Resumption of meiosis induced by meiosis-activating sterol has a different signal transduction pathway than spontaneous resumption of meiosis in denuded mouse oocytes cultured in vitro.

The sterol 4,4-dimethyl-5-cholesta-8,14,24-trien-3-ol (follicular fluid meiosis-activating sterol [FF-MAS]) isolated from human follicular fluid induces resumption of meiosis in mouse oocytes cultured in vitro. The purpose of this study was to examine the hypothesis that differential signal transduction mechanisms exist for FF-MAS-induced and spontaneous in vitro resumption of meiosis in mouse oocytes. Mouse oocytes were dissected from ovaries originating from mice primed with FSH 48 h before oocyte collection. Mechanically denuded germinal vesicle (GV) oocytes were in vitro matured in medium supplemented with hypoxanthine and FF-MAS or allowed to mature spontaneously; both groups were exposed to individual compounds known to inhibit specific targets in the cell. After 20-22 h of in vitro maturation, resumption of meiosis was assessed as the frequency of oocytes in GV breakdown (GVBD) stage. Pertussis toxin (2.5 microg/ml) did not influence resumption of meiosis in either group. Dibutyryl cyclic GMP (320 microM) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD, whereas the subtype 5 phosphodiesterase-inhibitor zaprinast (50 microM) inhibited GVBD in both groups. Microinjection of the catalytic subunit of cAMP-dependent protein kinase into oocytes inhibited spontaneous GVBD, but not FF-MAS-induced GVBD. An inhibitor of cytoplasmic polyadenylation, cordycepin (80 microM), inhibited or retarded spontaneous GVBD to a further extent than it did FF-MAS-induced GVBD. Spontaneous GVBD was more sensitive to the histone H1 kinase-inhibitor olomoucine (250 microM) than was FF-MAS-induced GVBD. Addition of the mitogen-activated protein kinase (MAPK)-inhibitor PD 98059 (50 microM), phospholipase C-inhibitor U-73122 (10 microM), p21(ras)-inhibitor lovastatine (250 microM), and the src-like kinase inhibitor PP2 (20 microg/ml) inhibited FF-MAS-induced GVBD, but not spontaneous GVBD. Both MAPKs, extracellular regulated kinase (ERK) 1 and ERK2, were phosphorylated under FF-MAS-induced meiotic resumption, in contrast to spontaneous meiotic resumption, in which ERK1 and ERK2 phosphorylation occurred 2 h after GVBD. In the present study, we show that FF-MAS acts through an MAPK-dependent pathway, and we suggest that src-like kinase, p21(ras), and phosphoinositide signaling lie upstream of MAPK in the FF-MAS-activated signaling pathway. Clearly, striking pathway differences are present between spontaneous versus FF-MAS-induced meiotic resumption.

Nuclear ultrastructure in bovine oocytes after inhibition of meiosis by chemical and biological inhibitors.

Various components of the ovarian follicle as well as different chemicals can suppress the resumption of meiosis in cumulus-oocyte complexes (COCs). In this study the nuclear ultrastructure of bovine COCs was assessed after 8 h of meiotic inhibition with 50 microM roscovitine (ROSC), 50 microM butyrolactone (BL-I), 2 mM 6-DMAP, 2 microM cycloheximide (CX), or a theca cell monolayer (TC). COCs were recovered according to standard in vitro methods, cultured in a simple and defined medium, and processed for transmission electron microscopy. Control COCs were processed before onset of culture and multiple oocytes were evaluated for each treatment. In all groups, the oocyte nucleus presented a dense fibrillar nucleolus consisting of a fibrillar sphere with a fibrillar center. In TC and 6-DMAP inhibited COCs condensed chromatin adhered to the nucleolus while in all other groups the perinuclear chromatin was separated from the nucleolus. In ROSC inhibited COCs, the nuclear envelope presented only slight small amplitude undulation. The BL-I-inhibited COCs presented an intermediate level of low amplitude undulation of the NE. In CX, 6-DMAP, and TC inhibited COCs the nuclear envelope presented extensively low amplitude undulations. In ROSC inhibited COCs, electron-dense granules formed ring-shaped structures. In some of the BL-I inhibited COCs multiple stellate crystal-like structures were found, and in these COCs the nuclear envelope and the perinuclear cisternae appeared less distinct than in the other BL-I inhibited COCs. In 6-DMAP inhibited COCs interchromatin-like granule clusters were present. In conclusion, the oocyte nuclei in all COCs presented a dense fibrillar nucleolus resembling that in control COCs. However, variations were observed in 1) the nuclear envelope morphology; 2) the chromatin location in relation to the nucleolus; and 3) the presence of different populations of intranuclear granules. Although all treatments inhibited oocyte nucleus breakdown, the mechanisms underlying these effects are different and require further characterization. Mol. Reprod. Dev. 59: 459-467, 2001.

