Effect of Nitric Oxide on the Control of Ovarian functions

一氧化氮（NO）最近已被牵连在各种生理过程，包括生殖功能。本研究采用针吸法从猪中等卵泡中分离培养颗粒细胞，观察NO对颗粒细胞类固醇合成的影响。在预孵育后，将细胞与NO合酶（NOS）的竞争性阻断剂NNG-单甲基-L-精氨酸（LNMMA）和NO供体NOC 18单独孵育或在FSH（200 ng/ml）/ hCG（5IU/ml）存在下孵育。LNMMA（0.01- 1 mM）显著（P<0.05）促进FSH刺激的PGC基础E_2释放，并呈剂量依赖性地促进E_2和P的释放。LNMMA也显著（P<0.001）抑制培养基中NO^-浓度，通过化学发光测量。NOC 10（0.1 - 1 mM）显著抑制PGC培养2 h后E_2的释放（P<0.01）。用ABC免疫组化染色，免疫组化法检测细胞内组成型NOS和诱导型NOS的分布。此外，我们评估了cGMP和芳香化酶活性对NO诱导的类固醇生成抑制的可能参与。cGMP类似物8-bromo-cGMP对PGC培养24小时内E_2和P的积累无明显影响。NOC 18（0.1 - 1 mM）可显著抑制基础及促性腺激素刺激的PGC芳香化酶活性（P<0.01），表明NO不依赖于cGMP而直接抑制中成熟PGC芳香化酶活性，从而抑制E_2分泌，提示NO是卵泡发育的重要局部调节因子。

Nitric oxide (NO) has been recently implicated in a variety of physiological precesses, including reproductive functions. The present study was undertaken to investigate whether NO might significantly induce any change in steroidogenesis in cultured porcine granulosa cells (PGC).Cells were obtained from medium follicles by the needle aspiration method. After pre-incubation, cells were incubated with N^G-monomethyl-L-arginine (LNMMA), a competitive blocker of NO synthase (NOS), and the NO donor, NOC 18, either alone or in the presence of FSH (200ng/ml) / hCG (5IU/ml). Estradiol (E_2) and progesterone (P) concentrations in media were measured by EIA.LNMMA (0.01- 1 mM) significantly (P<0.05) stimulated the basal E_2 release and dosedependently enhanced the E_2 and P release from FSH-stimulated PGC for 24-h culture. LNMMA also significantly (P<0.001) suppressed the NO^- concentrations in media, measured by chemiluminescence. NOC 10 (0.1 - 1 mM) significantly (P<0.01) suppressed the E_2 release from basal and gonadotropin stimulated PGC for 2-h culture. Cells were immunostained with an avidin-biotin-peroxidase (ABC) kit, and the localization of constitutive (c) NOS and inducible (i) NOS was immunohistochemically comfirmed. Additionnally, we evaluated the possible involvement of cGMP and aromatase activity on NO-induced inhibition of steroidogenesis. The cGMP analog, 8-bromo-cGMP,had no significant effect on the accumulation of E_2 and P in basal and gonadotropin simulated PGC during 24-h culture. However, NOC 18 (0.1 - 1 mM) significantly (P<0.01) inhibited the aromatase activity of basal and gonadotropin stimulated PGC,measured by a modified tritiated water method.These results show that no inhibits E_2 secretion independent of cGMP by directly inhibiting aromatase activity in moderately mature PGC,and suggest that NO is an important local modulator of the follicular development.