Protein synthesis during meiotic maturation of mouse oocytes in vitro: Synthesis and phosphorylation of a protein localized in the germinal vesicle

Isolated fully grown mouse oocytes, arrested in dictyate of the first meiotic prophase, synthesize a protein with an apparent molecular weight of 28,000 which is localized in the germinal vesicle of the oocyte (germinal vesicle-associated protein; GVAP). Analyses of the distribution of GVAP have been carried out on SDS-polyacrylamide gels using oocytes cultured in vitro in the presence of [³⁵S]methionine or [³H]lysine and germinal vesicles isolated individually from these cultured oocytes. The results of such analyses show that GVAP contains only about 2% of the total radiolabel incorporated into mouse oocyte proteins, but as much as 40% of the total radiolabel incorporated into proteins associated with isolated germinal vesicles. These measurements indicate that GVAP is at least 1000-fold more concentrated in the germinal vesicle than in the cytoplasm of the oocyte. Furthermore, the synthesis and phosphorylation of GVAP are apparently terminated at a time which coincides with germinal vesicle breakdown during spontaneous meiotic maturation of mouse oocytes in vitro. Although the exact nature of GVAP is not known as yet, it appears to be an example of a protein that is selectively sequestered in the germinal vesicle of the oocyte during oogenesis and whose synthesis and modification are dependent upon the presence of an intact germinal vesicle.     

A possible role for Mg2+ ions in the induction of meiotic maturation of Xenopus oocyte

Progesterone induces in vitro the meiotic cell division of Xenopus full-grown oocytes. Microinjection into oocyte of a solution containing Mg2+ (20 mM) facilitates by one order of magnitude the dose of progesterone which induces 50% of germinal vesicle breakdown. Microinjected in the absence of hormone, Mg2+ and also Mn2+ can induce maturation with efficiencies of, respectively, 24% (SEM = 8; n = 13) and 70% (SEM = 6; n = 23). The dose-response curves of cation-induction of maturation show an optimum of 20 mM for Mg2+ and 15 mM for Mn2+ (pipet concentration); higher doses were less active. Cation-induction of maturation is inhibited when oocytes are preincubated with cholera toxin (500 ng/ml); nevertheless, it cannot be interpreted at the level of cAMP, since both Mg2+ and Mn2+ microinjections provoke an increase in the oocyte cAMP content. Mg2+ induction of maturation is more efficient when oocytes are incubated in trimethylamine at pH 8.2, which is known to increase intracellular pH suggesting an action at the level of alkali pH-sensitive enzymes. Altogether, our results indicate a positive role for Mg2+ ions in the induction of oocyte maturation and raise an attractive hypothesis about the respective roles of cAMP and Mg2+ changes involved in the mechanism of progesterone action. Our results also show that co-injection of 2-glycerophosphate and Mg2+ ions, which are both commonly used in the preparation of the MPF mitotic factors from dividing cells, induces oocyte maturation more efficiently than Mg2+ alone and drastically shortens the kinetics of germinal vesicle breakdown to 1 h 30 min to 2 h 30 min.     

Requirement for glucose in ligand-stimulated meiotic maturation of cumulus cell-enclosed mouse oocytes.

