Perturbation of sheep ovarian surface epithelial cells by ovulation: evidence for roles of progesterone and poly(ADP-ribose) polymerase in the restoration of DNA integrity

Abstract

Ovarian cells within the area of impending follicular rupture in the sheep exhibit evidence of DNA fragmentation. Most cells undergo a degenerative process indicative of apoptosis and are deleted before ovulation. However, some ovarian surface epithelial cells located along the margins of ruptured follicles persist (with damaged DNA) into the ensuing luteal phase. It is conceivable that a genetically-altered progenitor cell that has survived a sub-lethal insult at ovulation (i.e. with unrepaired DNA but not committed to death) could give rise to a malignant phenotype that is propagated during the postovulatory ovarian surface wound-repair process. It was hypothesized that progesterone derived from the formative corpus luteum reestablishes genomic integrity among ovarian surface epithelial cells contiguous with the ovulatory site and that this response is mediated by the DNA repair/apoptosis-activating enzyme poly(ADP-ribose) polymerase (PARP). Ovarian surface epithelial cells immediately surrounding the ovulatory stigma of sheep follicles were recovered on luteal days 1, 2 or 4; control cells were obtained from an ipsilateral ovarian region removed from the point of ovulation. Immunofluorescent staining of end-linked digoxigenin or poly(ADP-ribose) was used to detect fragmented DNA or PARP-modified proteins within individual cells. Approximately 25% of surface epithelial cells that bordered the ovarian rupture site contained damaged DNA on days 1 and 2; there was a dramatic decrease by day 4 (< 5%). The decline in DNA-compromised cells was negated by in vivo inhibition of lutein progesterone production invoked by ovarian perivascular injection (day 1) of the 3 beta-hydroxysteroid dehydrogenase inhibitor isoxazol; this effect was reversed by exogenous progesterone. There was a positive relationship on day 2 between the level of ovarian progesterone and immunostaining of ADP-ribose polymers in surface epithelial cells associated with the ovulatory site. Evidence of DNA fragmentation or PARP activity in control cells was low and not affected by time of sample collection or treatments. The progesterone receptor antagonist RU486 and the transcriptional inhibitor actinomycin D blocked increases in PARP in ovarian surface epithelial cells incubated with progesterone. It is suggested that DNA damage to ovarian surface epithelium that is inflicted at ovulation is (normally) reconciled on a localized basis by progesterone/PARP-mediated repair or dedicated apoptotic cell death thereby conferring protection against clonal transformation.