Intracellular signals trigger ultrastructural events characteristic of meiotic maturation in oocytes of Xenopus laevis

Summary Oocytes of Xenopus laevis were treated with agents which induce individual intracellular signals normally evoked during the process of meiotic maturation. Ultrastructural analysis of these oocytes allowed identification of specific second messengers that individually trigger single ultrastructural changes characteristic of the meiotic maturation process: Manipulation of intracellular cAMP levels induced changes in cortical granule position. Cytoplasmic alkalinization triggered a disruption of the annulate lamellae, a specialized organelle in the periphery of oocytes. Activation of protein kinase C caused rapid formation of a cortical endoplasmic reticulum and subsequent disruption of cortical granules. Manipulation of transmembrane calcium flux had varied results dependent upon the agent employed. Two of the treatments, Verapamil and zero external calcium, induced a reorganization in the oocyte periphery. The results indicate that these ultrastructural events are under the control of specific intracellular signals known to be elicited during meiotic maturation.     

Ion current activity and molecules modulating maturation and growth stages of ascidian (Ciona intestinalis) oocytes

Electrophysiological techniques were used to study ion currents in the ascidian Ciona intestinalis oocyte plasma membranes during different stages of growth and meiosis. Three stages (A, B, C) of immature oocytes were discriminated in the ovary, with the germinal vesicle (GV) showing specific different features of growth and maturation. Stage A (pre‐vitellogenic) oocytes exhibited the highest L‐type Ca²⁺current activity, and were incompetent for meiosis resumption. Stage B (vitellogenic) oocytes showed Na⁺ currents that remained high during the maturation, up to the post‐vitellogenic stage C oocytes. The latter had acquired meiotic competence, undergoing spontaneous maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation plays a specific role in embryo development. Spontaneous maturation was inhibited at low pH whereas trypsin was able to trigger germinal vesicle breakdown (GVBD) regardless of pH; in addition spontaneous maturation was not affected by removal of follicle cells or by inhibiting junctional communication between oocyte and follicle cells. Taken together these results imply: (i) Ca²⁺ and Na⁺ currents are involved in meiotic progression, growth, and acquisition of meiotic competence; (ii) trypsin‐like molecules may have a role as candidates for providing the physiological stimulus to resume meiosis. Finally, we provide evidence that follicle cells in Ciona are not involved in triggering GVBD as it occurs in other ascidians. Mol. Reprod. Dev. 76: 1084–1093, 2009. © 2009 Wiley‐Liss, Inc.     

Haploid maternal genome derived from early diplotene oocytes can substitute for the female pronucleus in preimplantation mouse development

We describe the preimplantation development of mouse embryos that have received the haploid maternal genome derived from early diplotene nuclei of primordial oocytes (PO). Two generations of recipient egg-cells were used. Induction of two meiotic divisions of the PO nucleus and the reduction of the number of chromosomes to the haploid level were achieved in preovulatory oocytes (primary recipients). The developmental potential of the obtained haploid genome was examined in zygotes (secondary recipients). The nuclei of PO obtained from newborn mice were transferred by cell electrofusion to in vitro maturing (IVM) and enucleated preovulatory mouse oocytes. The reconstructed oocytes which had completed maturation, i.e., reached metaphase II, were artificially activated (8% ethanol + CHX). Activated oocytes were used as donors of haploid pronuclei of PO origin which were transferred (by karyoplast fusion) to partially enucleated zygotes containing only the male pronucleus. Thus, reconstituted zygotes were transplanted to the ligated oviducts of the cycling mice and 27% of them developed to the blastocyst stage. Our experiments demonstrate that 1) the nucleus of PO can be induced to premature meiotic divisions in an IVM enucleated preovulatory oocyte; 2) in the presence of a normal male pronucleus, the haploid pronucleus of PO origin can substitute for a female pronucleus during preimplantation development. Mol. Reprod. Dev. 48:488–495, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Errata

The pH-dependent control of the rate of protein synthesis in the fertilized sea urchin egg has suggested that pH may have an important role in cell activation at fertilization. We looked for similar changes of intracellular pH during meiotic maturation of the Xenopus oocyte. The basal pH of the oocyte is in the range 7.4–7.8. The higher values were found mostly in oocytes from animals with recent hormonal stimulation, suggesting a correlation with the elevated metabolism of such oocytes. Regardless of basal pH, progesterone-induced maturing oocytes alkalize an average of 0.18 pH unit. Beginning shortly before germinal vesicle breakdown, intracellular pH then decreases to near the original value. The same program is observed when maturation is induced by an injection of “maturation promoting factor.” Maturation is delayed or inhibited if intracellular pH is driven acidic. It is induced in the absence of progesterone by trimethylamine, a weak base which may act via an imposed alkalization. However, maturation still occurs when net alkalization is prevented. These data suggest that the alkalization during maturation is a form of metabolic “insurance” and that there may be both pH-dependent and pH-independent pathways for maturation. There is some evidence suggesting that pH changes are related to movement of other ions.     

