Zackrisson U, Mikuni M, Wallin A, Delbro D, Hedin L, Brännström M
Department of Obstetrics and Gynaecology, Göteborg University, Sahlgrenska sjukhuset, Sweden.
Hum Reprod. 1996 Dec;11(12):2667-73. doi: 10.1093/oxfordjournals.humrep.a019189.
Nitric oxide (NO) has emerged as one of several important intraovarian regulatory factors. In particular, NO has been implicated in the processes of ovulation and atresia-related apoptosis. The aim of the present study was to investigate the presence and distribution of the NO-generating nitric oxide synthase (NOS) enzymes in the ovary during follicular development, ovulation and luteal formation of the equine chorionic gonadotrophin (ECG)/human chorionic gonadotrophin (HCG)-primed rat. NADPH diaphorase activity was used as a histochemical marker for NOS within the ovary. Diaphorase reactivity was most abundant in the stroma (S) of the ovary and in the theca (T) layer of the follicle. In luteinized ovaries, weaker diaphorase reactivity was present within the corpora lutea (CL). Two different isoforms of NOS, the constitutively expressed endothelial NOS (eNOS) and the inducible isoform of NOS (iNOS), were immunolocalized in ovaries of immature rats and in ECG/HCG-primed rats during the periovulatory period from HCG injection until 2 days after ovulation. In addition, ovarian concentrations of eNOS and iNOS were quantified by immunoblotting. Immunoblotting with a monoclonal anti-eNOS antibody demonstrated the presence of eNOS mainly in the residual ovary (ROV) during the periovulatory period. In luteinized ovaries, higher concentrations of eNOS were seen in CL, while those in the ROV at this stage were lower than in the periovulatory ovary. Immature ovaries contained diminutive amounts of eNOS, detectable mostly in the ROV compartment. In contrast, iNOS was barely detectable during follicular development to the preovulatory stage. A slight elevation of iNOS was observed in the granulosa cells at 6 h after the HCG injection. The levels of iNOS during the luteal phase were also low. Immunohistochemical analysis using polyclonal eNOS and iNOS antibodies revealed the localization of these two isoforms primarily in the S and the T of the periovulatory ovary. In luteinized ovaries, positive immunoreactivity was also seen within the CL. With a monoclonal antibody against eNOS, intense immunoreactivity was observed in the S, T and within CL. There was a particularly strong staining in blood vessels. These data demonstrate the presence of an intraovarian NO-generating system. The localization of this system to the S, T and CL suggests a role for NO in the ovulatory process and in the regulation of CL function.
一氧化氮（NO）已成为卵巢内几种重要的调节因子之一。特别是，NO已被认为参与排卵和闭锁相关的细胞凋亡过程。本研究的目的是调查在马绒毛膜促性腺激素（ECG）/人绒毛膜促性腺激素（HCG）预处理的大鼠卵泡发育、排卵和黄体形成过程中，卵巢中产生NO的一氧化氮合酶（NOS）酶的存在和分布。NADPH黄递酶活性被用作卵巢内NOS的组织化学标记物。黄递酶反应性在卵巢基质（S）和卵泡的卵泡膜（T）层中最为丰富。在黄体化的卵巢中，黄体（CL）内的黄递酶反应性较弱。两种不同的NOS同工型，即组成性表达的内皮型NOS（eNOS）和诱导型NOS（iNOS），在未成熟大鼠的卵巢以及在从HCG注射到排卵后2天的排卵前期的ECG/HCG预处理大鼠的卵巢中进行了免疫定位。此外，通过免疫印迹法对卵巢中eNOS和iNOS的浓度进行了定量。用单克隆抗eNOS抗体进行免疫印迹显示，在排卵前期eNOS主要存在于残留卵巢（ROV）中。在黄体化的卵巢中，CL中eNOS的浓度较高，而此时ROV中的浓度低于排卵前期的卵巢。未成熟卵巢中eNOS含量极少，大多在ROV区域可检测到。相反，在卵泡发育到排卵前期阶段，iNOS几乎检测不到。在HCG注射后6小时，颗粒细胞中iNOS略有升高。黄体期iNOS的水平也很低。使用多克隆eNOS和iNOS抗体进行免疫组织化学分析显示，这两种同工型主要定位于排卵前期卵巢的S和T中。在黄体化的卵巢中，CL内也可见阳性免疫反应性。用抗eNOS单克隆抗体观察到，S、T和CL内有强烈的免疫反应性。血管中有特别强烈的染色。这些数据证明卵巢内存在一个产生NO的系统。该系统定位于S、T和CL表明NO在排卵过程和CL功能调节中起作用。
