mitosis and meiosis, respectively. This study provides insight into the relationship between the structures and functions of RSK isoforms. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Two p90 Ribosomal S6 Kinase Isoforms Are Involved in the Regulation of Mitotic and Meiotic Arrest in Artemia*

    PubMed Central

    Duan, Ru-Bing; Zhang, Li; Chen, Dian-Fu; Yang, Fan; Yang, Jin-Shu; Yang, Wei-Jun

    2014-01-01

    There are multiple isoforms of p90 ribosomal S6 kinase (RSK), which regulate diverse cellular functions such as cell growth, proliferation, maturation, and motility. However, the relationship between the structures and functions of RSK isoforms remains undetermined. Artemia is a useful model in which to study cell cycle arrest because these animals undergo prolonged diapauses, a state of obligate dormancy. A novel RSK isoform was identified in Artemia, which was termed Ar-Rsk2. This isoform was compared with an RSK isoform that we previously identified in Artemia, termed Ar-Rsk1. Ar-Rsk2 has an ERK-docking motif, whereas Ar-Rsk1 does not. Western blot analysis revealed that Ar-Rsk1 was activated by phosphorylation, which blocked meiosis in oocytes. Knockdown of Ar-Rsk1 reduced the level of phosphorylated cdc2 and thereby suppressed cytostatic factor activity. This indicates that Ar-Rsk1 regulates the cytostatic factor in meiosis. Expression of Ar-Rsk2 was down-regulated in Artemia cysts in which mitosis was arrested. Knockdown of Ar-Rsk2 resulted in decreased levels of cyclin D3 and phosphorylated histone H3, and the production of pseudo-diapause cysts. This indicates that Ar-Rsk2 regulates mitotic arrest. PLK and ERK RNAi showed that Ar-Rsk2, but not Ar-Rsk1, could be activated by PLK-ERK in Artemia. This is the first study to report that RSK isoforms with and without an ERK-docking motif regulate mitosis and meiosis, respectively. This study provides insight into the relationship between the structures and functions of RSK isoforms. PMID:24755224

  8. DNA double-strand breaks disrupted the spindle assembly in porcine oocytes.

    PubMed

    Wang, HaiYang; Luo, YiBo; Zhao, Ming-Hui; Lin, ZiLi; Kwon, Jeongwoo; Cui, Xiang-Shun; Kim, Nam-Hyung

    2016-02-01

    We used etoposide (25-100 µg/mL) to induce DNA double-strand breaks (DSBs) in porcine oocytes at the germinal vesicle (GV) stage to determine how such damage affects oocyte maturation. We observed that DNA damage did not delay the rate of germinal vesicle breakdown (GVBD), but did inhibit the final stages of maturation, as indicated by the failure to extrude the first polar body. Oocytes with low levels of DSBs failed to effectively activate ataxia telangiectasia-mutated (ATM) kinase, while those with severe DNA DSBs failed to activate checkpoint kinase 1 (CHK1)--the two regulators of the DNA damage response pathway--indicating that porcine oocytes lack an efficient G2/M phase checkpoint. DSBs induced spindle defects and chromosomal misalignments, leading to the arrest of these oocytes at meiotic metaphase I. The activity of maturation-promoting factor also did not increase appropriately in oocytes with DNA DSBs, although its abundance was sufficient to promote GVBD and chromosomal condensation. Following parthenogenetic activation, embryos from etoposide-treated oocytes formed numerous micronuclei. Thus, our results indicate that DNA DSBs do not efficiently activate the ATM/CHK1-dependent DNA-damage checkpoint in porcine oocytes, allowing these DNA-impaired oocytes to enter M phase. Oocytes with DNA damage did, however, arrest at metaphase I in response to spindle defects and chromosomal misalignments, which limited the ability of these oocytes to reach meiotic metaphase II.

  9. A constitutional complex chromosome rearrangement involving meiotic arrest in an azoospermic male: case report.

    PubMed

    Coco, R; Rahn, M I; Estanga, P García; Antonioli, G; Solari, A J

    2004-12-01

    Complex chromosome rearrangements are rare aberrations that frequently lead to reproductive failure and that may hinder assisted reproduction. A 25-year-old azoospermic male was studied cytogenetically with synaptonemal complex analysis of spermatocytes from a testicular biopsy and fluorescence in situ hybridization (FISH) of lymphocytes. The spermatocytes showed a pentavalent plus a univalent chromosome. Cell death occurred mainly at advanced pachytene stages. The sex chromosomes were involved in the multiple, as shown by their typical axial excrescences. Two autosomal pairs, including an acrocentric chromosome (15), were also involved in the multiple. FISH allowed the definite identification of all the involved chromosomes. An inverted chromosome 12 is translocated with most of one long arm of chromosome 15, while the centromeric piece of this chromosome 15 is translocated with Yqh, forming a small marker chromosome t(15;Y). The euchromatic part of the Y chromosome is joined to the remaining piece of chromosome 12, forming a neo-Y chromosome. The patient shows azoospermia and a normal phenotype. The disruption of spermatogenesis is hypothetically due to the extent of asynaptic segments and to sex-body association during pachytene. This CCR occurred 'de novo' during paternal spermatogenesis. Meiotic analysis and FISH are valuable diagnostic tools in these cases.

  10. Discovery of tMAC: a Drosophila testis-specific meiotic arrest complex paralogous to Myb–Muv B

    PubMed Central

    Beall, Eileen L.; Lewis, Peter W.; Bell, Maren; Rocha, Michael; Jones, D. Leanne; Botchan, Michael R.

    2007-01-01

    The Drosophila Myb–Muv B (MMB)/dREAM complex regulates gene expression and DNA replication site-specifically, but its activities in vivo have not been thoroughly explored. In ovarian amplification-stage follicle cell nuclei, the largest subunit, Mip130, is a negative regulator of replication, whereas another subunit, Myb, is a positive regulator. Here, we identified a mutation in mip40 and generated a mutation in mip120, two additional MMB subunits. Both mutants were viable, but mip120 mutants had many complex phenotypes including shortened longevity and severe eye defects. mip40 mutant females had severely reduced fertility, whereas mip120 mutant females were sterile, substantiating ovarian regulatory role(s) for MMB. Myb accumulation and binding to polytene chromosomes was dependent on the core factors of the MMB complex. In contrast to the documented mip130 mutant phenotypes, both mip40 and mip120 mutant males were sterile. We purified Mip40-containing complexes from testis nuclear extracts and identified tMAC, a new testis-specific meiotic arrest complex that contained Mip40, Caf1/p55, the Mip130 family member, Always early (Aly), and a Mip120 family member, Tombola (Tomb). Together, these data demonstrate that MMB serves diverse roles in different developmental pathways, and members of MMB can be found in alternative, noninteracting complexes in different cell types. PMID:17403774

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

    PubMed

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

    2014-01-01

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

  12. 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.

  13. 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

  14. Effect of vitrification on meiotic maturation, mitochondrial distribution and glutathione synthesis in immature silver fox cumulus oocyte complexes.

    PubMed

    Cao, Xinyan; Li, Jingchun; Xue, Hailong; Wang, Shiyong; Zhao, Weigang; Du, Zhanyu; Yang, Yifeng; Yue, Zhigang

    2017-03-15

    The present study was designed to investigate the effects of vitrifying oocytes obtained from silver foxes on nuclear maturation, mitochondrial distribution and glutathione (GSH) synthesis after in vitro culture for 72 h. Immature oocytes were randomly divided into three groups: (1) fresh GV (germinal vesicle) oocytes (Control group), (2) exposure to the equilibration and vitrification solution but without being plunged into liquid nitrogen (exposed group), and (3) vitrification by the cryoloop method (vitrified-warmed group). The number of survival oocytes was not decreased by either being exposed to the cryoprotectant or being vitrified-warmed compared with the control group (P > 0.05). After IVM, the percentage of resumption of meiosis for vitrified-warmed oocytes (41.9%) was significantly lower than in the control (81.2%) and exposed (79.1%) groups (P < 0.05). However, the proportion of oocytes reaching the metaphase II (MII) stage was similar among the different groups (11.4%, 9.3% and 5.2%, respectively, P > 0.05). The translocation of active mitochondria during fox oocyte maturation was revealed using MitoTracker Red staining and confocal laser microscopy. For fresh oocytes at the GV stage, active mitochondria were distributed around the entire cortex with small granulations and various-sized cavities (no MitoTracker signals). After IVM, the mitochondria formed large granulations and clumps throughout the cytoplasm. Vitrification significantly decreased the proportion of MII oocytes with normal mitochondrial distribution compared with the control and exposed groups (35.4%, 71.9% and 59.2%, respectively, P < 0.05). Similarly, the GSH content was significantly lower in vitrified-warmed oocytes compared with the control and exposed oocytes after IVM (3.4, 5.7 and 4.7 pM/oocyte, respectively, P < 0.05). However, no significant difference was observed between the cryoprotectant exposed and control groups with regard to the normal mitochondrial

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

    PubMed

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

    2011-01-01

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

  16. Arrest at metaphase of meiosis I in starfish oocytes in the ovary is maintained by high CO2 and low O2 concentrations in extracellular fluid.

    PubMed

    Moriwaki, Kei; Nakagawa, Takako; Nakaya, Fumio; Hirohashi, Noritaka; Chiba, Kazuyoshi

    2013-11-01

    During the spawning process in starfish, oocytes are arrested at metaphase of meiosis I (MI) within the ovary, and reinitiate meiosis only after they have been released into the seawater. However, this arrest does not occur if the ovary is removed from the animal. As the pH of the coelomic fluid is buffered by CO2/H(+)/HCO3(-), we investigated the involvement of gas concentrations in MI arrest. In vivo, the CO2 level in the coelomic fluid was high (∼1.5% vs. 0.04% in air) and the O2 level was low (0.1-1.0% vs. ∼20% in air). When these gas conditions were reproduced in isolated coelomic fluid or seawater, ovarian oocytes arrested at MI, just as in vivo. Isolated oocytes from the ovary required the similar high CO2 and low O2 level to remain arrested in MI and had an intracellular pH of ∼6.9. Intracellular pH increased to ∼7.3 when oocytes were transferred to seawater equilibrated with air, a condition that mimics that of spawning. We used ammonium acetate to clamp intracellular pH at different levels and found that MI arrest occurred when intracellular pH was ∼6.9. Our results support the idea that high CO2 and low O2 in the ovarian environment lead to low intracellular pH and MI arrest, while spawning into the seawater with low CO2 and high O2 results in high intracellular pH and release from MI arrest. The biological significance of MI arrest is that oocytes are spawned into seawater at the optimal physiological state of MI when the least polyspermy occurs.

  17. Continuous exposure to bisphenol A during in vitro follicular development induces meiotic abnormalities.

    PubMed

    Lenie, Sandy; Cortvrindt, Rita; Eichenlaub-Ritter, Ursula; Smitz, Johan

    2008-03-12

    Bisphenol A (BPA), a widely used environmental contaminant, may exert weak estrogenic, anti-androgenic and anti-thyroidic activities. BPA is suspected to possess aneugenic properties that may affect somatic cells and mammalian oocytes. Oocyte growth and maturation depend upon a complex bi-directional signaling between the oocyte and its companion somatic cells. Consequently, disturbances in oocyte maturation may originate either from direct effects of BPA at the level of the oocyte or from indirect influences at the follicular level, such as alterations in hormonal homeostasis. This study aimed to analyze the effects of chronic BPA exposure (3 nM to 30 microM) on follicle-enclosed growth and maturation of mouse oocytes in vitro. Oocytes were cultured and their spindle and chromosomes were stained by alpha-tubulin immunofluorescence and ethidium homodimer-2, respectively. Confocal microscopy was utilized for subsequent analysis. Only follicles that were exposed to 30 microM BPA during follicular development showed a slightly reduced granulosa cell proliferation and a lower total estrogen production, but they still developed and formed antral-like cavities. However, 18% of oocytes were unable to resume meiosis after stimulation of oocyte maturation, and 37% arrested after germinal vesicle breakdown, significantly different from controls (p<0.05). Only 45% of the oocytes extruded a first polar body (p < 0.05). 30 microM BPA led also to a significant increase in meiosis I-arrested oocytes with unaligned chromosomes and spindle aberrations. Oocytes that were able to progress beyond meiosis I, frequently arrested at an abnormal telophase I. Additionally, in many oocytes exposed to low chronic BPA that matured to meiosis II chromosomes failed to congress at the spindle equator. In conclusion, mouse follicle culture reveals non-linear dose-dependent effects of BPA on the meiotic spindle in mouse oocytes when exposure was chronic throughout oocyte growth and maturation.

  18. Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF.

    PubMed

    Keefe, David; Liu, Lin; Wang, Wei; Silva, Celso

    2003-01-01

    Meiotic spindles tether the chromosomes of oocytes and have been found to be structurally abnormal in older women. Conventional methods to image the meiotic spindle, such as immunostaining or transmission electron microscopy, require prior fixation, so they cannot be used clinically, and their utility in developmental studies is limited. Spindles can also be imaged non-invasively based on their birefringence, an inherent optical property of highly ordered molecules, such as microtubules, as they are illuminated with polarized light. Polarized light microscopy has been gainfully applied to embryology for decades, but recently a digital, orientation-independent polarized light microscope, the polscope, has demonstrated the exquisite sensitivity needed to image the low levels of birefringence exhibited by mammalian spindles. Its use of nearly circularly polarized light also produces orientation-independent measures of spindle birefringence, thus providing a method to quantify spindle architecture in living oocytes. The safety and utility of polscope imaging has been demonstrated in mammalian oocytes, including those from women undergoing ICSI. Spindle imaging with the polscope provides structural information closely related to the more invasive immunostaining method, and also enables study of the dynamic architecture of spindles. Profound effects of cooling on meiotic spindles have also been shown, and polscope imaging has been used to optimize thermodynamic stability of oocytes during ICSI. It has been shown that embryos derived from oocytes with normal, intact meiotic spindles exhibit superior development after fertilization and in-vitro culture. The mechanisms underlying age-related disruption of meiotic spindles in women remain unclear, but may relate to factors residing within the chromosomes themselves, since mice engineered to shorten their telomeres exhibit structurally abnormal spindles in their oocytes, and their embryos undergo cell cycle arrest and

  19. 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

  20. Activation of ribosomal protein S6 phosphorylation during meiotic maturation of Xenopus laevis oocytes: in vitro ordered appearance of S6 phosphopeptides.

    PubMed Central

    Martin-Pérez, J; Rudkin, B B; Siegmann, M; Thomas, G

    1986-01-01

    During meiotic maturation of Xenopus laevis stage 6 oocytes into unfertilized eggs, 40S ribosomal protein S6 undergoes multiple phosphorylation. Extracts prepared from unfertilized eggs are up to 10-fold more efficient in phosphorylating S6 than those prepared from immature oocytes. When analyzed by DEAE chromatography the S6 kinase activity elutes as a single peak. If extracts from unfertilized eggs are prepared in the absence of beta-glycerol phosphate, a putative phosphatase inhibitor, there is a severe reduction in recovered S6 kinase activity. Under optimal conditions, incubation of unfertilized egg extracts with 40S ribosomes in the presence of ATP leads to the average incorporation of 3.5 mol of phosphate/mol of S6. Prior incubation of these extracts with the cAMP-dependent protein kinase inhibitor does not inhibit S6 phosphorylation indicating that another kinase is responsible. Analysis of the in vitro phosphorylated peptides demonstrates that they migrate to the equivalent position of those observed previously in vivo and in vitro. More strikingly, if each of the increasingly phosphorylated derivatives of S6 is analyzed independently, it is found that the phosphopeptides appear in a specific order. Images Fig.1. Fig.4. Fig.5. Fig.6. PMID:3709523

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

    PubMed

    Maziero, R R D; Guaitolini, C R F; 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. © 2016 Blackwell Verlag GmbH.

  2. Calcium currents correlate with oocyte maturation during the reproductive cycle in Octopus vulgaris.

    PubMed

    Cuomo, Annunziata; Di Cristo, Carlo; Paolucci, Marina; Di Cosmo, Anna; Tosti, Elisabetta

    2005-03-01

    Using the whole-cell voltage clamp technique, we have studied the Ca2+ currents and the steady-state conductance during different oocyte growth stages and during the reproductive cycle of the female of Octopus vulgaris. Evidence is presented that L-type Ca2+ currents are high in small pre-vitellogenic oocytes (80-150 microm diameter) and significantly lower in early vitellogenic oocytes (180-300 microm diameter). Similarly, a significant decrease of the steady-state conductance occurred from the pre to early- vitellogenic oocytes. Octopus oocytes showed larger Ca2+ currents in the reproductive rather than non-reproductive periods. These data indicates that ion and L-type Ca2+ currents play a role in oocyte growth and cytoplasmic maturation, and possibly in preparing the plasma membrane to the interaction with the spermatozoon. By using fluorescent microscopy, we show that oocytes from 80 to 400 microm diameter have the large germinal vesicle characteristic of the immature oocytes. In subsequent stages of growth (up to 1000 microm diameter) the nucleus is no more visible and the metaphase spindle appears. These data demonstrate that Octopus vulgaris oocytes are arrested in the first meiotic prophase up to the early-vitellogenic stage and resume meiosis at this stage up to a second block presumably in metaphase I. We discuss a possible role for progesterone as the hormonal stimulus for the first prophase-metaphase meiotic transition.

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

    PubMed

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

    2011-08-28

    Mature mammalian oocytes are poised for completing meiosis II (MII) on fertilization by positioning the spindle close to an actomyosin-rich cortical cap. Here, we show that the Arp2/3 complex localizes to the cortical cap in a Ran-GTPase-dependent manner and nucleates actin filaments in the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 activity leads to rapid dissociation of the spindle from the cortex. Live-cell imaging and spatiotemporal image correlation spectroscopy analysis reveal that actin filaments flow continuously away from the Arp2/3-rich cortex, driving a cytoplasmic streaming expected to exert a net pushing force on the spindle towards the cortex. Arp2/3 inhibition not only diminishes this actin flow and cytoplasmic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, moving the spindle away from the cortex. Thus, the asymmetric MII spindle position is dynamically maintained as a result of balanced forces governed by the Arp2/3 complex.

  4. 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

    2016-08-02

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

  5. Further studies of the effects of follicular fluid and membrana granulosa cells on the spontaneous maturation of pig oocytes.

    PubMed

    Racowsky, C; McGaughey, R W

    1982-11-01

    Liberated cumulus-enclosed pig oocytes were cultured either alone in follicular fluid or with membrana granulosa cells in a complex serum based medium. After 24 h, oocytes were air-dried for cytogenetic analysis, meiotic stage was scored, and viability of granulosa cells was determined. Neither the release from meiotic arrest nor the progression of maturation to metaphase II was significantly inhibited by either of these follicular components. Co-culture of membrana granulosa cells and oocytes significantly stimulated maturation in one experimental series, while viability of the somatic cells was maintained in all experiments. These results do not support the concept of a stable oocyte maturation inhibitor of granulosa cell origin in follicular fluid.

  6. 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.

  7. The RNA-Binding Protein, ZFP36L2, Influences Ovulation and Oocyte Maturation

    PubMed Central

    Ball, Christopher B.; Rodriguez, Karina F.; Stumpo, Deborah J.; Ribeiro-Neto, Fernando; Korach, Kenneth S.; Blackshear, Perry J.; Birnbaumer, Lutz; Ramos, Silvia B. V.

    2014-01-01

    ZFP36L2 protein destabilizes AU-rich element-containing transcripts and has been implicated in female fertility. In the C57BL/6NTac mouse, a mutation in Zfp36l2 that results in the decreased expression of a form of ZFP36L2 in which the 29 N-terminal amino acid residues have been deleted, ΔN-ZFP36L2, leads to fertilized eggs that arrest at the two-cell stage. Interestingly, homozygous ΔN-Zfp36l2 females in the C57BL/6NTac strain release 40% fewer eggs than the WT littermates (Ramos et al., 2004), suggesting an additional defect in ovulation and/or oocyte maturation. Curiously, the same ΔN-Zfp36l2 mutation into the SV129 strain resulted in anovulation, prompting us to investigate a potential problem in ovulation and oocyte maturation. Remarkably, only 20% of ΔN-Zfp36l2 oocytes in the 129S6/SvEvTac strain matured ex vivo, suggesting a defect on the oocyte meiotic maturation process. Treatment of ΔN-Zfp36l2 oocytes with a PKA inhibitor partially rescued the meiotic arrested oocytes. Furthermore, cAMP levels were increased in ΔN-Zfp36l2 oocytes, linking the cAMP/PKA pathway and ΔN-Zfp36l2 with meiotic arrest. Since ovulation and oocyte maturation are both triggered by LHR signaling, the downstream pathway was investigated. Adenylyl cyclase activity was increased in ΔN-Zfp36l2 ovaries only upon LH stimulation. Moreover, we discovered that ZFP36L2 interacts with the 3′UTR of LHR mRNA and that decreased expression levels of Zfp36l2 correlates with higher levels of LHR mRNA in synchronized ovaries. Furthermore, overexpression of ZFP36L2 decreases the endogenous expression of LHR mRNA in a cell line. Therefore, we propose that lack of the physiological down regulation of LHR mRNA levels by ZFP36L2 in the ovaries is associated with anovulation and oocyte meiotic arrest. PMID:24830504

  8. Effect of co-culture with theca interna on nuclear maturation of horse oocytes with low meiotic competence, and subsequent fusion and activation rates after nuclear transfer.

    PubMed

    Choi, Young-Ho; Shin, Taeyoung; Love, Charley C; Johnson, Cindy; Varner, Dickson D; Westhusin, Mark E; Hinrichs, Katrin

    2002-02-01

    We conducted this study to examine whether or not co-culture with theca cells improves the maturation rate of horse oocytes with compact cumuli and to evaluate the cytoplasmic competence of oocytes after maturation by assessing fusion, activation and cleavage rates after nuclear transfer. We collected oocytes by scraping follicles from slaughterhouse-derived ovaries and classified them as having an expanded or a compact cumulus. Expanded oocytes were matured in M199 supplemented with 10% FBS and 5 microU/ml FSH for 24 h: compact oocytes were cultured in the same medium, or they were co-cultured in the same medium with theca interna explants, for 24 or 42 h. Oocytes were held with or without 10 microg/ml cytochalasin B, before washing and micromanipulation. and they were fused with donor fibroblasts by electrical pulse. Fused oocytes were activated with Ca ionophore/cycloheximide, cultured for 5 days, and stained with Hoechst to assess nuclear development. We considered oocytes with an enlarged nucleus, or having cleavage with multiple nuclei, to be activated. There was no significant difference in overall maturation rate between compact oocytes cultured with theca and compact controls. When these two groups were combined, there was a significant increase in the proportion of oocytes in MII between 24 and 42 h (P < 0.05). Expanded oocytes had a significantly higher rate of maturation than did compact oocytes (64% versus 25-30%; P < 0.001). There were no significant differences in rates of successful enucleation, fusion, activation or cleavage between compact control and compact + theca oocytes, nor between compact and expanded oocytes; however, expanded oocytes treated with cytochalasin B had a significantly higher survival rate after enucleation than did untreated expanded oocytes (P < 0.05). Three embryos developed from recombined oocytes, with maximum cleavage to 10 cells. The results of this study indicate that co-culture with theca cells does not increase

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

    PubMed

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

    2014-05-01

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

  10. JMY is required for asymmetric division and cytokinesis in mouse oocytes.

    PubMed

    Sun, Shao-Chen; Sun, Qing-Yuan; Kim, Nam-Hyung

    2011-05-01

    JMY is a transcriptional co-factor of p53. Latest work has revealed that JMY is also an actin nucleation factor that regulates new filament assembly and activates Arp2/3 complex in somatic cells; however, roles of JMY in mouse oocyte are unknown. Here we showed the expression and functions of JMY during mouse oocyte meiotic maturation. JMY mRNA is expressed largely from germinal vesicle to metaphase I stage, and gradually decreased during anaphase I, telophase I (TI) and metaphase II (MII) stages. Immunostaining results showed that JMY localized at the spindle and cytoplasm of oocytes. Depletion of JMY by RNAi resulted in symmetric division, failure of spindle migration and cytokinesis during oocyte meiotic maturation, showing a 2-cell-like MII oocyte and TI stage arrest. Actin cap and cortical granules-free domain formation were also disrupted after JMY RNAi, indicating the failure of spindle migration. JMY antibody injection results were consistent with those of JMY RNAi, further confirming the involvement of JMY in oocyte polarity. Our data indicate that JMY is required for spindle migration, asymmetric division and cytokinesis during mouse oocyte maturation.

  11. PUF-8 Functions Redundantly with GLD-1 to Promote the Meiotic Progression of Spermatocytes in Caenorhabditis elegans.

