Diseases of the Reproductive System
(W201) EXPRESSION OF FSH INHIBITORS AND APOPTOTIC PROTEINS IS INCREASED IN MURINE CYSTIC OVARIAN FOLLICLES INDUCED BY NEONATAL ESTRADIOL TREATMENT.
Kelkar, Radhika1, Mahale, Smita1, Shinde, Gayatri1, Nandedkar, Tarala 1, 1 National Institute for Research in Reproductive Health, Mumbai, Maharashtra, India
ABSTRACT- Follicular development and ovulation is a cyclic process regulated by FSH and LH and involves an interplay of steroids and intraovarian factors. Discrepancies in the same could cause disorders such as Polycystic Ovaries that lead to infertility. In the present study, the expression of intraovarian FSH inhibitors has been studied with a view to delineate the role of these proteins in abnormal folliculogenesis, using mouse models for atresia and cystic ovaries. Normal and atretic ovarian follicles were obtained by treating 21–day old mice with eCG, for 48h and 72h, respectively. Cystic follicles were induced by administration of 60g 17Estradiol to 5–day old mice and autopsied at 12 weeks, while controls were injected with vehicle. Neonatal estradiol treatment induced anovulation and ovarian cysts with reduced granulosa cell (GC) layers and thecal cell hyperplasia. An increase in Androgen Receptor expression detected by immunohistochemistry (IHC) confirmed the cystic attribute of these follicles. An increase in the expression of inhibin – a negative regulator of FSH was noted in atretic and cystic GC by IHC. Similar results were noted in case of FSH Binding Inhibitor (FSHBI) – an intraovarian protein that inhibits FSH binding to GC in vitro. In view of the ability of both inhibin and FSHBI to induce follicular atresia, the expression of apoptotic proteins was investigated in cystic follicles. An increase in Fas, Fas-Associated Death Domain and Caspase 8 was noted by IHC in cystic GC as compared to normal and atretic GC. Interestingly, localization of Cytochrome c and Caspase 9 in cystic GC and alteration in mitochondrial membrane potential indicated by a decrease in the staining for rhodamine123 dye suggested an involvement of the mitochondrial pathway as well in cystogenesis. Flow cytometric analysis of active caspase 3 revealed an increase in the percentage of GC bearing the terminal protease in the cystic population as compared to the normal and atretic population. Further, DNA fragmentation in cystic GC was confirmed by Terminal transferase-mediated dUTP Nick End Labelling (TUNEL). Our results indicate that 1) granulosa cell apoptosis is enhanced during cystogenesis as compared to normal follicular development and atresia, and 2) both the mitochondrial and the death receptor pathway are involved in the same.
KEY WORDS: ovary, apoptosis, atresia, polycystic