Requirement for phosphatidylinositol-3 '-kinase in cytokine-mediated germ cell survival during fetal oogenesis in the mouse

Apoptosis is responsible for primordial germ cell (PGC) attrition in the de veloping fetal ovary. In monolayer cultures of murine PGC, stem cell factor (SCF) and leukemia inhibitory factor (LIF) independently promote survival in vitro; however, the relevance of these data to fetal ovarian oogonium an d oocyte survival, as well as the intracellular events involved in transduc ing the antiapoptotic actions of these cytokines in germ cells, remain to b e elucidated. In this report, we investigated the effects of SCF and LIF, a lone and in combination, on the survival of oogonia and oocytes, and elabor ated on components of the signal transduction pathway used by these molecul es, after validating a method of culturing fetal mouse ovaries. We further employed this system to also test the hypothesis that insulin-like growth f actor-I (IGF-I), a classic antiapoptotic molecule, and transforming growth factor-beta (TGF-beta), a classic pro-apoptotic molecule, interact with the SCF/LIF pathway and function in a reciprocal fashion to precisely regulate germ cell numbers during fetal oogenesis. Freshly isolated embryonic day 1 3.5 ovaries contained nonapoptotic germ cells, as determined by histologic analysis of cellular morphology and in situ 3'-end-labeling of DNA integrit y. In vitro culture of fetal ovaries without tropic support for 24, 48, and 72 h resulted in a time-dependent induction of germ cell apoptosis, such t hat most oogonia and oocytes present after 72 h were apoptotic. Morphometri c analysis of serially sectioned ovaries indicated that the numbers of nona poptotic germ cells remaining after 24, 48, and 72 h of culture were 78%, 3 8%, and 10%, respectively, of the number present before culture (P < 0.05 f or all time points vs. 0 h). Inclusion of SCF (100 ng/ml) together with LIF (100 ng/ml) in the culture medium significantly attenuated germ cell apopt osis, with the SCF/LIF-treated ovaries retaining 5.5-fold more oogonia and oocytes after 72 h of culture as compared with control ovaries deprived of tropic support (P < 0.05). However, SCF or LIF, when added separately, had no (SCF)or little (LIF) inhibitory effect on germ cell apoptosis. Provision of 50 ng/ml TGF-I maintained survival of approximately two-thirds of the g erm cells in cultured ovaries (P < 0.05), whereas a combination of all thre e growth factors (SCF, LIF, IGF-I) completely preserved the fetal ovary in culture to that resembling a freshly-isolated gonad. Cotreatment with 25 ng /ml TGF-beta partially reversed the survival actions of IGF-I or SCF/LIF, s uch that only one-third of the starting number of oogonia/oocytes remained after 72 h of culture (P < 0.05). Lastly, the antiapoptotic effects of SCF/ LIF or IGF-I were almost entirely eliminated by cotreatment of fetal ovarie s with either one of two inhibitors of phosphatidylinositol-3'-kinase (PI3K ), LY294002 (5 mu M) or wortmannin (50 nM), whereas cotreatment with an inh ibitor of p70 S6 kinase (rapamycin, 25 ng/ml) was without effect. These dat a indicate that the combined actions of SCF, LIF, and IGF-I are required fo r maximal inhibition of apoptosis in germ cells of fetal mouse ovaries, and that the PI3K signaling pathway is an essential component of cytokine-medi ated female germ cell survival. Moreover, TGF-beta can partially override t he antiapoptotic actions of SCF/LIF or IGF-I in oogonia and oocytes, sugges ting the existence of a complex signaling network that ultimately determine s fetal ovarian germ cell fate.