Meiosis Arrest Research Articles

CNP/NPRB PLAYS AN IMPORTANT ROLE IN FOLLICULAR DEVELOPMENT AND OOCYTE MATURATION

Natriuretic peptide receptor B (NPRB) is the specific receptor for Ctype natriuretic peptides (CNP) expressed in various tissues including bones, brains, kidney, and male and female reproductive organs. NPRB transduce extracellular CNP signals to intracellular production of second messenger, cyclic GMP (cGMP). Although CNP and NPRB are highly expressed in ovaries, the role of CNP/NPRB in ovarian function remains unclear. CN (achondroplasia) is mutant mouse strain with loss of function mutation in the gene encoding NPRB showing chondrodyspalstic phenotype and both male and female infertility. The infertility of the CN female mice suggested an important role of CNP/NPRB in female reproductive system. In the present study, we investigated ovarian phenotypes of the CN mouse to reveal the possible functions of CNP/NPRB in female reproductive system. The CN female mice showed irregular estrous cycle and number of ovulated oocytes was significant less than that of normal mice. While ovulated oocytes were surrounded cumulus oophorus in normal mice, all ovulated oocytes of the CN mice at 0.5dpc were completely devoid of cumulus oophorus and no oocyte of the CN mouse developed to two-cell stage at 1.5dpc, indicating impaired ability of fertilization. Furthermore, no metaphase chromosomes and spindle structure was observed in the ovulated oocytes of the CN mouse while those of normal mice showed metaphase chromosomes and spindles which indicate arrest of meiosis at metaphase II. Histological analysis of the CN ovary showed reduced thickness and incomplete expansion of cumulus oophorus in antral follicles. Furthermore, most oocytes in antral follicle of the CN ovary showed lack of germinal vesicles and some of them showed metaphase chromosomes and denatured and fragmented cytoplasm indicating premature resumption of the meiotic arrest at the prophase of meiosis. These findings suggested that the infertile phenotypes of the CN mice is caused by defect of cumulus cell proliferation and expansion and/or premature resumption of meiosis during oogenesis, as well as essential role of CNP/NPRB signal in follicular development and oocyte maturation. (poster)

Mutations of SYCP3 are rare in infertile Spanish men with meiotic arrest

No functional SYCP3 exonic SNP was found in infertile Spanish patients with meiosis arrest, suggesting that SYCP3 mutations very likely are not relevant in Spain in genetic susceptibility to meiosis arrest, just as in other studied European populations.

Phosphorylation of Erp1 by p90rsk is required for cytostatic factor arrest in Xenopus laevis eggs

Until fertilization, the meiotic cell cycle of vertebrate eggs is arrested at metaphase of meiosis II by a cytoplasmic activity termed cytostatic factor (CSF), which causes inhibition of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets mitotic cyclins-regulatory proteins of meiosis and mitosis-for degradation. Recent studies indicate that Erp1/Emi2, an inhibitor protein for the APC/C, has an essential role in establishing and maintaining CSF arrest, but its relationship to Mos, a mitogen-activated protein kinase (MAPK) kinase kinase that also has an essential role in establishing CSF arrest through activation of p90 ribosomal S6 kinase (p90rsk), is unclear. Here we report that in Xenopus eggs Erp1 is a substrate of p90rsk, and that Mos-dependent phosphorylation of Erp1 by p90rsk at Thr 336, Ser 342 and Ser 344 is crucial for both stabilizing Erp1 and establishing CSF arrest in meiosis II oocytes. Semi-quantitative analysis with CSF-arrested egg extracts reveals that the Mos-dependent phosphorylation of Erp1 enhances, but does not generate, the activity of Erp1 that maintains metaphase arrest. Our results also suggest that Erp1 inhibits cyclin B degradation by binding the APC/C at its carboxy-terminal destruction box, and this binding is also enhanced by the Mos-dependent phosphorylation. Thus, Mos and Erp1 collaboratively establish and maintain metaphase II arrest in Xenopus eggs. The link between Mos and Erp1 provides a molecular explanation for the integral mechanism of CSF arrest in unfertilized vertebrate eggs.

