EXPRESSION AND FUNCTION OF A PROAPOPTOTIC BCL-2 FAMILY MEMBER BCL-XL BCL-2-ASSOCIATED DEATH PROMOTER (BAD) IN RAT OVARY/

Bcl-2-related anti-and proapoptotic proteins are important in the deci sion step of the intracellular death program upstream from the caspase proteases. Targeted overexpression of Bcl-2 in ovarian somatic cells of transgenic mice leads to decreased apoptosis of granulosa cells and is associated with higher ovulation rate, increased litter size, and ovarian teratoma formation. The ability of exogenous Bcl-2 proteins to promote follicle cell survival suggests that the trans gene can bind to endogenous ovarian Bcl-2 family members and modulate the intracellu lar apoptosis process in favor of cell survival. We used the yeast two -hybrid system to search for ovarian Bcl-2 interacting proteins. The s creening of an ovarian fusion complementary DNA library yielded severa l clones encoding for the death agonist Bcl-XL/Bcl-2-associated death promoter (BAD). Dimerization of Bcl-2-related proteins mediated by the consensus Bcl-2 homology (BH) domains is essential for their apoptosi s-regulating function. Consistent with these observations, yeast two-h ybrid assays indicated that the interaction of Bcl-2 with BAD is depen dent on both BH4 and BH2 domains of Bcl-2. Northern blot analysis show ed a wide distribution of BAD messenger RNA (mRNA) in diverse tissues with highest levels in the lung, ovary, uterus, and brain. In situ hyb ridization analysis indicated BAD mRNA expression in granulosa cells o f different sizes of follicles and also in the theca and interstitial cells. BAD mRNA was expressed in the ovaries between postnatal 15-27 d ays and did not alter during the developmentally occurring apoptosis f ound about postnatal day 18 when the first group of early antral folli cles were formed. Similarly, BAD mRNA levels did not change during fol licle atresia induced by estrogen withdrawal in immature rats. To stud y the role of BAD in the ovary, BAD complementary DNA was transfected into primary cultures of granulosa cells and in a gonadal tumor cell l ine. Overexpression of BAD induced apoptosis in both cell types, and t he effect of BAD was reversed by a membrane-permeable caspase inhibito r, indicating that BAD induces apoptosis via the activation of caspase cysteine proteases. In summary, the death agonist BAD was identified as a Eel-a-interacting protein in the ovary, and BAD mRNA is constitut ively expressed in granulosa cells, suggesting that BAD is an essentia l part of the ovarian cell death process. Because BAD overexpression i n granulosa cells leads to apoptosis, future studies on ovarian BAD bi nding proteins and hormonal regulation of the interactions among diffe rent Bcl-2 family members could provide a better understanding of the cellular mechanism of ovarian follicle atresia.