In this study, the effect of different energy sources used in Eagle's minimum essential medium on the meiotic maturation of mouse oocytes in culture was examined. The effects of glucose (5.5 mmol 1(-1)), pyruvate (0.23 mmol 1(-1)) and glutamine (2 mmol 1(-1)) in different combinations were tested on the maturation of denuded oocytes in the presence or absence of 300 mumol dibutyryl cAMP 1(-1) during 17-18 h of culture. In the absence of cyclic nucleotide, only oocytes from those groups containing pyruvate resumed maturation at a high frequency (99-100% germinal vesicle breakdown); all other combinations resulted in < or = 54% germinal vesicle breakdown. When dibutyryl cAMP was introduced, all pyruvate-containing groups exhibited maturation frequencies of about 50%, whereas maturation in all other groups was negligible (< or = 10% GVB). Pyruvate was also important for the maintenance of viability in denuded oocytes (> or = 86% viability in pyruvate-containing medium; < or = 35% viability in pyruvate-free groups). When cumulus cell-enclosed oocytes were cultured in medium without inhibitor, all combinations of energy substrates supported high frequencies of maturation (> or = 89% germinal vesicle breakdown) and viability (> or = 91%). The addition of dibutyryl cAMP resulted in inhibition of meiotic maturation (5-33% germinal vesicle breakdown) in all cultures except the pyruvate-alone group (97% germinal vesicle breakdown). Viability in cumulus cell-enclosed oocytes was greatest when two or more energy substrates were present in the medium. Follicle-stimulating hormone (FSH) produced a stimulation of meiotic maturation in all cultures of meiotically arrested cumulus cell-enclosed oocytes, but maximal induction of germinal vesicle breakdown was dependent upon D-glucose. Concanavalin A (ConA)-induced meiotic maturation was also dependent upon D-glucose. Uptake and metabolism of D-glucose by the cumulus cells is important in mediating the stimulatory effects of these ligands on oocyte maturation because (1) both FSH and ConA stimulated uptake of D-glucose and 2-deoxyglucose but not 3-O-methylglucose; (2) phloretin prevented the stimulatory action of FSH and ConA on germinal vesicle breakdown at a concentration that suppressed ligand-induced uptake of D-glucose; (3) 2-deoxyglucose, a hexose that suppresses glycolysis, prevented the induction of meiotic maturation by FSH and ConA and (4) D-mannose, a glycolysable sugar, was as effective as D-glucose in supporting the ligand effects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induced formation of asters and cleavage furrows in oocytes of Xenopus laevis during in vitro maturation

We have previously reported that injection of purified basal bodies or sperm into unfertilized eggs of Xenopus laevis induced the formation of asters and irregular cleavage furrows. Fully grown oocytes were found to be unable to form asters or cleavage furrows. In this paper we show that the oocyte acquires the ability to form asters upon basal body injection at the time of germinal vesicle breakdown during in vitro maturation. Our evidence indicates that aster formation requires progesterone-stimulated changes in the oocyte and mixing of cytoplasm and germinal vesicle plasm. The ability of the oocyte to form cleavage furrows arises six to eight hours after germinal vesicle breakdown. We infer that some maturational change in the cell cortex occurs to enable the egg surface to furrow. Experiments on the relationship of aster formation to furrow initiation indicates that asters stimulate furrow formation. However, some furrowing could be induced without aster formation in mature oocytes and unfertilized eggs by an activation stimulus, showing that asters are not essential for cleavage initiation. The significance of these observations are discussed in the light of our current understanding of meiotic maturation, cell cleavage and aster growth.     

Inhibition of oocyte maturation in the mouse: Participation of cAMP,steroid hormones,and a putative maturation-inhibitory factor