    PubMed

    Priti, Agarwal; Subramaniam, Kuppuswamy

    2015-06-10

    Successful meiotic progression of germ cells is crucial for gametogenesis. Defects in this process affect proper genetic transmission and sometimes lead to tumor formation in the germline. In Caenorhabditis elegans, the RNA-binding protein GLD-1 is essential for the meiotic development of oocytes. However, its role during spermatogenesis has not been understood. Here, we show that GLD-1 functions redundantly with the PUF family protein PUF-8 to ensure proper meiotic development of spermatocytes. When grown at 20°-the standard laboratory temperature for C. elegans growth-primary spermatocytes in both gld-1 and puf-8 single-mutant males and hermaphrodites complete the meiotic divisions normally. By contrast, some of the gld-1; puf-8 double-mutant spermatocytes exit meiosis and form germ cell tumors in both sexes. During larval development, gld-1; puf-8 double-mutant germ cells begin to express the meiotic marker HIM-3, lose P granules, and form the sperm-specific membranous organelle, which are characteristics of developing spermatocytes. However, some of these cells quickly lose HIM-3 and form germ cell tumors that lack membranous organelle but contain P granules. Mutations that block meiotic progression at late pachytene or diakinetic stage fail to arrest the tumorigenesis, suggesting that the gld-1; puf-8 double-mutant spermatocytes exit meiosis prior to the completion of pachytene. Together, results presented here uncover a novel function for gld-1 in the meiotic development of spermatocytes in both hermaphrodites and males.

  12. PUF-8 Functions Redundantly with GLD-1 to Promote the Meiotic Progression of Spermatocytes in Caenorhabditis elegans

    PubMed Central

    Priti, Agarwal; Subramaniam, Kuppuswamy

    2015-01-01

    Successful meiotic progression of germ cells is crucial for gametogenesis. Defects in this process affect proper genetic transmission and sometimes lead to tumor formation in the germline. In Caenorhabditis elegans, the RNA-binding protein GLD-1 is essential for the meiotic development of oocytes. However, its role during spermatogenesis has not been understood. Here, we show that GLD-1 functions redundantly with the PUF family protein PUF-8 to ensure proper meiotic development of spermatocytes. When grown at 20°—the standard laboratory temperature for C. elegans growth—primary spermatocytes in both gld-1 and puf-8 single-mutant males and hermaphrodites complete the meiotic divisions normally. By contrast, some of the gld-1; puf-8 double-mutant spermatocytes exit meiosis and form germ cell tumors in both sexes. During larval development, gld-1; puf-8 double-mutant germ cells begin to express the meiotic marker HIM-3, lose P granules, and form the sperm-specific membranous organelle, which are characteristics of developing spermatocytes. However, some of these cells quickly lose HIM-3 and form germ cell tumors that lack membranous organelle but contain P granules. Mutations that block meiotic progression at late pachytene or diakinetic stage fail to arrest the tumorigenesis, suggesting that the gld-1; puf-8 double-mutant spermatocytes exit meiosis prior to the completion of pachytene. Together, results presented here uncover a novel function for gld-1 in the meiotic development of spermatocytes in both hermaphrodites and males. PMID:26068572

  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. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

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

    1989-08-01

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

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

    SciTech Connect

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

    1991-08-01

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

  16. Influence of meiotic inhibition by butyrolactone-I during germinal vesicle stage on the ability of porcine oocytes to be activated by electric stimulation after nuclear maturation.

    PubMed

    Hirao, Yuji; Nishimoto, Namiko; Kure-bayashi, Satoshi; Takenouchi, Naoki; Yamauchi, Nobuhiko; Masuda, Hiroshi; Nagai, Takashi

    2003-08-01

    Butyrolactone-I (BL-I) is a specific inhibitor of cyclin-dependent kinases and prevents germinal vesicle breakdown (GVBD) in porcine oocytes. This study first focused on the effect of BL-I on the time course of GVBD and progression to metaphase II (MII) in oocytes after the removal of BL-I. When porcine oocytes were treated with 20 microM BL-I for 28 h, the intervals taken to undergo GVBD and progress to MII were 4-8 h and 16-20 h after washing out BL-I, respectively. These intervals were both approximately 8 h behind those of the control oocytes. When the BL-I treatment was performed for 20 h, instead of 28 h, there were no differences in the timing or frequency of progression to MII between the BL-I treatment and control groups. To determine whether the cytoplasmic maturation was the same, the ability of oocytes to form a female pronucleus in response to an electric stimulus was examined. When oocytes were stimulated at 28 h after the removal of BL-I, the rate of pronucleus formation was significantly lower in oocytes treated with BL-I than in untreated oocytes, despite the fact that both groups reached MII at a similar time. When the electric stimulus was given at 36 h after the removal of BL-I, oocytes had a pronucleus formation rate comparable to that of control oocytes. Therefore, the findings suggest that BL-I treatment of porcine oocytes at the germinal vesicle stage affects not only the progression of meiosis, but also the events involved in cytoplasmic maturation.

  17. 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.

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

    PubMed

    Yang, Min; Hall, Justin; Fan, Zhiqiang; Regouski, Misha; Meng, Qinggang; Rutigliano, Heloisa M; Stott, Rusty; Rood, Kerry A; Panter, Kip E; Polejaeva, Irina A

    2016-12-01

    Reduced developmental competence after IVF has been reported using oocyte derived from small follicles in several species including cattle, sheep, and goats. No information is currently available about the effect of follicle size of the cytoplast donor on in vivo development after somatic cell nuclear transfer (SCNT) in goats. Oocytes collected from large (≥3 mm) and small follicles (<3 mm) were examined for maturation and in vivo developmental competence after SCNT. Significantly greater maturation rate was observed in oocytes derived from large follicles compared with that of small follicles (51.6% and 33.7%, P < 0.05). Greater percent of large follicle oocytes exhibited a low glucose-6-phosphate dehydrogenase activity at germinal vesicle stage compared with small follicle oocytes (54.9% and 38.7%, P < 0.05). Relative mRNA expression analysis of 48 genes associated with embryonic and fetal development revealed that three genes (MATER, IGF2R, and GRB10) had higher level of expression in metaphase II oocytes from large follicles compared with oocytes from small follicles. Nevertheless, no difference was observed in pregnancy rates (33.3% vs. 47.1%) and birth rates (22.2% vs. 16.7%) after SCNT between the large and small follicle groups). These results indicate that metaphase II cytoplasts from small and large follicles have similar developmental competence when used in goat SCNT. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. 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.

  20. cyclic GMP Mediated Inhibition of Spontaneous Germinal Vesicle Breakdown Both with and without Cumulus in Mouse Oocyte.

    PubMed

    Hwang, Heekyung; Cheon, Yong-Pil

    2016-12-01

    Intact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT-8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest.

  1. cyclic GMP Mediated Inhibition of Spontaneous Germinal Vesicle Breakdown Both with and without Cumulus in Mouse Oocyte

    PubMed Central

    Hwang, Heekyung; Cheon, Yong-Pil

    2016-01-01

    ABSTRACT Intact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT-8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest. PMID:28144640

  2. Effect of holding technique and culture drop size in individual or group culture on blastocyst development after ICSI of equine oocytes with low meiotic competence.

    PubMed

    Choi, Y H; Love, L B; Varner, D D; Hinrichs, K

    2007-11-01

    The effect of medium-to-embryo ratio on blastocyst development of equine embryos from oocytes with compact cumuli was evaluated in the present experiment. In addition, two methods for holding oocytes before in vitro maturation were compared. In Experiment 1, oocytes cultured with roscovitine for 16-18h before maturation were fertilized by intracytoplasmic sperm injection and cultured individually in 2.5, 5, 10 or 50microl droplets. In Experiment 2, oocytes were either cultured with roscovitine or held in a modified M199 with 20% serum at room temperature (EH treatment) for 16-18h, then matured, fertilized and cultured in groups at 5microl medium per embryo. In Experiment 3, oocytes were held in the EH treatment, then were matured and fertilized. In Study 3.1, injected oocytes were cultured individually in drop sizes as for Experiment 1; in Study 3.2, groups of 2-7 oocytes were cultured in fixed drop sizes of 5 or 50microl. Blastocyst development rates of individually-cultured embryos were not significantly different among drop sizes in either Experiment 1 or 3 (15-29%). In Experiment 2, blastocyst rates were not significantly different between holding treatments (17-23%). In Experiment 3, for group-cultured oocytes, blastocyst development was not significantly different between 5 and 50microl drops (39 and 27%, respectively). In conclusion, compact-cumulus oocytes may be effectively held in the EH treatment before maturation, and single culture of equine embryos yields acceptable blastocyst development. The greatest blastocyst rate (39%) was obtained with group culture in a 5microl droplet.

  3. Evidence that carbonyl stress by methylglyoxal exposure induces DNA damage and spindle aberrations, affects mitochondrial integrity in mammalian oocytes and contributes to oocyte ageing.

    PubMed

    Tatone, Carla; Heizenrieder, Tanja; Di Emidio, Giovanna; Treffon, Patrick; Amicarelli, Fernanda; Seidel, Thorsten; Eichenlaub-Ritter, Ursula

    2011-07-01

    )/glutathion disulfide (GSSG)-dependent redox potential. Part A revealed that mRNA for glyoxalases decreases during meiotic maturation. Importantly, cumulus from aged mice in CEO obtained from stimulated cycles does not protect oocytes efficiently from MG-induced meiotic arrest during in vitro maturation. Part B showed that the MG-induced meiotic delay or arrest is associated with significant rises in spindle aberrations, chromosome congression failure and aberrant telophase I in oocytes. MG exposure of meiotically arrested GV-stage oocytes significantly increases the numbers of γ-H2AX spots in the nucleus suggesting increased DNA damage, while MG exposure during maturation affects chromatin condensation and induces chromosome lagging at anaphase I. Moreover, Part C revealed that carbonyl stress by chronic exposure to MG is associated with delays in changes in mitochondrial distribution and altered inner-mitochondrial GSH/GSSG redox potential, which might be particularly relevant for cytoskeletal dynamics as well as processes after fertilization. Sensitivity to a meiotic block by MG appears dependent on the genetic background. The sensitivity to carbonyl stress by MG appears to increase with maternal age. Since MG-exposure induces DNA damage, meiotic delay, spindle aberrations, anaphase I lagging and epimutation, aged oocytes are particularly at risk for such disturbances in the absence of efficient protection by cumulus. Furthermore, disturbances in mitochondrial distribution and redox regulation may be especially critical for fertilization and developmental competence of oocytes exposed to MG and carbonyl stress before or during maturation, for instance, in aged females, or in PCOS or diabetic patients, in agreement with recent suggestions of correlations between poor follicular and embryonic development, lower pregnancy rate and presence of toxic AGEs in serum, irrespective of age.

  4. 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

  5. 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

  6. In vivo effects of microinjected alkaline phosphatase and its low molecular weight substrates on the first meiotic cell division in Xenopus laevis oocytes.

    PubMed Central

    Hermann, J; Mulner, O; Bellé, R; Marot, J; Tso, J; Ozon, R

    1984-01-01

    Xenopus laevis oocytes were microinjected with low molecular weight phosphoesters such as 2-glycerophosphate, phosphotyrosine, phosphoserine, phosphothreonine, 4-nitrophenyl phosphate, and orthophosphate. These compounds were able to induce a considerable reduction in the time course of progesterone-induced maturation, with 2-glycerophosphate being the most effective. The basal level of cAMP and its drop during maturation were not affected by the microinjection of 2-glycerophosphate. The injection of alkaline phosphatase (EC 3.1.3.1.) from calf intestine at a low concentration (10 ng per oocyte) was able to decrease or abolish the effect of 2-glycerophosphate. At higher concentration (25 ng per oocyte) this enzyme totally blocked progesterone- or maturation-promoting factor-induced maturation. Alkaline phosphatase might behave in vivo as a phosphoprotein phosphatase active towards phosphotyrosine-containing proteins. In addition, our results indicate that phosphate or phosphoester-containing buffers should be avoided in the course of maturation-promoting factor purification. PMID:6089179

  7. Immunophotoaffinity labeling of the binding proteins for 1-methyladenine, an oocyte maturation-inducing hormone of starfish.

    PubMed

    Kida, Tetsuo; Matsuda, Shinjiro; Kuyama, Atsushi; Toraya, Tetsuo

    2014-01-01

    Starfish oocytes are naturally arrested at the prophase stage of the first meiotic division and resume meiosis in response to 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative receptors for 1-MeAde have not yet been characterized biochemically, although the specific binding of 1-MeAde to the isolated cortices of starfish oocytes was reported so far. Based on the structure-activity relationship of 1-MeAde analogs, we have designed a photoaffinity labeling reagent. The photoaffinity labeling of oocyte membrane fractions, followed by immunoblotting analysis with anti-1-MeAde antibody, results in the detection of an almost single protein band. This 1-MeAde-binding protein might be a possible candidate of the maturation-inducing hormone receptor of starfish.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  9. Inhibitory phosphorylation of Cdk1 mediates prolonged prophase I arrest in female germ cells and is essential for female reproductive lifespan

    PubMed Central

    Adhikari, Deepak; Busayavalasa, Kiran; Zhang, Jingjing; Hu, Mengwen; Risal, Sanjiv; Bayazit, Mustafa Bilal; Singh, Meenakshi; Diril, M Kasim; Kaldis, Philipp; Liu, Kui

    2016-01-01

    A unique feature of female germ cell development in mammals is their remarkably long arrest at the prophase of meiosis I, which lasts up to 50 years in humans. Both dormant and growing oocytes are arrested at prophase I and completely lack the ability to resume meiosis. Here, we show that the prolonged meiotic arrest of female germ cells is largely achieved via the inhibitory phosphorylation of Cdk1 (cyclin-dependent kinase 1). In two mouse models where we have introduced mutant Cdk1T14AY15F which cannot be inhibited by phosphorylation (Cdk1AF) in small meiotically incompetent oocytes, the prophase I arrest is interrupted, leading to a premature loss of female germ cells. We show that in growing oocytes, Cdk1AF leads to premature resumption of meiosis with condensed chromosomes and germinal vesicle breakdown followed by oocyte death, whereas in dormant oocytes, Cdk1AF leads to oocyte death directly, and both situations damage the ovarian reserve that maintains the female reproductive lifespan, which should be around 1 year in mice. Furthermore, interruption of the inhibitory phosphorylation of Cdk1 results in DNA damage, which is accompanied by induction of the Chk2 (checkpoint kinase 2)-p53/p63-dependent cell death pathway, which eventually causes global oocyte death. Together, our data demonstrate that the phosphorylation-mediated suppression of Cdk1 activity is one of the crucial factors that maintain the lengthy prophase arrest in mammalian female germ cells, which is essential for preserving the germ cell pool and reproductive lifespan in female mammals. PMID:27767095

  10. Cytostatic Factor Proteins Are Present in Male Meiotic Cells and β-Nerve Growth Factor Increases Mos Levels in Rat Late Spermatocytes

    PubMed Central

    Perrard, Marie-Hélène; Chassaing, Emeric; Montillet, Guillaume; Sabido, Odile; Durand, Philippe

    2009-01-01

    Background In co-cultures of pachytene spermatocytes with Sertoli cells, β-NGF regulates the second meiotic division by blocking secondary spermatocytes in metaphase (metaphase II), and thereby lowers round spermatid formation. In vertebrates, mature oocytes are arrested at metaphase II until fertilization, because of the presence of cytostatic factor (CSF) in their cytoplasm. By analogy, we hypothesized the presence of CSF in male germ cells. Methodology/Principal Findings We show here, that Mos, Emi2, cyclin E and Cdk2, the four proteins of CSF, and their respective mRNAs, are present in male rat meiotic cells; this was assessed by using Western blotting, immunocytochemistry and reverse transcriptase PCR. We measured the relative cellular levels of Mos, Emi2, Cyclin E and Cdk2 in the meiotic cells by flow cytometry and found that the four proteins increased throughout the first meiotic prophase, reaching their highest levels in middle to late pachytene spermatocytes, then decreased following the meiotic divisions. In co-cultures of pachytene spermatocytes with Sertoli cells, β-NGF increased the number of metaphases II, while enhancing Mos and Emi2 levels in middle to late pachytene spermatocytes, pachytene spermatocytes in division and secondary spermatocytes. Conclusion/Significance Our results suggest that CSF is not restricted to the oocyte. In addition, they reinforce the view that NGF, by enhancing Mos in late spermatocytes, is one of the intra-testicular factors which adjusts the number of round spermatids that can be supported by Sertoli cells. PMID:19802389

  11. Smc1β is required for activation of SAC during mouse oocyte meiosis.

    PubMed

    Miao, Yilong; Zhou, Changyin; Cui, Zhaokang; Dai, Xiaoxin; Zhang, Mianqun; Lu, Yajuan; Xiong, Bo

    2017-03-19

    Smc1β is a meiosis-specific cohesin subunit that is essential for sister chromatid cohesion and DNA recombination. Previous studies have shown that Smc1β-deficient mice in both sexes are sterile. Ablation of Smc1β during male meiosis leads to the blockage of spermatogenesis in pachytene stage, and ablation of Smc1β during female meiosis generates a highly error-prone oocyte although it could develop to metaphase II stage. However, the underlying mechanisms regarding how Smc1β maintains the correct meiotic progression in mouse oocytes have not been clearly defined. Here, we find that GFP-fused Smc1β is expressed and localized to the chromosomes from GV to MII stages during mouse oocyte meiotic maturation. Knockdown of Smc1β by microinjection of gene-specific morpholino causes the impaired spindle apparatus and chromosome alignment which are highly correlated with the defective kinetochore-microtubule attachments, consequently resulting in a prominently higher incidence of aneuploid eggs. In addition, the premature extrusion of polar bodies and escape of metaphase I arrest induced by low dose of nocodazole treatment in Smc1β-depleted oocytes indicates that Smc1β is essential for activation of spindle assembly checkpoint (SAC) activity. Collectively, we identify a novel function of Smc1β as a SAC participant beyond its role in chromosome cohesion during mouse oocyte meiosis.

  12. Cdk1, but not Cdk2, is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes.

    PubMed

    Adhikari, Deepak; Zheng, Wenjing; Shen, Yan; Gorre, Nagaraju; Ning, Yao; Halet, Guillaume; Kaldis, Philipp; Liu, Kui

    2012-06-01

    Mammalian oocytes are arrested at the prophase of meiosis I during fetal or postnatal development, and the meiosis is resumed by the preovulatory surge of luteinizing hormone. The in vivo functional roles of cyclin-dependent kinases (Cdks) during the resumption of meiosis in mammalian oocytes are largely unknown. Previous studies have shown that deletions of Cdk3, Cdk4 or Cdk6 in mice result in viable animals with normal oocyte maturation, indicating that these Cdks are not essential for the meiotic maturation of oocytes. In addition, conventional knockout of Cdk1 and Cdk2 leads to embryonic lethality and postnatal follicular depletion, respectively, making it impossible to study the functions of Cdk1 and Cdk2 in oocyte meiosis. In this study, we generated conditional knockout mice with oocyte-specific deletions of Cdk1 and Cdk2. We showed that the lack of Cdk1, but not of Cdk2, leads to female infertility due to a failure of the resumption of meiosis in the oocyte. Re-introduction of Cdk1 mRNA into Cdk1-null oocytes largely resumed meiosis. Thus, Cdk1 is the sole Cdk that is essential and sufficient to drive resumption of meiosis in mouse oocytes. We also found that Cdk1 maintains the phosphorylation status of protein phosphatase 1 and lamin A/C in oocytes in order for meiosis resumption to occur.

  13. Short-term hypertonic exposure enhances in vitro follicle growth and meiotic competence of enclosed oocytes while modestly affecting mRNA expression of aquaporin and steroidogenic genes in the domestic cat model.

    PubMed

    Songsasen, N; Thongkittidilok, C; Yamamizu, K; Wildt, D E; Comizzoli, P

    2017-03-01

    antral stage while Fshr was only affected in the former compared to the non-cultured control. Pre-incubating follicles in 350 mOsm medium for 24 h enhanced (P < 0.05) Star and Aqp7 while decreasing (P < 0.05) Aqp1 expression compared to the control in secondary follicles, but not in the early antral stage. In summary, short-term hypertonic exposure promoted cat follicle development in vitro (including the meiotic competence of the enclosed oocyte) possibly through a mechanism that does not involve water transport genes.

  14. Immunophotoaffinity labeling of binders of 1-methyladenine, the oocyte maturation-inducing hormone of starfish.

    PubMed

    Toraya, Tetsuo; Kida, Tetsuo; Kuyama, Atsushi; Matsuda, Shinjiro; Tanaka, Seiichi; Komatsu, Yo; Tsurukai, Taro

    2017-03-25

    Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary and resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative 1-MeAde receptors on the oocyte surface have been suggested, but not yet been biochemically characterized. Immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection, was attempted to detect unknown 1-MeAde binders including putative maturation-inducing hormone receptors in starfish oocytes. When the oocyte crude membrane fraction or its Triton X-100/EDTA extract was incubated with N(6)-[6-(5-azido-2-nitrobenzoyl)aminohexyl]carboxamidomethyl-1-methyladenine and then photo-irradiated, followed by western blotting with antibody that was raised against a 1-MeAde hapten, a single band with Mr of 47.5 K was detected. The band was lost when extract was heated at 100 °C. A similar 47.5 K band was detected in the crude membrane fraction of testis as well. Upon labeling with whole cells, this band was detected in immature and maturing oocytes, but only faintly in mature oocytes. As judged from these results, this 1-MeAde binder might be a possible candidate of the starfish maturation-inducing hormone receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. 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.

  16. 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

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

    PubMed

    Costache, Vlad; McDougall, Alex; Dumollard, Rémi

    2014-08-01

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

  18. Griseofulvin-induced aneuploidy and meiotic delay in male mouse germ cells: detected by using conventional cytogenetics and three-color FISH.

    PubMed

    Shi, Q; Schmid, T E; Adler, I

    1999-05-17

    Griseofulvin (GF) was tested in male mouse germ cells for the induction of meiotic delay and aneuploidy. Starved mice were orally treated with 500, 1000 and 2000 mg/kg of GF in corn oil and testes were sampled 22 h later for meiotic delay analysis and chromosome counting in spermatocytes at the second meiotic metaphase (MMII). A dose-related increase in meiotic delay by dose-dependently arresting spermatocytes in first meiotic metaphase (MMI) or/and prolonging interkinesis was observed. Hyperhaploid MMII cells were not significantly increased. Sperm were sampled from the Caudae epididymes 22 days after GF-treatment of the males for three-color fluorescence in situ hybridization (FISH). The frequencies of diploidies were 0.01-0.02% in sperm of the solvent control animals and increased dose-dependently to 0.03%, 0.068% and 0.091%, respectively, for 500, 1000 and 2000 mg/kg of GF. The frequencies of disomic sperm were increased significantly above the controls in all GF-treated groups but showed no dose response. The data for individual classes of disomic sperm indicated that MII was more sensitive than MI to GF-induced non-disjunction in male mice. A comparison of the present data from male mice and literature data from female mice suggests that mouse oocytes are more sensitive than mouse spermatocytes to GF-induced meiotic delay and aneuploidy.

  19. Meiosis in oocytes: predisposition to aneuploidy and its increased incidence with age.

    PubMed

    Jones, Keith T

    2008-01-01

    Mammalian oocytes begin meiosis in the fetal ovary, but only complete it when fertilized in the adult reproductive tract. This review examines the cell biology of this protracted process: from entry of primordial germ cells into meiosis to conception. The defining feature of meiosis is two consecutive cell divisions (meiosis I and II) and two cell cycle arrests: at the germinal vesicle (GV), dictyate stage of prophase I and at metaphase II. These arrests are spanned by three key events, the focus of this review: (i) passage from mitosis to GV arrest during fetal life, regulated by retinoic acid; (ii) passage through meiosis I and (iii) completion of meiosis II following fertilization, both meiotic divisions being regulated by cyclin-dependent kinase (CDK1) activity. Meiosis I in human oocytes is associated with an age-related high rate of chromosomal mis-segregation, such as trisomy 21 (Down's syndrome), resulting in aneuploid conceptuses. Although aneuploidy is likely to be multifactorial, oocytes from older women may be predisposed to be becoming aneuploid as a consequence of an age-long decline in the cohesive ties holding chromosomes together. Such loss goes undetected by the oocyte during meiosis I either because its ability to respond and block division also deteriorates with age, or as a consequence of being inherently unable to respond to the types of segregation defects induced by cohesion loss.

  20. Effect of protein synthesis inhibition before or during in vitro maturation on subsequent development of bovine oocytes.

