Characterization of the antiapoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) and the stimulation of its message by gonadotropins in the rat ovary

The majority of ovarian follicles undergo atresia mediated by apoptosis. Bc l-2-related proteins act as regulators of apoptosis via the formation of di mers with proteins inside and outside the Bcl-2 family. Previous studies ha ve identified BAD as a proapoptotic Bcl-2 family member expressed in the ov ary. It is known that BAD phosphorylation induced by survival factors leads to its preferential binding to 14-3-3 and suppression of the death-inducin g function of BAD. To identify ovarian binding partners for hypophosphoryla ted BAD, we performed a yeast two-hybrid screening of a rat ovary complemen tary DNA library using as bait a mutant BAD incapable of binding to 14-3-3. Screening of yeast transformants yielded positive clones encoding the rat ortholog of Mcl-1 (myeloid cell leukemia-1), an antiapoptotic Bcl-2 protein . Amino acid sequence analysis revealed that rat and human Mcl-1 showed a c omplete conservation of the Bcl-2 homology domains BH1, BH2, and BH3. In th e yeast two-hybrid system, Mcl-1 binds to the hypophosphorylated mutant of BAD and interacts preferentially with different proapoptotic (Bax, Bak, Bok , Bik, and BOD) compared with antiapoptotic Bcl-2 family members (Bcl-2, Bc l-xL, Bcl-w, Bfl-1, CED-9, and BHRF-1). Northern blot hybridization demonst rated expression of Mcl-1 transcripts of 2.3 and 3.7 kb in the ovary and di verse other rat tissues. In immature rats, PMSG treatment led to a transien t increase in the 2.3-kb Mcl-1 transcript, peaking at 6 h after injection a nd returning to baseline levels after 24 h. Moreover, the same transcript w as induced in the PMSG-primed preovulatory rat ovary 6 h after the administ ration of ovulatory doses of either hCG or FSH. In situ hybridization studi es revealed that the gonadotropin stimulation of ovarian Mel I message occu rs in both granulosa and thecal cells. In conclusion, rat Mcl-1 was identif ied as an ovarian BAD-interacting protein and the message for the antiapopt otic Mcl-1 protein was induced after treatment with gonadotropins in granul osa and thecal cells of growing follicles.