Maintenance of meiotic prophase arrest in vertebrate oocytes by a Gs protein-mediated pathway

Maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on an elevated level of cAMP in the oocyte. To investigate how the cAMP level is regulated, we examined whether the activity of an oocyte G protein of the family that stimulates adenylyl cyclase, Gs, is required to maintain meiotic arrest. Microinjection of a dominant negative form of Gs into Xenopus and mouse oocytes, or microinjection of an antibody that inhibits the Gs G protein into zebrafish oocytes, caused meiosis to resume. Together with previous studies, these results support the conclusion that Gs-regulated generation of cAMP by the oocyte is a common mechanism for maintaining meiotic prophase arrest in vertebrate oocytes.     

The APC/C activator FZR1 coordinates the timing of meiotic resumption during prophase I arrest in mammalian oocytes

FZR1, an activator of the anaphase-promoting complex/cyclosome (APC/C), is recognized for its roles in the mitotic cell cycle. To examine its meiotic function in females we generated an oocyte-specific knockout of the Fzr1 gene (Fzr1(Δ/Δ)). The total number of fully grown oocytes enclosed in cumulus complexes was 35-40% lower in oocytes from Fzr1(Δ/Δ) mice and there was a commensurate rise in denuded, meiotically advanced and/or fragmented oocytes. The ability of Fzr1(Δ/Δ) oocytes to remain prophase I/germinal vesicle (GV) arrested in vitro was also compromised, despite the addition of the phosphodiesterase milrinone. Meiotic competency of smaller diameter oocytes was also accelerated by Fzr1 loss. Cyclin B1 levels were elevated ~5-fold in Fzr1(Δ/Δ) oocytes, whereas securin and CDC25B, two other APC/C(FZR1) substrates, were unchanged. Cyclin B1 overexpression can mimic the effects of Fzr1 loss on GV arrest and here we show that cyclin B1 knockdown in Fzr1(Δ/Δ) oocytes affects the timing of meiotic resumption. Therefore, the effects of Fzr1 loss are mediated, at least in part, by raised cyclin B1. Thus, APC/C(FZR1) activity is required to repress cyclin B1 levels in oocytes during prophase I arrest in the ovary, thereby maintaining meiotic quiescence until hormonal cues trigger resumption.     

Continuous loss of oocytes throughout meiotic prophase in the normal mouse ovary

The number of germ cells reaches the maximum just prior to entry into meiosis, yet decreases dramatically by a few days after birth in the female mouse, rat, and human. Previous studies have reported a major loss at the pachytene stage of meiotic prophase during fetal development, leading to the hypothesis that chromosomal pairing abnormalities may be a signal for oocyte death. However, the identification as well as the quantification of germ cells in these studies have been questioned. A recent study using Mouse Vasa Homologue (MVH) as a germ cell marker reached a contradictory conclusion claiming that oocyte loss occurs in the mouse only after birth. In the present study, we established a new method to quantify murine germ cells by using Germ Cell Nuclear Antigen-1 (GCNA-1) as a germ cell marker. Comparison of GCNA-1 and MVH immunolabeling revealed that the two markers identify the same population of germ cells. However, nuclear labeling of GCNA-1 was better suited for counting germ cells in histological sections as well as for double labeling with the antibody against synaptonemal complex (SC) proteins in chromosome spreading preparations. The latter experiment demonstrated that the majority of GCNA-1-labeled cells entered and progressed through meiotic prophase during fetal development. The number of GCNA-1-positive cells in the ovary was estimated by counting the labeled cells retained in chromosome spreading preparations and also in histological sections by using the ratio estimation method. Both methods demonstrated a continuous decline in the number of GCNA-1-labeled cells during fetal development when the oocytes progress through meiotic prophase. These observations suggest that multiple causes are responsible for oocyte elimination.     

Apoptosis in mouse fetal and neonatal oocytes during meiotic prophase one

Background  

The vast majority of oocytes formed in the fetal ovary do not survive beyond birth. Possible reasons for their loss include the elimination of non-viable genetic constitutions arising through meiosis, however, the precise relationship between meiotic stages and prenatal apoptosis of oocytes remains elusive. We studied oocytes in mouse fetal and neonatal ovaries, 14.5–21 days post coitum, to examine the relationship between oocyte development and programmed cell death during meiotic prophase I.     

