Tao Yong, Fu Zhuo, Zhang Meijia, Xia Guoliang, Yang Jie, Xie Huirong
Animal Physiology and Biochemistry Department, College of Biological Sciences, China Agricultural University, 100094 Beijing, China.
Mol Cell Endocrinol. 2004 Jul 30;222(1-2):93-103. doi: 10.1016/j.mce.2004.04.014.
The present study is to investigate the immunolocalization of endothelial and inducible nitric oxide synthase (eNOS, iNOS) in porcine ovary and the effect of nitric oxide (NO) on antrum formation and oocyte meiotic resumption. In Experiment 1, preantral follicles (250-300 microm in diameter) were cultured in 0 (Control), 0.1, 0.3, 0.5 or 1 mM sodium nitroprusside (SNP), a NO donor. In Experiment 2, the cumulus-oocyte complexes (COCs) aspirated from medium follicles (3-6 mm in diameter) were incubated in 0.1mM SNP or two inhibitors for NOS, 10 mM aminoguanidine bicarbonate salt (AG) or 1 mM Nomega-nitro-l-arginine methyl ester (L-NAME), alone or concomitantly. In Experiment 3, ovarian tissues, corpus luteum (CL), corpus albican (CA) and COCs from small (1-2 mm in diameter), medium (3-6 mm) and large follicles (7-10 mm) were isolated, rinsed, fixed, paraffin embedded and stained by the conventional avidin-biotin complex method for the detection of eNOS and iNOS production. The results showed that 0.1mM SNP had no effect on antrum formation (P > 0.05) while 0.3, 0.5 or 1 mM significantly inhibited the antrum formation (P < 0.05). AG markedly inhibited porcine oocyte meiotic resumption (P < 0.05) while L-NAME inhibited first polar body (PB1) extrusion (P < 0.05). The immunoreactivity of eNOS in early antral follicles was restricted to oocyte and it increased from small, medium to large follicle-enclosed oocytes. Cumulus cells from large follicles showed weak eNOS immunoreactivity but those from small or medium follicles not. In CL, eNOS-positive staining was shown in granulosa lutein cells. In CA, it was in some parenchymal cells. In contrast, no immunoreactivity for iNOS was found in primordial, early antral follicle or the COCs aspirated from small and medium follicles. The large follicle-enclosed oocyte showed weak immunoreactivity. In CL, some granulosa lutein cells showed iNOS-positive cytoplasm. Such immunostaining was not found in CA. The results demonstrate that porcine ovaries have distinct cell-specific expression of both eNOS and iNOS, and that NO derived from both NOS is actively involved in meiotic resumption. Nitric oxide is not involved in the antrum formation of preantral follicles but exogenous NO inhibits the antrum formation. Endothelial nitric oxide synthase and inducible nitric oxide synthase might be differently functional in CL development and regression.
本研究旨在探讨内皮型和诱导型一氧化氮合酶（eNOS、iNOS）在猪卵巢中的免疫定位，以及一氧化氮（NO）对卵泡腔形成和卵母细胞减数分裂恢复的影响。在实验1中，将直径250 - 300微米的腔前卵泡培养于0（对照）、0.1、0.3、0.5或1 mM硝普钠（SNP，一种NO供体）中。在实验2中，从直径3 - 6毫米的中等卵泡中吸出的卵丘 - 卵母细胞复合体（COCs）分别单独或联合孵育于0.1 mM SNP或两种一氧化氮合酶抑制剂，即10 mM氨基胍碳酸氢盐（AG）或1 mM Nω-硝基-L-精氨酸甲酯（L-NAME）中。在实验3中，分离直径1 - 2毫米的小卵泡、直径3 - 6毫米的中等卵泡和直径7 - 10毫米的大卵泡中的卵巢组织、黄体（CL）、白体（CA）和COCs，冲洗、固定、石蜡包埋，并用传统的抗生物素蛋白 - 生物素复合物法染色，以检测eNOS和iNOS的产生。结果显示，0.1 mM SNP对卵泡腔形成无影响（P > 0.05），而0.3、0.5或1 mM则显著抑制卵泡腔形成（P < 0.05）。AG显著抑制猪卵母细胞减数分裂恢复（P < 0.05），而L-NAME抑制第一极体（PB1）排出（P < 0.05）。早期卵泡中eNOS的免疫反应性局限于卵母细胞，且从小卵泡、中等卵泡到被大卵泡包绕的卵母细胞，其免疫反应性增强。大卵泡的卵丘细胞显示出较弱的eNOS免疫反应性，而小卵泡或中等卵泡的卵丘细胞则未显示。在黄体中，颗粒黄体细胞显示eNOS阳性染色。在白体中，一些实质细胞呈阳性。相反，在原始卵泡、早期卵泡或从小卵泡和中等卵泡中吸出的COCs中未发现iNOS的免疫反应性。被大卵泡包绕 的卵母细胞显示出较弱的免疫反应性。在黄体中，一些颗粒黄体细胞的细胞质显示iNOS阳性。在白体中未发现这种免疫染色。结果表明，猪卵巢中eNOS和iNOS具有明显的细胞特异性表达，并表明两种一氧化氮合酶产生的NO均积极参与减数分裂恢复。一氧化氮不参与腔前卵泡的卵泡腔形成，但外源性NO抑制卵泡腔形成。内皮型一氧化氮合酶和诱导型一氧化氮合酶在黄体的发育和退化过程中可能具有不同的功能。
