Regulation for growth and differentiation of somatic cells by signals derived from oocytes

猪卵母细胞和颗粒细胞中均存在三种一氧化氮（NO）合成酶的内皮型（NOS-3），但其对卵母细胞中NO合成的调控尚不清楚。本研究旨在评价类固醇在卵母细胞NO合成过程中的控制作用。从未成熟猪卵巢的小卵泡中提取卵母细胞卵母细胞复合物（COCs），在缺乏雌激素的培养基中培养，研究类固醇或不含类固醇的猪卵泡液对卵母细胞释放NO的影响。从培养的COCs中分离卵母细胞，用1μM离子霉素孵育。采用NO检测器-高压液相色谱系统对NO代谢物进行鉴定。用10nM的17β-雌二醇（E_2）培养COCs卵母细胞，在离子霉素作用下释放NO，而黄体酮和睾酮对NO的合成影响不大。NOS抑制剂抑制NO的合成。E_2培养15h后，合成量达到最大值。然而，从窦卵泡新鲜获得的卵母细胞对离子霉素没有反应，E_2的作用被添加不含类固醇的卵泡液抑制。RT-PCR和Western blotting分析显示，E_2未增加NOS-3的表达。卵母细胞和卵丘细胞均检测到雌激素受体β，而雌激素受体a仅在卵丘细胞中检测到。上述结果提示，E_2可促进卵母细胞NOS-3合成NO，但不增加NOS-3的表达，但抑制NO的合成，至少在早期窦卵泡卵母细胞中是如此。

The endothelial type(NOS-3) of three isoforms of nitric oxide(NO) synthase occurs in porcine oocytes and granulose cells, but the regulation of NO synthesis in oocytes remains unknown. The present study was designed to evaluate steroid control in the process of oocyte NO synthesis. Cumulus-oocyte complexes(COCs), obtained from small-sized antral follicles of immature porcine ovaries, were cultured in estrogen-deprived medium, and the effect of steroids or steroid-free porcine follicular fluids on the NO release from oocytes was investigated. Oocytes that were isolated from cultured COCs were incubated with 1μM ionomycin. The NO metabolites were identified using a NO detector-high-pressure liquid chromatography system. Oocytes from COCs cultured with 10nM 17β-estradiol(E_2) released NO in response to ionomycin, whereas progesterone and testosterone had little effect on the synthesis of NO. An inhibitor of NOS suppressed the synthesis of NO. The maximal synthesis was observed after a 15 h-culture with E_2. However, oocytes freshly obtained from antral follicles did not response to ionomycin, and the E_2 action was suppressed by the addition of steroid-free follicular fluids. Analyses of RT-PCR and Western blotting showed that E_2 did not increase NOS-3 expression. In addition, estrogen receptor β was detected in oocytes and cumulus cells, and estrogen receptor a was detected only in cumulus cells. These findings suggest that oocyte NOS-3 is promoted for the synthesis of NO by E_2 without increases in NOS-3 expression, but the synthesis of NO is suppressed, at least in the oocytes of early antral follicles.