Protein kinase C and intracellular calcium are involved in follicle-stimulating hormone-mediated meiotic resumption of cumulus cell-enclosed porcine oocytes in hypoxanthine-supplemented medium

The present experiments were conducted to examine the hypothesis that folli cle-stimulating hormone (FSH) can stimulate the hydrolysis of phosphoinosit ide, generating the intracellular second messengers to activate protein kin ase C and mobilizing intracellular calcium, thus inducing oocyte meiotic re sumption. Pig cumulus cell-enclosed oocytes (CEO) were cultured for 24 hr i n 4 mM hypoxanthine (HX)-supplemented medium and treated with different age nts in the following designs: (1) CEO were treated with neomycin (an inhibi tor of phosphoinositide hydrolysis) in the presence of FSH or only treated with 7,12-dimethylbenzin(a) anthracene (DMBA, a tumor promoter which can ca use phosphorylation of phospholipase C (PLC), formation of inositol triphop hate, and mobilization of intracellular calcium) to mimic the direct activa tion of PLC; (2) CEO were challenged by FSH, together with sphingosine or s taurosporine (two kinds of PKC inhibitors); or treated with phorbol myrista te acetate (PMA, an activator of PKC) separately; (3) CEO were primed with BAPTA/AM (an intracellular calcium chelator) or BAPTA/AM +FSH for 60 min, a nd then transferred into a new culture medium supplemented with FSH but wit hout BAPTA/AM; total culture time was 24 hr. At the end of the culture, the incidence of germinal vesicle breakdown (GVBD) was calculated. The results showed that: (1) FSH (100 U/liter) could stimulate pig CEO to override the arrest of HX and resume meiosis; DMBA (10(-8)-10(-5) M) itself also had su ch a kind of effect; whereas neomycin, at the level of 10-20 mM, could dram atically inhibit the stimulatory effect of FSH. (2) Staurosporine (10(-9)-1 0(-6) M) or sphingosine (10(-8)-10(-5) M) could also inhibit the effect of FSH in a dose-dependent manner on stimulating CEO to resume meiosis. Howeve r, PMA (10(-8)-10(-5) M) alone had a dual effect on the meiotic resumption of pig CEO. PMA, at the level of 10(-8)-10(-6) M, could stimulate CEO to re sume meiosis, and at high concentration of 10(-5) M, it could even enhance the inhibitory effect of HX. (3) Priming CEO with BAPTA/AM only or BAPTA/AM + FSH for 60 min could significantly inhibit the effect of FSH in a dose-d ependent manner. These results indicate that in the process of ligand-media ted meiotic resumption of pig CEO, FSH can stimulate the hydrolysis of phos phoinositide leading to the activation of PKC and mobilization of intracell ular calcium; and suggest that multiple signaling pathways and signal inter action are involved in this process. (C) 1999 Wiley-Liss, Inc.