Induction of maturation-promoting factor during Xenopus oocyte maturation uncouples Ca2+ store depletion from store-operated Ca2+ entry

Department of Physiology and Biophysics, University of Arkansas Medical Science, Little Rock, AR 72205 During oocyte maturation, eggs acquire the ability to generate specialized Ca2+ signals in response to sperm entry. Such Ca2+ signals are crucial for egg activation and the initiation of embryonic development. We examined the regulation during Xenopus oocyte maturation of store-operated Ca2+ entry (SOCE), an important Ca2+ influx pathway in oocytes and other nonexcitable cells. We have previously shown that SOCE inactivates during Xenopus oocyte meiosis. SOCE inactivation may be important in preventing premature egg activation. In this study, we investigated the correlation between SOCE inactivation and the Mos–mitogen-activated protein kinase (MAPK)–maturation-promoting factor (MPF) kinase cascade, which drives Xenopus oocyte maturation. SOCE inactivation at germinal vesicle breakdown coincides with an increase in the levels of MAPK and MPF. By differentially inducing Mos, MAPK, and MPF, we demonstrate that the activation of MPF is necessary for SOCE inactivation during oocyte maturation. In contrast, sustained high levels of Mos kinase and the MAPK cascade have no effect on SOCE activation. We further show that preactivated SOCE is not inactivated by MPF, suggesting that MPF does not block Ca2+ influx through SOCE channels, but rather inhibits coupling between store depletion and SOCE activation.

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Antioxidants Stimulate Germinal Vesicle Breakdown, But Inhibit Chromosome Formation And Spindle Assembly In Porcine Oocytes

Microscopy and Microanalysis ◽

10.1017/s1431927600037314 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 964-965

Author(s):

Qing-Yuan Sun ◽

Randall S. Prather ◽

Heide Schatten

Keyword(s):

Oocyte Maturation ◽

Germinal Vesicle ◽

Spindle Assembly ◽

Mouse Oocyte ◽

Germinal Vesicle Breakdown ◽

Oocyte Meiosis ◽

First Polar Body ◽

Porcine Oocyte ◽

Chromosome Formation

Mammalian oocytes are arrested at the diplotene stage of the first meiotic division. Release of oocytes from their follicles induces meiotic resumption characterized by germinal vesicle breakdown (GVBD), followed by the chromosome formation and metaphase I spindle organization and finally the extrusion the first polar body. Recently it was shown that cellpermeant antioxidants significantly inhibit spontaneous resumption of meiosis in mouse oocytes, which may indicate a role of oxygen radicals in oocyte maturation. The regulation of mouse oocyte meiosis resumption is different from that of large domestic animals in that GVBD is independent of Ca2+ and protein synthesis. The present study investigated the influence of two cell-permeant antioxidants, 2(3)-ter-butyl-4-hydroxyanisole (BHA) and nordihydroguaiaretic acid (NDGA), on porcine oocyte meiosis resumption, chromatin behavior and spindle assembly. Our findings revealed a different role of antioxidants in porcine oocyte meiosis resumption than in mouse oocyte maturation.

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259 Cdk7 AND CYCLIN H, BUT NOT Mat1, ARE INVOLVED IN MEIOTIC RESUMPTION OF PORCINE IMMATURE OOCYTE

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab259 ◽

2011 ◽

Vol 23 (1) ◽

pp. 227

Author(s):

W. Fujii ◽

T. Nishimura ◽

K. Kano ◽

K. Naito

Keyword(s):