Critical roles for Dicer in the female germline

Dicer is an essential component of RNA interference (RNAi) pathways, which have broad functions in gene regulation and genome organization. Probing the consequences of tissue-restricted Dicer loss in mice indicates a critical role for Dicer during meiosis in the female germline. Mouse oocytes lacking Dicer arrest in meiosis I with multiple disorganized spindles and severe chromosome congression defects. Oogenesis and early development are times of significant post-transcriptional regulation, with controlled mRNA storage, translation, and degradation. Our results suggest that Dicer is essential for turnover of a substantial subset of maternal transcripts that are normally lost during oocyte maturation. Furthermore, we find evidence that transposon-derived sequence elements may contribute to the metabolism of maternal transcripts through a Dicer-dependent pathway. Our studies identify Dicer as central to a regulatory network that controls oocyte gene expression programs and that promotes genomic integrity in a cell type notoriously susceptible to aneuploidy.

Translational Unmasking of Emi2 Directs Cytostatic Factor Arrest in Meiosis II

Cytostatic factor (CSF) arrests unfertilized vertebrate eggs in metaphase of meiosis II by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from mediating cyclin destruction. The APC/C inhibitor Emi2/XErp1 satisfies a number of historical criteria for the molecular identification of CSF, but the mechanism by which CSF is activated selectively in meiosis II is the remaining unexplained criterion. Here we provide an explanation by showing that Emi2 is expressed specifically in meiosis II through translational de-repression or “unmasking” of its mRNA. We find that Emi2 protein is undetectable in immature, G2/prophase-arrested Xenopus oocytes and accumulates ~90 minutes after germinal vesicle breakdown. The 3’ untranslated region of Emi2 mRNA contains cytoplasmic polyadenylation elements that directly bind the CPEB protein and confer temporal regulation of Emi2 polyadenylation and translation. Our results demonstrate that cytoplasmic polyadenylation and translational unmasking of Emi2 directs meiosis II-specific CSF arrest.

Regulation of meiotic maturation1

Mammalian oocytes are arrested at prophase of the first meiotic division before induction of maturation by the preovulatory LH surge. In vitro, oocyte maturation occurs spontaneously. The first meiotic arrest is characterized by a large nucleus called the germinal vesicle. One important signaling molecule for resumption of meiosis is cyclic AMP (cAMP). High levels of cAMP block spontaneous meiotic resumption. Research investigating the regulation of oocyte cAMP has led to the discovery of new receptors, guanosine 5'-triphosphate-binding (G) proteins, cyclases, and phosphodiesterases. Leydig insulin-like 3, a polypeptide growth factor of the insulin family, is expressed in thecal cells. Leydig insulin-like 3 activates the Leu-rich, repeat-containing, G protein-coupled receptor 8, which is expressed in the oocyte. Coupled to the inhibitory GTP binding protein, this receptor leads to a decrease in cAMP production. Treatment with Leydig insulin-like 3 polypeptide initiates meiotic progression of oocytes in preovulatory follicles, demonstrating the importance of cAMP management for meiotic resumption. Furthermore, microinjection of an antibody against stimulatory G protein (Gs) into mouse oocytes results in meiotic resumption, suggesting that meiotic arrest of the oocyte is dependent on Gs activity. The orphan Gs-linked receptor, GPR3, is expressed in the oocyte. The oocytes of GPR3-null mice resume meiosis when still in their follicles, suggesting that GPR3 is involved in the control of cAMP production and thus meiotic arrest. Cyclic nucleotides are synthesized by cyclases and degraded by phosphodiesterases. Mouse and rat oocytes express isoform 3 of adenylyl cyclase. In the mouse, the null mutation results in approximately 50% of the oocytes resuming meiosis, demonstrating the importance of the synthesis of cAMP in controlling nuclear maturation. The null mutation of the major phosphodiesterase expressed in mouse oocytes results in female sterility due to ovulation of meiotically arrested oocytes that cannot be fertilized. Maintenance of meiotic arrest is explained by constitutive cAMP signaling associated with undetectable cAMP-phosphodiesterase activity. Collectively, these results are beginning to illuminate the key signaling molecules involved in the control of intraoocyte cAMP levels, thus regulating the arrest and resumption of meiosis.