The G-protein-coupled receptors GPR3 and GPR12 are involved in cAMP signaling and maintenance of meiotic arrest in rodent oocytes

In mammalian and amphibian oocytes, the meiotic arrest at the G2/M transition is dependent on cAMP regulation. Because genetic inactivation of a phosphodiesterase expressed in oocytes prevents reentry into the cell cycle, suggesting autonomous cAMP synthesis, we investigated the presence and properties of the G-protein-coupled receptors (GPCRs) in rodent oocytes. The pattern of expression was defined using three independent strategies, including microarray analysis of GV oocyte mRNAs, EST database scanning, and RT-PCR amplification with degenerated primers against transmembrane regions conserved in the GPCR superfamily. Clustering of the GPCR mRNAs from rat and mouse oocytes indicated the expression of the closely related Gpr3, Gpr12, and Edg3, which recognize sphingosine and its metabolites as ligands. Expression of these mRNAs was confirmed by RT-PCR with specific primers as well as by in situ hybridization. That these receptors are involved in the control of cAMP levels in oocytes was indicated by the finding that expression of the mRNA for Gpr3 and Gpr12 is downregulated in Pde3a-deficient oocytes, which have a chronic elevation of cAMP levels. Expression of GPR3 or GPR12 in Xenopus laevis oocytes prevented progesterone-induced meiotic maturation, whereas expression of FSHR had no effect. A block in spontaneous oocyte maturation was also induced when Gpr3 or Gpr12 mRNA was injected into mouse oocytes. Downregulation of GPR3 and GPR12 caused meiotic resumption in mouse and rat oocytes, respectively. However, ablation of the Gpr12 gene in the mouse did not cause a leaky meiotic arrest, suggesting compensation by Gpr3. Incubation of mouse oocytes with the GPR3/12 ligands SPC and S1P delayed spontaneous oocyte maturation. We propose that the cAMP levels required for maintaining meiotic arrest in mouse and rat oocytes are dependent on the expression of Gpr3 and/or Gpr12.     

Regulation of the meiotic prophase I to metaphase I transition in mouse spermatocytes

The meiotic prophase I to metaphase I transition (G2/MI) involves disassembly of synaptonemal complex (SC), chromatin condensation, and final compaction of morphologically distinct MI bivalent chromosomes. Control of these processes is poorly understood. The G2/MI transition was experimentally induced in mouse pachytene spermatocytes by okadaic acid (OA), and kinetic analysis revealed that disassembly of the central element of the SC occurred very rapidly after OA treatment, before histone H3 phosphorylation on Ser10. These events were followed by relocalization of SYCP3 and final condensation of bivalents. Enzymatic control of these G2/MI transition events was studied using small molecule inhibitors: butyrolactone I (BLI), an inhibitor of cyclin-dependent kinases (CDKs) and ZM447439 (ZM), an inhibitor of aurora kinases (AURKs). The formation of highly condensed MI bivalents and disassembly of the SC are regulated by both CDKs and AURKs. AURKs also mediate phosphorylation of histone H3 in meiosis. However, neither BLI nor ZM inhibited disassembly of the central element of the SC. Thus, despite evidence that the metaphase promoting factor is a universal regulator of the onset of cell division, desynapsis, the first and key step of the G2/MI transition, occurs independently of BLI-sensitive CDKs and ZM-sensitive AURKs.     

Meiotic arrest in human oocytes is maintained by a Gs signaling pathway

In mammalian oocytes, the maintenance of meiotic prophase I arrest prior to the surge of LH that stimulates meiotic maturation depends on a high level of cAMP within the oocyte. In mouse and rat, the cAMP is generated in the oocyte, and this requires the activity of a constitutively active, Gs-linked receptor, GPR3 or GPR12, respectively. To examine if human oocyte meiotic arrest depends on a similar pathway, we used RT-PCR and Western blotting to look at whether human oocytes express the same components for maintaining arrest as rodent oocytes. RNA encoding GPR3, but not GPR12, was expressed. RNA encoding adenylate cyclase type 3, which is the major adenylate cyclase required for maintaining meiotic arrest in the mouse oocyte, was also expressed, as was Galphas protein. To determine if this pathway is functional in the human oocyte, we examined the effect of injecting a function-blocking antibody against Galphas on meiotic resumption. This antibody stimulated meiotic resumption of human oocytes that were maintained at the prophase I stage using a phosphodiesterase inhibitor. These results demonstrate that human oocytes maintain meiotic arrest prior to the LH surge using a signaling pathway similar to that of rodent oocytes.     