Western Blot ◽

Oocyte Maturation ◽

Kinase Activity ◽

Germinal Vesicle ◽

Immature Oocyte ◽

Culture Period ◽

Regulatory Subunit ◽

Rt Pcr ◽

Germinal Vesicle Breakdown ◽

Maturation Promoting Factor

The complex kinase Cdk-activating kinase (CAK) consists of the catalytic subunit Cdk7, regulatory subunit Cyclin H, and assembly factor Mat1. The CAK is essential for maturation-promoting factor (MPF) activation by phosphorylating threonine 161 (T161) of Cdc2 in mitosis. Although it is known that meiotic resumption of oocytes is regulated by MPF activity, the role of CAK in meiosis is still unclear. In the present study, we attempted to confirm the involvement of CAK in meiotic resumption of porcine immature oocyte. Cumulus–oocyte complexes (COC) were collected from antral follicles of gilts and cultured up to 48 h in TYH medium containing 20% porcine follicular fluid, 3.2 mg/mL of BSA, and 1.0 IU mL–1 of pregnant mare serum gonadotropin. The T161 phosphorylation level of Cdc2 in cultured oocytes was analysed by Western blot analysis. The transcripts were collected from noncultured or cultured oocytes, and Cdk7, Cyclin H, and Mat1 expression were detected by RT-PCR. Overexpression of Cdc2 or inhibition of Cdk7, Cyclin H, and Mat1 during oocyte maturation was performed by microinjection of mRNA or antisense RNA into ooplasm of immature COC and verified by Western blot or semiquantitative RT-PCR. Maturation-promoting factor kinase activity was assayed by Histone H1 kinase activity assay. Statistical analyses in this study were carried out by Student’s t-test. The T161 phosphorylation of Cdc2 was found during the culture period from 18 h to 48h, which was after germinal vesicle breakdown (GVB). Overexpression of Cdc2 increased the incidence of GVB at 18 h, but overexpression of mutant Cdc2 (replaced T161 by alanine) had no influence on GVB. These results indicate that T161 phosphorylation of Cdc2 is important for meiotic resumption. Next, we attempted to confirm the CAK function during oocyte maturation. Transcripts of Cdk7, Cyclin H, and Mat1 were detectable throughout the culture period. Inhibition of Cdk7 and Cyclin H caused a decrease in T161 phosphorylation and MPF activity, and the incidence of GVB was significantly lower than in nontreated oocytes. In contrast, Mat1-inhibited oocytes resumed meiosis and developed to the metaphase II stage, and the incidence was not different between Mat1-inhibited oocytes and nontreated oocytes. These results suggest that Cdk7 and Cyclin H are working as CAK and activate Cdc2 by T161 phosphorylation, although Mat1 is dispensable during oocyte maturation.

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The potential roles of c-Jun N-terminal kinase (JNK) during the maturation and aging of oocytes produced by a marine protostome worm

Zygote ◽

10.1017/s0967199417000533 ◽

2017 ◽

Vol 25 (6) ◽

pp. 686-696 ◽

Cited By ~ 2

Author(s):

Stephen A. Stricker ◽

Niharika Ravichandran

Keyword(s):

Oocyte Maturation ◽

Germinal Vesicle ◽

Germinal Vesicle Breakdown ◽

Specific Antibodies ◽

Immature Oocytes ◽

Jnk Inhibitors ◽

Jnk Activation ◽

Mode Of Death ◽

Maturation Promoting Factor

SummaryPrevious investigations have indicated that c-Jun N-terminal kinase (JNK) regulates the maturation and aging of oocytes produced by deuterostome animals. In order to assess the roles of this kinase in a protostome, oocytes of the marine nemertean worm Cerebratulus were stimulated to mature and subsequently aged before being probed with phospho-specific antibodies against active forms of JNK and maturation-promoting factor (MPF). Based on blots of maturing oocytes, a 40-kD putative JNK is normally activated during germinal vesicle breakdown (GVBD), which begins at 30 min post-stimulation with seawater, whereas treating immature oocytes with JNK inhibitors downregulates both the 40-kD JNK signal and GVBD, collectively suggesting a 40-kD JNK may facilitate oocyte maturation. Along with this JNK activity, mature oocytes also exhibit high levels of MPF at 2 h post-stimulation. However, by ~6–8 h post-GVBD, mature oocytes lose the 40-kD JNK signal, and at ~20–30 h of aging, an ~48-kD phospho-JNK band arises as oocytes deactivate MPF and begin to lyse during a necroptotic-like mode of death. Accordingly, JNK inhibitors reduce the aging-related 48-kD JNK phosphorylation while maintaining MPF activity and retarding oocyte degradation. Such findings suggest that a 48-kD JNK may help deactivate MPF and trigger death. Possible mechanisms by which JNK activation either together with, or independently of, protein neosynthesis might stimulate oocyte degradation are discussed.

