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Characterization of Prohibitin in a Newly Established Rat Ovarian Granulosa Cell Line

Department of Obstetrics and Gynecology (W.E.T., A.B.), Department of Anatomy and Neurobiology (J.A.W., K.T.), Department of Biochemistry (D.L.), Morehouse School of Medicine, Atlanta, Georgia 30310; Department of Obstetrics and Gynecology (M.Z.), Boston University School of Medicine, Boston, Massachusetts 02118; and Department of Biochemistry (K.E.M.), Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208

Address all correspondence and requests for reprints to: Dr. Winston E. Thompson, Department of Obstetrics and Gynecology, Morehouse School of Medicine, 720 Westview Drive Southwest, Atlanta, Georgia 30310. E-mail: thompsw{at}msm.edu

Prohibitin is an evolutionary conserved protein that is associated with cellular differentiation, atresia, and luteolysis in the rat ovary. However, the specific cellular location and function of prohibitin in ovarian cells has not been clearly elucidated. To characterize the expression of prohibitin during cell proliferation, differentiation, and cell death, we have successfully established a temperature-sensitive granulosa cell line, designated RGA-1. At a permissive temperature of 33 C, RGA-1 cells proliferate, but revert to a differentiated phenotype at a nonpermissive temperature of 39 C. Significant inductions of prohibitin mRNA and protein expression were observed in the differentiated phenotype when compared with proliferating cells. Differentiated RGA-1 cells were found to express inhibin - and ß-transcripts, as well as steroidogenic acute regulatory protein and peripheral-type benzodiazepine receptor proteins in a manner reminiscent of steroidogenic functional responses observed in primary differentiated granulosa cells. Prohibitin expression correlated well with the expression of these steroidogenic proteins. At 39 C, RGA-1 cells also displayed increases in p53 protein levels, indicative of growth arrest in the nonproliferating cells. Confocal and electron microscopic examinations revealed increased prohibitin localization to the mitochondria at 39 C, along with changes in mitochondrial size and shape. These changes were accompanied by marked reductions in cytochrome c oxidase subunit II levels and in unit mitochondrial transmembrane potential. In addition, cell fractionation studies demonstrated that the prohibitin protein was mainly localized to the mitochondrial membrane. Collectively, these findings suggest a role for prohibitin in mitochondrial structure and function during growth and differentiation in ovarian granulosa cells. Prohibitin expression may also be indicative of mitochondrial destabilization during apoptosis-related events.

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