Null Oocytes Research Articles

Oocyte-Specific Deletion of Slc6a9 Encoding the GLYT1 Glycine Transporter Eliminates Glycine Transport in Mouse Preimplantation Embryos and Their Ability to Counter Hypertonic Stress.

Early preimplantation mouse embryos are sensitive to increased osmolarity, which can block their development. To overcome this, they accumulate organic osmolytes to maintain cell volume. The main organic osmolyte used by early mouse embryos is glycine. Glycine is transported during the mature egg and 1-cell to 4-cell embryo stages by a transporter identified as GLYT1, encoded by the Slc6a9 gene. Here, we have produced an oocyte-specific knockout of Slc6a9 by crossing mice that have a segment of the gene flanked by LoxP elements with transgenic mice expressing iCre driven by the oocyte-specific Gdf9 promoter. Slc6a9 null oocytes failed to develop glycine transport activity during meiotic maturation. However, females with these oocytes were fertile. When enclosed in their cumulus-oocyte complex, Slc6a9 null oocytes could accumulate glycine via GLYT1 transport in their coupled cumulus cells, which may support female fertility in vivo. In vitro, embryos derived from Slc6a9 null oocytes displayed a clear phenotype. While glycine rescued complete preimplantation development of wild type embryos from increased osmolarity, embryos derived from null oocytes failed to develop past the 2-cell stage even with glycine. Thus, Slc6a9 is required for glycine transport and protection against increased osmolarity in mouse eggs and early embryos.

The CRL4-DCAF13 ubiquitin E3 ligase supports oocyte meiotic resumption by targeting PTEN degradation.

Cullin ring-finger ubiquitin ligase 4 (CRL4) has multiple functions in the maintenance of oocyte survival and meiotic cell cycle progression. DCAF13, a novel CRL4 adaptor, is essential for oocyte development. But the mechanisms by which CRL4-DCAF13 supports meiotic maturation remained unclear. In this study, we demonstrated that DCAF13 stimulates the meiotic resumption-coupled activation of protein synthesis in oocytes, partially by maintaining the activity of PI3K signaling pathway. CRL4-DCAF13 targets the polyubiquitination and degradation of PTEN, a lipid phosphatase that inhibits PI3K pathway as well as oocyte growth and maturation. Dcaf13 knockout in oocytes caused decreased CDK1 activity and impaired meiotic cell cycle progression and chromosome condensation defects. As a result, chromosomes fail to be aligned at the spindle equatorial plate, the spindle assembly checkpoint is activated, and most Dcaf13 null oocytes are arrested at the prometaphase I. The DCAF13-dependent PTEN degradation mechanism fits in as a missing link between CRL4 ubiquitin E3 ligase and PI3K pathway, both of which are crucial for translational activation during oocyte GV-MII transition.

Evaluation of members of the TGFß superfamily as candidates for the oocyte factors that control mouse cumulus expansion and steroidogenesis

Oocytes secrete factors that control cumulus and granulosa functions, including cumulus expansion and steroid hormone production. Some members of the transforming growth factor ß (TGFß) superfamily influence these activities, yet it is still not determined conclusively whether any of these superfamily members are the previously reported oocyte-secreted factors. The aim of this study was to examine the effects of TGFß1 and growth differentiation factor 9 (GDF-9) on cumulus expansion and progesterone production by mouse oocytectomized (OOX) complexes in culture. TGFß1 mimics the effects of oocytes by both enabling cumulus expansion and inhibiting progesterone production; however, neutralizing antibodies to TGFß1 in cultures of cumulus-oocyte complexes (COCs) or in co-cultures of OOX complexes failed to inhibit the ability of oocytes to enable cumulus expansion or inhibit progesterone production. Activin A had no effect on progesterone production by OOX complexes. In experiments using oocytes obtained from mice with deficient expression of GDF-9, OOX complexes cultured in the presence of heterozygous oocytes were capable of full expansion, whereas OOX complexes cultured with oocytes from GDF-9 null mice did not expand. Similarly, GDF-9 null oocytes failed to suppress FSH-induced progesterone production by OOX complexes. These results support the hypothesis that GDF-9 is the cumulus expansion enabling factor produced by mouse oocytes and that GDF-9 also inhibits cumulus progesterone production; however, the possibility remains that loss of GDF-9 may indirectly affect the ability of oocytes to produce the factors that regulate cumulus cell activity.