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Ca2+cyt negatively regulates the initiation of oocyte maturation

The Journal of Cell Biology ◽

10.1083/jcb.200309138 ◽

2004 ◽

Vol 165 (1) ◽

pp. 63-75 ◽

Cited By ~ 35

Author(s):

Lu Sun ◽

Khaled Machaca

Keyword(s):

Cell Cycle ◽

Oocyte Maturation ◽

Cell Cycle Progression ◽

Germinal Vesicle ◽

Germinal Vesicle Breakdown ◽

Cycle Progression ◽

Nuclear Maturation ◽

Oocyte Meiosis ◽

Kinase Cascade ◽

Cell Cycle Machinery

Ca2+ is a ubiquitous intracellular messenger that is important for cell cycle progression. Genetic and biochemical evidence support a role for Ca2+ in mitosis. In contrast, there has been a long-standing debate as to whether Ca2+ signals are required for oocyte meiosis. Here, we show that cytoplasmic Ca2+ (Ca2+cyt) plays a dual role during Xenopus oocyte maturation. Ca2+ signals are dispensable for meiosis entry (germinal vesicle breakdown and chromosome condensation), but are required for the completion of meiosis I. Interestingly, in the absence of Ca2+cyt signals oocytes enter meiosis more rapidly due to faster activation of the MAPK-maturation promoting factor (MPF) kinase cascade. This Ca2+-dependent negative regulation of the cell cycle machinery (MAPK-MPF cascade) is due to Ca2+cyt acting downstream of protein kinase A but upstream of Mos (a MAPK kinase kinase). Therefore, high Ca2+cyt delays meiosis entry by negatively regulating the initiation of the MAPK-MPF cascade. These results show that Ca2+ modulates both the cell cycle machinery and nuclear maturation during meiosis.

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Activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are not required for the global histone deacetylation observed after germinal vesicle breakdown (GVBD) in porcine oocytes

Reproduction ◽

10.1530/rep.1.00924 ◽

2006 ◽

Vol 131 (3) ◽

pp. 439-447 ◽

Cited By ~ 12

Author(s):

Tsutomu Endo ◽

Kunihiko Naito ◽

Sachi Kume ◽

Yukio Nishimura ◽

Koji Kashima ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Protein Kinase ◽

Germinal Vesicle ◽

Histone Deacetylation ◽

Mitogen Activated Protein Kinase ◽

Germinal Vesicle Breakdown ◽

Immature Oocytes ◽

Mitogen Activated Protein ◽

Maturation Promoting Factor

The acetylation of nuclear core histone has been suggested to work as an epigenetic mark for transmitting gene expression patterns to daughter cells. Global histone deacetylations, presumably involved in the reprogramming of the gene expression, have been observed after germinal vesicle breakdown (GVBD) in a cell cycle-dependent manner during meiotic maturation of mouse and porcine oocytes, although the regulation mechanism of histone deacetylation has not been studied well. In the present study, we examined the involvement of a crucial cell-cycle-regulator, maturation-promoting factor (MPF), and a meiosis-related kinase, mitogen-activated protein kinase (MAPK), in the global histone deacetylation during porcine oocyte maturation. In order to know whether the activities of MPF and MAPK were required, or the breakdown of GV membrane was sufficient, for the global histone deacetylation observed after GVBD, we artificially destroyed the GV membrane of the porcine immature oocytes. The artificial GV destruction (AGVD) induced histone deacetylation without the activation of MPF and MAPK. This deacetylation after AGVD was not affected by an MPF inhibitor, roscovitine, or an inhibitor of protein synthesis, cycloheximide, but was completely prevented by an inhibitor of histone deactylases (HDACs), trichostatine A. HDAC1 was present in the GV of the immature oocytes and localized on chromosomes after GVBD and AGVD. These results suggest that the MPF and MAPK activities were dispensable and the breakdown of the GV membrane was sufficient for the global histone deacetylation, which was catalyzed by HDAC activity

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Pervasive 3′-UTR Isoform Switches During Mouse Oocyte Maturation

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.727614 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yuanlin He ◽

Qiuzhen Chen ◽

Jing Zhang ◽

Jing Yu ◽

Meng Xia ◽

...