Pyrethroids cypermethrin,deltamethrin and fenvalerate have different effects on in vitro maturation of pig oocytes at different stages of growth

Pesticides can significantly harm reproduction in animals and people. Pyrethroids are often used as insecticides, and their toxicity for mammals is considered to be low. However, cypermethrin, deltamethrin and fenvalerate – as potent specific inhibitors of protein phosphatase calcineurin – can influence the meiosis of mammalian oocytes. The objective of this study was to evaluate the effects of these pyrethroids on the in vitro maturation of pig oocytes at different levels of meiotic competence. Under the tested concentrations, cypermethrin, deltamethrin and fenvalerate neither had a significant effect on the viability of oocytes nor did they induce significant degeneration of oocytes. However, these pyrethroids significantly affected meiotic maturation. The effects depended on the stage of meiotic competence of the oocytes. Maturation of growing pig oocytes with partial meiotic competence was induced. On the other hand, in fully grown pig oocytes with full meiotic competence, maturation in vitro was delayed. The specificity of these effects was further supported by the same effect of non-pyrethroidal inhibitors of calcineurin – cyclosporin A or hymenistatin I – on the maturation of oocytes with different levels of meiotic competence. However, pyrethroids, which do not inhibit calcineurin – allethrin or permethrin – had no effect on pig oocyte maturation. We demonstrated a significant effect of pyrethroids on the maturation of mammalian oocytes under in vitro conditions. This indicates that exposure to these substances could affect the fertility of people or animals.     

Meiotic maturation of vitrified immature chousingha (Tetracerus quadricornis) oocytes recovered postmortem

The ability to recover and cryopreserve oocytes from postmortem ovaries of endangered or wildlife species holds tremendous potential for conservation using assisted reproductive technologies. The objective of this study was to assess the in vitro meiotic maturation of chousingha (four-horned antelope) oocytes following vitrification using open pulled straw (OPS) method. The average number of oocytes recovered per ovary was 65.6. The proportion of oocytes that matured was significantly lower in vitrified oocytes (29.4%) when compared with fresh oocytes (69.3%). The study provides evidence that it is possible to cryopreserve immature oocytes by vitrification collected from the ovaries of chousingha at postmortem and also demonstrates that these cryopreserved oocytes retain their potential to undergo in vitro meiotic maturation.     

Stage specific effects of carbendazim (MBC) on meiotic cell cycle progression in mouse oocytes

The effects of the pesticide carbendazim (MBC) on the in vitro meiotic maturation of mouse oocytes were evaluated using conventional and confocal fluorescence microscopy. The response of oocytes exposed to 0, 3, 10, or 30 μM MBC during meiotic maturation was analyzed with respect to chromosome organization, meiotic spindle microtubules, and cortical actin using fluorescent labels for each of these structures. Continuous exposure to MBC during the resumption of meiosis resulted in a dose-dependent inhibition of meiotic cell cycle progression at metaphase of meiosis-1. Drug exposure at the metaphase-anaphase transition of meiosis-1 did not interfere with cell cycle progression to metaphase-2 except at high concentrations (30 μM). At the level of spindle microtubule organization, MBC caused a loss of nonacetylated microtubules and a decrease in spindle size at 3 or 10 μM concentrations. Thirty μM MBC prevented spindle assembly when added at the beginning of meiotic maturation or caused spindle pole disruption and fragmentation when added to preformed spindles. Spindle disruption involved a loss of phosphoprotein epitopes, as monitored by MPM-2 staining, and resulted in the appearance of dispersed chromosomes that retained a metaphase-plate location on spindle fragments associated with the oocyte cortex. Polar body extrusion was impaired by MBC, and abnormal polar bodies were observed in most treated oocytes. The results suggest that MBC disrupts cell cycle progression in mouse oocytes by altering meiotic spindle microtubule stability and spindle pole integrity. Mol. Reprod. Dev. 46:351–362, 1997. © 1997 Wiley-Liss, Inc.     