The hypothesis that cumulus cells inhibit oocyte maturation by a cAMP-dependent process was tested (R. M. Schultz, R. Montgomery, P. F. Ward-Bailey, and J. J. Eppig (1983). Dev. Biol.95, 294–304.). Treatment of isolated cumulus cell-oocyte complexes with follicle-stimulating hormone (FSH) resulted in a dose-dependent increase in both cumulus cell cAMP levels and in the extent of inhibition of germinal vesicle breakdown (GVBD), the first morphological manifestation of oocyte maturation. Furthermore, it was found that concentrations of a membrane-permeable analog of cAMP, dibutyryl cAMP (dbcAMP), that were below those required for complete meiotic inhibition had a greater inhibitory effect on cumulus cell-enclosed oocytes than on denuded oocytes. Cumulus cell-enclosed and denuded oocytes matured at the same time in the absence of dbcAMP. Ablation of the gap junctions that couple cumulus cells to the oocyte abolished the maturation-inhibitory action of cumulus cells that was promoted either by FSH or low concentrations of dbcAMP. These results are consistent with the hypothesis that inhibition of oocyte maturation is mediated by a factor of granulosa/cumulus cell origin, other than cAMP, which requires cAMP for its activity and/or generation, and an intact intercellular coupling pathway between cumulus cells and the oocyte. A variety of steroid hormones potentiated the FSH-induced inhibition of maturation in cumulus cell-enclosed oocytes. In addition, steroid hormones inhibited maturation in denuded oocytes, but only when oocyte cAMP levels were elevated by cAMP analogs or forskolin. Steroids alone did not inhibit maturation of either cumulus cell-enclosed or denuded oocytes. Moreover, the steroids alone or in combination with FSH did not affect metabolic coupling between the cumulus cells and oocytes, nor did testosterone affect the forskolin-induced level of cAMP in denuded oocytes. Therefore, it is proposed that the oocyte is a site for the synergistic activity of steroid hormones with a cAMP-dependent process in inhibiting maturation. Results of these studies are discussed in terms of the roles of intercellular communication, cAMP, a putative maturation-inhibiting factor, and steroid hormones in the inhibition of maturation of mouse oocytes.     

Role of germinal vesicle material in producing maturation-promoting factor in starfish oocyte

In starfish, oocyte maturation is induced by 1-methyladenine (1-MeAde). 1-MeAde acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which in turn brings about germinal vesicle breakdown and subsequent process of oocyte maturation. The participation of germinal vesicle material in the production of MPF was investigated with oocytes of the starfish, Asterina pectinifera. When enucleated oocytes or oocyte fragments without germinal vesicles were treated with 1-MeAde, MPF was found to be produced. However, the amount of MPF produced was small as compared with that in the case of intact oocytes with germinal vesicles. The capacity of the enucleated oocytes to produce MPF was restored when germinal vesicle material was injected. On the other hand, it has been known that the amount of MPF increases when MPF is injected into intact oocytes (amplification of MPF). However, in the case of enucleated oocytes such increase of MPF was no longer observed, suggesting that germinal vesicle material is required for MPF amplification.     

Conservation of RNA polymerase during maturation of the Rana pipiens oocyte.

DNA-dependent RNA polymerase was extracted from oocytes of the frog, Rana pipiens. The bulk of the enzyme activity was present in the germinal vesicle and the amounts of each major form of such activity did not significantly change during oocyte maturation. Therefore, either nuclear polymerase activity is conserved after breakdown of the oocyte nucleus during maturation or, alternatively, de novo synthesis of the enzymes must occur during oocyte maturation concomitant with degradation. We have measured rates of protein synthesis in oocytes and determined a maximum rate of synthesis for RNA polymerases. Our kinetic studies show that no more than 20, 10, and 5% of RNA polymerases type I, IIa, and IIb, respectively, could be synthesized during steroid-induced oocyte maturation. These results thus show that the bulk of RNA polymerase accumulates in the germinal vesicle during oogenesis, is dispersed into the cytoplasm during maturation, and, since only limited synthesis seems to be occurring, the polymerase is available during embryogenesis.     

The role of the germinal vesicle in producing maturation-promoting factor (MPF) as revealed by the removal and transplantation of nuclear material in starfish oocytes