    PubMed

    Lonergan, P; Fair, T; Khatir, H; Cesaroni, G; Mermillod, P

    1998-08-01

    The overall objective of this study was to assess the effect of maintaining meiotic arrest in bovine oocytes in vitro on developmental competence. In Experiment 1 the effect of inhibition of meiotic resumption using cycloheximide (CX), on subsequent was examined. Immature cumulus oocyte complexes (COCs, n = 804) were cultured in the absence (24 h) or presence of CX for 6, 12, 18 or 24 h. The control was inseminated 24 h later, while CX-treated oocytes were cultured for a further 24 h before insemination. In Experiment 2 the effect of exposing the oocyte (n = 1239) during meiotic arrest to putative stimulatory substances (pFSH and FCS) was examined. In Experiment 3, to study the importance of protein synthesis during maturation, synthesis was blocked for a 6-h period at various times (6, 12, 18 h) after start of culture (n = 1117). In Experiment 1, there was no difference in cleavage rate between treatments. However, the percentage of 5 to 8 cell embryos at 72 h post insemination was significantly lower after CX treatment (64 vs 42 to 51%; P < 0.05). This was reflected in a lower rate of blastocysts at Day 6 (9 to 15 vs 31%, P < 0.002). While the blastocyst rate at Day 8 was lower in CX-treated oocytes, the effect was only significant when CX was present for longer than 12 h. A marked decrease in development was noted following inhibition for 18 h or more compared with the control (17 to 19 vs 40%; P < 0.0002). In Experiment 2, addition of either FSH or FCS to oocytes in the presence of CX had no effect on any of the parameters studied, even though there was a positive effect in control oocytes. In Experiment 3, treatment with CX after the oocytes had matured for varying periods resulted in decreased blastocyst rates at Days 6 and 8 of culture. The most significant drop in development occurred when oocytes were cultured for 12 h before exposure to CX (15 vs 40%; P < 0.0001). In conclusion, CX-blocked oocytes retained their developmental competence, although final

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

    PubMed

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

    2016-08-01

    Oocytes develop the competence for meiosis and early embryogenesis during their growth. Setdb1 is a histone H3 lysine 9 (H3K9) methyltransferase required for post-implantation development and has been implicated in the transcriptional silencing of genes and endogenous retroviral elements (ERVs). To address its role in oogenesis and pre-implantation development, we conditionally deleted Setdb1 in growing oocytes. Loss of Setdb1 expression greatly impaired meiosis. It delayed meiotic resumption, altered the dynamics of chromatin condensation, and impaired kinetochore-spindle interactions, bipolar spindle organization and chromosome segregation in more mature oocytes. The observed phenotypes related to changes in abundance of specific transcripts in mutant oocytes. Setdb1 maternally deficient embryos arrested during pre-implantation development and showed comparable defects during cell cycle progression and in chromosome segregation. Finally, transcriptional profiling data indicate that Setdb1 downregulates rather than silences expression of ERVK and ERVL-MaLR retrotransposons and associated chimearic transcripts during oogenesis. Our results identify Setdb1 as a newly discovered meiotic and embryonic competence factor safeguarding genome integrity at the onset of life. © 2016. Published by The Company of Biologists Ltd.

  2. 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

  3. In vitro maturation of immature human oocytes for clinical application.

    PubMed

    Chian, Ri-Cheng; Cao, Yun-Xia

    2014-01-01

    Meiotic progression in the oocyte is defined as oocyte maturation from reinitiation of the first meiotic division from the germinal vesicle (GV) stage to the metaphase-II (M-II) stage (Fig. 1), (Cha and Chian, Hum Reprod Update 4:103-120, 1998). Priming with FSH or HCG prior to immature oocyte retrieval improves oocyte maturation and pregnancy rates. The size of follicles may be an important feature for IVM treatment. The combination of natural-cycle IVF with immature oocyte retrieval followed by IVM is an attractive treatment for young women with all types of infertility without recourse to ovarian stimulation with an acceptable pregnancy rate.

  4. Meiotic abnormalities

    SciTech Connect

    1993-12-31

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

  5. Affinity chromatography of a binder of 1-methyladenine, the maturation-inducing hormone for starfish oocytes.

    PubMed

    Toraya, Tetsuo; Kuyama, Atsushi; Tanaka, Seiichi; Yamamoto, Masatoyo; Ohmiya, Tadamasa; Saito, Yuri; Tanabe, Tomoko

    2017-05-13

    Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary. They resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the maturation-inducing hormone for starfish oocytes. Putative 1-MeAde receptors have been suggested to be present on the oocyte surface, but not yet been characterized biochemically. As reported recently (T. Toraya, T. Kida, A. Kuyama, S. Matsuda, S. Tanaka, Y. Komatsu, T. Tsurukai, Biochem. Biophys. Res. Commun. 485 (2017) 41-46), it became possible to detect unknown 1-MeAde binders of starfish oocytes by immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection. We designed and synthesized water-soluble and insoluble polymer-bound 1-MeAde derivatives. A water-soluble polymer-bound 1-MeAde derivative, in which 1-MeAde is bound to dextran through an N(6)-substituent, triggered the germinal-vesicle breakdown toward follicle-free oocytes, dejellied oocytes, and denuded oocytes. This is consistent with the idea that putative 1-MeAde receptors are located on the cell surface of starfish oocytes. A water-insoluble polymer-bound 1-MeAde derivative, in which 1-MeAde is bound to Sepharose 4B through an N(6)-substituent, served as an effective affinity adsorbent for the partial purification of a 1-MeAde binder with Mr of 47.5 K that might be a possible candidate of the maturation-inducing hormone receptors of starfish oocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. 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.

  7. 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

  8. 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.

  9. Characterization of fission yeast meiotic mutants based on live observation of meiotic prophase nuclear movement.

    PubMed

    Hiraoka, Y; Ding, D Q; Yamamoto, A; Tsutsumi, C; Chikashige, Y

    2000-01-01

    We characterized four meiotic mutants of the fission yeast Schizosaccharomyces pombe by live observation of nuclear movement. Nuclei were stained with either the DNA-specific fluorescent dye Hoechst 33342 or jellyfish green fluorescent protein (GFP) fused with the N-terminal portion of DNA polymerase alpha. We first followed nuclear dynamics in wild-type cells to determine the temporal sequence of meiotic events: nuclear fusion in the conjugated zygote is immediately followed by oscillatory nuclear movements that continue for 146 min; then, after coming to rest, the nucleus remains in the center of the cell for 26 min before the first meiotic division. Next we examined nuclear dynamics in four meiotic mutants: mei1 (also called mat2), mei4, dhc1, and taz1. Mei1 and mei4 both arrest during meiotic prophase; our observations, however, show that the timing of mei1 arrest is quite different from that of mei4: the mei1 mutant arrests after nuclear fusion but before starting the oscillatory nuclear movements, while the mei4 mutant arrests after the nucleus has completed the oscillatory movements but before the first meiotic division. We also show examples of the dynamic phenotypes of dhc1 and taz1, both of which complete meiosis but exhibit impaired nuclear movement and reduced frequencies of homologous recombination: the dhc1 mutant exhibits no nuclear movement after nuclear fusion, while the taz1 mutant exhibits severely impaired nuclear movement after nuclear fusion.

  10. Meiotic process and aneuploidy

    SciTech Connect

    Grell, R.F.

    1985-01-01

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

  11. Effect of guaianolides in the meiosis reinitiation of amphibian oocytes.

    PubMed

    Zapata-Martínez, J; Sánchez-Toranzo, G; Chaín, F; Catalán, C A N; Bühler, M I

    2017-02-01

    Sesquiterpene lactones (STLs) are a large and structurally diverse group of plant metabolites generally found in the Asteraceae family. STLs exhibit a wide spectrum of biological activities and it is generally accepted that their major mechanism of action is the alkylation of the thiol groups of biological molecules. The guaianolides is one of various groups of STLs. Anti-tumour and anti-migraine effects, an allergenic agent, an inhibitor of smooth muscle cells and of meristematic cell proliferation are only a few of the most commonly reported activities of STLs. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under stimulus with progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. There are previous records of the inhibitory effect of dehydroleucodin (DhL), a guaianolide lactone, on the progression of meiosis. It has been also shown that DhL and its 11,13-dihydroderivative (2H-DhL; a mixture of epimers at C-11) act as blockers of the resumption of meiosis in fully grown ovarian oocytes from the amphibian Rhinella arenarum (formerly classified as Bufo arenarum). The aim of this study was to analyze the effect of four closely related guaianolides, i.e., DhL, achillin, desacetoxymatricarin and estafietin as possible inhibitors of meiosis in oocytes of amphibians in vitro and discuss some structure-activity relationships. It was found that the inhibitory effect on meiosis resumption is greater when the lactone has two potentially reactive centres, either a α,β-α',β'-diunsaturated cyclopentanone moiety or an epoxide group plus an exo-methylene-γ-lactone function.

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

    PubMed

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

    2015-06-01

    Inhibitors of cyclin-dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor-free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (-) at 38.5°C and 5% CO2 . At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/-) exhibited total expansion while in both Rosco groups (+/-) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/-). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression. © 2015 Blackwell Verlag GmbH.

  13. 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.

  14. RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes.

    PubMed

    Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

    2016-10-01

    Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.

  15. 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.

  16. The effect of pre-maturation culture using phosphodiesterase type 3 inhibitor and insulin, transferrin and selenium on nuclear and cytoplasmic maturation of bovine oocytes.

    PubMed

    Guimarães, A L S; Pereira, S A; Kussano, N R; Dode, M A N

    2016-04-01

    This study aims to evaluate if a pre-maturation culture (PMC) using cilostamide as a meiotic inhibitor in combination with insulin, transferrin and selenium (ITS) for 8 or 24 h increases in vitro embryo production. To evaluate the effects of PMC on embryo development, cleavage rate, blastocyst rate, embryo size and total cell number were determined. When cilostamide (20 μM) was used in PMC for 8 or 24 h, 98% of oocytes were maintained in germinal vesicles. Although the majority of oocytes resumed meiosis after meiotic arrest, the cleavage and blastocyst rates were lower than the control (P 0.05) to the control. The deleterious effect of 20 μM cilostamide treatment for 24 h on a PMC was confirmed by lower cumulus cell viability, determined by trypan blue staining, in that group compared with the other groups. A lower concentration (10 μM) and shorter exposure time (8 h) minimized that effect but did not improve embryo production. More studies should be performed to determine the best concentration and the arresting period to increase oocyte competence and embryo development.

  17. Nitric oxide synthase (NOS) inhibition during porcine in vitro maturation modifies oocyte protein S-nitrosylation and in vitro fertilization.

    PubMed

    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.

  18. Kinetics of nuclear status and kinase activities during in vitro maturation of canine oocytes.

    PubMed

    Suzukamo, Chika; Hoshina, Mariko; Moriya, Hiromi; Hishiyama, Nobuya; Nakamura, Shigeru; Kawai, Fumie; Sato, Harumi; Ariga, Makoto; Ito, Junya; Kashiwazaki, Naomi

    2009-04-01

    In contrast to those of other mammals, canine oocytes are ovulated at the germinal vesicle (GV) stage and then progress to the metaphase II (MII) stage in the oviduct. In other species, oocytes at the MII are widely used for in vitro fertilization or as recipients in somatic cell nuclear transfer. Many researchers have tried to improve the in vitro maturation (IVM) of canine oocytes. However, the proportion of MII oocytes remains low, resulting in poor efficiency of embryogenesis in vitro. This leads us to the possibility that the in vitro cytoplasmic maturation of canine oocytes is insufficient. Furthermore, the optimal culture period for IVM of canine oocytes is controversial, and physiological evaluation is required to improve canine IVM. We show here the time-dependent changes in mitogen-activated protein kinase (MAPK) and p34(cdc2) kinase activities in canine oocytes during IVM, since it is well known that both MAPK and p34(cdc2) kinase are activated following meiotic progression and show high activities in the MII stage in other species. Immediately after collection from ovaries, most oocytes were arrested at the GV stage, which was maintained until 24 h of culture. At 48 h of culture, more than half of the oocytes had progressed beyond the MI stage. A higher proportion of MII oocytes were observed with 72 h of culture compared with other culture periods. MAPK activity was found to increase in a time-dependent manner and reached a plateau at 72 h of culture. The level of p34(cdc2) kinase activity also increased in a time-dependent manner, with its maximal level observed after 72 h of culture. Activity was decreased with 96 h of culture, although there was no significant difference in the proportion of MII oocytes between 72 and 96 h. Our data thus show that the optimal culture period for IVM of canine oocytes is 72 h because both MAPK and p34(cdc2) kinase showed high activities at that time.

  19. Towards a new understanding on the regulation of mammalian oocyte meiosis resumption.

    PubMed

    Sun, Qing-Yuan; Miao, Yi-Liang; Schatten, Heide

    2009-09-01

    Mammalian oocytes reach prophase of first meiosis around the time of birth, and remain at this stage for months or years, depending on the species. Only after puberty will the fully-grown oocytes begin to resume meiosis which is stimulated by gonadotropin surge. It has long been known that a high level of intra-oocyte cyclic adenosine 3',5'-monophosphate (cAMP) prevents oocyte meiosis resumption as indicated by germinal vesicle breakdown (GVBD). Recently, guanosine triphosphate-binding (G) protein-coupled receptors/G proteins/adenyl cyclase pathway endogenous to the oocyte as well as cAMP diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that prevent oocytes from resuming meiosis. Another second messager molecule, guanosine 3',5'-cyclic monophosphate (cGMP), has also recently been found to play important roles in maintaining oocyte meiosis arrest. cGMP in the follicular somatic cells diffuses into the oocyte and causes an increase in oocyte cAMP, presumably by acting on phosphodiesterase 3 (PDE3). The cGMP level in the somatic compartment of the follicle decreases in response to luteinizing hormone (LH), and this change may be mediated through the epidermal growth factor (EGF)-like factors and specific cGMP-phosphodiesterase subtype activity. It is well known that gonadotropic stimulation of meiotic resumption depends on mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle; recent studies show that LH, through cAMP/protein kinase A (PKA) and protein kinase C (PKC) pathways, induces the synthesis of paracine factors such as EGF-like facors and meiosis activating sterol (MAS) to regulate oocyte GVBD via the MAPK pathway in follicle cells. A recent granulosa cell-specific knockout study has for the first time provided in vivo evidence for the important role of extracellular regulated kinase 1 and 2 (ERK1/2), two main forms of MAPK, and their downstream molecules in

  20. Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia.

    PubMed

    Vendrell, J M; García, F; Veiga, A; Calderón, G; Egozcue, S; Egozcue, J; Barri, P N

    1999-02-01

    The incidence of meiotic abnormalities and their relationship with different spermatogenic parameters was assessed in 103 male patients with presumably idiopathic severe oligoasthenozoospermia (motile sperm concentration < or = 1.5 x 10(6)/ml). Meiosis on testicular biopsies was independently evaluated by two observers. Meiotic patterns included normal meiosis and two meiotic abnormalities, i.e. severe arrest and synaptic anomalies. A normal pattern was found in 64 (62.1%), severe arrest in 21 (20.4%) and synaptic anomalies in 18 (17.5%). The overall rate of meiotic abnormalities was 37.9%. Most (66.7%) meiotic abnormalities occurred in patients with a sperm concentration < or = 1 x 10(6)/ml. In this group, total meiotic abnormalities were found in 57.8% of the patients; of these, 26.7% had synaptic anomalies. When the sperm concentration was < or = 0.5 x 10(6)/ml, synaptic anomalies were detected in 40% of the patients. In patients with increased follicle stimulating hormone (FSH) concentrations, total meiotic abnormalities occurred in 54.8% (synaptic anomalies in 22.6%). There were statistically significant differences among the three meiotic patterns in relation to sperm concentration (P < 0.001) and serum FSH concentration (P < 0.05). In the multivariate analysis, sperm concentration < or = 1 x 10(6)/ml and/or FSH concentration > 10 IU/l were the only predictors of meiotic abnormalities.

  1. Cytological techniques to study human female meiotic prophase.

    PubMed

    Roig, Ignasi; Garcia-Caldés, Montserrat

    2009-01-01

    Most of the human aneuploidies have a maternal origin. This feature makes the study of human female meiosis a fundamental topic to understand the reasons leading to this important social problem. Unfortunately, due to sample collection difficulties, not many studies have been performed on human female meiotic prophase. In this chapter we present a comprehensive collection of protocols that allows the study of human female meiotic prophase through different technical approaches using both spread and structurally preserved oocytes.

  2. 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

  3. 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. © 2016 by the Society for the Study of Reproduction, Inc.

  4. Cumulus Cell Transcripts Transit to the Bovine Oocyte in Preparation for Maturation1

    PubMed Central

    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

    2015-01-01

    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

  5. Conserved functions for Mos in eumetazoan oocyte maturation revealed by studies in a cnidarian.

    PubMed

    Amiel, Aldine; Leclère, Lucas; Robert, Lucie; Chevalier, Sandra; Houliston, Evelyn

    2009-02-24

    The kinase Mos, which activates intracellularly the MAP kinase pathway, is a key regulator of animal oocyte meiotic maturation. In vertebrate and echinoderm models, Mos RNA translation upon oocyte hormonal stimulation mediates "cytostatic" arrest of the egg after meiosis, as well as diverse earlier events [1-5]. Our phylogenetic survey has revealed that MOS genes are conserved in cnidarians and ctenophores, but not found outside the metazoa or in sponges. We demonstrated MAP kinase-mediated cytostatic activity for Mos orthologs from Pleurobrachia (ctenophore) and Clytia (cnidarian) by RNA injection into Xenopus blastomeres. Analyses of endogenous Mos in Clytia with morpholino antisense oligonucleotides and pharmacological inhibition demonstrated that Mos/MAP kinase function in postmeiotic arrest is conserved. They also revealed additional roles in spindle formation and positioning, strongly reminiscent of observations in starfish, mouse, and Xenopus. Unusually, cnidarians were found to possess multiple Mos paralogs. In Clytia, one of two maternally expressed paralogs accounted for the majority MAP kinase activation during maturation, whereas the other may be subject to differential translational regulation and have additional roles. Our findings indicate that Mos appeared early during animal evolution as an oocyte-expressed kinase and functioned ancestrally in regulating core specializations of female meiosis.

  6. The transcriptome of human oocytes

    PubMed Central

    Kocabas, Arif Murat; Crosby, Javier; Ross, Pablo J.; Otu, Hasan H.; Beyhan, Zeki; Can, Handan; Tam, Wai-Leong; Rosa, Guilherme J. M.; Halgren, Robert G.; Lim, Bing; Fernandez, Emilio; Cibelli, Jose Bernardo

    2006-01-01

    The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis, folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This report provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes may expand our knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis. PMID:16968779

  7. Meiotic DNA double-strand breaks and chromosome asynapsis in mice are monitored by distinct HORMAD2-independent and -dependent mechanisms.

    PubMed

    Wojtasz, Lukasz; Cloutier, Jeffrey M; Baumann, Marek; Daniel, Katrin; Varga, János; Fu, Jun; Anastassiadis, Konstantinos; Stewart, A Francis; Reményi, Attila; Turner, James M A; Tóth, Attila

    2012-05-01

    Meiotic crossover formation involves the repair of programmed DNA double-strand breaks (DSBs) and synaptonemal complex (SC) formation. Completion of these processes must precede the meiotic divisions in order to avoid chromosome abnormalities in gametes. Enduring key questions in meiosis have been how meiotic progression and crossover formation are coordinated, whether inappropriate asynapsis is monitored, and whether asynapsis elicits prophase arrest via mechanisms that are distinct from the surveillance of unrepaired DNA DSBs. We disrupted the meiosis-specific mouse HORMAD2 (Hop1, Rev7, and Mad2 domain 2) protein, which preferentially associates with unsynapsed chromosome axes. We show that HORMAD2 is required for the accumulation of the checkpoint kinase ATR along unsynapsed axes, but not at DNA DSBs or on DNA DSB-associated chromatin loops. Consistent with the hypothesis that ATR activity on chromatin plays important roles in the quality control of meiotic prophase, HORMAD2 is required for the elimination of the asynaptic Spo11(-/-), but not the asynaptic and DSB repair-defective Dmc1(-/-) oocytes. Our observations strongly suggest that HORMAD2-dependent recruitment of ATR to unsynapsed chromosome axes constitutes a mechanism for the surveillance of asynapsis. Thus, we provide convincing evidence for the existence of a distinct asynapsis surveillance mechanism that safeguards the ploidy of the mammalian germline.

  8. Meiosis, Balbiani body and early asymmetry of Thermobia oocyte.

    PubMed

    Tworzydlo, Waclaw; Marek, Magdalena; Kisiel, Elzbieta; Bilinski, Szczepan M

    2017-03-01

    The meiotic division guarantees maintenance of a genetic diversity; it consists of several stages, with prophase I being the longest and the most complex. We decided to follow the course of initial stages of meiotic division in ovaries of Thermobia domestica using modified techniques of squash preparations, semithin sections, and electron microscopy. We show that germaria contain numerous germline cells that can be classified into three categories: cystoblasts, meiotic oocytes, and growing previtellogenic oocytes. The cystoblasts are located most apically. The meiotic oocytes occupy the middle part of the germarium, and the previtellogenic oocytes can be found in the most basal part, near the vitellarium. Analyses of the semithin sections and squash preparations show that post leptotene meiotic chromosomes gather in one region of the nucleoplasm where they form the so-called bouquet. The telomeres of the bouquet chromosomes are attached to a relatively small area (segment) of the nuclear envelope. Next to this envelope segment, the nucleolar organizers are also located. We show that in concert to sequential changes inside the oocyte nuclei, rearrangement of organelles within the ooplasm (oocyte cytoplasm) takes place. This leads to the formation of the Balbiani body and consequent asymmetry of the ooplasm. These early nuclear and cytoplasmic asymmetries, however, are transient. During diplotene, the chromosome bouquet disappears, while the Balbiani body gradually disperses throughout the ooplasm. Finally, our observations indicate the presence of lampbrush chromosomes in the nuclei of previtellogenic oocytes. In the close vicinity to lampbrush chromosomes, characteristic spherical nuclear bodies are present.

  9. Polarized light microscopy in mammalian oocytes.

    PubMed

    Caamaño, J N; Muñoz, M; Diez, C; Gómez, E

    2010-06-01

    The meiotic spindle structure plays a key role in normal chromosome alignment and segregation during meiosis. Polarized light microscopy (PLM) allows non-invasive evaluation of the meiotic spindle of metaphase oocytes from different animal species. The purpose of this article is to review the use of PLM in animal reproduction, mainly in the assessment of the meiotic spindle in oocytes. A brief overview of the methods to assess the meiotic spindle is presented as well as the principles behind the PLM. The use of PLM to evaluate oocyte quality and spindle morphology is discussed and the results on the viability of the oocytes after being exposed to PLM are presented. Several researchers showed that PLM could be successfully implemented on cryopreservation, nuclear transfer and intracytoplasmic sperm injection procedures as a tool to improve the outcome of these procedures. In addition, PLM can be used to develop studies on oocyte maturation and spindle dynamics. However, the information on the practical use of this technology in farm animals is very limited and further studies are needed to assess the importance of PLM in animal reproduction.

  10. Involvement of estradiol-17β and its membrane receptor, G protein coupled receptor 30 (GPR30) in regulation of oocyte maturation in zebrafish, Danio rerio

    PubMed Central

    Pang, Yefei; Thomas, Peter

    2009-01-01

    The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20β-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17β, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes. PMID:18952087

  11. 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.

  12. 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.

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

    PubMed

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

    2015-01-01

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

  14. Chromosome malsegregation and embryonic lethality induced by treatment of normally ovulated mouse oocytes with nocodazole.

    PubMed

    Generoso, W M; Katoh, M; Cain, K T; Hughes, L A; Foxworth, L B; Mitchell, T J; Bishop, J B

    1989-02-01

    The mouse egg is ovulated with its nucleus arrested at the metaphase-II stage of meiosis. Sperm entry triggers the completion of the second meiotic division. It has been speculated that damage to the meiotic spindle of normally ovulated eggs at around the time of sperm entry could result in chromosome malsegregation and the death of conceptuses with numerical chromosome anomalies. This hypothesis was tested using nocodazole, a microtubule inhibitor. Nocodazole was administered either to maturing preovulatory oocytes or to normally ovulated eggs at one of the following stages: (1) the time of sperm entry, (2) early pronuclear stage, (3) pronuclear DNA synthesis, (4) prior to first cleavage division, (5) early 2-cell stage, or (6) prior to the second cleavage division. Little or no effect was observed for treatment times other than the time of sperm entry, when the egg is being activated to complete the second meiotic division. Remarkably high frequencies of embryonic lethality, expressed at around the time of implantation, were induced at this stage. Cytogenetic analysis of first cleavage metaphases of zygotes treated at the time of sperm entry revealed a high incidence of varied numerical chromosome anomalies, with changes in ploidy being predominant.

  15. 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

  16. Cell cycle control by daf-21/Hsp90 at the first meiotic prophase/metaphase boundary during oogenesis in Caenorhabditis elegans.