Maternal gene Ooep may participate in homologous recombination-mediated DNA double-strand break repair in mouse oocytes.

DNA damage in oocytes can cause infertility and birth defects. DNA double-strand breaks (DSBs) are highly deleterious and can substantially impair genome integrity. Homologous recombination (HR)-mediated DNA DSB repair plays dominant roles in safeguarding oocyte quantity and quality. However, little is known regarding the key players of the HR repair pathway in oocytes. Here, we identified oocyte-specific gene Ooep as a novel key component of the HR repair pathway in mouse oocytes. OOEP was required for efficient ataxia telangiectasia mutated (ATM) kinase activation and Rad51 recombinase (RAD51) focal accumulation at DNA DSBs. Ooep null oocytes were defective in DNA DSB repair and prone to apoptosis upon exogenous DNA damage insults. Moreover, Ooep null oocytes exhibited delayed meiotic maturation. Therefore, OOEP played roles in preserving oocyte quantity and quality by maintaining genome stability. Ooep expression decreased with the advance of maternal age, suggesting its involvement in maternal aging.

ALADIN is required for the production of fertile mouse oocytes.

Asymmetric cell divisions depend on the precise placement of the spindle apparatus. In mammalian oocytes, spindles assemble close to the cell's center, but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphase. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygously null for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages, including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is compromised due to problems in spindle orientation and anchoring at the first meiotic anaphase. ALADIN null oocytes that mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions.

Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras.

ABSTRACTThe mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1−/− null mouse embryos die but a previous study showed that some homozygous Gpi1−/− null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1−/−↔Gpi1c/c chimaera with functional Gpi1−/− null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1−/− null cells in adult Gpi1−/−↔Gpi1c/c chimaeras and determine if Gpi1−/− null germ cells are functional. Analysis of adult Gpi1−/−↔Gpi1c/c chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1−/− null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1−/− null oocytes in one female Gpi1−/−↔Gpi1c/c chimaera were functional and provided preliminary evidence that one male putative Gpi1−/−↔Gpi1c/c chimaera produced functional spermatozoa from homozygous Gpi1−/− null germ cells. Although the male chimaera was almost certainly Gpi1−/−↔Gpi1c/c, this part of the study is considered preliminary because only blood was typed for GPI. Gpi1−/− null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1−/− null germ cells, it successfully identified functional Gpi1−/− null oocytes and revealed that some Gpi1−/− null cells could survive in many adult tissues.

ERK1/2 Activities Are Dispensable for Oocyte Growth but Are Required for Meiotic Maturation and Pronuclear Formation in Mouse

Previous studies revealed that extracellular regulated kinase-1 and -2 (ERK1/2) cascade plays pivotal roles in regulating oocyte meiotic cell cycle progression. However, most knowledge about the in vivo function of ERK1/2 in mammalian oocytes was indirectly obtained from analyzing the phenotypes of Mos knockout mice. In this study, we knocked out Erk1 and Erk2 in mouse oocytes as early as the primordial follicle stage using the well-characterized Gdf9-Cre mouse model, and for the first time directly investigated the in vivo function of ERK1/2 in mouse oocytes. In this novel mouse model, we observed that ERK1/2 activities in oocyte are dispensable for primordial follicle maintenance, activation and follicle growth. Different from the Mos null oocytes, the ERK1/2-deleted oocytes had well-assembled spindles at metaphase I (MI), extruded polar body-1 (PB1) with normal sizes, and did not undergo a full parthenogenetic activation characterized for pronuclear formation. However, the ovulated ERK1/2-deleted oocytes had poorly-assembled metaphase II (MII) spindles, spontaneously released polar body-2 (PB2), and were arrested at another metaphase called metaphase III (MIII). In addition, ERK1/2 deletion prevented male pronuclear formation after fertilization, and caused female infertility. In conclusion, these results indicate that ERK1/2 activities are required for not only MII-arrest maintenance, but also efficient pronuclear formation in mouse oocytes.