In starfish, oocytes are released from prophase block by a hormone, which has been identified as 1-methyladenine. The action of 1-methyladenine is indirect in inducing oocyte maturation: it acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), the direct trigger of germinal vesicle breakdown (GVBD). Less than 5 min after hormone addition, thus about 10 min before appearance of the cytoplasmic maturation-promoting factor, a factor appears in the germinal vesicle, which triggers the production of cytoplasmic MPF, GVBD, and the subsequent events of meiotic maturation when transferred in the cytoplasm of any fully grown oocyte of the starfishes Marthasterias glacialis and Asterias rubens. Before hormone action, the germinal vesicle also contains a factor capable of inducing meiosis reinitiation in recipient oocytes, but in contrast with nuclear MPF, this factor acts exclusively when transferred in the cytoplasm of a special category of oocytes (the “competent” oocytes). In contrast to other oocytes (the “incompetent” oocytes) the competent oocytes are capable of producing MPF to some extent after enucleation, upon hormonal stimulation. Transfer of either nuclear or cytoplasmic MPF initially produced in hormone-treated maturing oocytes triggers the production of both cytoplasmic and nuclear MPF in non-hormone-treated recipient oocytes of both categories.     

New insight into the role of phosphodiesterase 3A in porcine oocyte maturation

Background  

The ovulatory surge of gonadotropins triggers oocyte maturation and rupture of the ovarian follicle. The resumption of nuclear maturation in the oocyte from the prophase stage is characterized by germinal vesicle breakdown (GVBD). It has previously been shown that specific inhibition of cAMP degradation by PDE3 prevents the resumption of oocyte meiosis. However, no report has characterized the activity of PDE3 in the porcine oocyte, or the implication of the cAMP-PDE3 pathway in the entire nuclear maturation process. In this study, PDE3 activity in the oocyte was assessed during in vitro maturation (IVM) and the possible roles of the cAMP-PDE3 pathway in the resumption and progression of meiosis were investigated in terms of different models of oocyte maturation.     

Preimplantation embryonic development of spontaneous mouse parthenotes after oocyte meiotic maturation in vitro

Of eggs ovulated in LT/Sv mice, 10–20% undergo spontaneous parthenogenetic activation, and 40–50% of the parthenotes develop to blastocysts when cultured in simple defined medium from the one-cell stage. Similar percentages of oocytes isolated from Graafian follicles undergo parthenogenetic activation after spontaneous maturation in simple defined medium, but embryonic development proceeds no further than the two-cell stage. The simple defined medium that supported preimplantation development of ovulated eggs and spontaneous maturation of extrafollicular oocytes contained no serum, free amino acids, or vitamins. The present experiments were conducted to determine what conditions during spontaneous maturation of extrafollicular oocytes could promote the ability of oocytes to develop to blastocysts after parthenogenetic activation and mimic the environment of preovulatory follicles. Cumulus-enclosed oocytes that were matured in simple medium supplemented with fetal bovine serum (FBS) developed to blastocysts after spontaneous parthenogenetic activation. Furthermore, minimum essential medium (MEM), a complex medium containing free amino acids and vitamins, could substitute completely for FBS for maturing oocytes from (C57BL/6J × LT/Sv)F₁ mice, and to a lesser extent for maturing LT/Sv oocytes. Therefore, even though germinal vesicle breakdown in mouse oocytes and preimplantation development of mouse eggs can occur in the absence of an exogenous supply of free amino acids and vitamins, a complete, or normal, mouse oocyte maturation cannot. These results also demonstrated that gonadotropins are not necessary during oocyte meiotic maturation for parthenogenetically activated eggs to develop through the preimplantation stages. Luteinizing hormone or 17β-estradiol in MEM during oocyte maturation had no effect on the subsequent development of parthenotes. In contrast, follicle stimulating hormone (FSH) and progesterone in the maturation medium decreased the number of ova that subsequently cleaved, and FSH decreased the number of cleaved eggs that developed to blastocysts.     

