HORMONAL-REGULATION OF NITRIC-OXIDE SYNTHASES AND THEIR CELL-SPECIFICEXPRESSION DURING FOLLICULAR DEVELOPMENT IN THE RAT OVARY

Nitric oxide (NO) has emerged as a novel regulator of several ovarian events, such as ovulation. steroidogenesis, and apoptotic cell death. The NO synthases (NOS) are a family of enzymes that catalyze the oxida tion of L-arginine to NO and L-citrulline. The purpose of the present study was to localize NOS isoforms in the rat ovary and to examine the ir hormonal regulation. We conducted immunohistochemistry and Western blot analysis using isoform-specific antibodies against brain NOS, end othelial NOS (eNOS), and inducible NOS (iNOS). Immature rats were supe rovulated by injecting PMSG (10 IU sc) followed by an injection of hum an CG (hCG; 10 IU sc) 48 h later. Ovaries were obtained from control r ats (no PMSG), 24 h and 48 h after PMSG treatment and 2 h, 8 h, 12 h, 20 h or 6 days and 10 days after hCG injection (n = 3-5 rats/group). R at ovaries were clearly devoid of brain NOS staining at any of the tim e points studied. In control ovaries, eNOS was detected in theca cell layer, ovarian stroma, and on the surface of oocytes. During follicula r development, eNOS staining was still expressed in the theca cell lay er and was also present in mural granulosa cells. After ovulation, hom ogenous eNOS staining was observed within cells of the corpus luteum ( CL), Western blots of ovarian homogenates demonstrated that during PMS G-induced follicle growth, eNOS levels increased by 2.5-fold relative to control rats (P < 0.05). eNOS levels were further increased 12 h an d 20 h after hCG injection (5-fold and 7-fold, respectively, relative to control; P < 0.05). The greatest amount of eNOS was observed in ova ries 10 days after hCG injection (15-fold relative to control; P < 0.0 5). We also detected expression of iNOS in the ovary, but the pattern and cell-specific staining differed from that observed for eNOS. In im mature ovaries and during follicular development, iNOS staining was fo und within the theca cell layer and stroma. After ovulation, iNOS stai ning was present only in the external layers of the developing CL, but in the degenerating CL (10 days post-hCG), strong staining in nonpare nchymal cells was observed within the entire CL. Western blots showed no changes in levels of ovarian iNOS protein during follicular develop ment, but a significant increase (6-fold relative to control; P < 0.05 ) was observed after an ovulatory dose of hCG. The highest level of iN OS was observed in ovaries 10 days after hCG injection (10-fold relati ve to control; P < 0.05). Our data demonstrate that ovarian eNOS and i NOS show distinct cell-specific expression patterns and are differenti ally regulated during follicular and luteal development.