Kasson B G, Meidan R, Davoren J B, Hsueh A J
Endocrinology. 1985 Sep;117(3):1027-34. doi: 10.1210/endo-117-3-1027.
Ovarian granulosa cells from small follicles have generally been considered to comprise a homogeneous cell population; however, stratified arrangements of hormone receptors have been found in antral and mural granulosa cells of Graafian follicles. Using cultured granulosa cells derived from immature, hypophysectomized, estrogen-treated rats, we have previously shown that vasoactive intestinal peptide (VIP) as well as FSH stimulates steroid production by these cells. Dose-response analysis indicated that the actions of these hormones were additive, suggesting the presence of subpopulations of granulosa cells. Using a continuous (0-30%) Metrizamide density gradient, we have identified three populations of granulosa cells with different sedimentation properties. After centrifugation for 15 min at 1500 X g, cells sedimented at Metrizamide concentrations of 13%, 18%, and 20% (peaks A, B, and C, respectively). The subpopulation with lowest density (peak A) comprised 5% of the total cells, whereas the remaining cells were distributed approximately equally in the other two peaks. The profiles of estrogen and progesterone production by these cells in response to FSH and VIP indicated that FSH preferentially stimulated steroid production in cells with the highest density (peak C), whereas VIP mainly induced steroidogenic responses in cells of intermediate density (peak B). In contrast, cells with the lowest density (peak A) were unresponsive to either hormone. Treatment with forskolin, a universal adenylate cyclase activator, induced steroid production in both subpopulations B and C. Further studies demonstrated that LH/human CG receptors were induced by FSH and forskolin in cells from peak C, whereas VIP treatment did not induce LH/human CG receptors in cells from peak B. In unfractionated cultured cells, GnRH potently antagonized FSH- but not VIP-induced steroidogenesis. Upon density-gradient fractionation, the profile of GnRH receptor content correlated well with GnRH effects since FSH-responsive cells (peak C) contained the majority of GnRH receptors. The present results demonstrate that granulosa cells from immature follicles are heterogeneous and consist of two major subpopulations of cells with differential responsiveness to FSH and VIP. These findings provide the basis for further morphological and biochemical analysis of subpopulations of granulosa cells during follicular development.
来自小卵泡的卵巢颗粒细胞通常被认为是一个同质细胞群体；然而，在格拉夫卵泡的窦状和壁层颗粒细胞中发现了激素受体的分层排列。利用从未成熟、垂体切除、雌激素处理的大鼠中分离培养的颗粒细胞，我们先前已表明血管活性肠肽（VIP）以及促卵泡激素（FSH）能刺激这些细胞产生类固醇。剂量反应分析表明这些激素的作用是相加的，提示存在颗粒细胞亚群。利用连续的（0 - 30%）甲泛葡胺密度梯度，我们鉴定出了具有不同沉降特性的三个颗粒细胞群体。在1500×g离心15分钟后，细胞在甲泛葡胺浓度为13%、18%和20%时沉降（分别为峰A、B和C）。密度最低的亚群（峰A）占细胞总数的5%，而其余细胞在另外两个峰中大致均匀分布。这些细胞对FSH和VIP反应时雌激素和孕酮产生的情况表明，FSH优先刺激密度最高的细胞（峰C）产生类固醇，而VIP主要诱导中等密度细胞（峰B）产生类固醇生成反应。相反，密度最低的细胞（峰A）对这两种激素均无反应。用福斯可林（一种通用的腺苷酸环化酶激活剂）处理可诱导B和C亚群细胞产生类固醇。进一步研究表明，FSH和福斯可林在峰C的细胞中诱导了促黄体生成素/人绒毛膜促性腺激素（LH/hCG）受体，而VIP处理未在峰B的细胞中诱导LH/hCG受体。在未分级的培养细胞中，促性腺激素释放激素（GnRH）强烈拮抗FSH诱导的类固醇生成，但不拮抗VIP诱导的类固醇生成。经密度梯度分级后，GnRH受体含量的情况与GnRH的作用密切相关，因为对FSH有反应的细胞（峰C）含有大多数的GnRH受体。目前的结果表明，来自未成熟卵泡的颗粒细胞是异质的，由对FSH和VIP反应不同的两个主要细胞亚群组成。这些发现为卵泡发育过程中颗粒细胞亚群的进一步形态学和生物化学分析提供了基础。
