HORMONAL-REGULATION OF THECAL CELL-FUNCTION DURING ANTRAL FOLLICLE DEVELOPMENT IN BOVINE OVARIES
HORMONAL-REGULATION OF THECAL CELL-FUNCTION DURING ANTRAL FOLLICLE DEVELOPMENT IN BOVINE OVARIES
复制标题
DOI：
10.1210/endo-127-6-2907
复制
发表时间：
1990-12-01
期刊：
影响因子：
4.8
通讯作者：
SKINNER, MK
中科院分区：
文献类型：
--
作者：
ROBERTS, AJ;SKINNER, MK
用在窦卵泡发育的不同阶段分离的细胞研究了卵泡膜细胞功能的激素调节。牛卵泡膜细胞分离自小窦、中窦和大Graffian卵泡（小、中和大卵泡）。建立了卵泡膜细胞的无血清培养物，并可存活至少6-8天的培养。膜细胞群的纯度进行了细胞化学表征，发现含有少于5%的内皮细胞和/或颗粒细胞污染。通过分析雄激素和孕酮的产生，研究了这种纯化的卵泡膜细胞在无血清培养中的类固醇生成能力。雄激素的生产是高的，在前3天的文化，然后下降到检测不到的水平。雄烯二酮的产量大约是睾酮产量的10倍。在整个8天的培养期间，孕酮的产生保持相对恒定。发现hCG在培养的第1-3天刺激雄激素产生，但对孕酮产生的影响可忽略不计。相比之下，hCG在培养的第3-6天刺激孕酮产生，但对雄激素产生的影响可以忽略不计。胰岛素刺激孕激素的生产在第3-6天的文化，但没有影响雄激素或孕激素的生产在第1-3天的文化。刺激卵泡膜细胞类固醇生成所需的hCG和胰岛素的最低有效浓度范围为约1-10 ng/ml。向培养物中加入血清降低了雄激素的产生并抑制了细胞的激素反应性。培养中的卵泡膜细胞似乎改变了它们的类固醇生成能力，从雄激素产生细胞变为孕酮产生细胞。卵泡膜细胞功能的发育调节分析表明，雄激素的产生和激素反应性是相对恒定的小，中，大卵泡。相反，孕酮的产生和激素反应性在小卵泡中最高，在中等卵泡中居中，在大卵泡中最低。对卵泡膜细胞功能的发育调节进行了更全面的分析，检查了放射性标记蛋白的分泌。膜细胞分泌大量放射性标记的蛋白质，分子量范围为5-500 kDa。有趣的是，胰岛素和hCG对从所检查的任何发育阶段分离的细胞的蛋白质分泌没有重大影响。放射性标记的分泌蛋白的主要发育变化是随着卵泡发育的进展，来自小卵泡的卵泡膜细胞分泌的大于100 kDa的蛋白质的丰富性下降。综合结果表明：1)培养的牛卵泡膜细胞似乎改变了类固醇生成能力，这反映在雄激素向孕酮的主要分泌的转变中。2)hCG/LH在刺激卵泡膜细胞类固醇生成中起主要作用，接近生理浓度的胰岛素可调节孕酮的产生。3)当一个小的窦状卵泡发育成一个大的Graffian卵泡时，卵泡膜细胞产生雄激素的能力保持相对恒定，而产生孕酮和几种分泌蛋白的能力下降。牛卵泡膜细胞的无血清培养有望用于阐明卵泡膜细胞在卵巢生理的激素和发育调节中的重要性。
The hormonal regulation of thecal cell function was investigated with cells isolated at various stages of antral follicle development. Bovine thecal cells were isolated from small antral, medium antral, and large Graffian follicles (small, medium, and large ovarian follicles). Serum-free cultures of thecal cells were established and viable for a minimum of 6-8 days of culture. The purity of the thecal cell population was characterized cytochemically and was found to contain less than 5% endothelial cell and/or granulosa cell contamination. The steroidogenic capacity of this purified population of thecal cells in serum-free culture was examined through an analysis of androgen and progesterone production. Androgen production was high during the first 3 days of culture, then declined to undetectable levels. Production of androstenedione was approximately 10-fold higher than production of testosterone. Progesterone production remained relatively constant throughout the 8-day culture period. hCG was found to stimulate androgen production during days 1-3 of culture, but had a negligible effect on progesterone production. In contrast, hCG stimulated progesterone production during days 3-6 of culture, but had a negligible effect on androgen production. Insulin stimulated progesterone production during days 3-6 of culture, but had no effect on androgen or progesterone production during days 1-3 of culture. The minimum effective concentrations of hCG and insulin required to stimulate steroidogenesis of the thecal cells ranged from approximately 1-10 ng/ml. Addition of serum to the cultures decreased androgen production and suppressed the hormone responsiveness of the cells. Thecal cells in culture appear to alter their steroidogenic capacity from an androgen-producing cell to a progesterone-producing cell. Analysis of the developmental regulation of thecal cell function revealed that androgen production and hormone responsiveness were relatively constant in small, medium, and large follicles. In contrast, progesterone production and hormone responsiveness were highest in small follicles, intermediate in medium follicles, and lowest in large follicles. A more general analysis of the developmental regulation of thecal cell function examined the secretion of radiolabeled proteins. A large number of radiolabeled proteins were secreted by thecal cells, ranging in molecular mass from 5-500 kDa. Interestingly, insulin and hCG had no major effect on secretion of proteins by cells isolated from any of the stages of development examined. The major developmental change in radiolabeled secreted proteins was a decline in the abundant secretion of proteins greater than 100 kDa by thecal cells from small follicles as follicle development progressed. Combined results suggest the following. 1) Bovine thecal cells in culture appear to alter steroidogenic capacity, reflected in a switch in predominant secretion of androgen to progesterone. 2) hCG/LH has a major role in the stimulation of thecal cell steroidogenesis, and insulin at nearly physiological concentrations may regulate progesterone production. 3) As a small antral follicle develops into a large Graffian follicle the ability of the thecal cells to produce androgen remains relatively constant, while the ability to produce progesterone and several secreted proteins declines. The serum-free culture of bovine thecal cells is anticipated to be useful to elucidate the importance of thecal cells in the hormonal and developmental regulation of ovarian physiology.