The Drosophila Calcipressin Sarah Is Required for Several Aspects of Egg Activation

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.

Effect of gonadotropins during in vitro maturation of feline oocytes on oocyte–cumulus cells functional coupling and intracellular concentration of glutathione

Information about the mechanisms of meiotic arrest and resumption of meiosis in feline oocytes is still limited. The aim of this study was to investigate the effect of the presence of gonadotropins during IVM, on meiotic progression in relation to the status of gap junction mediated communications between oocyte and cumulus cells, to the cAMP intracellular content, and to the intra-oocyte concentration of glutathione (GSH) in feline oocytes. Our results indicated that about 50% of cumulus–oocyte complexes (COCs) showed functionally open communications at the time of collection, while the remainder were partially or totally closed. After 3 h of culture, the percentage of COCs with functional gap junctions was significantly greater in the group matured in the presence of gonadotropins than in those matured without them, where an interruption of communications was observed. Moreover, this precocious uncoupling was associated with a moderate increase of cAMP concentration in the oocyte, lower than in the group exposed to gonadotropins. Intra-oocyte glutathione levels decreased significantly after 24 h of IVM, whether gonadotropins were present or absent during the culturing process. The presence of thiol compounds in the IVM medium induced an intra-oocyte GSH concentration significantly higher than that found in oocytes cultured without these compounds, and similar to the GSH content of immature oocytes. Moreover, the intracellular GSH concentration increased as meiosis progressed. The present study suggests that in feline oocytes, gonadotropins affect the dynamic changes in communications between oocyte and cumulus cells during IVM. However, the intracellular concentration of GSH is not influenced by the gonadotropin stimulation. Moreover, the presence of gonadotropins and thiol compounds results in an increase of GSH levels along with meiotic progression of the oocytes.

117. Meiosis arrest in a new animal model - a mutation on chromosome 5

Many causes of male infertility are currently unknown and there is a great need for the identification of new genes involved in spermatogenesis. The chemical mutagen, N-ethyl-N-nitrosourea (ENU) was utilized to induce random point mutations in the germline of C57Bl-6J mice in order to generate models of male infertility. Three generation breeding programs produced mice homozygous for ~3–4 point mutations. Following a phenotype-based screen of 1650 G3 males from 155 pedigrees, 15 lines were identified with abnormal male fertility parameters consistent with a recessive mutation. One infertile line, ENU23, exhibited a complete meiosis arrest with no germ cells ever proceeding to become spermatozoa. No infertile females have been observed. Light microscopic examination of Bouin’s fixed paraffin embedded testis sections showed that the arrest occurred post-prophase and prior to the completion of metaphase I, since normal metaphase was not observed. In cells beyond the prophase stage of development, spermatocytes appeared condensed, became enlarged and subsequently underwent cell death by apoptosis confirmed by TUNEL analysis. Electron microscopy visualized normal synaptonemal complexes. The degenerating primary spermatocytes exhibited condensed chromosomes irregularly arranged on a poorly formed microtubular spindle, indicating an abortive attempt to complete metaphase. The percentage of tubules with diplotene/metaphase-like cells was calculated for 3 abnormal and 3 normal ENU23 mice and showed 10.8% compared to 1.37%, respectively, indicating that prior to cell death the cells undergo a lag period. To identify the ENU23 causal mutation, the mutation was bred onto a mixed C57Bl-6J/CBA background and linkage analysis was performed, identifying a region on chromosome 5 between microsatellite markers D5Mit7 and D5Mit32. Candidate genes within this region are currently being sequenced in the search for the ENU-induced mutation causing the ENU23 phenotype. We believe this model of metaphase arrest is unique and will provide insights into the male specific events of meiosis.

