PERTURBATION OF SHEEP OVARIAN SURFACE EPITHELIAL-CELLS BY OVULATION -EVIDENCE FOR ROLES OF PROGESTERONE AND POLY(ADP-RIBOSE) POLYMERASE INTHE RESTORATION OF DNA INTEGRITY

Ovarian cells within the area of impending follicular rupture ill the sheep exhibit evidence of DNA fragmentation. Most cells undergo a dege nerative process indicative of apoptosis and are deleted before ovulat ion. However, some ovarian surface epithelial cells located along the margins of ruptured follicles persist (with damaged DNA) into the ensu ing luteal phase. It is conceivable that a genetically-altered progeni tor 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 mali gnant phenotype that is propagated during the postovulatory ovarian su rface wound-repair process. It was hypothesized that progesterone deri ved from the formative corpus luteum reestablishes genomic integrity a mong ovarian surface epithelial cells contiguous with the ovulatory si te and that this response is mediated by the DNA repair/apoptosis-acti vating enzyme poly(ADP-ribose) polymerase (PARP). Ovarian surface epit helial cells immediately surrounding the ovulatory stigma of sheep fol licles were recovered on luteal days 1, 2 or 4; control cells were obt ained from an ipsilateral ovarian region removed from the point of ovu lation. Immunofluorescent staining of end-linked digoxigenin or poly(A DP-ribose) was used to detect fragmented DNA or PARP-modified proteins within individual cells. Approximately 25% of surface epithelial cell s 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 pro gesterone production invoked by ovarian perivascular injection (day 1) of the 3 beta-hydroxysteroid dehydrogenase inhibitor isoxazol; this e ffect was reversed by exogenous progesterone. There was a positive rel ationship on day 2 between the level of ovarian progesterone and immun ostain-ng of ADP-ribose polymers in surface epithelial cells associate d with the ovulatory site. Evidence of DNA fragmentation or PARP activ ity in control cells was low and not affected by time of sample collec tion 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 su ggested that DNA damage to ovarian surface epithelium that is inflicte d at ovulation is (normally) reconciled on a localized basis by proges terone/PARP-mediated repair or dedicated apoptotic cell death, thereby conferring protection against clonal transformation.