EXPRESSION OF INHIBITOR OF APOPTOSIS PROTEINS (IAPS) IN RAT GRANULOSA-CELLS DURING OVARIAN FOLLICULAR DEVELOPMENT AND ATRESIA

The inhibitor of apoptosis proteins (IAPs) constitute a family of high ly conserved apoptosis suppressor proteins that was originally identif ied in baculoviruses. Although IAP homologs have been recently identif ied and demonstrated to suppress apoptosis in mammalian cells, their e xpression and role during follicular development and atresia are unkno wn. The present study was conducted to address these questions. Using established in vivo models for the induction of follicular development and atresia in immature rats, it was possible to compare the immunolo calization of X-link inhibitor of apoptosis protein (Xiap) and human i nhibitor of apoptosis protein-2 (Hiap-2), two members of the IAP famil y, at defined stages of follicular maturation and to relate the differ ences observed with those of follicular cell proliferation and apoptos is [as determined by proliferating cell nuclear antigen (PCNA) immunoh istochemistry and in. situ terminal deoxynucleotidyl transferase-media ted dUTP-biotin end labeling (TUNEL), respectively]. In addition, gran ulosa cell DNA and proteins were assessed for apoptotic fragmentation by 3'-end labeling/agarose gel electrophoresis (DNA ladder) and Hiap-2 and Xiap protein content by Western blot analysis, respectively. Hiap -2 and Xiap expression in both granulosa and theca cells increased wit h follicular maturation, reaching maximal levels at the antral stage o f development. The immunoreactivity for PCNA, Xiap, and Hiap-2 decreas ed markedly in atretic (TUNEL-positive) follicles at the small to medi um sized antral stage of development, suggesting follicular atresia ma y be associated with decreased granulosa cell LAP protein content and decreased proliferation. Atresia was also associated with a change in the intracellular distribution of IAPs in granulosa cells. Biochemical analysis of DNA fragmentation (DNA ladder) in granulosa cells from pr eantral and early antral follicles indicates extensive apoptosis that was associated with minimal IAP protein content. Gonadotropin treatmen t increased Hiap-2 and Xiap protein content and suppressed apoptosis i n granulosa cells, resulting in the development of follicles to the an tral and preovulatory stages. In addition, gonadotropin withdrawal ind uced apoptotic DNA fragmentation in granulosa cells in early antral an d antral follicles, which is accompanied by a marked decrease in Hiap- 2 and Xiap expression. These data suggest that IAPs may be involved in the suppression of granulosa cell apoptosis by gonadotropin in small to medium-sized antral follicles and play an important role in determi ning the fate of the cells, and thus also the eventual follicular dest iny (atresia us. ovulation).