Metabolism of glucose,pyruvate, and glutamine during the maturation of oocytes derived from pre‐pubertal and adult cows

The aim of the study was to compare the energy metabolism of oocytes from pre‐pubertal (2 to 3 months) and adult cows during maturation, to identify the cause of poor developmental potential in many pre‐pubertal oocytes. The metabolism of [5‐³H] glucose, [2‐¹⁴C] pyruvate, and [G‐³H] glutamine was measured at 0 hr, 12 hr, and 24 hr maturation. Oxidative metabolism was important during maturation of oocytes from both pre‐pubertal and adult cows, with pyruvate metabolism peaking at 12 hr and glutamine metabolism increasing linearly and peaking at 24 hr. Peak oxidative metabolism was significantly lower in oocytes from pre‐pubertal animals, for both pyruvate and glutamine (P < 0.05). Glucose metabolism increased significantly during oocyte maturation in both groups (0hr to 24 hr). Glucose metabolism was significantly lower in oocytes from pre‐pubertal cows at 12 hr (P < 0.05). Oocytes from pre‐pubertal animals were significantly smaller than oocytes from adult cows at 0 hr, 12 hr, and 24 hr maturation (P < 0.05). When metabolic rates were corrected for oocyte volume, there were no significant differences in substrate metabolism between oocytes from pre‐pubertal and adult cows. There was however, a delay in the increase in glucose metabolism in pre‐pubertal oocytes 0 hr to 12 hr maturation. Germinal vesicle breakdown was slower in oocytes from pre‐pubertal animals with more oocytes still at the germinal vesicle stage approximately 5 hr post‐aspiration, compared to oocytes from adult cows (P < 0.05). By 24 hr, development to metaphase II was equivalent for pre‐pubertal and adult oocytes. This study identified differences in energy metabolism, oocyte size, and meiotic progression between the oocytes from pre‐pubertal and adult cows that may account for the poor developmental potential of many pre‐pubertal oocytes. Mol. Reprod. Dev. 54:92–101, 1999. © 1999 Wiley‐Liss, Inc.     

Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis

Mouse oocytes are reversibly inhibited from resuming meiotic maturation in vitro by cAMP phosphodiesterase inhibitors such as 3-isobutyl-1-methyl xanthine (IBMX) and cAMP analogs such as dibutyryl cAMP (dbcAMP). Oocytes cultured in IBMX-containing medium were transferred to and cultured in IBMX-free medium for various periods of time prior to their return to either IBMX- or dbcAMP-containing medium. Results from these experiments defined a period of time in which oocytes became committed to resuming meiosis. Forskolin, which elevated the intracellular oocyte cAMP concentration, transiently inhibited oocytes from resuming meiosis. Levels of cAMP were determined in oocytes incubated in medium that allows resumption of meiosis. The level of oocyte cAMP decreased significantly during the time in which oocytes become committed to resuming meiosis. This decrease in oocyte cAMP was not observed in oocytes inhibited from resuming meiosis by IBMX. In addition, cAMP levels were determined in preovulatory antral follicles, cumulus cell-oocyte complexes, and oocytes during gonadotropin-induced resumption of meiosis in vivo. A decrease in oocyte cAMP preceded resumption of meiosis as manifested by germinal vesicle breakdown (GVBD). This decrease apparently occurred before or during a period of time in which follicle and cumulus cell cAMP were increasing. Associated with commitment to resume meiosis was a characteristic set of changes in oocyte phosphoprotein metabolism that preceded GVBD. These changes are, to date, some of the first reported biochemical changes that precede GVBD. Results from these experiments are discussed in terms of a possible role cAMP may play in regulation of resumption of meiosis in mammals.     

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.     

Increased ornithine decarboxylase activity during meiotic maturation in xenopus laevis oocytes

The activity of ornithine decarboxylase (ornithine carboxylyase E.C. 4.1.1.17) was studied during meiotic maturation induced in vitro by progesterone in follicle cell-free oocytes. Enzyme activity increased 4–6 fold during maturation, preceding germinal vesicle breakdown. The increase in ornithine decarboxylase activity was inhibited by cholera toxin, an agent that blocks meiotic maturation and increases cAMP levels within the cell. It was also prevented by cycloheximide but not by actinomycin D. Treatment of oocytes with D,L-α-difluoromethyl-ornithine, an irreversible inhibitor of ornithine decarboxylase and of putrescine synthesis, effectively abolished enzyme activity without preventing germinal vesicle breakdown. These observations show that the progesterone-induced increase in ornithine decarboxylase activity is not required for completion of meiotic division of the oocyte.     