The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes

In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.

A serotonin receptor antagonist induces oocyte maturation in both frogs and mice: Evidence that the same G protein‐coupled receptor is responsible for maintaining meiosis arrest in both species

Accumulating evidence has indicated that vertebrate oocytes are arrested at late prophase (G2 arrest) by a G protein coupled receptor (GpCR) that activates adenylyl cyclases. However, the identity of this GpCR or its regulation in G2 oocytes is unknown. We demonstrated that ritanserin (RIT), a potent antagonist of serotonin receptors 5-HT2R and 5-HT7R, released G2 arrest in denuded frog oocytes, as well as in follicle-enclosed mouse oocytes. In contrast to RIT, several other serotonin receptor antagonists (mesulergine, methiothepine, and risperidone) had no effect on oocyte maturation. The unique ability of RIT, among serotonergic antagonists, to induce GVBD did not match the antagonist profile of any known serotonin receptors including Xenopus 5-HT7R, the only known G(s)-coupled serotonin receptor cloned so far in this species. Unexpectedly, injection of x5-HT7R mRNA in frog oocytes resulted in hormone-independent frog oocyte maturation. The addition of exogenous serotonin abolished x5-HT7R-induced oocyte maturation. Furthermore, the combination of x5-HT7R and exogenous serotonin potently inhibited progesterone-induced oocyte maturation. These results provide the first evidence that a G-protein coupled receptor related to 5-HT7R may play a pivotal role in maintaining G2 arrest in vertebrate oocytes.

Cadmium at a non-toxic dose alters gene expression in mouse testes

The testes are important targets of cadmium (Cd)-induced toxicity and carcinogenicity in rodents. Exposure to Cd at environmentally relevant low levels is a significant human health concern, but the effects of Cd on the rodent testes at doses that do not cause overt lesions are poorly defined. We used cDNA microarray and quantitative real-time RT-PCR assays to determine gene expression profiles in the testes of CD-1 mice 12–72 h after a single s.c. injection of 5 μmol/kg CdCl 2. This dose of Cd did not produce overt histopathological changes, but clearly altered the expression of some genes that are likely to be important in toxicity responses. The most significant changes in gene expression occurred 24 h after treatment, corresponding to when the highest level of Cd was detected in the testes. Increased expression of the C- myc and Egr1 genes strongly suggests acute stress responses. Repressed expression of cell cycle-regulated cyclin B1 and CDC2 proteins indicates a potential for causing G2/M arrest and disturbance of meiosis. Decreased expression of pro-apoptotic genes, particularly Casp3, and DNA repair genes possibly contributes to Cd-induced carcinogenesis. These results indicate that changes in gene expression occur well before overt effects of Cd-induced testicular toxicity and carcinogenicity are apparent.

Function and interaction of maturation-promoting factor and mitogen-activated protein kinase during meiotic maturation and fertilization of oocyte*

Abstract Mitogen-activated protein kinase (MAP kinase) cascade and maturation-promoting factor (MPF) play very important roles during meiotic maturation and fertilization of oocyte. Interaction between MAP kinase and MPF influences meiotic maturation and fertilization of oocyte throughout the animal kingdom, including stimulation of germinal vesicle breakdown (GVBD), suppression of DNA replication, control of meiotic chromosome segregation, maintenance of metaphase II arrest, and resumption and completion of second meiosis. This review focuses on the function and interaction of MAP kinase and MPF during meiotic maturation and fertilization of oocyte.

A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis.

