Initiation Of Oocyte Maturation Research Articles

In vitro induction of catfish, Clarias batrachus, oocyte maturation by conspecific vitellogenin 1 (CFVg1).

Present study demonstrates that conspecific vitellogenin1 (CFVg1) induces oocyte maturation in the catfish, Clarias batrachus. CFVg1 is able to develop fertilizable eggs in the Clarias batrachus. Therefore, different in vitro oocyte culture experiments were designed to see whether CFVg1 has efficacy of oocyte maturation and its pathway. In in vitro oocyte culture experiment, CFVg1 showed a dose- and time-dependent response and 64% maturation was obtained at the dose level of 10µg/ml or more. CFVg1 induction of oocyte maturation was confirmed by co-incubating CFVg1 with CFVg1-antiserum (a-CFVg1), which inhibited the CFVg1-induced oocyte maturation. To answer issues lead to the understanding of the mechanism of vitellogenin (Vg) on oocyte maturation, trypsin digested CFVg1 and Indian major carp Cirhinus mrigala Vg HAI (Hydroxy appetite peak I) also showed significant level of maturation. Actinomycin-D and cycloheximide blocked the effect of CFVg1, indicating that CFVg1 acts through transcription and translation. Theophylline, the phosphodiesterase inhibitor, and cAMP also inhibited the stimulatory effect of CFVg1 on oocyte maturation, indicating indirectly that CFVg1-induced oocyte maturation by decreasing the intracellular cAMP possibly by activating the phosphodiesterase enzyme. Trilostane, the 3β-HSD-blocker, did not inhibit the CFVg1-induced oocyte maturation but wortmannin and Ly294002 two mechanistically different specific inhibitors of PI3 kinase blocked the oocyte maturation. The results thus indicate that oocyte maturation in catfish by Vg may be regulated by two pathways: (1) through decreasing the intraoocyte cAMP level by activating the cAMP-PKA pathway and (2) by cAMP-dependent PI3K/Akt pathway. Therefore, there might be role of vitellogenin itself in initiation of oocyte maturation.

Formation of mos RNA granules in the zebrafish oocyte that differ from cyclin B1 RNA granules in distribution, density and regulation

Many translationally repressed mRNAs are deposited in the oocyte cytoplasm for progression of the meiotic cell cycle and early development. mos and cyclin B1 mRNAs encode proteins promoting oocyte meiosis, and translational control of these mRNAs is important for normal progression of meiotic cell division. We previously demonstrated that cyclin B1 mRNA forms RNA granules in the zebrafish and mouse oocyte cytoplasm and that the formation of RNA granules is crucial for regulating the timing of translational activation of the mRNA. However, whether the granule formation is specific to cyclin B1 mRNA remains unknown. In this study, we found that zebrafish mos mRNA forms granules distinct from those of cyclin B1 mRNA. Fluorescent in situ hybridization analysis showed that cyclin B1 RNA granules were assembled in dense clusters, while mos RNA granules were distributed diffusely in the animal polar cytoplasm. Sucrose density gradient ultracentrifugation analysis showed that the density of mos RNA granules was partly lower than that of cyclin B1 mRNA. Similar to cyclin B1 RNA granules, mos RNA granules were disassembled after initiation of oocyte maturation at the timing at which the poly(A) tail was elongated. However, while almost all of the granules of cyclin B1 were disassembled simultaneously, a fraction of mos RNA granules firstly disappeared and then a large part of them was disassembled. In addition, while cyclin B1 RNA granules were disassembled in a manner dependent on actin filament depolymerization, certain fractions of mos RNA granules were disassembled independently of actin filaments. These results suggest that cytoplasmic regulation of translationally repressed mRNAs by formation of different RNA granules is a key mechanism for translational control of distinct mRNAs in the oocyte.

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 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-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 17alpha,20beta-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.

Non-dependence of cyclin E/Cdk2 kinase activity on the initiation of oocyte maturation in goldfish.

Cdk2 kinase activity increases during oocyte maturation but neither cyclin A nor B is associated with Cdk2 in mature oocytes in goldfish. As a potential Cdk2 partner in meiosis, a cyclin E homolog was isolated from a goldfish oocyte cDNA library. A monoclonal antibody was raised against bacterially produced full-length goldfish cyclin E. Both cyclin E and Cdk2 were already present in immature oocytes and their protein levels did not change remarkably during oocyte maturation. Cyclin E formed a complex mainly with Cdk2 just at the time of germinal vesicle breakdown (GVBD) in association with the increase in Cdk2 kinase activity, although a fraction of cyclin E bound to Cdk(s) other than Cdk2 and Cdc2. Ectopic activation of cyclin E/Cdk2 by the injection of cyclin E messenger RNA (mRNA) into immature oocytes did not induce maturation-promoting factor (MPF) activation and GVBD. Furthermore, inhibition of cyclin E/Cdk2 kinase activity by the injection of p21SDI1 into the oocytes treated with 17alpha,20beta-dihydroxy-4-pregnen-3-one had no effect on MPF activation and GVBD. These results indicate that cyclin E/Cdk2 kinase activity is insufficient and unnecessary for initiating goldfish oocyte maturation.

Differential Surface Ultrastructural Characteristics and Volumetric Dynamics of Bovine Oocytes during Maturation In Vivo Versus In Vitro.