Cyclic assembly-disassembly of cortical microtubules during maturation and early development of starfish oocytes

An extensive array of cortical microtubules in oocytes of the starfish Pisaster ochraceus undergoes multiple cycles of disappearance and reappearance during maturation and early development. These events were studied in isolated fragments of the oocyte cortex stained with antitubulin antibodies for indirect immunofluorescence. The meshwork of long microtubules is present in the cortex (a) of immature oocytes, i.e., before treatment with the maturation-inducing hormone 1-methyladenine, (b) for 10-20 min after treatment with 1-methyladenine, (c) after formation of the second polar body (in reduced numbers in unfertilized oocytes), and (d) in the intermitotic period between first and second cleavage divisions. The array of cortical microtubules is absent in oocytes (a) undergoing germinal vesicle breakdown, (b) during the two meiotic divisions (polar body divisions), and (c) during mitosis of the first and, perhaps, subsequent cleavage divisions. The cycle of assembly-disassembly of cortical microtubules is synchronized to the cycle of nuclear envelope breakdown and reformation and to the mitotic cycle; specifically, cortical microtubules are present when a nucleus is intact (germinal vesicle, female pronucleus, zygote nucleus, blastomere nucleus) and are absent whenever a meiotic or mitotic spindle is present. These findings are discussed in terms of microtubule organizing centers in eggs, possible triggers for microtubule assembly and disassembly, the eccentric location of the germinal vesicle, and the regulation of oocyte maturation and cell division.     

Nuclear-cytoplasmic interactions during ovine oocyte maturation

The present studies have been undertaken to investigate the interactions that occur between the nucleus and cytoplasm of ovine oocytes at various stages during meiotic maturation. We report that the nucleus of ovine fully grown dictyate stage oocytes can be efficiently removed by a microsurgical enucleation procedure. It is demonstrated that between the initiation of maturation and germinal vesicle breakdown certain newly synthesized polypeptides are selectively sequestered in the oocyte nucleus and the major sequestered polypeptide has a relative molecular mass of 28,000, which represent at least 9% of the total labelled polypeptides transferred to the oocyte nucleus during the first 4 h of maturation. The experiments provide evidence that the removal of the oocyte nucleus at various times before germinal vesicle breakdown (GVBD) does not prevent the major series of changes in protein synthesis that occurs after entry into a metaphase. We conclude therefore that the mixing of the nucleoplasm and cytoplasm is not essential for the initiation or progression of the protein reprogramming process during maturation. In addition, the experiments show that the development of the ability to condense chromatin during ovine oocyte maturation is independent of the oocyte nucleus. The combined results strongly support the hypothesis that the extensive series of translational changes that occur in oocytes during maturation are controlled by cytoplasmic rather than nuclear factors.     

The role of oocyte maturation in the ontogeny of the fertilization site in the hydrozoan Hydractinia echinata

Summary In hydrozoans the sperm will fuse with the egg only at the site of polar body formation. The primary oocyte and maturing oocytes which have produced the first polar body cannot be fertilized even though maturing oocytes which have produced the first polar body attract sperm. These eggs do not acquire the ability to be fertilized until after second polar body formation. If either first or second polar body formation is inhibited or if first and second polar body formation do not take place in close proximity to each other, the fertilization site is not set up. Under normal circumstances the site of polar body formation takes place at the region on the maturing oocyte surface nearest the site where the germinal vesicle resided in the primary oocyte. When maturing oocytes are centrifuged prior to polar body formation, the site of polar body formation is frequently shifted so that it does not correspond to the site where it would be given off under normal circumstances. Under these conditions the shifted site of polar body formation is the only site where the egg can be fertilized, indicating that the fertilization site is selected during oocyte maturation.Oocyte maturation in these hydrozoans is mediated by a hormone released by the somatic cells of gonophores as a consequence of bringing dark adapted gonophores into the light. The hormone acts directly on the oocyte to induce maturation. The oocyte only has to be exposed to the hormone for the first few minutes of the maturation process in order to complete the process of maturation.Dedicated to Professor N.H. Verdonk of the Rijksuniversiteit Utrecht on his 65th birthday     