Autoradiographic localization of specific binding of meiosis-activating sterol to cumulus-oocyte complexes from marmoset, cow, and mouse.

The sterol, 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), isolated from human follicular fluid, can induce resumption of meiosis in denuded and cumulus-enclosed mouse oocytes inhibited by hypoxanthine, IBMX, or dibutyric cyclic adenosine monophosphate. In this study the distribution of FF-MAS binding sites in denuded oocytes and in cumulus-oocyte complexes (COCs) was studied using light microscopic (LM) and transmission electron microscopic (TEM) autoradiography in marmoset, cow, and mouse oocytes. Denuded (n = 39) and cumulus-enclosed (n = 28) marmoset, cow, and mouse oocytes were cultured in the presence of [3H]FF-MAS with and without excess of unlabeled FF-MAS, respectively. In denuded oocytes LM autoradiography demonstrated specific binding to the oolemma and zona pellucida and, to some extent, the cytoplasm. In the nucleus, no specific binding of [3H]FF-MAS was demonstrated. In some COCs the labeling was dispersed throughout the zona pellucida, the oolemma, and the cytoplasm as well as the cumulus cells; whereas in others, only the outer part of the cumulus cells were labeled. TEM autoradiograms of denuded cow oocytes (n = 6) demonstrated that specific [3H]FF-MAS binding was closely related to the oolemma and that a low level of [3H]FF-MAS binding to cumulus cell remnants was present. In conclusion, specific binding of FF-MAS is predominant at the oolemma of denuded oocytes, suggesting the existence of a plasma membrane-associated molecule with affinity for FF-MAS (i.e., a putative FF-MAS receptor).

Meiosis-activating sterol-mediated resumption of meiosis in mouse oocytes in vitro is influenced by protein synthesis inhibition and cholera toxin.

To explore the possible signaling pathways of meiosis-activating sterol (MAS)-induced oocyte maturation and to elucidate whether the MAS pathway involves transcription or translation, arrested immature mouse oocytes were cultured with either the protein synthesis inhibitor cycloheximide or the heteronuclear RNA inhibitors alpha-amanitin or actinomycin D, respectively. Moreover, the possible involvement of a G protein-coupled receptor mechanism in MAS-mediated oocyte maturation was explored by influencing oocyte maturation with cholera toxin (CT). MAS-induced oocyte maturation was completely blocked by the addition of 50 microg/ml cycloheximide 4 h before the addition of MAS. Simultaneous addition of MAS and the protein synthesis inhibitor also significantly reduced the meiotic resumption compared to that in MAS-treated controls. In contrast, neither of the treatment regimens to inhibit transcription of DNA to RNA was observed to have any effect on the MAS-induced resumption of meiosis. CT was observed to inhibit MAS-induced, but not spontaneous, oocyte maturation in vitro, suggesting a putative involvement of G protein-coupled receptor mechanism in the MAS mode of action. In conclusion, protein synthesis was found to be an essential requirement for maintaining the oocytes' responsiveness to MAS-induced resumption of meiosis, in contrast to transcription.