Segregation of retinoic acid effects on fetal ovarian germ cell mitosis versus apoptosis by requirement for new macromolecular synthesis

Retinoic acid (RA), a naturally occurring metabolite of vitamin A, plays an essential role in regulating cellular growth, differentiation, and death in a variety of tissues, particularly during fetal development. However, essentially nothing is known of the effects of RA on fetal gametogenesis. Using a recently validated system of culturing murine fetal ovaries, herein we sought to characterize the actions of RA on female germ cell proliferation and apoptosis during oogenesis. In the absence of trophic hormone support, approximately 90% of the oogonia and oocytes present in fetal ovaries at the start of culture underwent apoptosis over a 72 h culture period (P < 0.05), whereas provision of 0.01-1 mu M RA dose dependently maintained germ cell numbers. In fact, ovaries cultured with 0.1 mu M RA for 72 h possessed approximately 30% more oogonia and oocytes as compared with the preculture mean number (P < 0.05). Additional experiments, using in situ DNA 3'-end-labeling and cellular morphology to assess apoptosis coupled with 5-bromo-2'-deoxyuridine incorporation to assess proliferation, revealed that RA acts as both a mitogen and a survival factor for female germ cells. Furthermore, the ability of RA to stimulate germ cell proliferation in cultured fetal ovaries was completely suppressed (P < 0.05) by cotreatment with inhibitors of transcription (alpha-amanitin, 0.1 mu g/ml) or protein synthesis (cycloheximide, 1.0 mu g/ml), whereas RC1-mediated suppression of germ cell apoptosis was not affected by cotreatment with either macromolecular synthesis inhibitor (P > 0.05). Moreover, cotreatment of fetal ovaries with 5 mu M LY294002, an inhibitor of phosphatidylinositol 3'-kinase, had no effect on RA-promoted germ cell maintenance (P > 0.05). By comparison, the antiapoptotic effects of insulin-like growth factor I on germ cells in cultured fetal ovaries were significantly attenuated by cotreating ovaries with LY294002 (P < 0.05) but not with alpha-amanitin or cycloheximide (P > 0.05). Importantly, the effect of RA on the female germ line was also observed in vivo because a single oral administration of 100 mg/kg RA to timed-pregnant female mice resulted in a significantly (P < 0.05) larger endowment of primordial oocytes in female offspring. That these actions were mediated, at least in part, by specific retinoid receptors was demonstrated by the finding of retinoic acid receptor protein in fetal female gonocytes, as assessed by immunohistochemical localization experiments. Collectively, these data indicate that RA can function, in vitro and in, vivo, as a potent germ cell survival factor and mitogen during fetal oogenesis in the mouse.