Localization, regulation and possible consequences of apoptotic protease-activating factor-1 (Apaf-1) expression in granulosa cells of the mouse ovary

The recent characterization of apoptotic protease-activating factor-1 (Apaf -1) in vertebrates as a putative homolog of the Caenorhabditis elegans gene , ced-4, indicates that the third major arm of the C. elegans programmed ce ll death machinery has also been conserved through evolution. Although apop tosis is now known to be important for ovarian follicular atresia in verteb rates, nothing is known of the role of Apaf-1 in ovarian function. Herein w e show by immunohistochemical analysis that Apaf-1 is abundant in granulosa cells of early antral follicles whereas in vivo gonadotropin priming compl etely suppresses Apaf-1 expression and granulosa cell apoptosis. Western bl ot analysis of fractionated protein extracts prepared from granulosa cells before and after in vitro culture without hormonal support to induce apopto sis indicated that mitochondrial cytochrome c release, a biochemical step r equired for the activation of Apaf-1, occurs in granulosa cells cultured in vitro. Moreover, Western blot analysis of procaspase-3 processing, a princ ipal downstream event set in motion by activated Apaf-1, indicated that hea lthy granulosa cells possess almost exclusively the inactive (pro-) form of the enzyme whereas granulosa cells deprived of hormonal support rapidly pr ocess procaspase-3 to the active enzyme. Lastly, we show that serum-starved granulosa cells activate caspase-3-like enzymes both prior to and after nu clear pyknosis, as revealed by a single-cell fluorescent caspase activity a ssay. These data, combined with previous observations regarding the role of homologs of the two other C. elegans cell death regulatory genes, ced-9 (B cl-2 family members) and ced-3 (caspases), in atresia fully support the hyp othesis that granulosa cell apoptosis is precisely coordinated by all three major arms of a cell death program conserved through evolution.