Bcl-XLONG protein expression and phosphorylation in granulosa cells

Expression of Bcl-x protein was evaluated in hen ovarian follicles, relativ e 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 wit h a molecular mass of approximately 28 kDa; the apparent higher molecular m ass band of this doublet was determined to represent a phosphorylated form. Consistent with previous findings reported for bcl-x messenger RNA, only t he Bcl-XLONG (apoptosis-suppressing form of protein was detected in the hen granulosa cells, and highest levels of Bcl-XLONG protein (sum of the prote in doublet) expression were found in granulosa from preovulatory follicles together with tissues with immune function (e.g. spleen and bone mallow). E vidence for Bcl-XSHORT expression was found only in the theca and several n onovarian tissues. Immunocytochemical analysis of preovulatory vs, prehiera rchal follicles confirmed the comparatively greater expression of cytoplasm ic Bcl-XLONG, particularly in preovulatory follicle granulosa. Levels of Bc l-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 gra nulosa cells from both stages of follicle development. Such results are cor related with the protein's proposed function to protect against cell death in apoptosis-resistant, preovulatory follicle granulosa cells and are consi stent with the ability of this cAMP agonist to increase bcl-XLONG messenger RNA levels in cultured cells. Furthermore, several factors that have previ ously 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 l evels of phosphorylated Bcl-XLONG, compared with control cells, whereas an inhibitor of protein kinase A (H-89) blocked such phosphorylation. By compa rison, transforming growth factor a, a factor previously found to attenuate apoptosis and apoptosis-inducing agents (e.g. paclitaxel, C8-ceramide, dau norubicin, UV irradiation) failed to phosphorylate Bcl-XLONG. From these st udies, it is concluded that both the phosphorylation of Bcl-XLONG (a short- term response) and increased levels of Bcl-XLONG (a comparatively stower re sponse) in hen granulosa cells are promoted by gonadotropins via the adenyl yl 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 ultimate ly a resistance to ovarian follicle atresia in vivo.