Androgens control spermatogenesis, but germ cells themselves do not express a functional androgen receptor (AR). Androgen regulation is thought to be mediated by Sertoli and peritubular myoid cells, but their relative roles and the mechanisms involved remain largely unknown. Using Cre/loxP technology, we have generated mice with a ubiquitous knockout of the AR as well as mice with a selective AR knockout in Sertoli cells (SC) only. Mice with a floxed exon 2 of the AR gene were crossed with mice expressing Cre recombinase ubiquitously or selectively in SC (under control of the anti-Müllerian hormone gene promoter). AR knockout males displayed a complete androgen insensitivity phenotype. Testes were located abdominally, and germ cell development was severely disrupted. In contrast, SC AR knockout males showed normal testis descent and development of the male urogenital tract. Expression of the homeobox gene Pem, which is androgen-regulated in SC, was severely decreased. Testis weight was reduced to 28% of that in WT littermates. Stereological analysis indicated that the number of SC was unchanged, whereas numbers of spermatocytes, round spermatids, and elongated spermatids were reduced to 64%, 3%, and 0% respectively of WT. These changes were associated with increased germ cell apoptosis and grossly reduced expression of genes specific for late spermatocyte or spermatid development. It is concluded that cell-autonomous action of the AR in SC is an absolute requirement for androgen maintenance of complete spermatogenesis, and that spermatocyte/spermatid development/survival critically depends on androgens.

126.A repository of ENU mutant mouse lines and their potential for male fertility research

One in 25 western men are infertile and the causal factor is frequently unknown, although it is expected that many are genetic in origin. My project aims to identify genes critical to mouse spermatogenesis using ENU mutagenesis. A further aim was to develop a repository of mutant mice and data on their fertility parameters for use by the reproductive biology community. This research will aid the diagnosis and development of specific treatments for human infertility and the development of contraceptive agents. The potent mutagen N-ethyl-N-nitrosourea (ENU) was utilized to generate libraries of C57BL/6 mice with random point mutations throughout their genomes. A 3 generational breeding program produced mice that were homozygous for a number of mutations. I subsequently performed a number of large scale screens on 3rd generation males, identifying lines carrying recessive mutations specifically affecting male fertility. Thus far we have observed a wide range of abnormal testis phenotypes including Sertoli Cell only, hypospermatogenesis, meiosis arrest, abnormal sperm morphology and abnormal hormone levels. From these analyses a repository including all data and tissues collected from 1200 3rd generation male mice from 122 different lines has been developed and will become publicly available. This includes testis and epididymal histology and serum levels of FSH, LH, activin A and inhibin. Further, I have stored gDNA long term and cryopreserved sperm to enable regeneration of lines in the future. In addition, I have developed a high throughput mutation screening protocol for the detection of mutations within genes of interest using denaturing high performance liquid chromatography (DHPLC). Collectively, our repository and gene screening techniques can be used in conjunction with artificial reproductive technologies to generate mouse models reflective of human conditions and altered specific gene function.

Spindle checkpoint proteins Mad1 and Mad2 are required for cytostatic factor-mediated metaphase arrest.

In cells containing disrupted spindles, the spindle assembly checkpoint arrests the cell cycle in metaphase. The budding uninhibited by benzimidazole (Bub) 1, mitotic arrest-deficient (Mad) 1, and Mad2 proteins promote this checkpoint through sustained inhibition of the anaphase-promoting complex/cyclosome. Vertebrate oocytes undergoing meiotic maturation arrest in metaphase of meiosis II due to a cytoplasmic activity termed cytostatic factor (CSF), which appears not to be regulated by spindle dynamics. Here, we show that microinjection of Mad1 or Mad2 protein into early Xenopus laevis embryos causes metaphase arrest like that caused by Mos. Microinjection of antibodies to either Mad1 or Mad2 into maturing oocytes blocks the establishment of CSF arrest in meiosis II, and immunodepletion of either protein blocked the establishment of CSF arrest by Mos in egg extracts. A Mad2 mutant unable to oligomerize (Mad2 R133A) did not cause cell cycle arrest in blastomeres or in egg extracts. Once CSF arrest has been established, maintenance of metaphase arrest requires Mad1, but not Mad2 or Bub1. These results suggest a model in which CSF arrest by Mos is mediated by the Mad1 and Mad2 proteins in a manner distinct from the spindle checkpoint.

