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

内皮型（NOS-3）的三种亚型的一氧化氮（NO）合酶存在于猪卵母细胞和颗粒细胞，但在卵母细胞中的NO合成的调控尚不清楚。本研究旨在评价类固醇激素在卵母细胞NO合成过程中的作用。将未成熟猪卵巢小腔卵泡的卵丘-卵母细胞复合体（COCs）置于去雌激素培养液中培养，研究了类固醇和去类固醇猪卵泡液对卵母细胞NO释放的影响。将从培养的COCs中分离的卵母细胞与1μM离子霉素孵育。NO代谢产物采用NO检测器-高压液相色谱系统进行鉴定。用10 nM的17β-雌二醇（E_2）刺激卵母细胞后，卵母细胞对离子霉素产生NO反应，而孕酮和睾酮对NO的合成影响不大，一氧化氮合酶抑制剂可抑制卵母细胞NO的合成，E_2刺激15 h后，卵母细胞NO的合成达到最大。而从窦状卵泡中新鲜获得的卵母细胞对离子霉素无反应，且E_2的作用可被无类固醇卵泡液所抑制。RT-PCR和Western blotting分析表明，E_2不增加NOS-3的表达。此外，在卵母细胞和卵丘细胞中检测到雌激素受体β，仅在卵丘细胞中检测到雌激素受体a。这些结果表明，E_2促进卵母细胞NOS-3合成NO，而不增加NOS-3的表达，但抑制NO的合成，至少在早期有腔卵泡的卵母细胞中是如此。

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.