    PubMed

    Inoue, Tadao; Hirata, Kazumasa; Kuwana, Yuichiro; Fujita, Masahiro; Miwa, Johji; Roy, Richard; Yamaguchi, Yasunori

    2006-01-01

    DAF-21, a Caenorhabditis elegans homologue of Hsp90, is expressed primarily in germline cells. Although mutations in the daf-21 gene affect animal fertility, its cellular roles have remained elusive. To phenocopy daf-21 mutations, we impaired the daf-21 function by RNA interference (RNAi), and found that oocytes skipped the diakinesis arrest and displayed a defective diakinesis arrest, which led to the production of endomitotic oocytes with polyploid chromosomes (Emo phenotype). The same Emo phenotype was also observed with RNAi against wee-1.3. To identify a cause for Emo, we examined the CDK-1 (Cdc2) phosphorylation status in Emo animals, since CDK-1 is a key regulator of the prophase/metaphase transition and is kept inactivated by WEE-1.3 kinase during prophase. We immunostained both daf-21(RNAi) and wee-1.3(RNAi) animals with anti-phosphorylated-CDK-1 antibody and observed no detectable phosphates on CDK-1 in either of the animals. We also examined WEE-1.3 expression in daf-21(RNAi) and found a significant reduction of WEE-1.3. These results indicate that CDK-1 was not phosphorylated in either daf-21(RNAi) or wee-1.3(RNAi) animals, and suggest that daf-21 was necessary for producing functional WEE-1.3. Thus, all together, we propose that DAF-21 indirectly regulates the meiotic prophase/metaphase transition during oocyte development by ensuring the normal function of WEE-1.3.

  17. Effects of alphafetoprotein on isolated mouse oocytes.

    PubMed

    Lambert, J C; Seralini, G E; Stora, C; Vallette, G; Vranckx, R; Nunez, E A

    1986-01-01

    The supposition of an effect of alphafetoprotein (AFP) on female germinal cells is put forward. The spontaneous in vitro maturation of adult mouse oocytes is significantly inhibited when mouse AFP replaces albumin in culture medium. Furthermore, the very unusual degenerative appearance of the cells subjected to AFP seems to indicate that this meiotic inhibition is linked to a premature degeneration of the oocytes rather than to a blockage of the cells at an earlier stage of maturation. Accordingly AFP, perhaps through its ligands, may play a role in reducing the number of gonocytes during fetal and immediate post-natal life rather than in stopping oocyte meiosis at the diplotene stage.

  18. Human oocyte maturation in vitro.

    PubMed

    Coticchio, Giovanni; Dal-Canto, Mariabeatrice; Guglielmo, Maria-Cristina; Mignini-Renzini, Mario; Fadini, Rubens

    2012-01-01

    Oocytes from medium-sized antral follicles have already completed their growth phase and, if released from the follicular environment and cultured in vitro, are able to resume the meiotic process and mature. However, in vitro maturation (IVM) does not entirely support all the nuclear and cytoplasmic changes that occur physiologically as an effect of the ovulatory stimulus. Regardless, oocyte IVM is widely applied for the breeding of agriculturally important species. In assisted reproduction technology, IVM has been proposed as an alternative treatment to circumvent the drawbacks of standard ovarian stimulation regimens. Initially introduced to eliminate the risks of ovarian hyperstimulation syndrome afflicting women presenting with polycystic ovaries, subsequently IVM has been suggested to represent an additional approach suitable also for normovulatory patients. So far, in children born from IVM cycles, no doubts of an increased incidence of congenital abnormalities have been raised. Many more births would be achieved if novel IVM systems, currently dominated by empiricism, could be conceived according to more physiological criteria. Recent findings shedding new light on the control of meiotic progression, the support of cumulus cells to the oocyte cellular reorganization occurring during maturation, and the modulation of the stimulus that promotes oocyte maturation downstream the mid-cycle gonadotropin signal are likely to provide crucial hints for the development of more efficient IVM systems.

  19. 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

  20. DNA damage response during mouse oocyte maturation

    PubMed Central

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

    2016-01-01

    ABSTRACT 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

  1. Ultrastructure of in vitro Matured Human Oocytes.

    PubMed

    Shahedi, Abbas; Khalili, Mohammad Ali; Soleimani, Mehrdad; Morshedizad, Shekoufeh

    2013-12-01

    Approximately 20% of recovered oocytes are immature and discarded in intracytoplasmic sperm injection (ICSI) procedures. These oocytes represent a potential resource for both clinical and basic science application. The aim of this study was to evaluate the ultrastructure architecture of in vitro matured human oocytes using transmission electron microscopy (TEM). A total of 204 immature oocytes from infertile patients who underwent ICSI cycles were included in this prospective study. Immature oocytes were divided into two groups: (i) GV oocytes (n = 101); and (ii) MI oocytes (n = 103). Supernumerary fresh in vivo matured oocytes (n = 10) were used as control. The rates of maturations were 61.38% for GV and 73.78% for MI oocytes in IVM medium (P = 0.07). However, the rate of oocyte arrest was significant between groups (P <0 .05). Ultrastructurally; in vitro and in vivo matured oocytes appeared round, with a homogeneous cytoplasm, an intact oolemma and an intact zona pellucida. However, immature oocytes indicated numerous large mitochondria-vesicle complexes (M-VC). Ultrastructural changes of M-VC in IVM groups emphasize the need for further research in order to refine culture conditions and improve the implantation rate of in-vitro matured oocytes.

  2. An improved IVM method for cumulus-oocyte complexes from small follicles in polycystic ovary syndrome patients enhances oocyte competence and embryo yield.

    PubMed

    Sánchez, F; Lolicato, F; Romero, S; De Vos, M; Van Ranst, H; Verheyen, G; Anckaert, E; Smitz, J E J

    2017-10-01

    clinical protocol (Group I, n = 15). A second prospective study was performed in 15 women with polycystic ovaries, to characterize and optimize the PMC conditions (Group II, n = 15). The latter study involved the evaluation of oocyte meiotic arrest, the preservation of cumulus-oocyte transzonal projections (TZPs), the patterns of oocyte chromatin configuration and cumulus cells apoptosis following the 24 and 46 h PMC. Furthermore, oocyte developmental potential following PMC (24 and 46 h) + IVM was also evaluated. The first 20 good-quality blastocysts from PMC followed by IVM were analysed by next generation sequencing to evaluate their aneuploidy rate. PMC in presence of CNP followed by IVM using FSH and AREG increased the meiotic maturation rate per COC to 70%, which is significantly higher than routine standard IVM (49%; P ≤ 0.001). Hence, with the new system the proportion of COCs yielding transferable Day 3 embryos and good-quality blastocysts increased compared to routine standard IVM (from 23 to 43%; P ≤ 0.001 and from 8 to 18%; P ≤ 0.01, respectively). CNP was able to prevent meiosis resumption for up to 46 h. After PMC, COCs had preserved cumulus-oocyte TZPs. The blastocysts obtained after PMC + IVM did not show increased aneuploidy rates as compared to blastocysts from conventional ART. The novel IVM approach in PCOS patients was tested in oocytes derived from small antral follicles which have an intrinsically low developmental potential. Validation of the system would be required for COCs from different (larger) follicular sizes, which may involve further adjustment of PMC conditions. Furthermore, considering that this is a novel strategy in human IVM treatment, its global efficiency needs to be confirmed in large prospective randomized controlled trials. The further application in infertile patients without PCOS, e.g. cancer patients, remains to be evaluated. The findings of this pilot study suggest that the efficiency gap between IVM and conventional IVF

  3. Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein

    PubMed Central

    1992-01-01

    Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force. PMID:1740471

  4. Oocyte polarized light microscopy, assay of specific follicular fluid metabolites, and gene expression in cumulus cells as different approaches to predict fertilization efficiency after ICSI.

    PubMed

    Revelli, Alberto; Canosa, Stefano; Bergandi, Loredana; Skorokhod, Oleksii A; Biasoni, Valentina; Carosso, Andrea; Bertagna, Angela; Maule, Milena; Aldieri, Elisabetta; D'Eufemia, Maria Diletta; Evangelista, Francesca; Colacurci, Nicola; Benedetto, Chiara

    2017-06-23

    The complex relationship between oocyte morphology, specific follicular fluid metabolites, gene expression in cumulus granulosa cells, and oocyte competence toward fertilization and embryo development still needs further clarification. Forty-six oocytes retrieved from the largest pre-ovulatory follicle of patients undergoing intra-cytoplasmic sperm injection (ICSI) were considered assessing: (a) oocyte morphological characteristics at polarized light microscopy (PLM), (b) specific follicular fluid (FF) metabolites previously suggested to influence oocyte competence (AMH, markers of redox status and of cytotoxicity), (c) transcription of AMH and AMH type II receptor genes in cumulus cells. Data were analyzed using mono-parametric tests and multivariable logistic analysis in order to correlate morphological and biochemical data with fertilization. Comparing normally fertilized oocytes (n = 29, F group) with unfertilized (n = 17, nF group) we observed that: (a) the meiotic spindle area and major axis were significantly higher in nF group and in fertilized oocytes undergoing an early embryo development arrest; (b) AMH level in FF was comparable in F and nF groups; (c) the FF of nF group contained significantly higher levels of cytotoxicity (lactate dehydrogenase) and oxidative stress (Cu,Zn-superoxide dismutase, catalase, 4-hydroxynonenal-protein conjugates) markers; (d) cumulus cells of nF group showed significantly higher AMH receptor type II gene expression. Taken together, these observations suggest that an excessive cytotoxicity level can alter AMH signal transduction within cumulus cells, in turn leading to partial inhibition of aromatase activity, altered cytoplasmic maturation and increased oxidative stress, factors able to impair oocyte fertilization competence and embryo growth.

  5. A requirement for protein phosphorylation in regulating the meiotic and mitotic cell cycles in echinoderms.

    PubMed

    Néant, I; Charbonneau, M; Guerrier, P

    1989-04-01

    Populations of hormone-stimulated starfish oocytes and fertilized sea urchin eggs undergo synchronous meiotic and mitotic divisions. We have studied the requirement for protein phosphorylation during these events by testing the effects of 6-dimethylaminopurine (6-DMAP) upon the incorporation of [32P]orthophosphate. It was found that 6-DMAP blocked meiosis reinitiation and early cleavage and simultaneously inhibited protein phosphorylation, without changing the rate of [35S]methionine incorporation or pattern of protein synthesis. The protein, cyclin (54 kDa in starfish and 57 kDa in sea urchin), continues to be synthesized in the presence of 6-DMAP. This protein is destroyed at first and second cell cycles when 6-DMAP is added 30 min following fertilization but not when this drug is present before fertilization. Thus, cyclin breakdown does not depend on the completion of the nuclear events of M-phase, and its time of breakdown is set at an early step between fertilization and first cleavage. Using tubulin immunostaining, we found that 6-DMAP did not affect the cortical microtubules and resting female centrioles of prophase-arrested starfish oocytes, whereas it induced a precocious disappearance of spindle fibers when applied to hormone-stimulated oocytes. While an early addition of 6-DMAP precluded nuclear breakdown and spindle formation in both systems, a late treatment always allowed chromosome separation and centriole separation. Under these conditions pericentriolar tubulin persisted and could organize new spindles after the inhibitor was removed. It is suggested that (1) the assembly of cortical and centriolar-associated microtubules is not controlled by the same factors as spindle-associated tubulin; (2) specific proteins which are required for the cell to enter the following M-phase can become operative only via a process depending upon protein phosphorylation; (3) microtubule-associated kinases may play an important role in MPF function and spindle dynamics.

  6. Apoptosis Maintains Oocyte Quality in Aging Caenorhabditis elegans Females

    PubMed Central

    Andux, Sara; Ellis, Ronald E.

    2008-01-01

    In women, oocytes arrest development at the end of prophase of meiosis I and remain quiescent for years. Over time, the quality and quantity of these oocytes decreases, resulting in fewer pregnancies and an increased occurrence of birth defects. We used the nematode Caenorhabditis elegans to study how oocyte quality is regulated during aging. To assay quality, we determine the fraction of oocytes that produce viable eggs after fertilization. Our results show that oocyte quality declines in aging nematodes, as in humans. This decline affects oocytes arrested in late prophase, waiting for a signal to mature, and also oocytes that develop later in life. Furthermore, mutations that block all cell deaths result in a severe, early decline in oocyte quality, and this effect increases with age. However, mutations that block only somatic cell deaths or DNA-damage–induced deaths do not lower oocyte quality. Two lines of evidence imply that most developmentally programmed germ cell deaths promote the proper allocation of resources among oocytes, rather than eliminate oocytes with damaged chromosomes. First, oocyte quality is lowered by mutations that do not prevent germ cell deaths but do block the engulfment and recycling of cell corpses. Second, the decrease in quality caused by apoptosis mutants is mirrored by a decrease in the size of many mature oocytes. We conclude that competition for resources is a serious problem in aging germ lines, and that apoptosis helps alleviate this problem. PMID:19057674

  7. The mouse Spo11 gene is required for meiotic chromosome synapsis.

    PubMed

    Romanienko, P J; Camerini-Otero, R D

    2000-11-01

    The Spo11 protein initiates meiotic recombination by generating DNA double-strand breaks (DSBs) and is required for meiotic synapsis in S. cerevisiae. Surprisingly, Spo11 homologs are dispensable for synapsis in C. elegans and Drosophila yet required for meiotic recombination. Disruption of mouse Spo11 results in infertility. Spermatocytes arrest prior to pachytene with little or no synapsis and undergo apoptosis. We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis. Spo11 localizes to discrete foci during leptotene and to homologously synapsed chromosomes. Other mouse mutants that arrest during meiotic prophase (Atm -/-, Dmc1 -/-, mei1, and Morc(-/-)) showed altered Spo11 protein localization and expression. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis.

  8. Features of Programmed Cell death in Intact Xenopus Oocytes and Early Embryos Revealed by Near-Infrared Fluorescence and Real-time Monitoring

    PubMed Central

    Johnson, Carrie E.; Freel, Christopher D.; Kornbluth, Sally

    2009-01-01

    Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. Described here is a novel method for monitoring caspase activity in living Xenopus oocytes and early embryos. The approach, utilizing microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we demonstrate that brief caspase activity (ten minutes) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggests that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators. PMID:19730443

  9. Oocyte Activation and Fertilisation: Crucial Contributors from the Sperm and Oocyte.

    PubMed

    Yeste, Marc; Jones, Celine; Amdani, Siti Nornadhirah; Coward, Kevin

    2017-01-01

    This chapter intends to summarise the importance of sperm- and oocyte-derived factors in the processes of sperm-oocyte binding and oocyte activation. First, we describe the initial interaction between sperm and the zona pellucida, with particular regard to acrosome exocytosis. We then describe how sperm and oocyte membranes fuse, with special reference to the discovery of the sperm protein IZUMO1 and its interaction with the oocyte membrane receptor JUNO. We then focus specifically upon oocyte activation, the fundamental process by which the oocyte is alleviated from metaphase II arrest by a sperm-soluble factor. The identity of this sperm factor has been the source of much debate recently, although mounting evidence, from several different laboratories, provides strong support for phospholipase C ζ (PLCζ), a sperm-specific phospholipase. Herein, we discuss the evidence in support of PLCζ and evaluate the potential role of other candidate proteins, such as post-acrosomal WW-binding domain protein (PAWP/WBP2NL). Since the cascade of downstream events triggered by the sperm-borne oocyte activation factor heavily relies upon specialised cellular machinery within the oocyte, we also discuss the critical role of oocyte-borne factors, such as the inositol trisphosphate receptor (IP3R), protein kinase C (PKC), store-operated calcium entry (SOCE) and calcium/calmodulin-dependent protein kinase II (CaMKII), during the process of oocyte activation. In order to place the implications of these various factors and processes into a clinical context, we proceed to describe their potential association with oocyte activation failure and discuss how clinical techniques such as the in vitro maturation of oocytes may affect oocyte activation ability. Finally, we contemplate the role of artificial oocyte activating agents in the clinical rescue of oocyte activation deficiency and discuss options for more endogenous alternatives.

  10. Tristetraprolin functions in cytoskeletal organization during mouse oocyte maturation

    PubMed Central

    Liu, Xiaohui; Li, Xiaoyan; Ma, Rujun; Xiong, Bo; Sun, Shao-Chen; Liu, Honglin; Gu, Ling

    2016-01-01

    Tristetraprolin (TTP), a member of TIS11 family containing CCCH tandem zinc finger, is one of the best characterized RNA-binding proteins. However, to date, the role of TTP in mammalian oocytes remains completely unknown. In the present study, we report the altered maturational progression and cytokinesis, upon specific knockdown of TTP in mouse oocytes. Furthermore, by confocal scanning, we observe the failure to form cortical actin cap during meiosis of TTP-depleted oocytes. Loss of TTP in oocytes also results in disruption of meiotic spindle morphology and chromosome alignment. In support of these findings, incidence of aneuploidy is accordingly increased when TTP is abated in oocytes. Our results suggest that TTP as a novel cytoskeletal regulator is required for spindle morphology/chromosome alignment and actin polymerization in oocytes. PMID:27458159

  11. Evolution of the meiotic prophase and of the chromosome pairing process during human fetal ovarian development.

    PubMed

    Roig, I; Robles, P; Garcia, R; Martin, M; Egozcue, J; Cabero, Ll; Barambio, S; Garcia, M

    2005-09-01

    Studies on human oocytes in prophase I are limited due to the difficulty in obtaining the sample. However, a complete study of meiotic prophase evolution and the homologue pairing process is necessary to try to understand the implication of oogenesis in the origin of human aneuploidy. A complete analysis of meiotic prophase progression comprising the long developmental time period during which meiotic prophase takes place, based on the analysis of a total of 8603 oocytes in prophase I from 15 different cases is presented. The pairing process of chromosomes 13 and 18 is also described. The findings significantly relate for the first time the evolution of meiotic prophase to fetal development. Although for both chromosomes 13 and 18 a high pairing efficiency is found, pairing failure at the pachytene stage has been observed in 0.1% of oocytes. However, errors at the diplotene stage are substantially increased, suggesting that complete, premature disjunction of the homologues commonly occurs. Moreover, pre-meiotic errors are also described. Our findings show that homologous chromosomes pair very efficiently, but the high frequency of complete, premature homologue separation found at diplotene suggests that mechanisms other than the pairing process could be more likely to lead to the high aneuploidy rate observed in human oocytes.

  12. Bub3 is a spindle assembly checkpoint protein regulating chromosome segregation during mouse oocyte meiosis.

    PubMed

    Li, Mo; Li, Sen; Yuan, Ju; Wang, Zhen-Bo; Sun, Shao-Chen; Schatten, Heide; Sun, Qing-Yuan

    2009-11-02

    In mitosis, the spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes have been attached to the spindle microtubules and aligned correctly at the equatorial metaphase plate. The major checkpoint proteins in mitosis consist of mitotic arrest-deficient (Mad)1-3, budding uninhibited by benzimidazole (Bub)1, Bub3, and monopolar spindle 1(Mps1). During meiosis, for the formation of a haploid gamete, two consecutive rounds of chromosome segregation occur with only one round of DNA replication. To pull homologous chromosomes to opposite spindle poles during meiosis I, both sister kinetochores of a homologue must face toward the same pole which is very different from mitosis and meiosis II. As a core member of checkpoint proteins, the individual role of Bub3 in mammalian oocyte meiosis is unclear. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of Bub3 in mouse oocyte meiosis. Our data showed that overexpressed Bub3 inhibited meiotic metaphase-anaphase transition by preventing homologous chromosome and sister chromatid segregations in meiosis I and II, respectively. Misaligned chromosomes, abnormal polar body and double polar bodies were observed in Bub3 knock-down oocytes, causing aneuploidy. Furthermore, through cold treatment combined with Bub3 overexpression, we found that overexpressed Bub3 affected the attachments of microtubules and kinetochores during metaphase-anaphase transition. We propose that as a member of SAC, Bub3 is required for regulation of both meiosis I and II, and is potentially involved in kinetochore-microtubule attachment in mammalian oocytes.

  13. 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.

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

    PubMed

    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.

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

    PubMed

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

    2016-11-01

    The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP - inhibiting the hydrolysis of intra-oocyte cAMP - and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options. © 2016 Society for Reproduction and Fertility.

  16. Cellular, biochemical and molecular mechanisms regulating oocyte maturation.

    PubMed

    Dekel, Nava

    2005-04-29

    The original model for regulation of oocyte maturation proposed by us in 1978 postulated that gap junction-mediated transmission of cAMP from the follicle cells to the oocyte inhibits meiosis and that luteinizing hormone (LH) terminates the flux of the follicle cAMP to the oocyte. A decrease in oocyte cAMP below inhibitory threshold occurs since oocytes lack the ability to generate sufficient amounts of cAMP to compensate for the phosphodiesterase activity. Our previous studies provided evidence to support this model. More recent studies in our laboratory were directed at identification of the cellular biochemical and molecular events initiated within rat oocytes upon the relief of cAMP inhibition. These studies: (i) identified an oocyte specific A kinase anchoring protein (AKAP) that is phosphorylated in oocytes resuming meiosis, (ii) confirmed that cdc25B governs meiosis reinitiation and demonstrated that its expression is translationally regulated, (iii) substantiated the indispensable role of proteasomal degradation at completion of the first meiotic division in a mammalian system, (iv) elucidated the role of MPF reactivation in suppressing interphase between the two meiotic divisions and (v) provided evidence that mos translation is negatively regulated by a protein kinase A (PKA)-mediated action of cAMP and is dependent on an active MPF. A detailed account on each of these findings is presented in this chapter.

  17. A Role for Retrotransposon LINE-1 in Fetal Oocyte Attrition in Mice

    PubMed Central

    Malki, Safia; van der Heijden, Godfried W.; O'Donnell, Kathryn A.; Martin, Sandra L.; Bortvin, Alex

    2014-01-01

    SUMMARY Fetal oocyte attrition (FOA) is a conserved but poorly understood process of elimination of over two-thirds of meiotic prophase I (MPI) oocytes before birth. We now implicate retrotransposons LINE-1 (L1), activated during epigenetic reprogramming of the embryonic germline, in FOA in mice. We show that wild-type fetal oocytes possess differential nuclear levels of L1ORF1p, a L1-encoded protein essential for L1 ribonucleoprotein particle (L1RNP) formation and L1 retrotransposition. We demonstrate that experimental elevation of L1 expression correlates with increased MPI defects, FOA, oocyte aneuploidy and embryonic lethality. Conversely, reverse transcriptase (RT) inhibitor AZT has a profound effect on the FOA dynamics and meiotic recombination, and implicates an RT-dependent trigger in oocyte elimination in early MPI. We propose that FOA serves to select oocytes with limited L1 activity and therefore best suited for the next generation. PMID:24882376

  18. X chromosome effect on maternal recombination and meiotic drive in the mouse.

    PubMed Central

    de La Casa-Esperón, Elena; Loredo-Osti, J Concepción; Pardo-Manuel de Villena, Fernando; Briscoe, Tammi L; Malette, Jan Michel; Vaughan, Joe E; Morgan, Kenneth; Sapienza, Carmen

    2002-01-01

    We observed that maternal meiotic drive favoring the inheritance of DDK alleles at the Om locus on mouse chromosome 11 was correlated with the X chromosome inactivation phenotype of (C57BL/6-Pgk1(a) x DDK)F(1) mothers. The basis for this unexpected observation appears to lie in the well-documented effect of recombination on meiotic drive that results from nonrandom segregation of chromosomes. Our analysis of genome-wide levels of meiotic recombination in females that vary in their X-inactivation phenotype indicates that an allelic difference at an X-linked locus is responsible for modulating levels of recombination in oocytes. PMID:12196408

  19. Improvements in oocyte competence in superovulated mice following treatment with cilostazol: Ovulation of immature oocytes with high developmental rates.

    PubMed

    Taiyeb, Ahmed M; Muhsen-Alanssari, Saeeda A; Dees, William L; Hiney, Jill; Kjelland, Michael E; Kraemer, Duane C; Ridha-Albarzanchi, Mundhir T

    2017-08-01

    Exogenous administration of superovulatory hormones negatively affects oocyte competence in mammals. Phosphodiesterase 3A inhibitors were found to improve competence of oocytes matured in vitro in several species, including humans. This study was therefore designed to define oocyte maturation synchronization and competence, in vivo, using superovulated mice treated with cilostazol, a selective phosphodiesterase 3A inhibitor. Swiss Webster mice were superovulated and treated orally with 7.5mg cilostazol once or twice to result in ovulation of immature oocytes at the metaphase I (MI) or germinal vesicle (GV) stage, respectively. Control immature oocytes were recovered from preovulatory follicles of superovulated mice not treated with cilostazol. Treated GV oocytes had significantly higher rates of synchronized and advanced chromatin configuration and cortical granule distribution than did control GV oocytes. Treated GV oocytes had a moderate increase in cAMP levels and consequently higher rates of meiotic maturation, IVF, and blastocyst formation than did control GV oocytes (P<0.0001). Treated MI oocytes had higher rates of normal spindles and chromosomes aligned at the metaphase plate than did control MI oocytes (P<0.003). Treated mice ovulating MI oocytes produced litter sizes larger than those observed in control mice ovulating mature oocytes (P<0.002). This study reveals that synchronization of oocyte maturation in superovulated mice improves oocyte development and competence. The capability of cilostazol, a clinically approved medication, to improve mouse oocyte competence suggests the potential benefit of including this compound in ovarian hyperstimulation programs to improve in vitro fertilization outcomes in infertile women. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Coordinate developmental control of the meiotic cell cycle and spermatid differentiation in Drosophila males.

