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 decision 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 transgene can bind to endogenous ovarian Bcl-2 family members and modulate the intracellular apoptosis process in favor of cell survival. We used the yeast two-hybrid system to search for ovarian Bcl-2 interacting proteins. The screening of an ovarian fusion complementary DNA library yielded several 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 apoptosis-regulating function. Consistent with these observations, yeast two-hybrid assays indicated that the interaction of Bcl-2 with BAD is dependent on both BH4 and BH2 domains of Bcl-2. Northern blot analysis showed a wide distribution of BAD messenger RNA (mRNA) in diverse tissues with highest levels in the lung, ovary, uterus, and brain. In situ hybridization analysis indicated BAD mRNA expression in granulosa cells of different sizes of follicles and also in the theca and interstitial cells. BAD mRNA was expressed in the ovaries between postnatal 15-27 days and did not alter during the developmentally occurring apoptosis found about postnatal day 18 when the first group of early antral follicles were formed. Similarly, BAD mRNA levels did not change during follicle atresia induced by estrogen withdrawal in immature rats. To study the role of BAD in the ovary, BAD complementary DNA was transfected into primary cultures of granulosa cells and in a gonadal tumor cell line. Overexpression of BAD induced apoptosis in both cell types, and the effect of BAD was reversed by a membrane-permeable caspase inhibitor, indicating that BAD induces apoptosis via the activation of caspase cysteine proteases. In summary, the death agonist BAD was identified as a Bcl-2-interacting protein in the ovary, and BAD mRNA is constitutively expressed in granulosa cells, suggesting that BAD is an essential part of the ovarian cell death process. Because BAD overexpression in granulosa cells leads to apoptosis, future studies on ovarian BAD binding proteins and hormonal regulation of the interactions among different Bcl-2 family members could provide a better understanding of the cellular mechanism of ovarian follicle atresia.
Kaipia
A
A
Department of Gynecology and Obstetrics, Stanford University School of Medicine, California 94305-5317, USA.
Hsu
S Y
SY
Hsueh
A J
AJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005260:Female; D019254:Genes, bcl-2 / Q000502:physiology*; D006107:Granulosa Cells / Q000378:metabolism; D005810:Multigene Family / Q000502:physiology*; D010053:Ovary / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014407:Tumor Cells, Cultured / Q000378:metabolism; D051020:bcl-X Protein
C494813:Bcl2l1 protein, rat; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051020:bcl-X Protein