Essential Role for endogenous siRNAs during meiosis in mouse oocytes.

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

Dynamics of genomic 5-hydroxymethylcytosine during mouse oocyte growth.

Recent studies of the demethylation process in murine zygotes have shown that 5-methylcytosine (5mC) is first converted into 5-hydroxymethylcytosine (5hmC) or further-oxidized cytosines in the paternal genome by the maternal ten-eleven translocation 3 (TET3) enzyme. This process is crucial for normal embryogenesis, and our aim was to elucidate the effect of Tet3 on the maternal genome during female germ-line development. Immunofluorescence analysis showed that 5hmC was clearly present in fully grown oocytes but not in nongrowing and early growth-stage oocytes. The 5hmC in the maternal genome was clearly detectable in DNA methyltransferase 3-like enzyme (Dnmt3L)-null oocytes and their fertilized zygotes, although Dnmt3L is essential for DNA methylation in oocytes. An analysis using an enzyme digestion-based method showed that 5hmC was present in LTR retrotransposons from the late growth period of oocytes. Quantitative RT-PCR analysis showed that Tet3 expression was enhanced during oocyte growth and exhibited an approximately 40-fold increase between nongrowing and fully grown oocytes. Our results show that 5hmC is generated since the oocyte growth stage, accompanied by up-regulation of Tet3; 5hmC is located mainly in LTR retrotransposons, indicating that 5hmC generated in growth-stage oocytes is responsible for genomewide demethylation after fertilization.

Two-Cell Embryo Developmental Block Is Due to Lack of Cytoplasmic Lattices in Mouse Antral NSN Oocytes.

In most mammals, the antral compartment of the ovary contains two different kinds of oocytes, SN and NSN (Surrounded Nucleolus and Not Surrounded Nucleolus), whose main differentiating characteristic lies in their acquisition of developmental competence: still inexplicably only the SN (70% of the antral oocytes population) are able to develop to the blastocyst stage while the NSN arrest at the two-cell stage. Taking advantage of proteomics and microscopy techniques, we analyzed both SN and NSN oocytes, showing for the first time that NSN-derived embryo inability to develop to the blastocyst stage is due to the under-regulation of some maternal proteins together with a lack of cytoplasmic lattices (CPLs). Additionally, we analyzed the lipid droplets content, showing that NSN oocyte cytoplasm contains a significantly higher amount of lipid droplets than SN. The proteomic profile of 600 antral oocytes (300 SN and 300 NSN) showed up-regulation of 6 proteins in SN and 23 in NSN; in particular, when compared to SN oocytes, NSN showed a significant down-regulation of the proteins MATER and FILIA. MATER is the main constituent of the Subcortical Maternal Complex and it is considered one of the factors responsible for the acquisition of meiotic competence. In fact, it is already known that Matertm/tm oocytes, without any distinction based on chromatin organization, do not have cytoplasmic lattices and are not able to progress beyond the two-cell stage. Based on our proteomic results, we speculated that the Matertm/tm antral population should be composed mostly of NSN oocytes. To test this hypothesis, we analyzed Matertm/tm antral oocytes showing that 84% of these oocytes are of the NSN type. To confirm this data, we performed Transmission Electron Microscopy analysis on SN and NSN oocytes, revealing that NSN contain no or very few cytoplasmic lattices as compared to SN. For this reason, the absence of CPLs can be considered a NSN-specific molecular morphology feature. MATER has also been correlated with the anti-apoptotic pathway, so the accumulation of lipid droplets in Mater null oocytes could be related to apoptotic activity. Through the morphometric analysis of lipid droplet content, we demonstrated that NSN oocytes contain a significantly higher amount of lipid droplets than SN. This is further evidence of the role played by CPLs in the resumption of meiosis and of lipid droplets being good candidate markers for oocyte developmental competence. Our results provide the first molecular evidence that accounts for NSN-derived embryo's inability to progress beyond the two-cell stage and, thus, for some of the naturally occurring pre-implantation losses in mammals. These data will also give basic knowledge about the function of the miniature, wonderfully organized laboratory of molecular biology that is the oocyte.

