INHIBITION OF NITRIC-OXIDE - EFFECTS ON INTERLEUKIN-1-BETA-ENHANCED OVULATION RATE STEROID-HORMONES, AND OVARIAN LEUKOCYTE DISTRIBUTION AT OVULATION IN THE RAT

The ovulatory process resembles an inflammatory reaction with an infil tration of leukocytes, production of inflammatory mediators such as cy tokines, and a general edema and hyperemia. Nitric oxide (NO), a poten t vasodilator and the main mediator of macrophage tumoricidal and bact eriocidal activities, is known to participate in inflammatory reaction s and has been shown to mediate the interleukin-1 beta (IL-1 beta)-dir ected tissue-remodeling events within the ovary. The regulation by NO of ovulation rate, leukocyte distribution, and steroid release in the rat ovary was investigated through use of a combination of in vivo and in vitro models of ovulation and a competitive inhibitor, N-omega-nit ro-L-arginine methyl ester (L-NAME), of the NO synthase (NOS) enzyme. Subcutaneous L-NAME (1.5 x 10(-4) mol/kg) administration significantly reduced the in vivo ovulation rate of eCG/hCG-primed rats (L-NAME-tre ated: 10.6 +/- 1.8 [mean +/- SEM] oocytes per ovary [O/O], 11.0 +/- 1. 2 rupture sites per ovary [RS/O]; saline-treated: 18.0 +/- 1.8 O/O, 19 .4 +/- 1.1 RS/O; p < 0.01) at 20 h post-hCG. These results were reflec ted in vitro, where addition of L-NAME (3.5 x 10(-5) mol/L) to LH (0.1 mu g/ml)-perfused ovaries decreased ovulation rate from 8.2 +/- 1.6 t o 2.7 +/- 1 ovulations per ovary (p < 0.05) and simultaneously decreas ed nitrite accumulation at the completion of perfusions from 16.5 +/- 1.9 to 4.1 +/- 0.5 nmol/ml (p < 0.001). The addition of L-NAME to LH IL-1 beta (4 ng/ml)-perfused ovaries decreased ovulation rate from 15 .2 +/- 2.4 to 0.8 +/- 0.8 ovulations per ovary (p < 0.001) and simulta neously decreased nitrite accumulation at 22 h from 22.8 +/- 2.2 to 1. 9 +/- 0.6 nmol/ml (p < 0.001). Studies analyzing and manipulating perf usion flow rate indicated that the L-NAME effects on ovulation rate ar e primarily due to a reduction in flow rate resulting from inhibition of NO, which may be a consequence of the known vasoconstrictor effects of NOS inhibitors. The observed reduction of in vivo ovulation rate b y NO inhibition at 20 h post-hCG was associated with a significant red uction in thecal MCA149+ neutrophils at 12 h post-hCG, the expected ti me of ovulation (L-NAME-treated: 98.4 +/- 9.2 cells per thecal area; s aline-treated: 211.5 +/- 11.5 cells per thecal area; p < 0.001), while ED1+ monocytes/macrophages underwent similar but nonsignificant chang es. Plasma (20 h post-hCG) and perfusate progesterone were not differe nt with L-NAME treatment, while perfusate estradiol levels were marked ly reduced upon addition of L-NAME, suggesting a role for NO in ovulat ion but not in the process of luteinization. In summary, deprivation o f NO by use of the competitive inhibitor, L-NAME, led to fewer ovulati ons, reduced accumulation of nitrite, a decreased neutrophil count in the theca of preovulatory follicles, and reduced estradiol secretion, while progesterone release remained unaffected. The NO pathway may the refore play an important role in the regulation of ovulation and the m ediation of IL-1 beta's pro-ovulatory effects. There are likely to be primarily vascular effects, but also a nonvascular component, to the N O regulation of ovulation, with both components indirectly affecting o vulatory leukocyte distribution and steroid secretion.