Surface characteristics of the bovine cumulus-oocyte complex (COC) during maturation in vivo versus in vitro were compared by scanning electron microscopy (SEM). Oocyte diameter changes during maturation were also analyzed prior to oocyte fixation. In vivo matured oocytes were retrieved from live super-ovulated cows by transvaginal ultrasound-guided aspiration of all visible ovarian follicles (≥2 mm in diameter) 0, 12 and 24 h (n=85, 38 and 39, respectively) after hCG administration. In vitro matured (IVM) oocytes refer to those recovered from small antral follicles (2-6 mm in diameter) of slaughterhouse ovaries and then cultured in standard maturation conditions for 0 h (n=188), 12 h (n=138) or 24 h (n=228). SEM analysis showed that immature oocytes of both origins manifested compact COCs with smooth cumulus surface and minimal intercellular spaces among the cumulus cell mass. At 24 h of maturation, more IVM oocytes manifested cumulus expansion compared to in vivo matured oocytes (100%, n=228 vs 44%, n=39), but cumulus expansion was found much more prominently in the cumulus-expanded in vivo matured oocytes than in the IVM oocytes. The zona pellucida showed a fibrous, open mesh-like structure, irrespective of the maturation stage and the source of oocytes. The vitelline surface of the immature oocytes was characterized by distribution of large cellular tongue-shaped protrusions (TSPs) varying in density. These TSPs structures gradually changed to microvilli (MV)-predominant structures upon maturation of the oocytes. At 12 h of maturation, significantly more oocytes (p<0.05) manifested cumulus expansion and transition from TSPs- to MV-predominant vitelline surface structures in the in vitro group (100%, n=106) compared to only 11% (n=38) in the in vivo group. Oocyte diameter decreased gradually in the oocytes maturing in vitro tween 0 and 24 h of incubation, being 127 ± 1, 122 ± 1 and 116 ± 1 μm at 0, 12 and 24 h of incubation, respectively. The corresponding values for the oocytes maturing in vivo were 121 ± 2, 129 ± 2, and 101 ± 1 μm, respectively. In conclusion, we found in our study conditions that initiation of oocyte maturation in vivo after hCG administration seemed to be slower than in vitro after maturation culture. But, at 24 h after the onset of maturation more prominent and complete cumulus expansion and more dramatic volumetric changes were observed in the in vivo matured oocytes than in the IVM oocytes.

Inhibition of v-Mos kinase activity by protein kinase A.

We investigated the effect of cyclic AMP-dependent protein kinase (PKA ) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37v-mos in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Combined tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies indicated that Ser-56 is the major in vivo phosphorylation site on v-Mos. In vivo phosphorylation at Ser-56 correlated with slower migration of the v-Mos protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, even though Ser-56 was phosphorylated by PKA, this phosphorylation was not involved in the inhibition of v-Mos kinase. The alanine-for-serine substitution at residue 56 did not affect the ability of v-Mos to autophosphorylate in vitro or, more importantly, to activate MEK1 in transformed NIH 3T3 cells. We identified Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. From our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation in mice could be explained at least in part by its inhibition of Mos kinase.

Role of c-mos proto-oncogene product in the regulation of mouse oocyte maturation.

The c-mos proto-oncogene product (Mos) is essential for the initiation of oocyte maturation, for the suppression of DNA synthesis during meiosis, and for the second metaphase arrest in Xenopus. To clarify the function of Mos in mice, c-mos-deficient mice were generated by gene targeting. We cultured oocytes from c-mos-deficient females to determine the role of Mos in oocyte maturation. c-mos-deficient oocytes matured normally to the second metaphase, but were activated without fertilization. Thus, prevention of parthenogenetic activation might be an ultimate biological function of Mos in animal oocytes.

Analysis of the mechanism(s) of metaphase I arrest in maturing mouse oocytes.

Fully grown mouse oocytes are normally competent to progress from prophase I to metaphase II without interruption. However, growing mouse oocytes initially become only partially competent to undergo meiotic maturation. Meiotic maturation in these oocytes does not progress beyond metaphase I. In contrast to the oocytes of most strains of mice, most oocytes of strain LT/Sv mice become arrested at metaphase I even when they are fully grown. The initiation of oocyte maturation is correlated with an increase in p34cdc2 kinase activity that continues to rise until metaphase I. The transition into anaphase I is normally correlated with a decrease in p34cdc2 kinase activity. This study demonstrated that metaphase I arrest in both partially competent growing oocytes and fully grown LT/Sv oocytes is correlated with a sustained elevation of p34cdc2 kinase activity. In fact, p34cdc2 activity continued to increase during the time when activity normally decreased. In normally maturing oocytes, some, but not all, of the cyclin B, the regulatory protein associated with p34cdc2, became degraded in oocytes that entered anaphase I. In contrast, the amount of cyclin B present in the metaphase I-arrested oocytes continued to increase at the time when it was being degraded in normal oocytes progressing to metaphase II. These results suggest that the progression of meiosis is arrested at metaphase I in both groups of oocytes because of continued p34cdc2 kinase activity sustained, at least in part, by restricted degradation of cyclin B.(ABSTRACT TRUNCATED AT 250 WORDS)

Initiation of meiotic maturation in Xenopus laevis oocytes by lanthanum

PROGESTERONE can induce true maturation of isolated amphibian oocytes in vitro1. It has been found recently that the ionophore A23187, in the presence of high magnesium or calcium concentrations, can also promote meiosis in vitro2, and it has been observed that 0.5–1 mM pro-pranolol and some related compounds, are potent inducers of maturational events in Xenopus laevis oocytes3. This last result suggests that displacement of membrane calcium might be involved in maturation, because this is known to occur in other systems treated with high concentrations of propranolol4. We have therefore studied the action of La3+, since this ion is expected to act specifically at the superficial plasma membrane calcium sites5. We report here that maturation is induced by La3+, indicating strongly that calcium ion movements have a role in the initiation of oocyte maturation.