Clusterin protects granulosa cells from apoptotic cell death during follicular atresia

Clusterin expression is associated with programmed cell death (apoptosis) i n many cell types but its exact role has not yet been defined. This study w as carried out to determine the cellular localization of clusterin in the o vary and its functional role in the apoptotic cell death of ovarian follicl es. A homogenous population of healthy and atretic follicles was obtained b y treating immature rats with pregnant mare serum gonadotropin (PMSG). Apop totic cell death was evaluated by TUNEL. Clusterin expression in the health y and atretic follicles was examined by immunohistochemical and Western blo t analyses, and gene expression was examined by Northen blot analysis. Clus terin protein and its mRNA are only expressed in granulosa cells of atretic follicles obtained from PMSG-treated rats on day 5 of the treatment. Healt hy follicles from PMSG-treated rats on day 2 of the treatment do not expres s clusterin. Theca and stroma cells of both healthy and atretic follicles s howed no signs of apoptosis and did not express clusterin. Withdrawal of tr ophic support from granulosa cells in cultures to induce apoptosis resulted in a dramatic increase in the levels of clusterin and its mRNA compared to cells cultured in serum-supplemented medium. In an attempt to establish th e functional role of clusterin in the apoptotic cell death of ovarian folli cles, the biosynthesis of clusterin in granulosa cells of healthy follicles was blocked by treatment of cells with antisense oligonucleotide to its cD NA. Treatment of granulosa cells with the antisense oligonucleotide resulte d in an increase in the apoptotic cell death compared to the control. These findings indicate that depletion of clusterin can lead to the programmed c ell death in ovary, suggesting a functional role for this protein in follic ular atresia. (C) 2000 Academic Press.