Resumption of meiosis induced by meiosis-activating sterol has a differentsignal 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 mei osis-activating sterol [FF-MAS]) isolated from human follicular fluid induc es resumption of meiosis in mouse oocytes cultured in vitro. The purpose of this study was to examine the hypothesis that differential signal transduc tion mechanisms exist for FF-MAS-induced and spontaneous in vitro resumptio n of meiosis in mouse oocytes. Mouse oocytes were dissected from ovaries or iginating from mice primed with FSH 48 h before oocyte collection. Mechanic ally denuded germinal vesicle (GV) oocytes were in vitro matured in medium supplemented with hypoxanthine and FF-MAS or allowed to mature spontaneousl y; both groups were exposed to individual compounds known to inhibit specif ic 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) st age. Pertussis toxin (2.5 mug/ml) did not influence resumption of meiosis i n either group. Dibutyryl cyclic GMP (320 muM) inhibited FF-MAS-induced GVB D, but not spontaneous GVBD, whereas the subtype 5 phosphodiesterase-inhibi tor zaprinast (50 muM) 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 muM), inhibited or retarded spontaneous GVB D to a further extent than it did FF-MAS-induced GVBD. Spontaneous GVBD was more sensitive to the histone HI kinase-inhibitor olomoucine (250 muM) tha n was FF-MAS-induced GVBD. Addition of the mitogen-activated protein kinase (MAPK)-inhibitor PD 98059 (50 muM), phospholipase C-inhibitor U-73122 (10 muM), p21(ras)-inhibitor lovastatine (250 muM), and the src-like kinase inh ibitor PP2 (20 mug/ml) inhibited FF-MAS-induced GVBD, but not spontaneous G VBD. Both MAPKs, extracellular regulated kinase (ERK) 1 and ERK2, were phos phorylated under FF-MAS-induced meiotic resumption, in contrast to spontane ous 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 phos phoinositide signaling lie upstream of MAPK in the FF-MAS-activated signali ng pathway. Clearly, striking pathway differences are present between spont aneous versus FF-MAS-induced meiotic resumption.