    PubMed

    Lin, T Y; Viswanathan, S; Wood, C; Wilson, P G; Wolf, N; Fuller, M T

    1996-04-01

    Wild-type function of four Drosophila genes, spermatocyte arrest, cannonball, always early and meiosis I arrest, is required both for cell-cycle progression through the G2/M transition of meiosis I in males and for onset of spermatid differentiation. In males mutant for any one of these meiotic arrest genes, mature primary spermatocytes with partially condensed chromosomes accumulate and postmeiotic cells are lacking. The arrest in cell-cycle progression occurs prior to degradation of cyclin A protein. The block in spermatogenesis in these mutants is not simply a secondary consequence of meiotic cell-cycle arrest, as spermatid differentiation proceeds in males mutant for the cell cycle activating phosphatase twine. Instead, the arrest of both meiosis and spermiogenesis suggests a control point that may serve to coordinate the male meiotic cell cycle with the spermatid differentiation program. The phenotype of the Drosophila meiotic arrest mutants is strikingly similar to the histopathological features of meiosis I maturation arrest infertility in human males, suggesting that the control point may be conserved from flies to man.

  1. Regulation of Intracellular pH by p90Rsk-dependent Activation of an Na+/H+ Exchanger in Starfish Oocytes*

    PubMed Central

    Harada, Kaori; Fukuda, Eriko; Hirohashi, Noritaka; Chiba, Kazuyoshi

    2010-01-01

    Starfish oocytes arrest at metaphase of the first meiotic division (MI arrest) in the ovary and resume meiosis after spawning into seawater. MI arrest is maintained by lower intracellular pH (pHi) and release from arrest by cellular alkalization. To elucidate pHi regulation in oocytes, we cloned the starfish (Asterina pectinifera) Na+/H+ exchanger 3 (ApNHE3) expressed in the plasma membrane of oocytes. The cytoplasmic domain of ApNHE3 contains p90 ribosomal S6 kinase (p90Rsk) phosphorylation sites, and injection of a constitutively active p90Rsk and the upstream regulator Mos to immature oocytes, stimulated an increase in pHi. This increase was blocked by 5-(N-ethyl-N-isopropyl)-amiloride, a NHE inhibitor, and SL0101, a specific Rsk inhibitor. The MAPK kinase (MEK) inhibitor U0126 blocked the Mos-induced, but not the p90Rsk-induced, pHi increase, suggesting that the Mos-MEK-MAPK-p90Rsk pathway promotes ApNHE3 activation. In a cell-free extract, the Mos-MEK-MAPK-p90Rsk pathway phosphorylates ApNHE3 at Ser-590, -606, and -673. When p90Rsk-dependent ApNHE3 phosphorylation was blocked by a dominant-negative C-terminal fragment, or neutralizing antibody, the p90Rsk-induced pHi increase was suppressed in immature oocytes. However, ApNHE3 is up-regulated via the upstream phosphatidylinositol 3-kinase pathway before MAPK activation and the active state is maintained until spawning, suggesting that the p90Rsk-dependent ApNHE3 phosphorylation is unlikely to be the primary regulatory mechanism involved in MI arrest exit. After meiosis is completed, unfertilized eggs maintain their elevated pHi (∼7.4) until the onset of apoptosis. We suggest that the p90Rsk/ApNHE3-dependent elevation of pHi increases fertilization success by delaying apoptosis initiation. PMID:20507995

  2. HORMAD1-dependent checkpoint/surveillance mechanism eliminates asynaptic oocytes.

    PubMed

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

    2012-06-01

    Meiotic pachytene checkpoints monitor the failure of homologous recombination and synapsis to ensure faithful chromosome segregation during gamete formation. To date, the molecular basis of the mammalian pachytene checkpoints has remained largely unknown. We here report that mouse HORMAD1 is required for a meiotic prophase checkpoint that eliminates asynaptic oocytes. Hormad1-deficient mice are infertile and show an extensive failure of homologous pairing and synapsis, consistent with the evolutionarily conserved function of meiotic HORMA domain proteins. Unexpectedly, Hormad1-deficient ovaries contain a normal number of oocytes despite asynapsis and consequently produce aneuploid oocytes, indicating a checkpoint failure. By the analysis of Hormad1/Spo11 double mutants, the Hormad1 deficiency was found to abrogate the massive oocyte loss in the Spo11-deficient ovary. The Hormad1 deficiency also causes the eventual loss of pseudo sex body in the Spo11-deficient ovary and testis. These results suggest the involvement of HORMAD1 in the repressive chromatin domain formation that is proposed to be important in the meiotic prophase checkpoints. We also show the extensive phosphorylation of HORMAD1 in the Spo11-deficient testis and ovary, suggesting an involvement of novel DNA damage-independent phosphorylation signaling in the surveillance mechanism. Our present results provide clues to HORMAD1-dependent checkpoint in response to asynapsis in mammalian meiosis.

  3. Bisphenol A and Related Alkylphenols Exert Nongenomic Estrogenic Actions Through a G Protein-Coupled Estrogen Receptor 1 (Gper)/Epidermal Growth Factor Receptor (Egfr) Pathway to Inhibit Meiotic Maturation of Zebrafish Oocytes1

    PubMed Central

    Fitzgerald, Amanda C.; Peyton, Candace; Dong, Jing; Thomas, Peter

    2015-01-01

    Xenobiotic estrogens, such as bisphenol A (BPA), disrupt a wide variety of genomic estrogen actions, but their nongenomic estrogen actions remain poorly understood. We investigated nongenomic estrogenic effects of low concentrations of BPA and three related alkylphenols on the inhibition of zebrafish oocye maturation (OM) mediated through a G protein-coupled estrogen receptor 1 (Gper)-dependent epidermal growth factor receptor (Egfr) pathway. BPA (10–100 nM) treatment for 3 h mimicked the effects of estradiol-17beta (E2) and EGF, decreasing spontaneous maturation of defolliculated zebrafish oocytes, an effect not blocked by coincubation with actinomycin D, but blocked by coincubation with a Gper antibody. BPA displayed relatively high binding affinity (15.8% that of E2) for recombinant zebrafish Gper. The inhibitory effects of BPA were attenuated by inhibition of upstream regulators of Egfr, intracellular tyrosine kinase (Src) with PP2, and matrix metalloproteinase with ilomastat. Treatment with an inhibitor of Egfr transactivation, AG1478, and an inhibitor of the mitogen-activated protein kinase (MAPK) 3/1 pathway, U0126, increased spontaneous OM and blocked the inhibitory effects of BPA, E2, and the selective GPER agonist, G-1. Western blot analysis showed that BPA (10–200 nM) mimicked the stimulatory effects of E2 and EGF on Mapk3/1 phosphorylation. Tetrabromobisphenol A, 4-nonylphenol, and tetrachlorobisphenol A (5–100 nM) also inhibited OM, an effect blocked by cotreatment with AG1478, as well as with the GPER antagonist, G-15, and displayed similar binding affinities as BPA to zebrafish Gper. The results suggest that BPA and related alkylphenols disrupt zebrafish OM by a novel nongenomic estrogenic mechanism involving activation of the Gper/Egfr/Mapk3/1 pathway. PMID:26490843

  4. Fyn Kinase Activity Is Required for Normal Organization and Functional Polarity of the Mouse Oocyte Cortex

    PubMed Central

    Luo, Jinping; Mcginnis, Lynda K.; Kinsey, William H.

    2014-01-01

    Summary The objective of the present study was to determine whether Fyn kinase participated in signaling events during sperm–egg interactions, sperm incorporation, and meiosis II. The functional requirement of Fyn kinase activity in these events was tested through the use of the protein kinase inhibitor SKI-606 (Bosutinib) and by analysis of Fyn-null oocytes. Suppression of Fyn kinase signaling prior to fertilization caused disruption of the functional polarity of the oocyte with the result that sperm were able to fuse with the oocyte in the immediate vicinity of the meiotic spindle, a region that normally does not allow sperm fusion. The loss of functional polarity was accompanied by disruption of the microvilli and cortical granule-free zone that normally overlie the meiotic spindle. Changes in the distribution of cortical granules and filamentous actin provided further evidence of disorganization of the oocyte cortex. Rho B, a molecular marker for oocyte polarity, was unaffected by suppression of Fyn activity; however, the polarized association of Par-3 with the cortex overlying the meiotic spindle was completely disrupted. The defects in oocyte polarity in Fyn-null oocytes correlated with a failure of the MII chromosomes to maintain a position close to the oocyte cortex which seemed to underlie the above defects in oocyte polarity. This was associated with a delay in completion of meiosis II, however, pronuclei eventually formed and subsequent mitotic cleavages and blastocyst formation occurred normally. PMID:19363790

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

    PubMed Central

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

    2015-01-01

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

  6. The catecholestrogen, 2-hydroxyestradiol-17beta, acts as a G protein-coupled estrogen receptor 1 (GPER/GPR30) antagonist to promote the resumption of meiosis in zebrafish oocytes.

    PubMed

    Chourasia, Tapan K; Pang, Yefei; Thomas, Peter

    2015-03-01

    Estradiol-17beta (E2) maintains high cAMP levels and meiotic arrest in zebrafish oocytes through activation of G protein-coupled estrogen receptor (GPER). The catecholestrogen 2-hydroxyestradiol-17beta (2-OHE2) has an opposite effect to that of E2 on oocyte maturation (OM) and cAMP levels in Indian catfish oocytes. We tested the hypothesis that 2-OHE2 is produced in zebrafish ovaries and promotes the resumption of oocyte meiosis through its action as a GPER antagonist. Ovarian 2-OHE2 production by estrogen-2-hydroxylase (EH) was up-regulated by gonadotropin treatment at the onset of OM, consistent with a physiological role for 2-OHE2 in regulating OM. The increases in EH activity and OM were blocked by treatment with CYP1A1 and CYP1B1 inhibitors. Expression of cyp1a, cyp1b1, and cyp1c mRNAs was increased by gonadotropin treatment, further implicating these Cyp1s in 2-OHE2 synthesis prior to OM. Conversely, aromatase activity and cyp19a1 mRNA expression declined during gonadotropin induction of OM. 2-OHE2 treatment significantly increased spontaneous OM in defolliculated zebrafish oocytes and reversed the inhibition of OM by E2 and the GPER agonist G-1. 2-OHE2 was an effective competitor of [(3)H]-E2 binding to recombinant zebrafish GPER expressed in HEK-293 cells. 2-OHE2 also antagonized estrogen actions through GPER on cAMP production in zebrafish oocytes, resulting in a reduction in cAMP levels. Stimulation of OM by 2-OHE2 was blocked by pretreatment of defolliculated oocytes with the GPER antibody. Collectively, the results suggest that 2-OHE2 functions as a GPER antagonist and promotes OM in zebrafish through blocking GPER-dependent E2 inhibition of the resumption of OM. © 2015 by the Society for the Study of Reproduction, Inc.

  7. FANCM limits meiotic crossovers.

    PubMed

    Crismani, Wayne; Girard, Chloé; Froger, Nicole; Pradillo, Mónica; Santos, Juan Luis; Chelysheva, Liudmila; Copenhaver, Gregory P; Horlow, Christine; Mercier, Raphaël

    2012-06-22

    The number of meiotic crossovers (COs) is tightly regulated within a narrow range, despite a large excess of molecular precursors. The factors that limit COs remain largely unknown. Here, using a genetic screen in Arabidopsis thaliana, we identified the highly conserved FANCM helicase, which is required for genome stability in humans and yeasts, as a major factor limiting meiotic CO formation. The fancm mutant has a threefold-increased CO frequency as compared to the wild type. These extra COs arise not from the pathway that accounts for most of the COs in wild type, but from an alternate, normally minor pathway. Thus, FANCM is a key factor imposing an upper limit on the number of meiotic COs, and its manipulation holds much promise for plant breeding.

  8. Meiotic sex chromosome inactivation.

    PubMed

    Turner, James M A

    2007-05-01

    X chromosome inactivation is most commonly studied in the context of female mammalian development, where it performs an essential role in dosage compensation. However, another form of X-inactivation takes place in the male, during spermatogenesis, as germ cells enter meiosis. This second form of X-inactivation, called meiotic sex chromosome inactivation (MSCI) has emerged as a novel paradigm for studying the epigenetic regulation of gene expression. New studies have revealed that MSCI is a special example of a more general mechanism called meiotic silencing of unsynapsed chromatin (MSUC), which silences chromosomes that fail to pair with their homologous partners and, in doing so, may protect against aneuploidy in subsequent generations. Furthermore, failure in MSCI is emerging as an important etiological factor in meiotic sterility.

  9. Meiotic chromosome abnormalities in human spermatogenesis.

    PubMed

    Martin, Renée H

    2006-08-01

    The last few years have witnessed an explosion in the information about chromosome abnormalities in human sperm and the meiotic events that predispose to these abnormalities. We have determined that all chromosomes are susceptible to nondisjunction, but chromosomes 21 and 22 and, especially, the sex chromosomes have an increased frequency of aneuploidy. Studies are just beginning on the effects of potential mutagens on the chromosomal constitution of human sperm. The effects of pesticides and cancer therapeutic agents have been reviewed. In the last decade, there has been a great impetus to study chromosome abnormalities in sperm from infertile men because the advent of intracytoplasmic sperm injection (ICSI) made it possible for these men to father pregnancies. A large number of studies have demonstrated that infertile men have an increased frequency of chromosomally abnormal sperm and children, even when they have a normal somatic karyotype. Meiotic studies on the pachytene stage of spermatogenesis have demonstrated that infertile men have impaired chromosome synapsis, a significantly decreased frequency of recombination, and an increased frequency of chromosomes completely lacking a recombination site. Such errors make these cells susceptible to meiotic arrest and the production of aneuploid gametes.

  10. Supplementation of l-carnitine during in vitro maturation improves embryo development from less competent bovine oocytes.

    PubMed

    Knitlova, Drahomira; Hulinska, Pavlina; Jeseta, Michal; Hanzalova, Katerina; Kempisty, Bartosz; Machatkova, Marie

    2017-10-15

    The present study was designed to define the impact of l-carnitine, supplemented during maturation, on bovine oocytes with different meiotic competence in terms of their IVF outcomes. Meiotically more competent (MMC) and less competent (MLC) oocytes were obtained separately from differently sized follicles at selected phases of folliculogenesis. The oocytes were matured with or without l-carnitine, fertilized and cultured to the blastocyst stage. The oocytes were examined for nuclear maturation, mitochondrial cluster formation, lipid consumption, fertilization and embryo development. The proportion of oocytes at metaphase II was significantly higher in the l-carnitine-treated MMC than that in the l-carnitine-treated MLC oocytes. However in comparison with the untreated controls, the proportion of MII oocytes with mitochondrial clusters was significantly higher only in the l-carnitine-treated MLC oocytes, which also showed a significantly lower mean lipid content. The l-carnitine-treated MLC oocytes showed significantly higher fertilization and syngamy rates than the untreated MLC oocytes. On the other hand, in the l-carnitine-treated MMC oocytes, the fertilization rate was similar to that of the untreated controls and the syngamy rate was significantly delayed. Although no significant differences in cleavage on Day 2 were found among all oocyte categories, l-carnitine treatment resulted in a significantly higher blastocyst yield in the MLC oocytes on Day 7 and Day 8 and a significantly higher proportion of expanded blastocysts in relation to the total number of blastocysts in MMC oocytes on Day 8. It can be concluded that l-carnitine supplementation during maturation improves the development of bovine embryos from meiotically less competent oocytes and accelerates blastocyst formation from more competent oocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2014-01-01

    In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte. PMID:25183874

  12. Meiotic silencing and fragmentation of the male germline restricted chromosome in zebra finch.

    PubMed

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

    2010-06-01

    During male meiotic prophase in mammals, X and Y are in a largely unsynapsed configuration, which is thought to trigger meiotic sex chromosome inactivation (MSCI). In avian species, females are ZW, and males ZZ. Although Z and W in chicken oocytes show complete, largely heterologous synapsis, they too undergo MSCI, albeit only transiently. The W chromosome is already inactive in early meiotic prophase, and inactive chromatin marks may spread on to the Z upon synapsis. Mammalian MSCI is considered as a specialised form of the general meiotic silencing mechanism, named meiotic silencing of unsynapsed chromatin (MSUC). Herein, we studied the avian form of MSUC, by analysing the behaviour of the peculiar germline restricted chromosome (GRC) that is present as a single copy in zebra finch spermatocytes. In the female germline, this chromosome is present in two copies, which normally synapse and recombine. In contrast, during male meiosis, the single GRC is always eliminated. We found that the GRC in the male germline is silenced from early leptotene onwards, similar to the W chromosome in avian oocytes. The GRC remains largely unsynapsed throughout meiotic prophase I, although patches of SYCP1 staining indicate that part of the GRC may self-synapse. In addition, the GRC is largely devoid of meiotic double strand breaks. We observed a lack of the inner centromere protein INCENP on the GRC and elimination of the GRC following metaphase I. Subsequently, the GRC forms a micronucleus in which the DNA is fragmented. We conclude that in contrast to MSUC in mammals, meiotic silencing of this single chromosome in the avian germline occurs prior to, and independent of DNA double strand breaks and chromosome pairing, hence we have named this phenomenon meiotic silencing prior to synapsis (MSPS).

  13. Noninvasive assays of in vitro matured human oocytes showed insignificant correlation with fertilization and embryo development.

    PubMed

    Ashourzadeh, Sareh; Khalili, Mohammad Ali; Omidi, Marjan; Mahani, Seyed Nooraldin Nematollahi; Kalantar, Seyed Mehdi; Aflatoonian, Abbas; Habibzadeh, Victoria

    2015-08-01

    Recently, the upgrading of in vitro maturation (IVM) of human oocytes as a promising strategy has emerged in assisted reproductive technology (ART). The goal was to evaluate the correlation of the in vitro matured oocytes selected on the basis of the zona pellucida (ZP) birefringence and meiotic spindles (MS) detection with fertilization and subsequent embryo development in ICSI program. A total of 168 immature oocytes [germinal vesicle (n = 140) and metaphase I (n = 28)] obtained from patients undergoing oocytes retrieval for ICSI. After in vitro culture for 24-40 h, 112 (67 %) oocytes reached to MII stage. Using a polarized microscopy, the presence of MS and ZP birefringence were assessed in matured oocytes, followed by ICSI performance. The rates of fertilization in oocytes with spindles (51.3 %) were similar to that of the oocytes without spindles (50.7 %; P = 1.00). Moreover, the fertilization rates in high birefringence (HB) oocytes was not statistically different than oocytes with low birefringence (LB) (P = 0.44). The findings also showed that 64.9 % of the fertilized oocytes developed to embryos, in which 33.3 % were derived from spindle-detected oocytes. Regarding the ZP birefringence, 35.5 % of the embryos were derived from HB oocytes. There were insignificant relationships between the MS detection and ZP birefringence score with the rates of fertilization and embryo development in IVM oocytes.

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

    PubMed

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

    2015-08-13

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

  15. 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

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

    PubMed

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

    2015-02-01

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

  17. Reversible meiotic abnormalities in azoospermic men with bilateral varicocele after microsurgical correction.

    PubMed

    North, M O; Lellei, I; Rives, N; Erdei, E; Dittmar, A; Barbet, J P; Tritto, G

    2004-05-01

    Because of a possible relationship between microenvironmental disturbances and meiotic abnormalities and of a straight relationship between lower-quality semen in patient carrying a varicocele and first meiotic non-disjunction, bilateral bipolar testicular biopsies are realized according the thermic differential gradient described in varicocele. Systematic meiotic studies of multiple testicular biopsies from 65 azoospermic men with bilateral varicocele were done in a multi-centric study on microsurgical correction of bilateral varicocele with microthermic intra-operative evaluation using minimally invasive thermal microsensors (Betatherm 10K3MCD2). In the present study abnormal temperature raising, histomorphometric abnormalities (spermatocyte arrest) and meiotic abnormalities (class IIC) are strongly correlated. In the ten patients submitted to another testicular biopsy procedure six months after surgery for TESE, normal thermal differential is registered and no meiotic abnormalities recurrences are found.

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

    PubMed Central

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

    2013-01-01

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

  19. 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

  20. Functions of the MRE11 complex in the development and maintenance of oocytes.

    PubMed

    Inagaki, Akiko; Roset, Ramon; Petrini, John H J

    2016-03-01

    The MRE11 complex (MRE11, RAD50, and NBS1) is a central component of the DNA damage response, governing both double-strand break repair and DNA damage response signaling. To determine the functions of the MRE11 complex in the development and maintenance of oocytes, we analyzed ovarian phenotypes of mice harboring the hypomorphic Mre11 (ATLD1) allele. Mre11 (ATLD1/ATLD1) females exhibited premature oocyte elimination attributable to defects in homologous chromosome pairing and double-strand break repair during meiotic prophase. Other aspects of meiotic progression, including attachment of telomeres to the nuclear envelope and recruitment of RAD21L, a component of the meiotic cohesin complex to the synaptonemal complex, were normal. Unlike Dmc1 (-/-) and Trp13 (Gt/Gt) mice which exhibit comparable defects in double-strand break repair and oocyte depletion by 5 days post-partum, we found that oocyte attrition occurred by 12 weeks in Mre11 (ATLD1/ATLD1) . Disruption of the oocyte checkpoint pathway governed by Chk2 gene further enhanced the survival of Mre11 (ATLD1/ATLD1) follicles. Together our data suggest that the MRE11 complex influences the elimination of oocytes with unrepaired meiotic double-strand breaks post-natally, in addition to its previously described role in double-strand break repair and homologous synapsis during female meiosis.

  1. The POU gene ceh-18 promotes gonadal sheath cell differentiation and function required for meiotic maturation and ovulation in Caenorhabditis elegans.

    PubMed

    Rose, K L; Winfrey, V P; Hoffman, L H; Hall, D H; Furuta, T; Greenstein, D

    1997-12-01

    In Caenorhabditis elegans, specialized contractile myoepithelial cells of the somatic gonad, the gonadal sheath cells, are closely apposed to oocytes and are required for normal meiotic maturation and ovulation. Previously we found that mutations in the ceh-18 gene, which encodes a POU-class homeoprotein expressed in sheath cells, result in oocyte defects. To determine the basis for these oocyte defects, we have used time-lapse video Nomarski microscopy to observe meiotic maturation, ovulation, and early embryogenesis in ceh-18 mutants. In ceh-18 mutants sheath cell contractions are weaker, less frequent, and uncoordinated throughout the sequence of ovulation events, and ovulation is defective. Defective ovulation can result in the formation of endomitotic oocytes in the gonad, the formation of haploid embryos, and reversals in embryonic polarity. ceh-18 mutant oocytes exhibit defects prior to nuclear envelope breakdown, suggesting that they are physiologically different from the wild type. We observed delays in meiotic maturation, as well as maturation out of the normal spatial and temporal sequence, suggesting that proximal sheath cells directly or indirectly promote and spatially restrict meiotic maturation. Analysis of sheath cell differentiation in ceh-18 mutants using antibodies to proteins of the contractile apparatus reveals that although contractile proteins are expressed, the sheath cells appear disorganized. Transmission electron microscopy reveals that ceh-18 mutant sheath cells are morphologically irregular and only loosely cover oocytes. Taken together, these observations indicate that ceh-18 is a crucial determinant of sheath cell differentiation, a function required for normal meiotic maturation and ovulation.

  2. Evaluation of equine oocyte developmental competence using polarized light microscopy.

    PubMed

    Bertero, A; Ritrovato, F; Evangelista, F; Stabile, V; Fortina, R; Ricci, A; Revelli, A; Vincenti, L; Nervo, T

    2017-06-01

    The purpose of this study was to observe in vitro-matured equine oocytes with an objective computerized technique that involves the use of a polarized light microscope (PLM) in addition to the subjective morphological evaluation obtained using a classic light microscope (LM). Equine cumulus-oocyte complexes (COCs, n = 922) were subjected to different in vitro maturation times (24, 36 or 45 h), however, only 36-h matured oocytes were analyzed using CLM. The 36-h matured oocytes that reached maturity were parthenogenetically activated to evaluate the quality and meiotic competence. Average maturation percentages per session in groups 1, 2 and 3 (24-, 36- and 45-h matured oocytes respectively) were 29.31 ± 13.85, 47.01 ± 9.90 and 36.62 ± 5.28%, whereas the average percentages of immature oocytes per session were 28.78 ± 20.17, 7.83 ± 5.51 and 22.36 ± 8.39% respectively. The zona pellucida (ZP) birefringent properties were estimated and correlated with activation outcome. ZP thickness and retardance of the inner layer of the zona pellucida (IL-ZP) were significantly increased in immature oocytes compared with mature oocytes (P < 0.001 and P < 0.01 respectively). The comparison between parthenogenetically activated and non-activated oocytes showed a significant increase in the area and thickness of the IL-ZP in parthenogenetically activated oocytes (P < 0.01). These results show that the 36-h in vitro maturation (IVM) protocol allowed equine oocytes to reach maturity, and PLM observation of ZP can be used to distinguish mature and immature oocytes as well as activated and non-activated oocytes. © 2017 Society for Reproduction and Fertility.