An essential role for putrescine biosynthesis during meiotic maturation of amphibian oocytes

The objective of this study was to investigate the role of polyamines during meiotic maturation of Xenopus oocytes. The results indicate a rapid and significant increase in the activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway, during the meiotic maturation induced by either progesterone or human chorionic gonadotropin (HCG). This increase in the enzyme activity was followed by an accumulation of putrescine without any effect on the levels of spermidine or spermine. The inhibition of ODC activity and the accumulation of putrescine levels by α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ODC, also resulted in the inhibition of maturation mediated by progesterone in Xenopus oocytes. DFMO caused an inhibition of both maturation and ovulation induced by HCG in ovarian fragments. This inhibition was readily reversible by exogenous supply of putrescine to the medium. These observations suggest that putrescine plays an important role during the meiotic maturation of amphibian oocytes.     

The Mos/MAPK pathway is involved in metaphase II arrest as a cytostatic factor but is neither necessary nor sufficient for initiating oocyte maturation in goldfish

Mos plays a crucial role in meiotic cell division in vertebrates. In Xenopus, Mos is involved in the initiation of oocyte maturation as an initiator and in the arrest at the metaphase II stage (MII) as a component of the cytostatic factor (CSF). The function of Mos is mediated by MAP kinase (MAPK). We investigated the function of the Mos/MAPK pathway during goldfish oocyte maturation induced by 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), a natural maturation-inducing hormone in fishes. Mos was absent in immature goldfish oocytes. It appeared before the onset of germinal vesicle breakdown (GVBD), increased to a maximum in mature oocytes arrested at MII and disappeared after fertilization. MAPK was activated after Mos synthesis but before maturation-promoting factor (MPF) activation, and its activity reached maximum at MII. Injection of either Xenopus or goldfish c-mos mRNA into one blastomere of 2-cell-stage Xenopus and goldfish embryos induced metaphase arrest, suggesting that goldfish Mos has a CSF activity. Injection of constitutively active Xenopus c-mos mRNA into immature goldfish oocytes induced MAPK activation, but neither MPF activation nor GVBD occurred. Conversely, the injection of goldfish c-mos antisense RNA inhibited both Mos synthesis and MAPK activation in the 17α,20β-DP-treated oocytes, but these oocytes underwent GVBD. These results indicate that the Mos/MAPK pathway is not essential for initiating goldfish oocyte maturation despite its general function as a CSF. We discuss the general role of Mos/MAPK during oocyte maturation, with reference to the difference in contents of inactive MPF (pre-MPF) stored in immature oocytes. Received: 10 February 2000 / Accepted: 25 April 2000     

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

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

ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

Proteomic screen for potential regulators of M-phase entry and quality of meiotic resumption in Xenopus laevis oocytes

The quality of oocytes depends largely on the capacity to resume meiotic maturation. In Xenopus laevis, only fully grown oocytes react to progesterone stimulation by resumption of meiotic maturation associated with the entry into the meiotic M-phase. Proteins involved in this process are poorly known. To identify novel proteins regulating M-phase entry, we performed a differential proteomic screen. We compared proteomes of fully grown stage VI oocytes characterized as poorly or highly responsive to progesterone treatment. The comparison of 2-D gels allowed us to identify several spots including two specifically present in highly responsive oocytes and two specifically present in poorly responsive ones. By mass spectrometry we identified the two proteins specifically present in highly responsive oocytes as inosine 5′monophosphate cyclohydrolase and YjgF homologues, and the two specifically present in poorly responsive oocytes as elongation factor 2 (EF2) and S-adenosyl-l-homocysteine hydrolase (SAHH). The proteins specifically expressed in highly responsive oocytes may participate in the stimulation of meiotic maturation and M-phase entry, while the proteins specifically present in poorly maturing oocytes may participate in the inhibition of meiotic resumption.     

alpha-Endosulfine is a conserved protein required for oocyte meiotic maturation in Drosophila

Meiosis is coupled to gamete development and must be well regulated to prevent aneuploidy. During meiotic maturation, Drosophila oocytes progress from prophase I to metaphase I. The molecular factors controlling meiotic maturation timing, however, are poorly understood. We show that Drosophila alpha-endosulfine (endos) plays a key role in this process. endos mutant oocytes have a prolonged prophase I and fail to progress to metaphase I. This phenotype is similar to that of mutants of cdc2 (synonymous with cdk1) and of twine, the meiotic homolog of cdc25, which is required for Cdk1 activation. We found that Twine and Polo kinase levels are reduced in endos mutants, and identified Early girl (Elgi), a predicted E3 ubiquitin ligase, as a strong Endos-binding protein. In elgi mutant oocytes, the transition into metaphase I occurs prematurely, but Polo and Twine levels are unaffected. These results suggest that Endos controls meiotic maturation by regulating Twine and Polo levels, and, independently, by antagonizing Elgi. Finally, germline-specific expression of the human alpha-endosulfine ENSA rescues the endos mutant meiotic defects and infertility, and alpha-endosulfine is expressed in mouse oocytes, suggesting potential conservation of its meiotic function.