Study on growth and differentiation of avian granulosa and theca cells.

已有研究表明，孕酮是鹌鹑颗粒细胞类固醇合成的最终产物，其产生受黄体生成素的刺激。在长期培养的发展过程中，我们发现颗粒细胞迅速失去了产生孕酮的能力。此外，随着培养时间的延长，培养液中的孕酮水平逐渐降低。本研究旨在研究颗粒细胞在长期培养过程中孕酮的转化。取排卵前10小时的颗粒细胞，在39℃、5%CO_2和95%空气中，用添加胰岛素、转铁蛋白和皮质酮的无血清培养液培养在纤维连接蛋白包被的孔中。为了研究孕酮代谢，在培养液中加入孕酮，继续培养3小时。然后，从介质中提取放射性类固醇，并进行薄层色谱分析。从薄层平板中回收的放射性代谢物是ide…。通过比较氧化或乙酰化后它们与正品的层析行为，以及晶体在与正品重复结晶过程中恒定的比放射性，进一步证实了这一点。新鲜分离的颗粒细胞没有表现出任何孕酮转化，但在培养3天后，颗粒细胞获得了将放射性孕酮转化为两种主要代谢物的能力，鉴定为5β-孕烷-3，20-二酮和3-β-羟基-5β-孕酮20-酮。在培养过程中，根据这些5β-还原代谢物的量计算出的5β-还原酶的活性增加。在培养液中加入抗氧化剂，可以有效地防止孕酮产生的自发下降，从而阻止5β-还原酶活性的增加。这些结果表明，在有氧培养条件下新诱导或激活的5β-还原酶可能是颗粒细胞培养过程中孕酮产生减少的原因之一。较少

It has been shown that progesterone is the end product of steroidogenesis in quail granulosa cells and its production is stimulated by LH. During the development of a long-term cultures, we found that the granulosa cells rapidly lost the ability to produce progesterone. Moreover, progesterone levels in the media progressively decreased with time of culture. The present study was conducted to study the conversion of progesterone by the granulosa cells during long-term culture.Granulosa cells, obtained from quail at 10 hrs before ovulation, were cultured in fibronectin-coated wells with serum-free medium supplemented with insulin, transferrin, and corticosterone at 39 C in 5% CO_2 and 95% air. To investigate progesterone metabolism, [ ^<14>C]progesterone was added to the medium and the culture was continued for 3 hrs. Then, radioactive steroids were extracted from the medium and analyzed by thin-layer chromatooraphy. The radioactive metabolites, recovered from thin-layer plates, were ide … More ntified by comparisons of their chromatographic behavior with that of authentic preparations after oxidation or acetylation, and by the constant specific radioactivities of crystals during repeated crystallization with the authentic preparation.Freshly isolated granulosa cells did not show any conversion of progesterone, but after 3 days in culture, the granulosa cells acquired the ability to convert radioactive progesterone into two major metabolites, identified as 5beta-pregnane-3, 20-dione and 3beta-hydroxy-5beta-pregnan20-one. Activity of 5beta-reductase, which was calculated from the amounts of these 5beta-reduced metabolites, increased during the culture. Inclusion of antioxidants to the medium, which was effective in preventing the spontaneous decrease in progesterone production during culture, prevented the increase of 5beta-reductase activity.These results suggest that 5beta-reductase, which is newly induced or activated under the aerobic culture conditions, might contribute to the decrease in progesterone production in the granulosa cells during culture. Less