Keyword(s):

Oocyte Maturation ◽

Molecular Mechanisms ◽

Developmental Stages ◽

Germinal Vesicle ◽

Differentially Expressed ◽

Sequencing Data ◽

Germinal Vesicle Breakdown ◽

Oocyte Meiosis ◽

Gene Level ◽

Individual Mouse

Oocyte maturation is the foundation for developing healthy individuals of mammals. Upon germinal vesicle breakdown, oocyte meiosis resumes and the synthesis of new transcripts ceases. To quantitatively profile the transcriptomic dynamics after meiotic resumption throughout the oocyte maturation, we generated transcriptome sequencing data with individual mouse oocytes at three main developmental stages: germinal vesicle (GV), metaphase I (MI), and metaphase II (MII). When clustering the sequenced oocytes, results showed that isoform-level expression analysis outperformed gene-level analysis, indicating isoform expression provided extra information that was useful in distinguishing oocyte stages. Comparing transcriptomes of the oocytes at the GV stage and the MII stage, in addition to identification of differentially expressed genes (DEGs), we detected many differentially expressed transcripts (DETs), some of which came from genes that were not identified as DEGs. When breaking down the isoform-level changes into alternative RNA processing events, we found the main source of isoform composition changes was the alternative usage of polyadenylation sites. With detailed analysis focusing on the alternative usage of 3′-UTR isoforms, we identified, out of 3,810 tested genes, 512 (13.7%) exhibiting significant switches of 3′-UTR isoforms during the process of moues oocyte maturation. Altogether, our data and analyses suggest the importance of examining isoform abundance changes during oocyte maturation, and further investigation of the pervasive 3′-UTR isoform switches in the transition may deepen our understanding on the molecular mechanisms underlying mammalian early development.

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A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK

Development ◽

10.1242/dev.129.9.2129 ◽

2002 ◽

Vol 129 (9) ◽

pp. 2129-2139 ◽

Cited By ~ 2

Author(s):

Marion Peter ◽

Jean-Claude Labbé ◽

Marcel Dorée ◽

Elisabeth Mandart

Keyword(s):

Protein Kinase ◽

Oocyte Maturation ◽

Xenopus Oocyte ◽

Xenopus Oocytes ◽

Mapk Pathway ◽

Germinal Vesicle ◽

Mapk Cascade ◽

Germinal Vesicle Breakdown ◽

Mapk Activation

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation, leading us to question what the Mos target might be if Mos is still required. We now demonstrate that Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF. We have found that Myt1 is likely to be the Mos target in this process, as Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. We propose that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes.

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Requirement for phosphorylation of cyclin B1 for Xenopus oocyte maturation.

Molecular Biology of the Cell ◽

10.1091/mbc.6.9.1111 ◽

1995 ◽

Vol 6 (9) ◽

pp. 1111-1124 ◽

Cited By ~ 40

Author(s):

J Li ◽

A N Meyer ◽

D J Donoghue

Keyword(s):

