CUMULUS CELLS OF OOCYTE-CUMULUS COMPLEXES SECRETE A MEIOSIS-ACTIVATING SUBSTANCE WHEN STIMULATED WITH FSH

The effect of the different follicular cell types on resumption of mei osis was studied during stimulation with FSH. Cumulus enclosed oocytes (CEO), denuded oocytes (DO), and cumulus and mural granulosa cells we re used. The resumption of meiosis and oocyte maturation were assessed by the determination of the germinal vesicle breakdown (GVBD) and pol ar body formation (PB) at the end of a 24 hr culture period in the pre sence of 4 mM hypoxanthine (HX). The effects of recombinant LH (r-LH) and hCG were also evaluated. Oocyte exposure to the gonadotrophins var ied from 5 min to 24 hr (i.e., priming time). Oocytes were obtained fr om immature gonadotrophin-stimulated and -unstimulated mice. 1. FSH (1 IU/L-75 IU/L) provoked a dose-dependent increase in GVBD and PB in CE O, but not in DO, in stimulated and unstimulated mice. Eight IU/L was sufficient for inducing resumption of meiosis. In contrast, LH and hCG (both 1 IU/L-1500 IU/L) were without effect on GVBD and PB in CEO and DO of oocytes from stimulated and unstimulated mice. A combination of 8IU/L FSH and 4-8 IU/L hCG produced an additive effect, whereas combi nations with LH and higher concentrations of hCG had no such effect. 2 . A 2 hr priming with FSH (8 IU/L-75 IU/L) induced a dose-dependent oo cyte maturation in CEO. Thirty minutes of priming with FSH (75 IU/L) w as sufficient for induction of meiotic resumption in CEO. 3. Priming C EO with FSH for 2 hr followed by the separation and repooling of oocyt es and cumulus cells induced oocyte maturation. GVBD of new, unprimed DO added to cumulus cells of primed CEO increased slightly but was sig nificant, whereas GVBD in DO isolated from the primed CEO only increas ed marginally. DO cocultured with FSH-primed cumulus masses seem to be prevented from resuming meiosis. 4. Priming a coculture of granulosa cells and DO with FSH for 2 hr caused a significant increase in GVBD c ompared to the control, evaluated after 24 hr. In contrast, a 24 hr FS H-priming of a coculture of granulosa cells and DO was without effect on GVBD. 5. A spent medium in which unstimulated cumulus cells or mura l granulosa cells had grown was without effect on GVBD in DO. However, a small fraction of the DO resumed meiosis after culture in a spent m edium derived from a 2 hr priming of CEO and spent media from 24 hr pr iming of CEO induced a 2-3 times higher GVBD frequency in the DO compa red to the controls. Heat treatment of spent media (70 degrees C, 30 m in) from a 24 hr FSH-priming of CEO still induced GVBD in naive DO. Th e results showed that FSH, in a concentration of as little as 8 IU/L, but not r-LH and hCG, induced within 30 minutes the cumulus cells to p roduce and after 2 hr to secrete a diffusible heat stable meiosis acti vating substance, This substance overcame, ina paracrine fashion, the inhibiting effect of HX and induced oocyte maturation directly in DO. The production of this substance, however, was dependent on the initia l connection between the cumulus cells and the oocyte, indicating an i mportant 2-way communication between these 2 cell types. The mural gra nulosa cells did not produce a meiosis inducing activity by stimulatio n with FSH, but significantly, more DO matured after coculture with th e nonstimulated granulosa cells for 24 hr than for 2 hr. It is propose d that the heat stable meiosis activating component of the spent media from the FSH-stimulated CEO belongs to the meiosis activating sterols , MAS, previously isolated from human follicular fluid and from adult bull testes, (C) 1997 Wiley-Liss, Inc.