Apoptotic and proliferative changes during induced atresia of pre-ovulatory follicles in the rat

Atresia, a degenerative process through which many follicles are removed fr om the growing pool, involves apoptotic changes in the follicular granulosa cells. To identify histochemical markers of early stages of atresia, an in -vivo rat model was used which allowed the study of atresia of pre-ovulator y follicles in a synchronized and chronological order. By blocking the pre- ovulatory luteinizing hormone surge with a gonadotrophin-releasing hormone (GnRH) antagonist, ovulation of the pre-ovulatory follicles is prevented, a fter which these follicles became atretic. The first morphological sign of atresia (pyknotic granulosa cell nuclei) was found 27 h after injection of GnRH antagonist. Since the pre-ovulatory follicles gradually become atretic in a synchronous fashion, this model provided an opportunity to study and define markers of future atresia in pre-ovulatory follicles, Atresia involv es apoptosis of granulosa cells, and therefore internucleosomal DNA fragmen tation was examined. Using the terminal deoxynucleotidyltransferase-mediate d dUTP-biotin nick end labelling (TUNEL) assay it was found that the first sign of internucleosomal DNA fragmentation in granulosa cells of pre-ovulat ory follicles was detectable 24 h after GnRH antagonist treatment. Ln order to find an upstream marker of atresia, the 5-bromo-deoxyuridine (BrdU) lab elling index was used as a measure of proliferation, Already at 14 h after GnRH antagonist treatment, when morphological signs of atresia were not yet present, a clear decrease in BrdU labelling index was found in the granulo sa cells.