Zinc Requirement During Meiosis I–Meiosis II Transition in Mouse Oocytes Is Independent of the MOS-MAPK Pathway1

Zinc is essential for many biological processes, including proper functioning of gametes. We recently reported that zinc levels rise by over 50% during oocyte maturation and that attenuation of zinc availability during this period could be achieved using the membrane-permeable heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). This zinc insufficiency resulted in formation of large polar bodies, failure to establish metaphase II arrest, and impaired establishment of cortical polarity. As these phenotypes resemble those of MOS null oocytes, we examined the impact of zinc insufficiency on the MOS-MAPK pathway. Reduced levels of both MOS protein and phosphorylation of MAP2K1/2 are observed in zinc-insufficient oocytes; however, these differences appear only after completion of the first meiotic division. In addition, activation of the downstream effector of the MOS pathway, MAPK3/1, is not affected by zinc insufficiency, and reduced MOS levels are observed only with the presence of TPEN after the first polar body extrusion. These data are inconsistent with the hypothesis that reduced MOS mediates the observed phenotype. Finally, MOS overexpression does not rescue the phenotype of zinc-insufficient oocytes, confirming that the observed disruption of asymmetric division and spindle abnormalities cannot be attributed to impaired MOS signaling. Zinc-insufficient oocytes do not increase maturation promoting factor (MPF) activity following the first meiotic division, and increasing MPF activity through expression of nondegradable cyclin B1 partially rescues the ability of zinc-insufficient oocytes to enter metaphase II. Although we have shown that zinc has a novel role in the meiotic cell cycle, it is not mediated through the MOS-MAPK pathway.

MicroRNA Function Is Globally Suppressed in Mouse Oocytes and Early Embryos

Dicer, which is required for the processing of both microRNAs (miRNAs) and small interfering RNAs (siRNAs), is essential for oocyte maturation [1, 2]. Oocytes express both miRNAs and endogenous siRNAs (endo-siRNAs) [3, 4]. To determine whether the abnormalities in Dicer knockout oocytes during meiotic maturation are secondary to the loss of endo-siRNAs and/or miRNAs, we deleted Dgcr8, which encodes an RNA-binding protein specifically required for miRNA processing. In striking contrast to Dicer, Dgcr8-deficient oocytes matured normally and, when fertilized with wild-type sperm, produced healthy-appearing offspring, even though miRNA levels were reduced to similar levels as Dicer-deficient oocytes. Furthermore, the deletion of both maternal and zygotic Dgcr8 alleles did not impair preimplantation development, including the determination of the inner cell mass and trophectoderm. Most surprisingly, the mRNA profiles of wild-type and Dgcr8 null oocytes were essentially identical, whereas Dicer null oocytes showed hundreds of misregulated transcripts. These findings show that miRNA function is globally suppressed during oocyte maturation and preimplantation development and that endo-siRNAs, rather than miRNAs, underlie the Dicer knockout phenotype in oocytes.

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

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.

Loss of surface EWI‐2 on CD9 null oocytes

CD9, a member of the tetraspanin family, associates with a variety of other proteins to form the tetraspanin web. CD9 forms direct and relatively stable associations with the immunoglobulin superfamily proteins EWI-2 and EWI-F. Deletion of the Cd9 gene results in female infertility since Cd9 null mice produce oocytes that fail to fuse. It is thought that the absence of CD9 causes the inability of the oocytes to fuse. In this study, we report that the expression level of EWI-2 on the Cd9(-/-) oocyte surface is <10% of the wild-type level. Hence, the severe reduction in EWI-2 activity may be responsible for the loss of fusion ability. An entirely different mutant of CD9, not a deletion but a depalmitoylated construct, does not affect in vivo female fertility suggesting that the palmitate modification of CD9 is not essential for its putative fusion function. Additionally, the level of EWI-2 on the surface of the oocytes from these females was comparable to the EWI-2 level on wild-type oocytes. We also found that soluble, recombinant EWI-2 binds preferentially to acrosome-reacted sperm but the bound EWI-2 does not inhibit sperm-oocyte fusion. Overall, the results indicate that deletion of CD9, which is known to have multiple associations, may have pleiotropic effects on function that will require further dissection.

