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 follicle-stimulating hormone (FSH) can stimulate the hydrolysis of phosphoinositide, generating the intracellular second messengers to activate protein kinase C and mobilizing intracellular calcium, thus inducing oocyte meiotic resumption. Pig cumulus cell-enclosed oocytes (CEO) were cultured for 24 hr in 4 mM hypoxanthine (HX)-supplemented medium and treated with different agents in the following designs: (1) CEO were treated with neomycin (an inhibitor of phosphoinositide hydrolysis) in the presence of FSH or only treated with 7,12-dimethylbenzin(a) anthracene (DMBA, a tumor promoter which can cause phosphorylation of phospholipase C (PLC), formation of inositol triphophate, and mobilization of intracellular calcium) to mimic the direct activation of PLC; (2) CEO were challenged by FSH, together with sphingosine or staurosporine (two kinds of PKC inhibitors); or treated with phorbol myristate 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, and then transferred into a new culture medium supplemented with FSH but without 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 such a kind of effect; whereas neomycin, at the level of 10-20 mM, could dramatically inhibit the stimulatory effect of FSH. (2) Staurosporine (10(-9)-10(-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. However, 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 resume 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-dependent manner. These results indicate that in the process of ligand-mediated meiotic resumption of pig CEO, FSH can stimulate the hydrolysis of phosphoinositide leading to the activation of PKC and mobilization of intracellular calcium; and suggest that multiple signaling pathways and signal interaction are involved in this process.
Su
Y Q
YQ
College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China.
Xia
G L
GL
Byskov
A G
AG
Fu
G D
GD
Yang
C R
CR
D000818:Animals; D002118:Calcium / Q000378:metabolism; D003470:Culture Media; D004789:Enzyme Activation; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism* / Q000494:pharmacology; D006868:Hydrolysis; D019271:Hypoxanthine / Q000378:metabolism* / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D008540:Meiosis / Q000502:physiology*; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism* / Q000502:physiology; D013552:Swine
D003470:Culture Media; D010716:Phosphatidylinositols; D019271:Hypoxanthine; D005640:Follicle Stimulating Hormone; D011493:Protein Kinase C; D002118:Calcium