Hormonal Regulation of Nitric Oxide Synthases and Their Cell-Specific Expression 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. TheNO synthases (NOS) are a family of enzymes that catalyze the oxidation 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 their hormonal regulation. We conducted immunohistochemistry andWestern blot analysis using isoform-specific antibodies against brain NOS, endothelial NOS (eNOS), and inducible NOS (iNOS). Immature rats were superovulated by injecting PMSG (10 IU sc) followed by an injection of human CG (hCG; 10 IU sc) 48 h later. Ovaries were obtained from control rats (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 5 3–5 rats/group). Rat ovaries were clearly devoid of brain NOS staining at any of the time points studied. In control ovaries, eNOS was detected in the theca cell layer, ovarian stroma, and on the surface of oocytes. During follicular development, eNOS staining was still expressed in the theca cell layer and was also present in mural granulosa cells. After ovulation, homogenous eNOS stainingwas observedwithin cells of the corpus luteum (CL). Western blots of ovarian homogenates demonstrated that during PMSG-induced follicle growth, eNOS levels increased by 2.5-fold relative to control rats (P , 0.05). eNOS levels were further increased 12 h and 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 ovaries 10 days after hCG injection (15-fold relative to control; P , 0.05). We also detected expression of iNOS in the ovary, but the pattern and cell-specific staining differed from that observed for eNOS. In immature ovaries and during follicular development, iNOS staining was found within the theca cell layer and stroma. After ovulation, iNOS stainingwas present only in the external layers of the developing CL, but in the degenerating CL (10 days post-hCG), strong staining in nonparenchymal cells was observed within the entire CL. Western blots showed no changes in levels of ovarian iNOS protein during follicular development, but a significant increase (6-fold relative to control; P , 0.05) was observed after an ovulatory dose of hCG. The highest level of iNOS was observed in ovaries 10 days after hCG injection (10-fold relative to control; P , 0.05). Our data demonstrate that ovarian eNOS and iNOS show distinct cell-specific expression patterns and are differentially regulated during follicular and luteal development. (Endocrinology 138: 460–468, 1997) N OXIDE (NO) is now acknowledged as an important product of mammalian cells with critical functions in the regulation of blood pressure, neurotransmission, and host defense (1, 2). It is synthesized from arginine by NO synthase (NOS), which exists in multiple isoforms (1, 2). To date, three genes encoding NOSs with different biochemical characteristics have been isolated (3, 4). Two constitutively expressed isoforms were first identified in the brain and in the endothelium. These isoforms require calcium and calmodulin for activity and respond to stimuli by producing small quantities of NO (3, 5). In contrast, an inducible NOS (iNOS) has been found in a variety of cells, including cytokine-stimulated cells of the immune system (4). The expression of this isoform is correlated with cytotoxic/cytostatic events and results in a sustained synthesis of NO over long periods (1, 2). NO exerts many of its functions by reacting with metaland thiol-containing proteins, which can result in both activation and inhibition of the target