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

    PubMed

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

    2017-10-01

    Canine oocytes ovulated at prophase complete meiosis and continue to develop in presence of a high progesterone concentration in the oviduct. Considering that meiotic competence of canine oocyte is accomplished in the oviductal environment, we postulate that hormonal milieu resulting from the circulating progesterone concentration may affect oocyte maturation and early development of embryos. From 237 oocyte donors, 2620 oocytes were collected and their meiotic status and morphology were determined. To determine optimal characteristics of the mature oocytes subjected to nuclear transfer, a proportion of the meiotic status of the oocytes were classified in reference to time post-ovulation as well as progesterone (P4) level. A high proportion of matured oocytes were collected from >126h (55.5%) post-ovulation or 40-50ngmL(-1) (46.4%) group compared to the other groups. Of the oocyte donors that provided mature oocytes in vivo, there was no correlation between serum progesterone of donors and time post ovulation, however, time post-ovulation were significantly shorter for <30ng/mL group (P<0.05). Using mature oocytes, 1161 cloned embryos were reconstructed and transferred into 77 surrogates. In order to determine the relationship between pregnancy performance and serum progesterone level, embryos were transferred into surrogates showing various P4 serum levels. The highest pregnancy (31.8%) and live birth cloning efficacy (2.2%) rates were observed when the embryos were transferred into surrogates with circulating P4 levels were from 40 to 50ngmL(-1). In conclusion, measurement of circulating progesterone of female dog could be a suitable an indicator of the optimal time to collect quality oocyte and to select surrogates for cloning. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. MEK inhibitors block AICAR-induced maturation in mouse oocytes by a MAPK-independent mechanism.

    PubMed

    LaRosa, Cean; Downs, Stephen M

    2005-02-01

    The present study was carried out to assess the possible role of mitogen-activated protein kinase (MAPK) in the meiosis-inducing action of the AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Cumulus cell-enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured 4 hr in Eagle's minimum essential medium containing dbcAMP plus increasing concentrations of AICAR or okadaic acid (OA). OA is a phosphatase inhibitor known to stimulate both meiotic maturation and MAPK activation and served as a positive control. Both OA and AICAR were potent inducers of meiotic resumption in mouse oocytes and brought about the phosphorylation (and thus, activation) of MAPK, but by different kinetics: MAPK phosphorylation preceded GVB in OA-treated oocytes, while that resulting from AICAR treatment appeared only after GVB. The MEK inhibitors, PD98059 and U0126, blocked the meiotic resumption induced by AICAR but not that induced by OA. Although the MEK inhibitors suppressed MAPK phosphorylation in both OA- and AICAR-treated oocytes, meiotic resumption was not causally linked to MAPK phosphorylation in either group. Furthermore, AICAR-induced meiotic resumption in Mos-null oocytes (which are unable to stimulate MAPK) was also abrogated by PD98059 treatment. A non-specific effect of the MEK inhibitors on AICAR accessibility to the oocyte was discounted by showing that they failed to suppress either nucleoside uptake or AICAR-stimulated phosphorylation of acetyl CoA carboxylase (ACC), a substrate of AMPK. The suppression of AICAR-induced maturation by MEK inhibitors must, therefore, be occurring by actions unrelated to MEK stimulation of MAPK; consequently, it would be prudent to consider this possible non-specific action of the inhibitors when they are used to block MAPK activation in mouse oocytes.

  5. Tet1 controls meiosis by regulating meiotic gene expression.

    PubMed

    Yamaguchi, Shinpei; Hong, Kwonho; Liu, Rui; Shen, Li; Inoue, Azusa; Diep, Dinh; Zhang, Kun; Zhang, Yi

    2012-12-20

    Meiosis is a germ-cell-specific cell division process through which haploid gametes are produced for sexual reproduction. Before the initiation of meiosis, mouse primordial germ cells undergo a series of epigenetic reprogramming steps, including the global erasure of DNA methylation at the 5-position of cytosine (5mC) in CpG-rich DNA. Although several epigenetic regulators, such as Dnmt3l and the histone methyltransferases G9a and Prdm9, have been reported to be crucial for meiosis, little is known about how the expression of meiotic genes is regulated and how their expression contributes to normal meiosis. Using a loss-of-function approach in mice, here we show that the 5mC-specific dioxygenase Tet1 has an important role in regulating meiosis in mouse oocytes. Tet1 deficiency significantly reduces female germ-cell numbers and fertility. Univalent chromosomes and unresolved DNA double-strand breaks are also observed in Tet1-deficient oocytes. Tet1 deficiency does not greatly affect the genome-wide demethylation that takes place in primordial germ cells, but leads to defective DNA demethylation and decreased expression of a subset of meiotic genes. Our study thus establishes a function for Tet1 in meiosis and meiotic gene activation in female germ cells.

  6. Tet1 controls meiosis by regulating meiotic gene expression

    PubMed Central

    Yamaguchi, Shinpei; Hong, Kwonho; Liu, Rui; Shen, Li; Inoue, Azusa; Diep, Dinh; Zhang, Kun; Zhang, Yi

    2012-01-01

    Meiosis is a germ cell-specific cell division process through which haploid gametes are produced for sexual reproduction1. Prior to initiation of meiosis, mouse primordial germ cells (PGCs) undergo a series of epigenetic reprogramming steps2,3, including global erasure of DNA methylation on the 5-position of cytosine (5mC) at CpG4,5. Although several epigenetic regulators, such as Dnmt3l, histone methyltransferases G9a and Prdm9, have been reported to be critical for meiosis6, little is known about how the expression of meiotic genes is regulated and how their expression contributes to normal meiosis. Using a loss of function approach, here we demonstrate that the 5mC-specific dioxygenase Tet1 plays an important role in regulating meiosis in mouse oocytes. Tet1 deficiency significantly reduces female germ cell numbers and fertility. Univalent chromosomes and unresolved DNA double strand breaks are also observed in Tet1-deficient oocytes. Tet1 deficiency does not greatly affect the genome-wide demethylation that takes place in PGCs but leads to defective DNA demethylation and decreased expression of a subset of meiotic genes. Our study thus establishes a function for Tet1 in meiosis and meiotic gene activation in female germ cells. PMID:23151479

  7. DAF-2 and ERK couple nutrient availability to meiotic progression during Caenorhabditis elegans oogenesis.

    PubMed

    Lopez, Andrew L; Chen, Jessica; Joo, Hyoe-Jin; Drake, Melanie; Shidate, Miri; Kseib, Cedric; Arur, Swathi

    2013-10-28

    Coupling the production of mature gametes and fertilized zygotes to favorable nutritional conditions improves reproductive success. In invertebrates, the proliferation of female germline stem cells is regulated by nutritional status. However, in mammals, the number of female germline stem cells is set early in development, with oocytes progressing through meiosis later in life. Mechanisms that couple later steps of oogenesis to environmental conditions remain largely undefined. We show that, in the presence of food, the DAF-2 insulin-like receptor signals through the RAS-ERK pathway to drive meiotic prophase I progression and oogenesis; in the absence of food, the resultant inactivation of insulin-like signaling leads to downregulation of the RAS-ERK pathway, and oogenesis is stalled. Thus, the insulin-like signaling pathway couples nutrient sensing to meiotic I progression and oocyte production in C. elegans, ensuring that oocytes are only produced under conditions favorable for the survival of the resulting zygotes.

  8. DAF-2 and ERK Couple Nutrient Availability to Meiotic Progression during Caenorhabditis elegans Oogenesis

    PubMed Central

    Lopez, Andrew L.; Chen, Jessica; Joo, Hyoe-Jin; Drake, Melanie; Shidate, Miri; Kseib, Cedric; Arur, Swathi

    2013-01-01

    Coupling the production of mature gametes and fertilized zygotes to favorable nutritional conditions improves reproductive success. In invertebrates, the proliferation of female germ line stem cells is regulated by nutritional status. But, in mammals the number of female germ line stem cells is set early in development, with oocytes progressing through meiosis later in life. Mechanisms that couple later steps of oogenesis to environmental conditions remain largely undefined. We show that in the presence of food, the DAF-2 insulin-like receptor signals through the RAS-ERK pathway to drive meiotic prophase I progression and oogenesis; in the absence of food, the resultant inactivation of insulin-like signaling leads to downregulation of RAS-ERK pathway, and oogenesis is stalled. Thus, the insulin-like signaling pathway couples nutrient sensing to meiotic I progression and oocyte production in C. elegans, ensuring that oocytes are only produced under conditions favorable for the survival of the resulting zygotes. PMID:24120884

  9. 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.

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

    PubMed

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

    2016-06-14

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

  11. In vitro inhibition of oocyte nuclear maturation in the bovine.

    PubMed

    Sirard, M A; First, N L

    1988-09-01

    Bovine follicular oocytes (N = 5991) were exposed to an analog of cyclic adenosine 3',5'-monophosphate (cAMP), dibutyryl cyclic AMP (db-cAMP) (2.5, 5, and 10 mM), the phosphodiesterase inhibitor isobutyl methyl xanthine (IBMX, 0.2 mM), or the purine, hypoxanthine (0.5, 1.0, 2.0 mM), and the nucleoside, adenosine (0.05, 0.1, 0.2 mM), for 6 or 21 h to assess their effects on oocyte nuclear maturation. Potential effects of bovine follicular fluid (BFF) were also evaluated after different preculture washing procedures. In a separate experiment, denuded oocytes were used to study the effect of cumulus removal on meiotic inhibition. Db-cAMP decreased the frequency of germinal vesicle breakdown (GVBD) at 6 h (88% for control and 51%, 45%, and 32% for 2.5, 5, and 10 mM concentrations, respectively). IBMX had a comparable effect with only 41% of the oocytes resuming meiosis. Hypoxanthine and adenosine alone or in combinations also decreased the number of oocytes undergoing GVBD at 6 h. Only 22% GVBD occurred when the combined highest concentration of both substances was used compared to 88% in controls. If oocytes were incubated in 50% BFF after a wash in control medium during processing, 56% would resume meiosis at 6 h vs. 35% if the washing procedure included inhibitors (db-cAMP + IBMX). Total BFF (100%) during washing and maturation prevented 72% of the oocytes from resuming meiosis. Db-cAMP and IMBX combined or BFF also inhibited meiotic resumption of denuded oocytes. At 21 h, the inhibitory effects were less pronounced, with most oocytes only delayed in completing the first reduction division.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Partial characterization of the factor in theca-cell conditioned medium that inhibits the progression of FSH-induced meiosis of bovine oocytes surrounded by cumulus cells connected to the membrana granulosa.

    PubMed

    van Tol, H T; Bevers, M M

    2001-11-01

    A factor, secreted by theca cells, inhibits FSH induced resumption of meiosis in bovine oocytes that are surrounded by cumulus cells which are attached to a piece of the membrana granulosa (COCGs). In order to characterize this factor, theca cell conditioned medium (CMt) was heat-treated, filtered through a 5 kD spin off filter, charcoal treated, chloroform extracted and protease treated. To investigate whether the meiosis inhibiting factor produced by theca cells was also present in follicular fluid (FF), the same treatments were done with 50% bovine follicular fluid (bFF). COCGs, originating from 2 to 8 mm follicles of bovine ovaries collected at a slaughterhouse, were cultured in groups of 15 per 600 microl medium supplemented with 0.05 IU ml FSH for 22 hr at 39 degrees C in a humidified atmosphere of 5% CO(2). After culture the oocytes were denuded, stained with orcein, and the nuclear status assessed. Heat treatment did not affect the meiosis arresting capacity of CMt since a similar proportion of the oocytes remained at the GV stage after 22 hr of culture in heat treated CMt as compared to the proportion of oocytes in the GV stage after culture in untreated CMt. Filtering through a 5 kD spin-off filter revealed that the meiosis inhibiting action was maintained in the <5 kD fraction, although there was a significant (P < 0.05) loss of inhibiting activity compared to nonfiltered CMt. No significant decrease was observed in the meiosis arresting capacity of the <5 kD fraction after charcoal or protease treatment. Extraction of the <5 kD fraction with chloroform also did not affect the theca cell produced factor. The effect of the theca cell factor on the progression of meiosis of the oocytes that resumed meiosis, as demonstrated by a very low percentage of the oocytes that matured up to the M2 stage, was not affected following any of the treatments. With regard to bFF, the results show a lower percentage of the oocytes in the GV stage after culture in 50% bFF as

  13. Effect of nicotine on in vitro maturation of bovine oocytes.

    PubMed

    Liu, Ying; Li, Guang-Peng; Rickords, Lee F; White, Kenneth L; Sessions, Benjamin R; Aston, Kenneth I; Bunch, Thomas D

    2008-01-15

    The putative effect of nicotine on maturation and the chromosomal complement of bovine oocytes were investigated in the present study. Cumulus-enclosed oocytes were incubated in maturation medium with 0, 0.5, 1.0, 2.5, 5.0, and 10.0 mmol concentrations of nicotine. The results indicated that: (1) nicotine affected cumulus cell expansion in a dose-dependent manner and the perivitelline space failed to form when concentrations were equal to or greater than 5.0 mmol; (2) oocytes treated with 0.5 and 1.0 mmol nicotine concentrations resulted in maturation rates (83.3% and 85.9%, respectively) which was similar to the control (86.2%), whereas treatment with 2.5 and 5.0 mmol concentrations significantly decreased maturation rates to 70.2% and 26.7%, respectively; (3) nicotine at or over 2.5 mmol caused extremely irregular meiotic spindles and interrupted microfilament organization; (4) chromosomal analyses of oocytes with PB1 showed that oocytes derived from 0.5 and 1.0 mmol nicotine groups had haploid complements similar to the control (87-90%), but when the concentrations were increased to 2.5 and 5.0 mmol the haploid state was significantly reduced to around 70%; (5) oocytes at GVBD (germinal vesicle breakdown) and metaphase I stages were less affected by nicotine at 5.0 and 10.0 mmol concentrations than GV-stage oocytes; (6) maturation rates of the short-term nicotine-treated oocytes could be improved when subsequently incubated in normal maturation medium. Prolonged culture of nicotine-pretreated oocytes resulted in self-activation and some oocytes formed 1 or 2 pronuclei. In conclusion, nicotine affects bovine oocyte cumulus cell expansion, maturation rate, and chromosomal complement in a dose-dependent and an oocyte-stage-dependent manner.

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

    PubMed Central

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

    2016-01-01

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

  15. 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.

  16. 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.

  17. Follicle-stimulating hormone accelerates mouse oocyte development in vivo.

    PubMed

    Demeestere, Isabelle; Streiff, Agathe K; Suzuki, João; Al-Khabouri, Shaima; Mahrous, Enas; Tan, Seang Lin; Clarke, Hugh J

    2012-07-01

    During folliculogenesis, oocytes grow and acquire developmental competence in a mutually dependent relationship with their adjacent somatic cells. Follicle-stimulating hormone (FSH) plays an essential and well-established role in the differentiation of somatic follicular cells, but its function in the development of the oocyte has still not been elucidated. We report here that oocytes of Fshb(-/-) mice, which cannot produce FSH, grow at the same rate and reach the same size as those of wild-type mice. Consistent with this observation, the granulosa cells of Fshb(-/-) mice express the normal quantity of mRNA encoding Kit ligand, which has been implicated in oocyte growth. Oocytes of Fshb(-/-) mice also accumulate normal quantities of cyclin B1 and CDK1 proteins and mitochondrial DNA. Moreover, they acquire the ability to complete meiotic maturation in vitro and undergo transition from non-surrounded nucleolus to surrounded nucleolus. However, these events of late oocyte development are significantly delayed. Following in vitro maturation and fertilization, only a small number of embryos derived from oocytes of Fshb(-/-) mice reach the blastocyst stage. Administration of equine chorionic gonadotropin, which provides FSH activity, 48 h before in vitro maturation increases the number of blastocysts obtained subsequently. These results indicate that FSH is not absolutely required for oocyte development in vivo but that this process occurs more rapidly in its presence. We suggest that FSH may coordinate the development of the germline and somatic compartments of the follicle, ensuring that ovulation releases a developmentally competent egg.

  18. 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

  19. Meiosis resumption of canine oocytes cultured in the isolated oviduct.

    PubMed

    Luvoni, G C; Chigioni, S; Allievi, E; Macis, D

    2003-10-01

    The aim of this study was to investigate the effects of culture in isolated oviducts relative to meiotic maturation, the time required to resume meiosis and the viability of the canine oocytes. For this purpose, cumulus-oocyte complexes and isthmus-ampullar tracts of the oviducts were collected from bitches undergoing ovariohysterectomies and destined to two experiments of culture. In experiment 1, the oocytes were cultured for 24 or 30 h: (1) in 100 micro l drops under oil; (2) on the mucosal epithelium of the open oviducts; (3) in the ligated oviducts. In experiment 2, oocytes were cultured in the ligated oviduct for 24, 30 and 48 h. A group of control oocytes was not cultured (0 h). The results showed that within 30 h of culture, a higher proportion of oocytes (p < 0.001) resumed meiosis in the ligated oviduct (63.8%) than in drop (20.4%) or in the open oviduct (27.1%). Moreover, 24 and 30 h of culture assured higher proportions of meiosis resumption than 48 h (69.2 and 59.1% vs 35.8%, p < 0.005). Oocyte resumption of meiosis was mainly determined by oocytes at meiotic stages preceding metaphase I, while stages between metaphase I and II in the ligated oviduct ranged between 12.5 and 31.9%. The extension of the culture time up to 48 h in the oviduct increased oocyte degeneration significantly (59.3%, p < 0.0001) compared with 24 and 30 h (18.7 and 27.3%, respectively) and the oviductal epithelium showed nuclear picnosis and degeneration following culture. The present study suggests that the close physical interaction between the canine oocytes and the oviductal tract positively affects oocyte maturation, and meiosis is resumed within 30 h of culture. Moreover, the oocyte survival is better preserved within 30 h in the ligated oviduct compared with the conventional culture in drop or to the culture in the open oviduct, but the ligated oviduct does not assure viability of the oocytes up to 48 h of culture.

  20. Specific inhibition of endogenous beta-tubulin synthesis in Xenopus oocytes by anti-messenger oligodeoxynucleotides.

    PubMed Central

    Jessus, C; Cazenave, C; Ozon, R; Hélène, C

    1988-01-01

    An oligodeoxynucleotide containing 27 nucleotides, complementary to a highly conserved sequence of beta-tubulin mRNAs, led to a nearly complete inhibition of beta-tubulin synthesis in Xenopus oocytes after microinjection. Inhibition persisted 24 hours post-injection and was specific for beta-tubulin as the synthesis of alpha-tubulin as well as that of other proteins from the oocyte was not affected. Complete inhibition of beta-tubulin synthesis did not prevent progesterone-induced meiotic maturation and formation of the chromosome spindle. This result indicates that the pool of endogenous tubulin already present in fully-grown oocytes is sufficient to allow normal meiotic maturation. This finding correlates with previous experiments showing that the turn-over of tubulin is very slow in the oocyte. Images PMID:3357774

  1. A light- and electron-microscope study of the oocyte nucleus during development of the antral follicle in the prepubertal mouse.

    PubMed

    Chouinard, L A

    1975-05-01

    with the dismantling of the nuclear envelope. The extranucleolar bodies increase noticeably in size during the preparatory and the maturative follicle stages; they shrink in size and undergo dissolution during the preovulatory stage of antral follicle development. An attempt is made to interpret these morphological changes in the light of current knowledge concerning the architectural and functional organization of the oocyte nucleus in general during meiotic prophase. The relevant observational evidence would be consistent with the view that, during antral follicle deveopment, the mouse oocyte nucleus is not, as too often assumed, in a period of arrested evolution; its formed components undergo structural, maturational and functional changes which are of significance not only for the resumption of the first meiotic prophase but also for the early development of the embryo.

  2. 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

  3. SPO11-independent DNA repair foci and their role in meiotic silencing.

    PubMed

    Carofiglio, Fabrizia; Inagaki, Akiko; de Vries, Sandra; Wassenaar, Evelyne; Schoenmakers, Sam; Vermeulen, Christie; van Cappellen, Wiggert A; Sleddens-Linkels, Esther; Grootegoed, J Anton; Te Riele, Hein P J; de Massy, Bernard; Baarends, Willy M

    2013-06-01

    In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11(YF/YF)), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.

  4. Kif4 Is Essential for Mouse Oocyte Meiosis.

    PubMed

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

    2017-01-01

    Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age.

  5. Kif4 Is Essential for Mouse Oocyte Meiosis

    PubMed Central

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

    2017-01-01

    Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age. PMID:28125646

  6. 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

  7. Confinement induces actin flow in a meiotic cytoplasm

    PubMed Central

    Pinot, Mathieu; Steiner, Villier; Dehapiot, Benoit; Yoo, Byung-Kuk; Chesnel, Franck; Blanchoin, Laurent; Kervrann, Charles; Gueroui, Zoher

    2012-01-01

    In vivo, F-actin flows are observed at different cell life stages and participate in various developmental processes during asymmetric divisions in vertebrate oocytes, cell migration, or wound healing. Here, we show that confinement has a dramatic effect on F-actin spatiotemporal organization. We reconstitute in vitro the spontaneous generation of F-actin flow using Xenopus meiotic extracts artificially confined within a geometry mimicking the cell boundary. Perturbations of actin polymerization kinetics or F-actin nucleation sites strongly modify the network flow dynamics. A combination of quantitative image analysis and biochemical perturbations shows that both spatial localization of F-actin nucleators and actin turnover play a decisive role in generating flow. Interestingly, our in vitro assay recapitulates several symmetry-breaking processes observed in oocytes and early embryonic cells. PMID:22753521

  8. Defying DNA Double-Strand Break-Induced Death during Prophase I Meiosis by Temporal TAp63α Phosphorylation Regulation in Developing Mouse Oocytes

    PubMed Central

    Kim, Dal-Ah

    2014-01-01

    The dichotomy in DNA damage sensitivity of developing mouse oocytes during female germ line development is striking. Embryonic oocytes withstand hundreds of programmed DNA double-strand breaks (DSBs) required for meiotic recombination. Postnatal immature oocytes fail to tolerate even a few DSBs induced by gamma radiation treatment. TAp63α, a p53 family member, undergoes phosphorylation and mediates postnatal immature oocyte death following gamma radiation treatment, which is thought important for germ line quality maintenance. Whether prenatal meiotic oocytes tolerate DNA DSBs simply because they lack TAp63α expression is not clear. We found a significant number of oocytes in newborn mice initiate TAp63α expression and simultaneously carry meiotic DNA DSBs. However, the risk of premature death appears unlikely, because newborn oocytes strongly abate TAp63α phosphorylation induction and resist normally lethal doses of ionizing radiation damage. A calyculin A-sensitive Ser/Thr phosphatase activity downregulates TAp63α phosphorylation and ATM kinase mediates phosphorylation. Possible alterations in the relative balance of these counteracting activities during development may first temper TAp63α phosphorylation and death induction during meiotic DNA DSB repair and recombination, and afterward, implement germ line quality control in later stages. Insights into inherent DNA DSB resistance mechanisms in newborn oocytes may help prevent infertility in women in need of radiation or chemotherapy. PMID:24515437

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

    PubMed

    Tsuji, Ai; Nakamura, Toshinobu; Shibata, Katsumi

    2015-01-01

    Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.

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

    PubMed

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

    2007-10-01

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

  11. Human oocyte cryopreservation in infertility and oncology.

    PubMed

    Porcu, Eleonora; Bazzocchi, Antonia; Notarangelo, Leonardo; Paradisi, Roberto; Landolfo, Chiara; Venturoli, Stefano

    2008-12-01

    To evaluate the present state of research and clinical application of human oocyte cryopreservation in infertility and oncology. Recent literature documents have an increasing interest in cryopreserving human eggs. A number of studies report on different freezing protocols and various types of clinical application. Increasing attention is paid to vitrification as an alternative to slow cooling for oocyte cryopreservation. Several studies cover the modification of meiotic spindle during cryopreservation in order to assess the less damaging cryopreservation system. The first births with cryopreserved oocytes in cancer patients are reported. Egg freezing may circumvent the ethical and legal concerns regarding embryo cryopreservation, increase assisted reproduction flexibility and be a concrete option to save fertility in women with cancer. Recently, egg survival and pregnancy rates improved, with the birth of more than 500 children. The birth rate per thawed oocyte is around 5-6%. As regards safety, data on birth defects seems to be reassuring so far but must be monitored by an international registry. Comparative studies between slow freezing and vitrification in the same patient population are needed to elucidate pros and cons of each technique.

