ALTERATIONS IN THE CELL-CYCLE OF MOUSE CUMULUS GRANULOSA-CELLS DURINGEXPANSION AND MUCIFICATION IN-VIVO AND IN-VITRO

The cell cycle characteristics of mouse cumulus granulosa cells were d etermined before, during and following their expansion and mucificatio n in vivo and in vitro. Cumulus-oocyte complexes (COG) were recovered from ovarian follicles or oviducts of prepubertal mice previously inje cted with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMS G and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigo rously controlled conditions, by flow cytometry. The proportion of cum ulus cells in three cell cycle-related populations (G(0)/G(1); S; G(2) /M) was calculated before and after exposure to various experimental c onditions in vivo or in vitro. About 30% of cumulus cells recovered fr om undifferentiated (compact) COC isolated 43-45 h after PMSG injectio ns were in S phase and 63% were in G(0)/G(1) (2C DNA content). Less th an 10% of the cells were in the G(2)/M population. Cell cycle profiles of cumulus cells recovered from mucified COC (oviducal) after PMSG+hC G-induced ovulation varied markedly from those collected before hCG in jection and were characterized by the relative absence of S-phase cell s and an increased proportion of cells in G(0)/G(1). Cell cycle profil es of cumulus cells collected from mucified COC recovered from mouse o varian follicles before ovulation (9-10 h after hCG) were also charact erized by loss of S-phase cells and an increased G(0)/G(1) population. Results suggest that changes in cell cycle parameters in vivo are pri marily mediated in response to physiological changes that occur in the intrafollicular environment initiated by the ovulatory stimulus. A si milar lack of S-phase cells was observed in mucified cumulus cells col lected 24 h after exposure in vitro of compact COC to dibutyryl cyclic adenosine monophosphate (DBcAMP), follicle-stimulating hormone or epi dermal growth factor (EGF). Additionally, the proportion of cumulus ce lls in G(2)/M was enhanced in COC exposed to DBcAMP, suggesting that c ell division was inhibited under these conditions. Thus, both the G(1) --> S-phase and G(2) --> M-phase transitions in the cell cycle appear to be amenable to physiological regulation. Time course studies revea led dose-dependent changes in morphology occurred within 6 h of exposu re in vitro of COC to EGF or DBcAMP. Results suggest that the disappea rance of the S-phase population is a consequence of a decline in the n umber of cells beginning DNA synthesis and exit of cells from the S ph ase following completion of DNA synthesis. Furthermore, loss of prolif erative activity in cumulus cells appears to be closely associated wit h COC expansion and mucification, whether induced under physiological conditions in vivo or in response to a range of hormonal stimuli in vi tro, The observations indicate that several signal-transducing pathway s mediate changes in cell cycle parameters during cumulus cell differe ntiation.