Can the presence of wild‐type oocytes during insemination rescue the fusion defect of CD9 null oocytes?

Can the presence of wild‐type oocytes during insemination rescue the fusion defect of CD9 null oocytes?

An Inside Job for siRNAs

Among the three main categories of small silencing RNAs in insects and mammals-siRNAs, miRNAs, and piRNAs-siRNAs were thought to arise primarily from exogenous sources, whereas miRNAs and piRNAs arise from endogenous loci. Recent work in flies and mice reveals several classes of endogenous siRNAs (endo-siRNAs) that contribute to functions previously reserved for miRNAs and piRNAs, including gene regulation and transposon suppression.

Generation of mouse oocytes defective in cAMP synthesis and degradation: Endogenous cyclic AMP is essential for meiotic arrest

Although it is established that cAMP accumulation plays a pivotal role in preventing meiotic resumption in mammalian oocytes, the mechanisms controlling cAMP levels in the female gamete have remained elusive. Both production of cAMP via GPCRs/Gs/adenylyl cyclases endogenous to the oocyte as well as diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that preclude maturation. Here we have used a genetic approach to investigate the different biochemical pathways contributing to cAMP accumulation and maturation in mouse oocytes. Because cAMP hydrolysis is greatly decreased and cAMP accumulates above a threshold, oocytes deficient in PDE3A do not resume meiosis in vitro or in vivo, resulting in complete female infertility. In vitro, inactivation of Gs or downregulation of the GPCR GPR3 causes meiotic resumption in the Pde3a null oocytes. Crossing of Pde3a−/− mice with Gpr3−/− mice causes partial recovery of female fertility. Unlike the complete meiotic block of the Pde3a null mice, oocyte maturation is restored in the double knockout, although it occurs prematurely as described for the Gpr3−/− mouse. The increase in cAMP that follows PDE3A ablation is not detected in double mutant oocytes, confirming that GPR3 functions upstream of PDE3A in the regulation of oocyte cAMP. Metabolic coupling between oocytes and granulosa cells was not affected in follicles from the single or double mutant mice, suggesting that diffusion of cAMP is not prevented. Finally, simultaneous ablation of GPR12, an additional receptor expressed in the oocyte, does not modify the Gpr3−/− phenotype. Taken together, these findings demonstrate that Gpr3 is epistatic to Pde3a and that fertility as well as meiotic arrest in the PDE3A-deficient oocyte is dependent on the activity of GPR3. These findings also suggest that cAMP diffusion through gap junctions or the activity of additional receptors is not sufficient by itself to maintain the meiotic arrest in the mouse oocyte.

Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution

Microvilli are found on the surface of many cell types, including the mammalian oocyte, where they are thought to act in initial contact of sperm and oocyte plasma membranes. CD9 is currently the only oocyte protein known to be required for sperm–oocyte fusion. We found CD9 is localized to the oocyte microvillar membrane using transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that CD9 null oocytes, which are unable to fuse with sperm, have an altered length, thickness and density of their microvilli. One aspect of this change in morphology was quantified using TEM by measuring the radius of curvature at the microvillar tips. A small radius of curvature is thought to promote fusibility and the radius of curvature of microvillar tips on CD9 wild-type oocytes was found to be half that of the CD9 null oocytes. We found that oocyte CD9 co-immunoprecipitates with two Ig superfamily cis partners, EWI-2 and EWI-F, which could have a role in linking CD9 to the oocyte microvillar actin core. We also examined latrunculin B-treated oocytes, which are known to have reduced fusion ability, and found altered microvillar morphology by SEM and TEM. Our data suggest that microvilli may participate in sperm–oocyte fusion. Microvilli could act as a platform to concentrate adhesion/fusion proteins and/or provide a membrane protrusion with a low radius of curvature. They may also have a dynamic interaction with the sperm that serves to capture the sperm cell and bring it into close contact with the oocyte plasma membrane.

Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation

cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.

Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20

Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which we term endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.