  12. Y-autosome translocation interferes with meiotic sex inactivation and expression of autosomal genes: a case study in the pig.

    PubMed

    Barasc, H; Mary, N; Letron, R; Calgaro, A; Dudez, A M; Bonnet, N; Lahbib-Mansais, Y; Yerle, M; Ducos, A; Pinton, A

    2012-01-01

    Y-autosome translocations are rare in humans and pigs. In both species, these rearrangements can be responsible for meiotic arrest and subsequent infertility. Chromosome pairing abnormalities on the SSCX, SSCY and SSC1 chromatin domains were identified by analyzing pachytene spermatocytes from a boar carrying a (Y;1) translocation by immunolocalization of specific meiotic protein combined with FISH. Disturbance of the meiotic sex chromosome inactivation (MSCI) was observed by Cot-RNA-FISH and analysis of ZFY gene expression by sequential RNA- and DNA-FISH on spermatocytes. We hypothesized that the meiotic arrest observed in this boar might be due to the silencing of critical autosomal genes and/or the reactivation of some sex chromosome genes.

  13. Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors

    PubMed Central

    Perkins, Adrienne T.; Das, Thomas M.; Panzera, Lauren C.; Bickel, Sharon E.

    2016-01-01

    In humans, errors in meiotic chromosome segregation that produce aneuploid gametes increase dramatically as women age, a phenomenon termed the “maternal age effect.” During meiosis, cohesion between sister chromatids keeps recombinant homologs physically attached and premature loss of cohesion can lead to missegregation of homologs during meiosis I. A growing body of evidence suggests that meiotic cohesion deteriorates as oocytes age and contributes to the maternal age effect. One hallmark of aging cells is an increase in oxidative damage caused by reactive oxygen species (ROS). Therefore, increased oxidative damage in older oocytes may be one of the factors that leads to premature loss of cohesion and segregation errors. To test this hypothesis, we used an RNAi strategy to induce oxidative stress in Drosophila oocytes and measured the fidelity of chromosome segregation during meiosis. Knockdown of either the cytoplasmic or mitochondrial ROS scavenger superoxide dismutase (SOD) caused a significant increase in segregation errors, and heterozygosity for an smc1 deletion enhanced this phenotype. FISH analysis indicated that SOD knockdown moderately increased the percentage of oocytes with arm cohesion defects. Consistent with premature loss of arm cohesion and destabilization of chiasmata, the frequency at which recombinant homologs missegregate during meiosis I is significantly greater in SOD knockdown oocytes than in controls. Together these results provide an in vivo demonstration that oxidative stress during meiotic prophase induces chromosome segregation errors and support the model that accelerated loss of cohesion in aging human oocytes is caused, at least in part, by oxidative damage. PMID:27791141

  14. Cytoskeleton-associated protein 5 and clathrin heavy chain binding regulates spindle assembly in mouse oocytes

    PubMed Central

    Wang, Dong-Hui; Han, Zhe; Kong, Xiang-Wei; Ma, Yu-Zhen; Yun, Zhi-Zhong; Liang, Cheng-Guang

    2017-01-01

    Mammalian oocyte meiotic maturation is the precondition of early embryo development. Lots of microtubules (MT)-associated proteins participate in oocyte maturation process. Cytoskeleton-associated protein 5 (CKAP5) is a member of the XMAP215 family that regulates microtubule dynamics during mitosis. However, its role in meiosis has not been fully studied. Here, we investigated the function of CKAP5 in mouse oocyte meiotic maturation and early embryo development. Western blot showed that CKAP5 expression increased from GVBD, maintaining at high level at metaphase, and decreased after late 1-cell stage. Confocal microscopy showed there is no specific accumulation of CKAP5 at interphase (GV, PN or 2-cell stage). However, once cells enter into meiotic or mitotic division, CKAP5 was localized at the whole spindle apparatus. Treatment of oocytes with the tubulin-disturbing reagents nocodazole (induces MTs depolymerization) or taxol (prevents MTs depolymerization) did not affect CKAP5 expression but led to a rearrangement of CKAP5. Further, knock-down of CKAP5 resulted in a failure of first polar body extrusion, serious defects in spindle assembly, and failure of chromosome alignment. Loss of CKAP5 also decreased early embryo development potential. Furthermore, co-immunoprecipitation showed that CKAP5 bound to clathrin heavy chain 1 (CLTC). Taken together, our results demonstrate that CKAP5 is important in oocyte maturation and early embryo development, and CKAP5 might work together with CLTC in mouse oocyte maturation. PMID:28177917

  15. Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors.

    PubMed

    Perkins, Adrienne T; Das, Thomas M; Panzera, Lauren C; Bickel, Sharon E

    2016-11-01

    In humans, errors in meiotic chromosome segregation that produce aneuploid gametes increase dramatically as women age, a phenomenon termed the "maternal age effect." During meiosis, cohesion between sister chromatids keeps recombinant homologs physically attached and premature loss of cohesion can lead to missegregation of homologs during meiosis I. A growing body of evidence suggests that meiotic cohesion deteriorates as oocytes age and contributes to the maternal age effect. One hallmark of aging cells is an increase in oxidative damage caused by reactive oxygen species (ROS). Therefore, increased oxidative damage in older oocytes may be one of the factors that leads to premature loss of cohesion and segregation errors. To test this hypothesis, we used an RNAi strategy to induce oxidative stress in Drosophila oocytes and measured the fidelity of chromosome segregation during meiosis. Knockdown of either the cytoplasmic or mitochondrial ROS scavenger superoxide dismutase (SOD) caused a significant increase in segregation errors, and heterozygosity for an smc1 deletion enhanced this phenotype. FISH analysis indicated that SOD knockdown moderately increased the percentage of oocytes with arm cohesion defects. Consistent with premature loss of arm cohesion and destabilization of chiasmata, the frequency at which recombinant homologs missegregate during meiosis I is significantly greater in SOD knockdown oocytes than in controls. Together these results provide an in vivo demonstration that oxidative stress during meiotic prophase induces chromosome segregation errors and support the model that accelerated loss of cohesion in aging human oocytes is caused, at least in part, by oxidative damage.

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

    PubMed

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

    2011-10-16

    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.

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

    PubMed Central

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

    2016-01-01

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

  18. Control of Oocyte Growth and Development by Intercellular Communication Within the Follicular Niche.

    PubMed

    El-Hayek, Stephany; Clarke, Hugh J

    2016-01-01

    In the mammalian ovary, each oocyte grows and develops within its own structural and developmental niche-the follicle. Together with the female germ cell in the follicle are somatic granulosa cells, specialized companion cells that surround the oocyte and provide support to it, and an outer layer of thecal cells that serve crucial roles including steroid synthesis. These follicular compartments function as a single physiological unit whose purpose is to produce a healthy egg, which upon ovulation can be fertilized and give rise to a healthy embryo, thus enabling the female germ cell to fulfill its reproductive potential. Beginning from the initial stage of follicle formation and until terminal differentiation at ovulation, oocyte and follicle growth depend absolutely on cooperation between the different cellular compartments. This cooperation synchronizes the initiation of oocyte growth with follicle activation. During growth, it enables metabolic support for the follicle-enclosed oocyte and allows the follicle to fulfill its steroidogenic potential. Near the end of the growth period, intra-follicular interactions prevent the precocious meiotic resumption of the oocyte and ensure its nuclear differentiation. Finally, cooperation enables the events of ovulation, including meiotic maturation of the oocyte and expansion of the cumulus granulosa cells. In this chapter, we discuss the cellular interactions that enable the growing follicle to produce a healthy oocyte, focusing on the communication between the germ cell and the surrounding granulosa cells.

  19. Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption.

    PubMed

    McGinnis, Lynda K; Luo, Jinping; Kinsey, William H

    2013-04-01

    Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

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

    PubMed

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

    2008-12-01

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

  1. Evidence for polar cytoplasm/nuage in rat oocytes.

    PubMed

    Young, J K; Allworth, A E; Baker, J H

    1999-07-01

    In many organisms oocytes contain dark-staining material, termed nuage, that is concentrated at one pole of the oocyte cytoplasm and that influences the further development of the oocyte after fertilization. In mammalian oocytes, ultrastructural studies have detected small patches of nuage-like material, but thus far no nuage-rich zone of polar cytoplasm has been reported. Here, we report that when large sections of rat ovary embedded in methacrylate resin are stained with toluidine blue and surveyed, many oocytes contain a narrow, sharply defined, basophilic zone of polar cytoplasm that appears analogous to the polar cytoplasm of Xenopus and other non-mammalian species. This basophilic polar cytoplasm was common in multilaminar follicles and was not visible in smaller, primordial follicles. In one out of five oocytes stimulated with hCG to complete the first meiotic division, a relatively faint region of cortical basophilia was detectable. Further studies will be needed to ascertain if this nuage-like material has an influence upon the development of oocytes similar to that seen in non-mammalian species.

  2. [Polarized light microscopy for evaluation of oocytes as a prognostic factor in the evolution of a cycle in assisted reproduction].

    PubMed

    González-Ortega, C; Cancino-Villarreal, P; Alonzo-Torres, V E; Martínez-Robles, I; Pérez-Peña, E; Gutiérrez-Gutiérrez, A M

    2016-04-01

    Identification of the best embryos to transfer is a key element for success in assisted reproduction. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems has allowed the visualization of the meiotic spindle and the different layers of the zona pellucida in human oocytes on the basis of birefringence in a non-destructive way. Conflicting results have been reported regarding the predictive value in ICSI cycles. To assess the predictive ability of meiotic spindle and zona pellucida of human oocytes to implant by polarized microscopy in ICSI cycles. Prospective and observational clinical study. 903 oocytes from 94 ICSI cycles were analyzed with polarized microscopy. Meiotic spindle visualization and zona pellucida birefringence values by polarized microscopy were correlated with ICSI cycles results. Meiotic spindle visualization and birefringence values of zona pellucida decreased in a direct basis with increasing age. In patients aged over the 35 years, the percentage of a visible spindle and mean zona pellucida birefringence was lower than in younger patients. Fertilization rate were higher in oocytes with visible meiotic spindle (81.3% vs. 64%; p < 0.0001), as well as embryo quality (47.4% vs. 39%; p=0.01). Fertilization rate was higher in oocytes with positive values of birefringence (77.5 % vs. 68.5% p=0.005) with similar embryo quality. Conception cycles showed oocytes with higher mean value of zona birefringence and visible spindle vs. no-conception cycles (p<0.05). Polarized light microscopy improves oocyte selection, which significantly impacts in the development of embryos with greater implantation potential. The use of polarized light microscopy with sperm selection methods, blastocyst culture and deferred embryo transfers will contribute to transfer fewer embryos without diminishing rates of live

  3. Deletion of Mylk1 in oocytes causes delayed morula-to-blastocyst transition and reduced fertility without affecting folliculogenesis and oocyte maturation in mice.

    PubMed

    Liang, Qiu-Xia; Zhang, Qing-Hua; Qi, Shu-Tao; Wang, Zhong-Wei; Hu, Meng-Wen; Ma, Xue-Shan; Zhu, Min-Sheng; Schatten, Heide; Wang, Zhen-Bo; Sun, Qing-Yuan

    2015-04-01

    The mammalian oocyte undergoes two rounds of asymmetric cell divisions during meiotic maturation and fertilization. Acentric spindle positioning and cortical polarity are two major factors involved in asymmetric cell division, both of which are thought to depend on the dynamic interaction between myosin II and actin filaments. Myosin light chain kinase (MLCK), encoded by the Mylk1 gene, could directly phosphorylate and activate myosin II. To determine whether MLCK was required for oocyte asymmetric division, we specifically disrupted the Mylk1 gene in oocytes by Cre-loxP conditional knockout system. We found that Mylk1 mutant female mice showed severe subfertility. Unexpectedly, contrary to previously reported in vitro findings, our data showed that oocyte meiotic maturation including spindle organization, polarity establishment, homologous chromosomes separation, and polar body extrusion were not affected in Mylk1(fl/fl);GCre(+) females. Follicular development, ovulation, and early embryonic development up to compact morula occurred normally in Mylk1(fl/fl);GCre(+) females, but deletion of MLCK caused delayed morula-to-blastocyst transition. More than a third of embryos were at morula stage at 3.5 Days Postcoitum in vivo. The delayed embryos could develop further to early blastocyst stage in vitro on Day 4 when most control embryos reached expanded blastocysts. Our findings provide evidence that MLCK is linked to timely blastocyst formation, though it is dispensable for oocyte meiotic maturation. © 2015 by the Society for the Study of Reproduction, Inc.

  4. 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

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

    PubMed

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

    2016-02-01

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

  6. Cardiac arrest

    MedlinePlus

    ... or low levels can cause cardiac arrest. Severe physical stress. Anything that causes a severe stress on your body can lead to cardiac arrest. This can include trauma, electrical shock, or major blood loss. Recreational drugs. Using certain drugs, such as cocaine ...

  7. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

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

    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.

  10. Aym1, a mouse meiotic gene identified by virtue of its ability to activate early meiotic genes in the yeast Saccharomyces cerevisiae.

    PubMed

    Malcov, Mira; Cesarkas, Karen; Stelzer, Gil; Shalom, Sarah; Dicken, Yosef; Naor, Yaniv; Goldstein, Ronald S; Sagee, Shira; Kassir, Yona; Don, Jeremy

    2004-12-01

    Our understanding of the molecular mechanisms that operate during differentiation of mitotically dividing spermatogonia cells into spermatocytes lags way behind what is known about other differentiating systems. Given the evolutionary conservation of the meiotic process, we screened for mouse proteins that could specifically activate early meiotic promoters in Saccharomyces cerevisiae yeast cells, when fused to the Gal4 activation domain (Gal4AD). Our screen yielded the Aym1 gene that encodes a short peptide of 45 amino acids. We show that a Gal4AD-AYM1 fusion protein activates expression of reporter genes through the promoters of the early meiosis-specific genes IME2 and HOP1, and that this activation is dependent on the DNA-binding protein Ume6. Aym1 is transcribed predominantly in mouse primary spermatocytes and in gonads of female embryos undergoing the corresponding meiotic divisions. Aym1 immunolocalized to nuclei of primary spermatocytes and oocytes and to specific type A spermatogonia cells, suggesting it might play a role in the processes leading to meiotic competence. The potential functional relationship between AYM1 and yeast proteins that regulate expression of early meiotic genes is discussed.

  11. 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

  12. Meiotic exchange and segregation in female mice heterozygous for paracentric inversions.

    PubMed Central

    Koehler, Kara E; Millie, Elise A; Cherry, Jonathan P; Schrump, Stefanie E; Hassold, Terry J

    2004-01-01

    Inversion heterozygosity has long been noted for its ability to suppress the transmission of recombinant chromosomes, as well as for altering the frequency and location of recombination events. In our search for meiotic situations with enrichment for nonexchange and/or single distal-exchange chromosome pairs, exchange configurations that are at higher risk for nondisjunction in humans and other organisms, we examined both exchange and segregation patterns in 2728 oocytes from mice heterozygous for paracentric inversions, as well as controls. We found dramatic alterations in exchange position in the heterozygotes, including an increased frequency of distal exchanges for two of the inversions studied. However, nondisjunction was not significantly increased in oocytes heterozygous for any inversion. When data from all inversion heterozygotes were pooled, meiotic nondisjunction was slightly but significantly higher in inversion heterozygotes (1.2%) than in controls (0%), although the frequency was still too low to justify the use of inversion heterozygotes as a model of human nondisjunction. PMID:15082541

  13. On the transition from the meiotic to mitotic cell cycle during early mouse development.

    PubMed

    Kubiak, Jacek Z; Ciemerych, Maria A; Hupalowska, Anna; Sikora-Polaczek, Marta; Polanski, Zbigniew

    2008-01-01

    Here, we outline the mechanisms involved in the regulation of cell divisions during oocyte maturation and early cleavages of the mouse embryo. Our interest is focused on the regulation of meiotic M-phases and the first embryonic mitoses that are differently tuned and are characterized by specifically modified mechanisms, some of which have been recently identified. The transitions between the M-phases during this period of development, as well as associated changes in their regulation, are of key importance for both the meiotic maturation of oocytes and the further development of the mammalian embryo. The mouse is an excellent model for studies of the cell cycle during oogenesis and early development. Nevertheless, a number of molecular mechanisms described here were discovered or confirmed during the study of other species and apply also to other mammals including humans.

  14. Inhibition of glycosphingolipid synthesis induces p34cdc2 activation in Xenopus oocyte.

    PubMed

    De Smedt, V; Rime, H; Jessus, C; Ozon, R

    1995-11-20

    In Xenopus prophase-blocked oocytes, it is assumed that progesterone interacts with the plasma membrane to initiate a signalling cascade that ultimately leads to MPF activation. Progesterone regulates negatively the cAMP pathway through an inhibition of adenylate cyclase. However, the mechanisms linking the initial action of the hormone with adenylate cyclase activity remain to be elucidated. Here, we demonstrate that PDMP, an inhibitor of glucosphingolipid synthesis, triggers oocyte meiotic maturation in a cAMP- and cycloheximide-dependent manner, whereas exogenous ceramide is unefficient. We propose that sphingolipid metabolism and targeting represent an important regulatory process of oocyte meiosis.

  15. [Patient's experience of topical anesthesia by lidocaine vaginal gel for oocyte retrieval].

    PubMed

    Guillaume, A; Schuller-Dufour, E; Faitot, V; Pirrello, O; Rongières, C; Ohl, J; Nisand, I; Bettahar, K

    2016-10-01

    A recent adverse effect of a paracervical block (cardiac arrest) occurred during an oocyte retrieval (OR), forcing us to reconsider our pain management during OR. Since then, we decided to use intravaginal lidocaine gel as analgesia during OR.

  16. Functional Topography of the Fully Grown Human Oocyte

    PubMed Central

    Monti, Manuela; Calligaro, Alberto; Behr, Barry; Pera, Renee Rejo; Redi, Carlo Alberto; Wossidlo, Mark

    2017-01-01

    In vivo maturation (IVM) of human oocytes is a technique used to increase the number of usable oocytes for in vitro fertilization (IVF) and represents a necessity for women with different ovarian pathologies. During IVM the oocytes progress from the germinal vesicle stage (GV) through the metaphase II and during this journey both nuclear and cytoplasmic rearrangements must be obtained to increase the probability to get viable and healthy zygotes/embryos after IVF. As the successful clinical outcomes of this technique are a reality, we wanted to investigate the causes behind oocytes maturation arrest. For obvious ethical reasons, we were able to analyze only few human immature oocytes discarded and donated to research by transmission electron microscopy showing that, as in the mouse, they have different chromatin and cytoplasmic organizations both essential for further embryo development. PMID:28348419

  17. Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11.

    PubMed

    Baudat, F; Manova, K; Yuen, J P; Jasin, M; Keeney, S

    2000-11-01

    Spo11, a protein first identified in yeast, is thought to generate the chromosome breaks that initiate meiotic recombination. We now report that disruption of mouse Spo11 leads to severe gonadal abnormalities from defective meiosis. Spermatocytes suffer apoptotic death during early prophase; oocytes reach the diplotene/dictyate stage in nearly normal numbers, but most die soon after birth. Consistent with a conserved function in initiating meiotic recombination, Dmc1/Rad51 focus formation is abolished. Spo11(-/-) meiocytes also display homologous chromosome synapsis defects, similar to fungi but distinct from flies and nematodes. We propose that recombination initiation precedes and is required for normal synapsis in mammals. Our results also support the view that mammalian checkpoint responses to meiotic recombination and/or synapsis defects are sexually dimorphic.

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

    PubMed

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

    2013-07-01

    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.

  19. 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.

  20. 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.

  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.

  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. 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.

  4. Nicotine alters bovine oocyte meiosis and affects subsequent embryonic development.

    PubMed

    Liu, Ying; Li, Guang-Peng; White, Kenneth L; Rickords, Lee F; Sessions, Benjamin R; Aston, Kenneth I; Bunch, Thomas D

    2007-11-01

    The effects of nicotine on nuclear maturation and meiotic spindle dynamics of bovine oocytes and subsequent embryonic development were investigated. Maturation rates (85%-94%) derived from nicotine treatments at 0.01 to 1.0 mM were similar to the control (86%), but significantly decreased at 2.0 to 6.0 mM. Haploid complements of metaphase II oocytes in 0.01 to 1.0 mM nicotine (approximately 90%) were similar to the control, while lower (ranged from 63% to 76%, P < 0.05 or P < 0.01) haploid oocytes were observed in the 2.0 to 6.0 mM nicotine groups. The majority of the PB1-free oocytes derived from 3.0 to 6.0 mM nicotine treatments were diploidy (2n = 60). Spindle microtubules changed from characteristically being asymmetrical in the controls to being equally distributed into two separate chromosome groups in the nicotine treatments. Nicotine disorganized the microfilament organization and inhibited the movement of anaphase or telophase chromosomes to the cortical area. The inhibited two chromosome groups became two spindles that either moved close in proximity or merged entirely together resulting in diploidy within the affected oocyte. Nicotine treatment significantly reduced the rate of cleavage and blastocyst development after parthenogenetic activation. Diploidy and cell number were drastically reduced in the resultant blastocysts. In conclusion, nicotine can alter the normal process of bovine oocyte meiosis and affects subsequent embryonic development.

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

    PubMed

    Grøndahl, Marie Louise; Borup, Rehannah; Vikeså, Jonas; Ernst, Erik; Andersen, Claus Yding; Lykke-Hartmann, Karin

    2013-09-01

    Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human oocyte in the quiescent state and at the pinnacle of maturity at ovulation. In silico bioinformatic comparisons were made between the transcriptome of human oocytes from dormant primordial follicles and that of human metaphase II (MII) oocytes and granulosa cells and unique gene expression profiles were identified as well as functional and pathway enrichments associated with the oocytes from the two developmental hallmarks. A total of 729 genes were highly enriched in oocytes from primodial follicles and 1456 genes were highly enriched in MII oocytes (>10-fold, P < 0.001) representing functional categories such as cell cycle regulation, DNA protection and epigenetics, with representative genes validated by qPCR analysis. Dominating canonical pathways in the oocytes from primordial follicles were androgen, estrogen receptor, glucocorticoid receptor and PI3K/AKT signaling (P < 0.001). In the MII, mitotic roles of polo-like kinases, estrogen receptor, JAK/Stat signaling (P < 0.001) and the ERK/MAPK (P < 0.01) signaling were enriched. Some of the highly differentially expressed genes were completely new in human reproduction (CDR1, TLC1A, UHRF2) while other genes [ABO, FOLR1 (folate receptor), CHRNA3 (nicotine receptor)] may relate to clinical observations as diverse as premature ovarian failure, folic acid deficiency and smoking affecting female fertility. The in silico analysis identified novel reproduction-associated genes and highlighted molecular mechanisms and pathways associated with the unique functions of the human oocyte in its two extremes during folliculogenesis. The data provides a fundamental basis for future functional studies in regulation of human oogenesis.

  6. Phosphodiesterase 3 inhibitors suppress oocyte maturation and consequent pregnancy without affecting ovulation and cyclicity in rodents.

    PubMed Central

    Wiersma, A; Hirsch, B; Tsafriri, A; Hanssen, R G; Van de Kant, M; Kloosterboer, H J; Conti, M; Hsueh, A J

    1998-01-01

    During each reproductive cycle, a preovulatory surge of gonadotropins induces meiotic maturation of the oocyte in the preovulatory follicle followed by ovulation. Although gonadotropins stimulate cAMP production in somatic cells of the follicle, a decrease in intra-oocyte cAMP levels is required for resumption of meiosis in oocytes. Based on the observed compartmentalization of the cAMP-degrading enzyme, phosphodiesterase, in follicular somatic and germ cells, inhibitors of phosphodiesterase 3 were used to block meiosis in ovulating oocytes in rodents. By this strategy, we demonstrated that fertilization and pregnancy could be prevented without disturbing follicle rupture and normal estrous cyclicity. In contrast to conventional contraceptive pills that disrupt ovarian steroidogenesis and reproductive cycles, the present strategy achieves effective contraception by selective blockage of oocyte maturation and development without alterations in ovulation and reproductive cyclicity. PMID:9691090

  7. Chromosome identification in human oocytes and polar bodies by spectral karyotyping.

    PubMed

    Márquez, C; Cohen, J; Munné, S

    1998-01-01

    Sixty unfertilized human oocytes and two fresh polar bodies were karyotyped by spectral karyotyping (SKY). The oocytes were provided by 29 women ranging from 30 to 42 yr of age. The mean hybridization efficiency for oocytes was 95.2% (60/63). Nondisjunction of bivalent chromosomes (13.3%) and predivision of sister chromatids at meiosis I (3.3%) were unequivocally determined by analysis first with SKY and then fluorescence in situ hybridization. Four oocytes (6.7%) were hyperhaploid, six (10.0%) were hypohaploid, one (1.7%) showed balanced predivision, and another (1.7%) was diploid. No specific structural rearrangements were detected. This study demonstrates that the SKY technique can be used successfully as an alternative method of karyotyping second meiotic metaphase chromosomes from human oocytes and polar bodies in appropriate spreads.

