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Regular Article |
a Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
ABSTRACT
Follicles from the hen ovary that have been selected into the preovulatory hierarchy are committed to ovulation and rarely become atretic under normal physiological conditions. In part, this is attributed to the resistance of the granulosa layer to apoptosis. The present studies were conducted to evaluate the role of the phosphatidylinositol (PI) 3-kinase/Akt signaling pathway in hen granulosa cell survival and, by implication, follicle viability. Cloning of the chicken akt2 homologue revealed a high degree of amino acid homology to its mammalian counterparts within the catalytic domain, plus complete conservation of the putative Thr308 and Ser474 phosphorylation sites. Treatment of granulosa cells from the three largest preovulatory follicles with insulin-like growth factor (IGF)-I and, to a lesser extent, transforming growth factor (TGF)-
induces rapid phosphorylation of Akt, and such phosphorylation is effectively blocked by the PI 3-kinase-inhibitor LY294006. Serum withdrawal from cultured cells for 33–44 h initiates oligonucleosome formation, an indicator of apoptotic cell death, whereas cotreatment with IGF-I prevents this effect. Moreover, treatment of cultured cells for 20 h with LY294006 induces apoptosis. The potential for nonspecific cell toxicity following LY294006 treatment is considered unlikely because of the ability of either LH or 8-bromo cAMP cotreatment to block LY294006-induced cell death. Finally, both IGF-I and TGF- also activate mitogen-activated protein (MAP) kinase signaling, at least in part, through the phosphorylation of Erk. However, treatment with neither U0126 nor PD98059 (inhibitors of MAP kinase kinase) induced cell death in cultured granulosa cells, despite the ability of each inhibitor to effectively block Erk phosphorylation. Taken together, these results provide evidence for a role of the Akt signaling pathway in promoting cell survival within the preovulatory follicle granulosa layer. In addition, the data indicate the importance of an alternative survival pathway mediated via gonadotropins and protein kinase A independent of Akt signaling.
First decision: 21 November 2000.
1 Supported by USDA 99-35203-7736 and NIH HD36095. Genbank accession number for the chicken akt2 cDNA is AF181260.
2 Correspondence: A.L. Johnson, Department of Biological Sciences, P.O. Box 369, University of Notre Dame, Notre Dame, IN 46556. FAX: 219 631 7413; johnson.128{at}nd.edu
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