Characterization of prohibitin in a newly established rat ovarian granulosa cell line

Prohibitin is an evolutionary conserved protein that is associated with cel lular differentiation, atresia, and luteolysis in the rat ovary. However, t he specific cellular location and function of prohibitin in ovarian cells h as not been clearly elucidated. To characterize the expression of prohibiti n during cell proliferation, differentiation, and cell death, we have succe ssfully established a temperature-sensitive granulosa cell line, designated RGA-1. At a permissive temperature of 33 C, RGA-1 cells proliferate, but r evert to a differentiated phenotype at a nonpermissive temperature of 39 C. Significant inductions of prohibitin mRNA and protein expression were obse rved in the differentiated phenotype when compared with proliferating cells . Differentiated RGA-1 cells were found to express inhibin alpha- and beta -transcripts, as well as steroidogenic acute regulatory protein and periphe ral-type benzodiazepine receptor proteins in a manner reminiscent of steroi dogenic functional responses observed in primary differentiated granulosa c ells. Prohibitin expression correlated well with the expression of these st eroidogenic 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 prohibiti n localization to the mitochondria at 39 C, along with changes in mitochond rial size and shape. These changes were accompanied by marked reductions in cytochrome c oxidase subunit II levels and in unit mitochondrial transmemb rane potential. In addition, cell fractionation studies demonstrated that t he prohibitin protein was mainly localized to the mitochondrial membrane. C ollectively, these findings suggest a role for prohibitin in mitochondrial structure and function during growth and differentiation in ovarian granulo sa cells. Prohibitin expression may also be indicative of mitochondrial des tabilization during apoptosis-related events.