Zinc maintains prophase I arrest in mouse oocytes through regulation of the MOS-MAPK pathway

Meiosis in mammalian females is marked by two arrest points, at prophase I and metaphase II, which must be tightly regulated in order to produce a haploid gamete at the time of fertilization. The transition metal zinc has emerged as a necessary and dynamic regulator of the establishment, maintenance, and exit from metaphase II arrest, but the roles of zinc during prophase I arrest are largely unknown. In this study, we investigate the mechanisms of zinc regulation during the first meiotic arrest. Disrupting zinc availability in the prophase I arrested oocyte by treatment with the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) causes meiotic resumption even in the presence of pharmacological inhibitors of meiosis. We further show that the MOS-MAPK pathway mediates zinc-dependent prophase I arrest, as the pathway prematurely activates during TPEN-induced meiotic resumption. Conversely, inhibition of the MOS-MAPK pathway maintains prophase I arrest. While prolonged zinc insufficiency ultimately results in telophase I arrest, early and transient exposure of oocytes to TPEN is sufficient to induce meiotic resumption and bypass the telophase I block, allowing the formation of developmentally competent eggs upon parthenogenetic activation. These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the first and second meiotic arrest points.     

Oocyte-specific expression of Gpr3 is required for the maintenance of meiotic arrest in mouse oocytes

The maintenance of meiotic prophase arrest in mouse oocytes within fully grown follicles, prior to the surge of luteinizing hormone (LH) that triggers meiotic resumption, depends on a high level of cAMP within the oocyte. cAMP is produced within the oocyte, at least in large part, by the G(s)-linked G-protein-coupled receptor, GPR3. Gpr3 is localized in the mouse oocyte but is also present throughout the follicle. To investigate whether Gpr3 in the follicle cells contributes to the maintenance of meiotic arrest, RNA interference (RNAi) was used to reduce the amount of Gpr3 RNA within follicle-enclosed oocytes. Follicle-enclosed oocytes injected with small interfering double-stranded RNA (siRNA) targeting Gpr3, but not control siRNAs, stimulated the resumption of meiosis in the majority of oocytes following a 3-day culture period. Reduction of RNA was specific for Gpr3 because an unrelated gene was not reduced by microinjection of siRNA. Meiotic resumption was stimulated in isolated oocytes injected with the same siRNA and cultured for 1 to 2 days, but at a much lower rate than in follicle-enclosed oocytes that could be cultured for longer. These results demonstrate that GPR3 specifically in the oocyte, rather than in the follicle cells, is responsible for maintenance of meiotic arrest in mouse oocytes. Furthermore, the method developed here for specifically reducing RNA in follicle-enclosed oocytes, which can be cultured for a sufficient time to reduce the level of endogenous protein, should be generally useful for targeting a wide range of other proteins that may be involved in meiotic arrest, the resumption of meiosis, fertilization, or early embryonic development.     

Incorporation of H3-thymidine into the oocytes of a sterlet in the early meiotic prophase]

DNA synthesis in meiotic oocytes of the sterlet (A. ruthenus) has been studied during early prophase stages using H3-thymidine. The pattern of H3-thymidine incorporation is similar to that in oocytes of Amphibia and Osteichthyes. In the oogonia as well as in the leptotene and zygotene oocytes, the label is predominantly localized over chromosomes. An intensive incorporation of H3-thymidine into the material of the heterochromatic "cap" has been observed during pachytene. Thus, the main synthesis of extra DNA in the sterlet oocytes occurs during pachytene. No DNA in synthesized by the diplotene oocytes.     

Developmental capacity of mouse oocytes following maintenance of meiotic arrest in vitro

