Bcl-Xlong protein expression and phosphorylation in granulosa cells

Expression of Bcl-x protein was evaluated in hen ovarian follicles, relative to stage of development, and in cultured granulosa cells after treatment with various apoptosis-suppressing or -inducing agents. Using Western blot analysis, Bcl-Xlong was most frequently observed migrating as a doublet with a molecular mass of approximately 28 kDa; the apparent higher molecular mass band of this doublet was determined to represent a phosphorylated form. Consistent with previous findings reported for bcl-x messenger RNA, only the Bcl-Xlong (apoptosis-suppressing) form of protein was detected in the hen granulosa cells, and highest levels of Bcl-Xlong protein (sum of the protein doublet) expression were found in granulosa from preovulatory follicles together with tissues with immune function (e.g. spleen and bone marrow). Evidence for Bcl-Xshort expression was found only in the theca and several nonovarian tissues. Immunocytochemical analysis of preovulatory vs. prehierarchal follicles confirmed the comparatively greater expression of cytoplasmic Bcl-Xlong, particularly in preovulatory follicle granulosa. Levels of Bcl-Xlong were significantly increased after 20 h of culture in the presence of 8-bromo-cAMP (8-br-cAMP; compared with culture in control medium) in granulosa cells from both stages of follicle development. Such results are correlated with the protein's proposed function to protect against cell death in apoptosis-resistant, preovulatory follicle granulosa cells and are consistent with the ability of this cAMP agonist to increase bcl-Xlong messenger RNA levels in cultured cells. Furthermore, several factors that have previously been demonstrated to suppress apoptosis in granulosa cells, in vitro, (e.g. 8-br-cAMP, LH, FSH) were found to rapidly (within 15 min) increase levels of phosphorylated Bcl-Xlong, compared with control cells, whereas an inhibitor of protein kinase A (H-89) blocked such phosphorylation. By comparison, transforming growth factor α, a factor previously found to attenuate apoptosis and apoptosis-inducing agents (e.g. paclitaxel, C8-ceramide, daunorubicin, UV irradiation) failed to phosphorylate Bcl-Xlong. From these studies, it is concluded that both the phosphorylation of Bcl-Xlong (a short-term response) and increased levels of Bcl-Xlong (a comparatively slower response) in hen granulosa cells are promoted by gonadotropins via the adenylyl cyclase/cAMP signaling pathway. Moreover, elevated levels of chicken Bcl-Xlong protein expression and its phosphorylated state are correlated with resistance to apoptotic cell death in granulosa cells in vitro and ultimately a resistance to ovarian follicle atresia in vivo.