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

    PubMed

    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.

  9. [Diagnosticum of abnormalities of plant meiotic division].

    PubMed

    Shamina, N V

    2006-01-01

    Abnormalities of plant meiotic division leading to abnormal meiotic products are summarized schematically in the paper. Causes of formation of monads, abnormal diads, triads, pentads, polyads, etc. have been observed in meiosis with both successive and simultaneous cytokinesis.

  10. Hormone-induced cortical maturation ensures the slow block to polyspermy and does not couple with meiotic maturation in starfish.

    PubMed

    Hirohashi, Noritaka; Harada, Kaori; Chiba, Kazuyoshi

    2008-06-01

    Meiotic progression in starfish oocytes is reinitiated by a maturation-inducing hormone called 1-methyladenine (1-MeAde). In addition to meiotic maturation, 1-MeAde induces cortical maturation in which cortical granules become competent to discharge in response to fusion of a single sperm, which results in the formation of the fertilization envelope. We found that subthreshold concentrations of 1-MeAde induce cortical maturation without germinal vesicle breakdown (GVBD). During cortical maturation, the IP3 sensitivity of calcium stores was increased as well as during meiotic maturation. When oocytes were exposed with 1-MeAde only on a hemisphere of oocytes, the IP3 sensitivity of the cortical region was increased only in the exposed hemisphere, suggesting that signals and components involved in cortical maturation do not readily spread in the cytoplasm. Although a specific inhibitor of phosphatidylinositol-3 kinase, LY294002 blocked both GVBD and cortical maturation, a Cdc2 kinase inhibitor, roscovitine did not block cortical maturation. Inhibition of Akt activation by injecting the competitors for Akt phosphorylation and membrane recruitment also blocked cortical maturation. These results suggest that the signaling pathway leading to Akt activation is common in cortical maturation and meiotic maturation, and Cdc2 activation was not required for cortical maturation.

  11. Fatty Acid Synthesis and Oxidation in Cumulus Cells Support Oocyte Maturation in Bovine

    PubMed Central

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

    2014-01-01

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

  12. Steroid hormones promote bovine oocyte growth and connection with granulosa cells.

    PubMed

    Makita, Miho; Miyano, Takashi

    2014-09-01

    E2 and A4 maintained the connections between oocytes and granulosa cells during in vitro growth culture of bovine oocytes for 14 days, resulting in the complete oocyte growth and the acquisition of meiotic competence in more than half the oocytes.

  13. 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.

  14. Chromosome choreography: the meiotic ballet.

    PubMed

    Page, Scott L; Hawley, R Scott

    2003-08-08

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

  15. Meiotic Development in Caenorhabditis elegans

    PubMed Central

    Lui, Doris Y.

    2013-01-01

    Caenorhabditis elegans has become a powerful experimental organism with which to study meiotic processes that promote the accurate segregation of chromosomes during the generation of haploid gametes. Haploid reproductive cells are produced through one round of chromosome replication followed by two successive cell divisions. Characteristic meiotic chromosome structure and dynamics are largely conserved in C. elegans. Chromosomes adopt a meiosis-specific structure by loading cohesin proteins, assembling axial elements, and acquiring chromatin marks. Homologous chromosomes pair and form physical connections though synapsis and recombination. Synaptonemal complex and crossover formation allow for the homologs to stably associate prior to remodeling that facilitates their segregation. This chapter will cover conserved meiotic processes as well as highlight aspects of meiosis that are unique to C. elegans. PMID:22872477

  16. Impact of histone H4K16 acetylation on the meiotic recombination checkpoint in Saccharomyces cerevisiae

    PubMed Central

    Cavero, Santiago; Herruzo, Esther; Ontoso, David; San-Segundo, Pedro A.

    2016-01-01

    In meiotic cells, the pachytene checkpoint or meiotic recombination checkpoint is a surveillance mechanism that monitors critical processes, such as recombination and chromosome synapsis, which are essential for proper distribution of chromosomes to the meiotic progeny. Failures in these processes lead to the formation of aneuploid gametes. Meiotic recombination occurs in the context of chromatin; in fact, the histone methyltransferase Dot1 and the histone deacetylase Sir2 are known regulators of the pachytene checkpoint in Saccharomyces cerevisiae. We report here that Sas2-mediated acetylation of histone H4 at lysine 16 (H4K16ac), one of the Sir2 targets, modulates meiotic checkpoint activity in response to synaptonemal complex defects. We show that, like sir2, the H4-K16Q mutation, mimicking constitutive acetylation of H4K16, eliminates the delay in meiotic cell cycle progression imposed by the checkpoint in the synapsis-defective zip1 mutant. We also demonstrate that, like in dot1, zip1-induced phosphorylation of the Hop1 checkpoint adaptor at threonine 318 and the ensuing Mek1 activation are impaired in H4-K16 mutants. However, in contrast to sir2 and dot1, the H4-K16R and H4-K16Q mutations have only a minor effect in checkpoint activation and localization of the nucleolar Pch2 checkpoint factor in ndt80-prophase-arrested cells. We also provide evidence for a cross-talk between Dot1-dependent H3K79 methylation and H4K16ac and show that Sir2 excludes H4K16ac from the rDNA region on meiotic chromosomes. Our results reveal that proper levels of H4K16ac orchestrate this meiotic quality control mechanism and that Sir2 impinges on additional targets to fully activate the checkpoint. PMID:28357333

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

    PubMed

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

    2014-01-01

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

  18. Effects of dimethyl sulfoxide on asymmetric division and cytokinesis in mouse oocytes

    PubMed Central

    2014-01-01

    Background Dimethyl sulfoxide (DMSO) is used extensively as a permeable cryoprotectant and is a common solvent utilized for several water-insoluble substances. DMSO has various biological and pharmacological activities; however, the effect of DMSO on mouse oocyte meiotic maturation remains unknown. Results In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell–like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap formation and spindle migration. These features are among the primary causes of abnormal symmetric division; however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each “blastomere” of the 2-cell–like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each “blastomere” and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection (ICSI). Furthermore, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division failure. Conclusion Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric division. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI. PMID:24953160

  19. Effects of dimethyl sulfoxide on asymmetric division and cytokinesis in mouse oocytes.

    PubMed

    Zhou, Dongjie; Shen, Xinghui; Gu, Yanli; Zhang, Na; Li, Tong; Wu, Xi; Lei, Lei

    2014-06-21

    Dimethyl sulfoxide (DMSO) is used extensively as a permeable cryoprotectant and is a common solvent utilized for several water-insoluble substances. DMSO has various biological and pharmacological activities; however, the effect of DMSO on mouse oocyte meiotic maturation remains unknown. In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell-like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap formation and spindle migration. These features are among the primary causes of abnormal symmetric division; however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each "blastomere" of the 2-cell-like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each "blastomere" and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection (ICSI). Furthermore, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division failure. Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric division. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI.

  20. 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.

  1. Genetic studies in mice directly link oocytes produced during adulthood to ovarian function and natural fertility.

    PubMed

    Wang, Ning; Satirapod, Chonthicha; Ohguchi, Yasuyo; Park, Eun-Sil; Woods, Dori C; Tilly, Jonathan L

    2017-08-30

    Multiple labs have reported that mammalian ovaries contain oogonial stem cells (OSCs), which can differentiate into oocytes that fertilize to produce offspring. However, the physiological relevance of these observations to adult ovarian function is unknown. Here we performed targeted and reversible ablation of premeiotic germ cells undergoing differentiation into oocytes in transgenic mice expressing the suicide gene, herpes simplex virus thymidine kinase (HSVtk), driven by the promoter of stimulated by retinoic acid gene 8 (Stra8), a germ cell-specific gene activated during meiotic commitment. Over a 21-day ablation phase induced by the HSVtk pro-drug, ganciclovir (GCV), oocyte numbers declined due to a disruption of new oocyte input. However, germ cell differentiation resumed after ceasing the ablation protocol, enabling complete regeneration of the oocyte pool. We next employed inducible lineage tracing to fate map, through Cre recombinase-mediated fluorescent reporter gene activation only in Stra8-expressing cells, newly-formed oocytes. Induction of the system during adulthood yielded a mosaic pool of unmarked (pre-existing) and marked (newly-formed) oocytes. Marked oocytes matured and fertilized to produce offspring, which grew normally to adulthood and transmitted the reporter to second-generation offspring. These findings establish that oocytes generated during adulthood contribute directly to ovarian function and natural fertility in mammals.

  2. Probing the meiotic mechanism of intergenomic exchanges by genomic in situ hybridization on lampbrush chromosomes of unisexual Ambystoma (Amphibia: Caudata).

    PubMed

    Bi, Ke; Bogart, James P

    2010-04-01

    The meiotic mechanism of unisexual salamanders in the genus Ambystoma was previously explained by observing lampbrush chromosomes (LBCs). In polyploid unisexual females, a pre-meiotic endomitotic event doubles the chromosome number so that, after meiotic reduction, the mature eggs have the same ploidy as the female. It was assumed that synapses during meiotic I prophase, which result in observed bivalents, join duplicated sister chromosomes. Previous studies also found LBC quadrivalents in some oocytes that could be explained by occasional synapses between homologs. The discovery of widespread intergenomic exchanges among unisexual populations has prompted new speculations on this meiotic mechanism. Synapses that involve homeologous chromosomes may be frequent during meiosis and could be responsible for intergenomic exchanges and the high embryonic mortality of unisexuals. Furthermore, LBC quadrivalents may be established by associations between homeologous rather than homologous chromosomes. The present study investigated these two important aspects pertaining to the mechanism of intergenomic exchanges: the frequency of homeologous synapses and the relationship between homeologous associations and meiotic quadrivalents. We applied genomic in situ hybridization (GISH) on LBCs from oocytes of 14 triploid and two tetraploid unisexual females. Homeologous bivalents were not observed, and all 13 LBC quadrivalents that we found were the result of homologous synapses and were not associated with any homeologous or exchanged LBCs. Intergenomic exchanges were used as markers to compare the same chromosomes at meiotic diplotene and mitotic metaphase stages. We conclude that contemporary intergenomic exchanges are very rare, and no direct link exists between intergenomic exchanges and high embryonic mortality. The actual mechanisms and evolutionary implications of intergenomic exchanges appear to be complicated and difficult to assess. The application of GISH-type molecular

  3. [Mitochondrial and oocyte development].

    PubMed

    Deng, Wei-Ping; Ren, Zhao-Rui

    2007-12-01

    Oocyte development and maturation is a complicated process. The nuclear maturation and cytoplasmic maturation must synchronize which can ensure normal oocyte fertilization and following development. Mitochondrial is the most important cellular organell in cytoplasm, and the variation of its distribution during oocyte maturation, the capacity of OXPHOS generating ATP as well as the content or copy number or transcription level of mitochondrial DNA play an important role in oocyte development and maturation. Therefore, the studies on the variation of mitochondrial distribution, function and mitochondrial DNA could enhance our understanding of the physiology of reproduction and provide new insight to solve the difficulties of assisted reproduction as well as cloning embryo technology.

  4. 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

  5. The cohesion stabilizer sororin favors DNA repair and chromosome segregation during mouse oocyte meiosis.

    PubMed

    Huang, Chun-Jie; Yuan, Yi-Feng; Wu, Di; Khan, Faheem Ahmed; Jiao, Xiao-Fei; Huo, Li-Jun

    2017-03-01

    Maintenance and timely termination of cohesion on chromosomes ensures accurate chromosome segregation to guard against aneuploidy in mammalian oocytes and subsequent chromosomally abnormal pregnancies. Sororin, a cohesion stabilizer whose relevance in antagonizing the anti-cohesive property of Wings-apart like protein (Wapl), has been characterized in mitosis; however, the role of Sororin remains unclear during mammalian oocyte meiosis. Here, we show that Sororin is required for DNA damage repair and cohesion maintenance on chromosomes, and consequently, for mouse oocyte meiotic program. Sororin is constantly expressed throughout meiosis and accumulates on chromatins at germinal vesicle (GV) stage/G2 phase. It localizes onto centromeres from germinal vesicle breakdown (GVBD) to metaphase II stage. Inactivation of Sororin compromises the GVBD and first polar body extrusion (PBE). Furthermore, Sororin inactivation induces DNA damage indicated by positive γH2AX foci in GV oocytes and precocious chromatin segregation in MII oocytes. Finally, our data indicate that PlK1 and MPF dissociate Sororin from chromosome arms without affecting its centromeric localization. Our results define Sororin as a determinant during mouse oocyte meiotic maturation by favoring DNA damage repair and chromosome separation, and thereby, maintaining the genome stability and generating haploid gametes.

  6. A role for retrotransposon LINE-1 in fetal oocyte attrition in mice.

    PubMed

    Malki, Safia; van der Heijden, Godfried W; O'Donnell, Kathryn A; Martin, Sandra L; Bortvin, Alex

    2014-06-09

    Fetal oocyte attrition (FOA) is a conserved but poorly understood process of elimination of more than two-thirds of meiotic prophase I (MPI) oocytes before birth. We now implicate retrotransposons LINE-1 (L1), activated during epigenetic reprogramming of the embryonic germline, in FOA in mice. We show that wild-type fetal oocytes possess differential nuclear levels of L1ORF1p, an L1-encoded protein essential for L1 ribonucleoprotein particle (L1RNP) formation and L1 retrotransposition. We demonstrate that experimental elevation of L1 expression correlates with increased MPI defects, FOA, oocyte aneuploidy, and embryonic lethality. Conversely, reverse transcriptase (RT) inhibitor AZT has a profound effect on the FOA dynamics and meiotic recombination, and it implicates an RT-dependent trigger in oocyte elimination in early MPI. We propose that FOA serves to select oocytes with limited L1 activity that are therefore best suited for the next generation. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    PubMed

    Lin, Zi-Li; Kim, Nam-Hyung

    2015-06-01

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

  8. Altered cohesin gene dosage affects Mammalian meiotic chromosome structure and behavior.

    PubMed

    Murdoch, Brenda; Owen, Nichole; Stevense, Michelle; Smith, Helen; Nagaoka, So; Hassold, Terry; McKay, Michael; Xu, Huiling; Fu, Jun; Revenkova, Ekaterina; Jessberger, Rolf; Hunt, Patricia

    2013-01-01

    Based on studies in mice and humans, cohesin loss from chromosomes during the period of protracted meiotic arrest appears to play a major role in chromosome segregation errors during female meiosis. In mice, mutations in meiosis-specific cohesin genes cause meiotic disturbances and infertility. However, the more clinically relevant situation, heterozygosity for mutations in these genes, has not been evaluated. We report here evidence from the mouse that partial loss of gene function for either Smc1b or Rec8 causes perturbations in the formation of the synaptonemal complex (SC) and affects both synapsis and recombination between homologs during meiotic prophase. Importantly, these defects increase the frequency of chromosomally abnormal eggs in the adult female. These findings have important implications for humans: they suggest that women who carry mutations or variants that affect cohesin function have an elevated risk of aneuploid pregnancies and may even be at increased risk of transmitting structural chromosome abnormalities.

  9. Meiotic functions of RAD18.

    PubMed

    Inagaki, Akiko; Sleddens-Linkels, Esther; Wassenaar, Evelyne; Ooms, Marja; van Cappellen, Wiggert A; Hoeijmakers, Jan H J; Seibler, Jost; Vogt, Thomas F; Shin, Myung K; Grootegoed, J Anton; Baarends, Willy M

    2011-08-15

    RAD18 is an ubiquitin ligase that is involved in replication damage bypass and DNA double-strand break (DSB) repair processes in mitotic cells. Here, we investigated the testicular phenotype of Rad18-knockdown mice to determine the function of RAD18 in meiosis, and in particular, in the repair of meiotic DSBs induced by the meiosis-specific topoisomerase-like enzyme SPO11. We found that RAD18 is recruited to a specific subfraction of persistent meiotic DSBs. In addition, RAD18 is recruited to the chromatin of the XY chromosome pair, which forms the transcriptionally silent XY body. At the XY body, RAD18 mediates the chromatin association of its interaction partners, the ubiquitin-conjugating enzymes HR6A and HR6B. Moreover, RAD18 was found to regulate the level of dimethylation of histone H3 at Lys4 and maintain meiotic sex chromosome inactivation, in a manner similar to that previously observed for HR6B. Finally, we show that RAD18 and HR6B have a role in the efficient repair of a small subset of meiotic DSBs.

  10. 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.

  11. 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

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

    PubMed Central

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

    2016-01-01

    Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule–chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation. PMID:26564158

  13. Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line.

    PubMed

    Jaramillo-Lambert, Aimee; Ellefson, Marina; Villeneuve, Anne M; Engebrecht, JoAnne

    2007-08-01

    The replication of chromosomes in meiosis is an important first step for subsequent chromosomal interactions that promote accurate disjunction in the first of two segregation events to generate haploid gametes. We have developed an assay to monitor DNA replication in vivo in mitotic and meiotic germline nuclei of the nematode Caenorhabditis elegans. Using mutants that affect the mitosis/meiosis switch, we show that meiotic S phase is at least twice as long as mitotic S phase in C. elegans germ cell nuclei. Furthermore, our assay reveals that different regions of the genome replicate at different times, with the heterochromatic-like X chromosomes replicating at a distinct time from the autosomes. Finally, we have exploited S-phase labeling to monitor the timing of progression through meiotic prophase. Meiotic prophase for oocyte production in hermaphrodites lasts 54-60 h. Further, we find that the duration of the pachytene sub-stage is modulated by the presence of sperm. On the other hand, meiotic prophase for sperm production in males is completed by 20-24 h. Possible sources for the sex-specific differences in meiotic prophase kinetics are discussed.

  14. 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.

  15. The fission yeast meiotic checkpoint kinase Mek1 regulates nuclear localization of Cdc25 by phosphorylation.

    PubMed

    Pérez-Hidalgo, Livia; Moreno, Sergio; San-Segundo, Pedro A

    2008-12-01

    In eukaryotic cells, fidelity in transmission of genetic information during cell division is ensured by the action of cell cycle checkpoints. Checkpoints are surveillance mechanisms that arrest or delay cell cycle progression when critical cellular processes are defective or when the genome is damaged. During meiosis, the so-called meiotic recombination checkpoint blocks entry into meiosis I until recombination has been completed, thus avoiding aberrant chromosome segregation and the formation of aneuploid gametes. One of the key components of the meiotic recombination checkpoint is the meiosis-specific Mek1 kinase, which belongs to the family of Rad53/Cds1/Chk2 checkpoint kinases containing forkhead-associated domains. In fission yeast, several lines of evidence suggest that Mek1 targets the critical cell cycle regulator Cdc25 to delay meiotic cell cycle progression. Here, we investigate in more detail the molecular mechanism of action of the fission yeast Mek1 protein. We demonstrate that Mek1 acts independently of Cds1 to phosphorylate Cdc25, and this phosphorylation is required to trigger cell cycle arrest. Using ectopic overexpression of mek1(+) as a tool to induce in vivo activation of Mek1, we find that Mek1 promotes cytoplasmic accumulation of Cdc25 and results in prolonged phosphorylation of Cdc2 at tyrosine 15. We propose that at least one of the mechanisms contributing to the cell cycle delay when the meiotic recombination checkpoint is activated in fission yeast is the nuclear exclusion of the Cdc25 phosphatase by Mek1-dependent phosphorylation.

  16. Nonrandom segregation of the mouse univalent X chromosome: evidence of spindle-mediated meiotic drive.

    PubMed Central

    LeMaire-Adkins, R; Hunt, P A

    2000-01-01

    A fundamental principle of Mendelian inheritance is random segregation of alleles to progeny; however, examples of distorted transmission either of specific alleles or of whole chromosomes have been described in a variety of species. In humans and mice, a distortion in chromosome transmission is often associated with a chromosome abnormality. One such example is the fertile XO female mouse. A transmission distortion effect that results in an excess of XX over XO daughters among the progeny of XO females has been recognized for nearly four decades. Utilizing contemporary methodology that combines immunofluorescence, FISH, and three-dimensional confocal microscopy, we have readdressed the meiotic segregation behavior of the single X chromosome in oocytes from XO females produced on two different inbred backgrounds. Our studies demonstrate that segregation of the univalent X chromosome at the first meiotic division is nonrandom, with preferential retention of the X chromosome in the oocyte in approximately 60% of cells. We propose that this deviation from Mendelian expectations is facilitated by a spindle-mediated mechanism. This mechanism, which appears to be a general feature of the female meiotic process, has implications for the frequency of nondisjunction in our species. PMID:11014823

  17. Synapsis and meiotic recombination analyses: MLH1 focus in the XY pair as an indicator.

    PubMed

    Codina-Pascual, Montserrat; Oliver-Bonet, Maria; Navarro, Joaquima; Campillo, Mercè; García, Ferran; Egozcue, Susana; Abad, Carlos; Egozcue, Josep; Benet, Jordi

    2005-08-01

    Anomalies in meiotic prophase I have been related to partial or total meiotic arrest. These anomalies include an abnormal synaptic process, resulting in disorders in meiotic recombination. In the present study, we analyse primary spermatocytes from 12 infertile men (four with non-obstructive azoospermia, six with oligoastenoteratozoospermia, one with astenoteratozoospermia and one normozoospermic) and five control fertile donors using immunocytological techniques for synaptonemal complex, meiotic recombination and centromeric proteins. Mean numbers of MLH1 foci per cell, frequencies of cells presenting an MLH1 focus in the XY pair and percentages of cells affected by abnormal synaptic patterns (gaps and splits) are reported for each of the infertile patients and control men. A positive correlation between the frequency of cells showing a recombination focus in the XY pair and the number of autosomal recombination foci per cell is found. Reduced recombination in the XY pair and an increased number of cells affected by gaps may explain some idiopathic male infertility cases. The results suggest that recombination in the XY pair could be an indicator for general recombination frequency and for a successful meiotic process.

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

    PubMed Central

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

    2016-01-01

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

  19. Mitochondrial biogenesis and degradation are induced by CCCP treatment of porcine oocytes.

    PubMed

    Itami, N; Shiratsuki, S; Shirasuna, K; Kuwayama, T; Iwata, H

    2015-08-01

    In this study, we investigated the mitochondrial quality control system in porcine oocytes during meiotic maturation. Cumulus cell oocyte complexes (COCs) collected from gilt ovaries were treated with 10  μM carbonyl cyanide-m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) for 2  h. The CCCP treatment was found to significantly reduce ATP content, increase the amount of phosphorylated AMP-activated protein kinase and elevate reactive oxygen species levels in oocytes. When the CCCP-treated COCs were cultured further for 44  h in maturation medium, the ATP levels were restored and the parthenogenetic developmental rate of oocytes to the blastocyst stage was comparable with that of untreated COCs. To examine the effects of CCCP treatment of oocytes on the kinetics of mitochondrial DNA copy number (Mt number), COCs treated with 0 or 10  μM CCCP were cultured for 44  h, after which the Mt number was determined by RT-PCR. CCCP treatment was found to increase the Mt number in the modified maturation medium in which mitochondrial degradation was inhibited by MG132, whereas CCCP treatment did not affect the Mt number in the maturation medium lacking MG132. The relative gene expression of TFAM was furthermore shown to be significantly higher in CCCP-treated oocytes than in untreated oocytes. Taken together, the finding presented here suggest that when the mitochondria are injured, mitochondrial biogenesis and degradation are induced, and that these processes may contribute to the recuperation of oocytes.

  20. Induction and activation of meiosis and subsequent parthenogenetic development of growing pig oocytes using calcium ionophore A23187.

    PubMed

    Sedmíková, Markéta; Burdová, Jana; Petr, Jaroslav; Etrych, Milan; Rozinek, Jirí; Jílek, Frantisek

    2003-12-01

    The pig ovary contains a large number of growing oocytes, which do not mature in vitro and cannot be readily used in various biotechnologies. This study was conducted to determine the possibility of inducing meiotic maturation in growing pig oocytes with an internal diameter of 110 microm, which had developed partial meiotic competence. Most of these oocytes spontaneously stopped maturation at the metaphase I stage (68%); a limited number proceeded to the metaphase II stage (26%). Treatment with calcium ionophore A23187 (50 microM for 5 or 10 min) after 24h in vitro culture overcame the block at the metaphase I stage, and treated growing pig oocytes matured to the metaphase II stage (66%). Oocytes in which maturation had been induced by calcium ionophore were again treated with calcium ionophore. Up to 58% of the treated oocytes were activated. Parthenogenetic development in oocytes treated with ionophore for meiosis induction and activation was very limited. The portion which reached morula stage did not exce