Effect of genistein alone and in combination with okadaic acid on the cell cycle resumption of mouse oocytes.

Our biopharmacological approach suggests that the now well-documented inhibitory effects of genistein on the maturation of mammalian oocytes do not seem to be related to its effect on tyrosine kinases. Indeed, we show that both tyrphostin B46 and Lavendustin A, two selective inhibitors of protein tyrosine kinases, fail to inhibit meiosis reinitiation. According to recent findings, the G2/M arrest induced by genistein could be due to inhibition of the kinase activity of cdc2. We were therefore mainly interested in dissecting the cytological effects of genistein on mouse primary and secondary oocytes. Genistein exerts the same cytological effects as IBMX on primary oocytes: their germinal vesicle is maintained in a central position, the cytoplasmic microtubule network is stabilized, the central GV immobilization is overcome by demecolcine and they complete normal maturation after their transfer to culture medium. The GV-arresting activity of genistein is also bypassed by OA but combination of both drugs results in a dramatic reorganization of the cytoskeleton leading to a huge membrane bulging, which is quite different to apoptotic-related blebbing. MAP Kinase activation is correlated with meiosis reinitiation. When applied after GVBD has taken place, genistein does not inhibit MAPK activation, metaphase spindle formation and metaphase-to-anaphase transition, but prevents the barrel-shaped MI spindle from undergoing its peripheral migration and the oocytes from extruding their first polar body. It may thus be concluded that the checkpoint control for anaphase onset is unaffected by the drug. On the contrary, our results suggest that spindle anaphase A to spindle anaphase B transition, spindle degradation, mid-body formation and cytokinesis are triggered by a genistein-sensitive mechanism that might be a mid-anaphase checkpoint. Finally, we confirm that genistein induces transition to interphase in metaphase II oocytes but never induces cortical granule exocytosis, the cytoplasmic hallmark of activation.