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

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

Synaptonemal complex defects in a man with nonobstructive azoospermia

Objective: We have previously demonstrated an association between decreased recombination and nondisjunction in sperm. The aim of this study was to analyze early stages of meiosis in infertile men to determine if any abnormalities in chromosome pairing or recombination could be discerned. Design:The stages of meiotic prophase, extent of chromosome pairing and the number of recombination foci were compared in a man with nonobstructive azoospermia and controls. Materials and Methods: Immunofluorescence methods were used to identify the synaptonemal complex in various stages of prophase. Antibodies were used to identify the synaptonemal complex (SCP1/SCP3), the centromere (CREST) and sites of recombination (MLH1). Results: Only 36 meiotic spreads could be recovered from the infertile man with nonobstructive azoospermia compared to hundreds of spreads available from controls. One-third of his cells were delayed in the early stages of meiosis (zygotene). The infertile man had a greatly reduced frequency of recombination with a mean of only 33.4% foci (range 1–67) compared to a mean of 50 foci (range 36–67) in controls. Also 34.8% of pachytene cells had at least one bivalent with no recombinant foci and 52.2% of cells had unpaired chromosome regions. Lack of chromosome pairing is known to cause meiotic arrest in humans. Conclusion:This is the first infertile man identified to have an arrest in meiosis because of an error in the processing of recombination foci or a defect in SCP 1 leading to incomplete chromosome pairing.

Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro.

In vitro use of arresters of meiosis could improve cytoplasmic maturation of immature oocytes by controlling the period of prophase I. Phosphodiesterases (PDE) are responsible for the breakdown and concomitant inactivation of the cyclic nucleotides cAMP and cGMP and are implicated in the regulation of oocyte meiotic maturation. Selective inhibitors of phosphodiesterase type 3 (PDE3) prevent meiotic resumption of mammalian oocytes. This study evaluated the impact of meiosis arrest by PDE3 inhibitor, Org 9935, on developmental competence of geminal vesicle (GV)-stage oocytes from small antral follicles. Cumulus-oocyte complexes (COC), retrieved from antral follicles 24 h after eCG exposure and cultured in the presence of PDE3 inhibitor (10 microM) for an additional 24 h, remained arrested in the meiotic prophase. The GV configuration of oocytes before and after the arrest by PDE3 inhibitor was examined. After the period of meiosis arrest, a significantly increased proportion of oocytes had acquired a nucleolus surrounded by a condensed chromatin rim at the GV, which is a morphological correlate of transcriptional repression. Removal of inhibitor resulted in 90.6% +/- 8.3% of oocytes with the first polar body extruded. Fertilization was significantly improved in oocytes that had been arrested compared with oocytes collected 24 h after eCG and undergoing in vitro maturation immediately. Embryonic preimplantation and live offspring rates of arrested oocytes were higher, although not significantly, than those of nonarrested oocytes. These results suggest that a temporal block of meiosis by PDE3 inhibitor promotes developmental competence of mice oocytes retrieved from small antral follicles.

Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH.

Reinitiation of meiosis in oocytes usually occurs as a two-step process during which release from the prophase block is followed by an arrest in metaphase of the first or second meiotic division [metaphase I (MI) or metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly understood, although it is a widely observed phenomenon in invertebrates. The blockage of fully grown starfish oocytes in prophase of meiosis I is released by the hormone 1-methyladenine. It has been believed that meiosis of starfish oocytes proceeds completely without MI or MII arrest, even when fertilization does not occur. Here we show that MI arrest of starfish oocytes occurs in the ovary after germinal vesicle breakdown. This arrest is maintained both by the Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH in the ovary before spawning. Immediately after spawning into seawater, activation of Na+/H+ antiporters via a heterotrimeric G protein coupling to a 1-methyladenine receptor in the oocyte leads to an intracellular pH increase that can overcome the MI arrest even in the presence of active MAP kinase.