Lee H T, Bahr J M
Department of Animal Sciences, University of Illinois, Urbana 61801.
Endocrinology. 1990 Feb;126(2):779-86. doi: 10.1210/endo-126-2-779.
We have found that androgens and estradiol-17 beta (E2) produced by theca cells suppress progesterone (P4) secretion by granulosa cells of the domestic hen in a dose-dependent manner. Furthermore, testosterone (T) and E2 inhibited the conversion of cholesterol to pregnenolone (P5) and of P5 to P4, respectively. The aim of this study was to determine if T and E2 suppress P4 biosynthesis by changing activities of the cytochrome P450 cholesterol side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) (Exp I) and the amount of P450scc (Exp II). Granulosa layers of the largest follicle of two to four hens were obtained 22 h before ovulation, pooled, and isolated granulosa cells were prepared. In Exp I, the specific activities of the P450scc and 3 beta-HSD were measured in mitochondrial and microsomal proteins of granulosa cells, respectively, in the presence of T or E2 (0-10 microM). Addition of T to mitochondrial proteins increased the Michaelis-Menten constant (Km) with no change in the maximum velocity (Vmax) of the P450scc, which suggests competitive inhibition (Ki = 30.9 microM), whereas E2 had no effect on Km and Vmax of the P450scc. Likewise, addition of E2 to microsomal proteins increased the Km with no change in the Vmax of the 3 beta-HSD, which suggests competitive inhibition (Ki = 15.1 microM), whereas T had no effect on Km and Vmax of the 3 beta-HSD. In Exp II, granulosa cells (3 x 10(5)/3 ml.tube) were incubated for 0-12 h in triplicate for each combined treatments of 25-OH-cholesterol (8 microM) and cyanoketone (10 microM), T, or E2 (0-10 microM) in the presence or absence of LH (25 ng). Protein content and P5 secretion were measured and the amount of P450scc was determined by Western blot analysis. Incubation of granulosa cells with T decreased the amount of the P450scc in granulosa cells cultured for 12 h and P5 secretion in granulosa cells cultured for 3 h or longer (P less than 0.05), without a change in protein content and cell viability. Our results suggest that P4 production by granulosa cells is suppressed by T and E2 acting as competitive inhibitors of the P450scc and 3 beta-HSD, respectively, and by T decreasing the amount of the P450scc. We conclude that steroidogenesis in the follicle of the chicken is regulated through the interaction of theca and granulosa layers.
我们发现，卵泡膜细胞产生的雄激素和雌二醇-17β（E2）以剂量依赖的方式抑制家鸡颗粒细胞分泌孕酮（P4）。此外，睾酮（T）和E2分别抑制胆固醇向孕烯醇酮（P5）的转化以及P5向P4的转化。本研究的目的是确定T和E2是否通过改变细胞色素P450胆固醇侧链裂解酶（P450scc）和3β-羟基类固醇脱氢酶（3β-HSD）的活性（实验I）以及P450scc的量（实验II）来抑制P4的生物合成。在排卵前22小时获取两到四只母鸡最大卵泡的颗粒层，合并后制备分离的颗粒细胞。在实验I中，分别在存在T或E2（0 - 10 microM）的情况下，测定颗粒细胞线粒体和微粒体蛋白中P450scc和3β-HSD的比活性。向线粒体蛋白中添加T增加了米氏常数（Km），而P450scc的最大反应速度（Vmax）没有变化，这表明存在竞争性抑制（抑制常数Ki = 30.9 microM），而E2对P450scc的Km和Vmax没有影响。同样，向微粒体蛋白中添加E2增加了Km，而3β-HSD的Vmax没有变化，这表明存在竞争性抑制（Ki = 15.1 microM），而T对3β-HSD的Km和Vmax没有影响。在实验II中，对于每种联合处理，将颗粒细胞（3×10⁵/3 ml试管）在25 - 羟基胆固醇（8 microM）和氰基酮（10 microM）、T或E2（0 - 10 microM）存在或不存在促黄体生成素（LH，25 ng）的情况下一式三份孵育0 - 12小时。测量蛋白含量和P5分泌，并通过蛋白质印迹分析确定P450scc的量。用T孵育颗粒细胞会降低培养12小时的颗粒细胞中P450scc的量以及培养3小时或更长时间的颗粒细胞中P5的分泌（P < 0.05），而蛋白含量和细胞活力没有变化。我们的结果表明，颗粒细胞产生P4受到T和E2的抑制，T和E2分别作为P450scc和3β-HSD的竞争性抑制剂，并且T会减少P450scc的量。我们得出结论，鸡卵泡中的类固醇生成是通过卵泡膜层和颗粒层的相互作用来调节的。