Serotonin inhibition of steroid-induced meiotic maturation in the teleost Fundulus heteroclitus: Role of cyclic AMP and protein kinases

The transduction of the serotonin (5-HT) signal in Fundulus heteroclitusovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation-inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) was investigated. Steroid-induced oocyte maturation was inhibited by 5-HT in a dose-dependent manner; maximum inhibition (90%) was observed with 10⁻⁴ M 5-HT. Groups of follicle-enclosed oocytes were cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and treated with increasing doses of 5-HT. Serotonin was found to slightly increase the levels of follicular 3′,5′-cyclic adenosine monophosphate (cAMP) in a dose-dependent manner; 10⁻⁴ M 5-HT induced approximately a 3-fold increase in cAMP with respect to the controls. The changes in cAMP were then evaluated in follicles treated with 17,20βP in IBMX-free culture media in the presence or absence of 10⁻⁴ M 5-HT. The exposure of follicles to 17,20βP alone produced a small and transient reduction in cAMP (40%) within 1–3 hr of steroid stimulation, and these early changes in cAMP appeared associated with a high incidence of germinal vesicle breakdown (80% GVBD) by 24 hr of incubation. Under these conditions, treatment of follicles with 5-HT also increased significantly the production of cAMP, and when 5-HT was combined with 17,20βP, the steroid-mediated reduction in cAMP was prevented and the levels of GVBD inhibited by 95%. Meiosis also was reinitiated with either the protein kinase A (PKA) inhibitor H8 or the protein kinase C (PKC) activator PMA, and the 5-HT inhibitory action on GVBD was found to be 100-fold reduced or completely ineffective, respectively. Preincubation of follicles with the PKC inhibitor GF109203x abolished PMA-induced GVBD in a dose-dependent manner, whereas this inhibitor had no effect on 17,20βP-triggered meiotic maturation, indicating that activation of PKC is apparently sufficient but not necessary to reinitiate meiosis. Taken together, these findings suggest that 5-HT may inhibit 17,20βP-induced meiotic reinitiation through the activation of a cAMP-PKA transduction pathway and that PKC possibly induces oocyte maturation by a different pathway than the steroid and thus is not affected by 5-HT. Mol. Reprod. Dev. 49:333–341, 1998. © 1998 Wiley-Liss, Inc.     

Changes in adenine nucleotide levels and respiration during 1-methyladenine induced maturation of starfish oocytes

The effects of 1-methyladenine on oxygen consumption and adenine nucleotide levels were examined in oocytes of Pisaster ochraceus and Patiria miniata. Oocytes of both genera to which 1-methyladenine was added consumed more oxygen than control oocytes beginning 1 to $\text{1}\text{1}\text{2}\text{h}$ after 1-methyladenine addition. The increase in oxygen consumption was correlated with maturation changes in the oocytes and particularly with germinal vesicle breakdown. Pre-fertilization oxygen consumption of eggs did not differ significantly from post-fertilization oxygen consumption of eggs in either genus for $\text{2}\text{1}\text{2}\text{h}$ after fertilization. ATP and AMP concentrations within the oocytes decreased during 1-methyladenine induced maturation, while the ADP concentration increased. It was suggested that increases in ADP concentration and decreases in ATP concentration within maturing starfish oocytes occurred in response to greater energy demands. The simultaneous increase in oocyte oxygen consumption was interpreted as an indicator of increased oxidative phosphorylation acting to restore initial nucleotide concentrations.