It was shown previously that the frequencies of fertilization and pre- and post-implantation embryonic development of mouse oocytes matured in vitro were similar to those of oocytes matured in vivo (Schroeder and Eppig, Dev Biol 102:493–497, 1984). The present study determined the developmental capacity of mouse oocytes after they had been maintained in meiotic arrest in vitro by substances thought to be important regulators of meiosis in vivo. Oocytes were maintained in meiotic arrest for 12 or 24 h in medium containing maturation inhibitor(s), washed free of inhibitor, and cultured 16 h in inhibitor-free (control) medium to permit meiotic maturation. Four different medium supplements were used to maintain meiotic arrest: (1) 100 μM dibutyryl cAMP plus 1 mM hypoxanthine; (2) 4 mM hypoxanthine plus 0.75 mM adenosine (H + AR); (3) 300 μM dibutyryl cAMP; and (4) 50 μM IBMX. Parallel groups of oocytes were treated to the same experimental protocol except that no inhibitory compounds were used; eg, oocytes were cultured a total of 28 or 40 h in control medium that permitted the resumption of maturation. These latter groups tested the effect of extended culture of mature oocytes on subsequent development. Control oocytes were cultured 16 h in control medium. Oocytes were inseminated and subsequently assessed for development to two-cell and blastocyst stages. When oocytes were first cultured 12 or 24 h in medium that maintained meiotic arrest, development to two-cells in all groups but one were within 10% of controls (70%). The 24 h H + AR group was the one exception (47% two-cells). By contrast, culturing oocytes for 28 or 40 h in inhibitor-free medium resulted in a precipitous decrease in development to two cells (27% and 7%, respectively). Blastocyst development followed the same pattern. When uridine (U) was added to H + AR medium, development to two cells was increased significantly. Also, the addition of FSH to the maturation medium significantly increased both two-cell and blastocyst development in the H + AR and H + AR + U groups. Transfer of compacted morulae from the H + AR + U/FSH group into pseudopregnant hosts produced live young 19 days postinsemination. These data demonstrate that prolonged culture of oocytes matured in vitro decreased their capacity to undergo normal development following insemination, but if oocytes were maintained in meiotic arrest during prolonged culture and then allowed to mature spontaneously, their developmental potential was significantly preserved. These results also lend support for a physiological role of cAMP and purines in the maintenance of meiotic arrest in vivo.     

The G protein coupled receptor 3 is involved in cAMP and cGMP signaling and maintenance of meiotic arrest in porcine oocytes

The arrest of meiotic prophase in mammalian oocytes within fully grown follicles is dependent on cyclic adenosine monophosphate (cAMP) regulation. A large part of cAMP is produced by the Gs-linked G-protein-coupled receptor (GPR) pathway. In the present study, we examined whether GPR3 is involved in the maintenance of meiotic arrest in porcine oocytes. Expression and distribution of GPR3 were examined by western blot and immunofluorescence microscopy, respectively. The results showed that GPR3 was expressed at various stages during porcine oocyte maturation. At the germinal vesicle (GV) stage, GPR3 displayed a maximal expression level, and its expression remained stable from pro-metaphase I (MI) to metaphase II (MII). Immunofluorescence staining showed that GPR3 was mainly distributed at the nuclear envelope during the GV stage and localized to the plasma membrane at pro-MI, MI and MII stages. RNA interference (RNAi) was used to knock down the GPR3 expression within oocytes. Injection of small interfering double-stranded RNA (siRNA) targeting GPR3 stimulated meiotic resumption of oocytes. On the other hand, overexpression of GPR3 inhibited meiotic maturation of porcine oocytes, which was caused by increase of cGMP and cAMP levels and inhibition of cyclin B accumulation. Furthermore, incubation of porcine oocytes with the GPR3 ligand sphingosylphosphorylcholine (SPC) inhibited oocyte maturation. We propose that GPR3 is required for maintenance of meiotic arrest in porcine oocytes through pathways involved in the regulation of cAMP and cGMP.     

Modulation of meiotic arrest in mouse oocytes by guanyl nucleotides and modifiers of G-proteins.

Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in cAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTP gamma S. This effect was specific for GTP gamma S, because GppNHp, GTP, and ATP gamma S were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF4-). Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF4- abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF4- alone were without effect. Both cholera toxin and AlF4- augmented the stimulatory action of FSH on cAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved at the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the alpha subunit of Gs and increased generation of cAMP, whereas AlF4- appears to act by antagonizing a cAMP-dependent step.     

Onset and progress of meiotic prophase in the oocytes in the B6.YTIR sex-reversed mouse ovary

When the Y chromosome of a Mus musculus domesticus male mouse (caught in Tirano, Italy) is placed on a C57BL/6J genetic background, approximately half of the XY (B6.YTIR) progeny develop into normal-appearing but infertile females. We have previously reported that the primary cause of infertility can be attributed to their oocytes. To identify the primary defect in the XY oocyte, we examined the onset and progress of meiotic prophase in the B6.YTIR fetal ovary. Using bromo-deoxyuridine incorporation and culture, we determined that the germ cells began to enter meiosis at the developmental ages and in numbers comparable to those in the control XX ovary. Furthermore, the meiotic prophase appeared to progress normally until the late zygotene stage. However, the oocytes that entered meiosis early in the XY ovary failed to complete the meiotic prophase. On the other hand, a considerable number of oocytes entered meiosis at late developmental stages and completed the meiotic prophase in the XY ovary. We propose that the timing of entry into meiosis and the XY chromosomal composition influence the survival of oocytes during meiotic prophase in the fetal ovary.     