Biological Activity ◽

Oocyte Maturation ◽

Xenopus Oocyte ◽

Biological Significance ◽

Germinal Vesicle ◽

Cyclin B1 ◽

Site Directed Mutagenesis ◽

Germinal Vesicle Breakdown ◽

Rapid Kinetics ◽

Phosphopeptide Mapping

Maturation-promoting factor, consisting of cdc2 protein kinase and a regulatory B-type cyclin, is a universal regulator of meiosis and mitosis in eukaryotes. In Xenopus, there are two subtypes of B-type cyclins, designated B1 and B2, both of which are phosphorylated. In this study, we have investigated the biological significance of this phosphorylation for Xenopus cyclin B1 during meiotic maturation. We have used a combination of site-directed mutagenesis and phosphopeptide-mapping to identify serine residues 2, 94, 96, 101, and 113 as presumptive phosphorylation sites, and together these sites account for all cyclin B1 phosphorylation in oocytes before germinal vesicle breakdown (GVBD). Single Ser-->Ala mutants as well as multiple site mutants have been constructed and characterized. Phosphorylation of cyclin B1 appears to be required for Xenopus oocyte maturation, based on the significantly diminished ability of the quintuple Ala mutant to induce oocyte maturation. Furthermore, partial phosphorylation of these five sites is sufficient to meet this requirement. Phosphorylation of cyclin B1 is not required for cdc2 kinase activity, for binding to cdc2 protein, for stability of cyclin B1 before GVBD, or for destruction of cyclin B1 after GVBD or after egg activation. A quintuple Glu mutant was also constructed, with serine residues 2, 94, 96, 101, and 113 mutated to Glu. In contrast to the quintuple Ala mutant, the quintuple Glu mutant was able to induce oocyte maturation efficiently, and with more rapid kinetics than wild-type cyclin B1. These data confirm that phosphorylation, as mimicked by Ser-->Glu mutations, confers enhanced biological activity to cyclin B1. Possible roles of cyclin B1 phosphorylation are discussed that might account for the increased biological activity of the quintuple Glu mutant.

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Nucleus, Cytoskeleton, and Mitogen-Activated Protein Kinase p38 Dynamics during In Vitro Maturation of Porcine Oocytes

Animals ◽

10.3390/ani9040163 ◽

2019 ◽

Vol 9 (4) ◽

pp. 163

Author(s):

Payungsuk Intawicha ◽

Li-Kuang Tsai ◽

Shih-Ying Yen ◽

Neng-Wen Lo ◽

Jyh-Cherng Ju

Keyword(s):

Oocyte Maturation ◽

Mammalian Cells ◽

In Vitro Maturation ◽

Germinal Vesicle ◽

Mitogen Activated Protein Kinase ◽

Meiotic Maturation ◽

Germinal Vesicle Breakdown ◽

Mitogen Activated Protein ◽

Porcine Oocyte

The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p < 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p < 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p < 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm.

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Involvement of microRNA-335-5p in cytoskeleton dynamics in mouse oocytes

Reproduction Fertility and Development ◽

10.1071/rd12138 ◽

2013 ◽

Vol 25 (4) ◽

pp. 691 ◽

Cited By ~ 20

Author(s):

Xiang-Shun Cui ◽

Shao-Chen Sun ◽

Yong-Kook Kang ◽

Nam-Hyung Kim

Keyword(s):

Germinal Vesicle ◽

Mitogen Activated Protein Kinase ◽

Germinal Vesicle Breakdown ◽

Oocyte Meiosis ◽

Extracellular Signal ◽

Multiple Processes ◽

Mitogen Activated Protein ◽

Actin Nucleator ◽

Cytoskeleton Dynamics ◽

Multiple Poles

MicroRNA is a short RNA molecule expressed in eukaryotic cells that is involved in multiple processes, including translational repression, target degradation and gene silencing. However, its specific role(s) in these processes remains largely unknown, especially in terms of germ cell development. The present study identified a microRNA, namely miR-335-5p, that is involved in mouse oocyte meiosis. MiR-335-5p was highly expressed in oocytes, but levels decreased markedly shortly after fertilisation. Microinjection of miR-335-5p or its inhibitor into oocytes resulted in a higher proportion of 2-cell-like MII oocytes and oocytes at the germinal vesicle breakdown and/or MI stage, indicating failure of asymmetric oocyte division. This may be due to regulation of actin because perturbation of miR-335-5p resulted in reduced expression of actin nucleator Daam1, a member of the Formin family. Moreover, injection of miR-335-5p or its inhibitor resulted in aberrant spindle morphology, namely an elongated spindle and multiple poles spindle. After injection of oocytes, levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) decreased, suggesting that miR-335-5p may regulate spindle formation via the mitogen-activated protein kinase pathway. Overexpression and inhibition of miR-335-5p had no effect on embryo development. Together, the results of the present study indicate that miR-335-5p is a novel regulator expressed in oocytes that is involved in cytoskeleton dynamics.

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