The Xenopus laevis isoform of G protein-coupled receptor 3 (GPR3) is a constitutively active cell surface receptor that participates in maintaining meiotic arrest in X. laevis oocytes

Oocytes are held in meiotic arrest in prophase I until ovulation, when gonadotropins trigger a subpopulation of oocytes to resume meiosis in a process termed "maturation." Meiotic arrest is maintained through a mechanism whereby constitutive cAMP production exceeds phosphodiesterase-mediated degradation, leading to elevated intracellular cAMP. Studies have implicated a constitutively activated Galpha(s)-coupled receptor, G protein-coupled receptor 3 (GPR3), as one of the molecules responsible for maintaining meiotic arrest in mouse oocytes. Here we characterized the signaling and functional properties of GPR3 using the more amenable model system of Xenopus laevis oocytes. We cloned the X. laevis isoform of GPR3 (XGPR3) from oocytes and showed that overexpressed XGPR3 elevated intraoocyte cAMP, in large part via Gbetagamma signaling. Overexpressed XGPR3 suppressed steroid-triggered kinase activation and maturation of isolated oocytes, as well as gonadotropin-induced maturation of follicle-enclosed oocytes. In contrast, depletion of XGPR3 using antisense oligodeoxynucleotides reduced intracellular cAMP levels and enhanced steroid- and gonadotropin-mediated oocyte maturation. Interestingly, collagenase treatment of Xenopus oocytes cleaved and inactivated cell surface XGPR3, which enhanced steroid-triggered oocyte maturation and activation of MAPK. In addition, human chorionic gonadotropin-treatment of follicle-enclosed oocytes triggered metalloproteinase-mediated cleavage of XGPR3 at the oocyte cell surface. Together, these results suggest that GPR3 moderates the oocyte response to maturation-promoting signals, and that gonadotropin-mediated activation of metalloproteinases may play a partial role in sensitizing oocytes for maturation by inactivating constitutive GPR3 signaling.     

Ultrastructural study on the meiotic prophase nucleus of rat oocytes.

Rat oocytes in the meiotic prophase are studied by means of classical techniques of electron microscopy, preferential staining methods for DNA and RNA and specific enzymatic hydrolysis. The axial cores in leptotene and the lateral arms in the pachytene synaptonemal complex are composed by fibrils that keep a positive contrast after the application of the ethylenediaminetetraacetic acid staining method. They disappear with RNAse treatment, which reveals the presence of chromatin fibrils in the zone occupied by the cores. Preferential staining for DNA corroborates this evidence. Medial arm and lateral-medial fibrils are formed by ribonucleoproteic filaments that form bridges between pairing homologues in the zygotene. In the advanced pachytene stage, the RNA becomes scarce in these structures. No DNA can be detected either in the lateral-medial fibrils or in the medial arm. During diplotene the synaptonemal complex loses its individually and the synaptic space becomes wider and irregular. At the same time, loss of chromatin and a large increase of RNA-containing particles occur. These processes lead to the typical interphasic arrangement of nuclear components seen in the dictyate stage.     

A G(s)-linked receptor maintains meiotic arrest in mouse oocytes, but luteinizing hormone does not cause meiotic resumption by terminating receptor-G(s) signaling

The maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on the activity of a G(s) G-protein that activates adenylyl cyclase and elevates cAMP, and in the mouse oocyte, G(s) 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-G(s) signaling, we examined the effect of LH on the localization of Galpha(s). G(s) activation in response to stimulation of an exogenously expressed beta(2)-adrenergic receptor causes Galpha(s) to move from the oocyte plasma membrane into the cytoplasm, whereas G(s) inactivation in response to inhibition of the beta(2)-adrenergic receptor causes Galpha(s) to move back to the plasma membrane. However, LH does not cause a change in Galpha(s) localization, indicating that LH does not act by terminating receptor-G(s) signaling.