Granulosa cell differentiation in vitro: effect of insulin on growth and functional integrity.
Granulosa cell differentiation in vitro: effect of insulin on growth and functional integrity.
复制标题
体外颗粒细胞分化：胰岛素对生长和功能完整性的影响。
DOI：
10.1095/biolreprod25.2.421
复制
发表时间：
1981
影响因子：
3.6
通讯作者：
Schomberg,DW
中科院分区：
文献类型：
--
作者：
May,JV;Schomberg,DW
胰岛素是在含血清培养基中维持的猪颗粒细胞单层中FSH介导的LH/hCG受体诱导的必要条件（May et al，1980）。在这份报告中，我们进一步描述了胰岛素在这一过程中的作用，以及对细胞单层性能的其他参数的作用，例如，接种效率、细胞生长、基础和促性腺激素刺激的孕酮产生以及芳香化酶活性。胰岛素不影响新鲜收获的活颗粒细胞的接种效率（>90%）。在培养的前2天，单层细胞含量在无胰岛素条件下略有增加。胰岛素并没有改变这种缓慢的细胞分裂速度。然而，在最初的2天后，与胰岛素处理的单层细胞相比，胰岛素去除的单层细胞的细胞和蛋白质含量显著降低（60%，P<0.001），而胰岛素处理的单层细胞的细胞和蛋白质含量相对于初始接种物保持或增加。在培养的前2天，胰岛素显著增强FSH和hCG刺激的孕酮产生（P<0.001）。FSH刺激的孕酮分泌与胰岛素呈剂量依赖性，FSH介导的LH/hCG受体诱导也是如此。胰岛素诱导的上皮样形态在体外，更成熟的细胞的特征。单独的LH和FSH不能改变成纤维细胞的形态，也不影响胰岛素引起的变化。因此，体外颗粒细胞的总体形态成熟与胰岛素介导的生化分化相关，例如，LH/hCG受体诱导和类固醇生成能力。虽然胰岛素增强了细胞分化的几个标志物，但它并没有改善培养过程中芳香化酶活性的丧失，这是该模型系统的一个特征。在培养的前2天，在睾酮的存在下，颗粒细胞单层分泌大量的雌激素。然而，这种能力随着培养时间的推移而迅速下降。无论是单独的胰岛素还是胰岛素与FSH的组合都不能急性刺激芳香化酶活性，这些治疗也不能防止芳香化酶活性的丧失。不能替代胰岛素作为FSH介导的LH/LH的必需品。hCG受体诱导或促性腺激素刺激孕酮分泌。我们的研究结果表明，胰岛素是维持猪颗粒细胞单层的几个功能特性的关键，因此应考虑在体外卵巢功能调节研究中常规使用。
Insulin is a requisite for the FSH-mediated induction of LH/hCG receptors in porcine granulosa cell monolayers maintained in serum-containing medium (May et al, 1980). In this report we describe further the role of insulin on this process and on other parameters of cell monolayer performance, e.g., plating efficiency, cell growth, basal and gonadotropin-stimulated progesterone production, and aromatase activity.Insulin did not affect the plating efficiency (>90%) of freshly harvested, viable granulosa cells. During the first 2 days of culture, monolayer cell content increased slightly under insulin-free conditions. Insulin did not alter this slow rate of cell division. After the first 2 days, however, cell and protein content of monolayers deprived of insulin decreased significantly (60%, P<0.001) relative to insulin-treated monolayers in which cell and protein content were either maintained or increased relative to the initial innoculum.Basal progesterone secretion declined with time in culture but was maintained or slightly increased under the influence of insulin. Both FSH- and hCG-stimulated progesterone production were significantly enhanced (P<0.001) by insulin after the first 2 days of culture. FSH-stimulated progesterone secretion was dose-dependent with respect to insulin as was FSH-mediated LH/hCG receptor induction.The immature granulosa cells used in these studies assumed a fibroblastic morphology in the absence of added hormones. Insulin induced an epithelioid morphology in vitro, characteristic of more mature cells. LH and FSH alone were incapable of altering the fibroblastic morphology and did not affect the change brought about by insulin. Thus, gross morphological maturation of granulosa cells in vitro correlated with insulin-mediated biochemical differentiation, e.g., LH/hCG receptor induction and steroidogenic capacity.Although insulin enhanced several markers of cell differentiation, it did not ameliorate the loss of aromatase activity during culture, a characteristic of this model system. During the first 2 days of culture and in the presence of testosterone, granulosa cell monolayers secreted significant amounts of estrogen. However, this capability declined rapidly with time in culture. Neither insulin alone nor insulin combined with FSH acutely stimulated aromatase activity, nor did these treatments protect against the loss of aromatase activity.Highly purified porcine relaxin and commercially available multiplication-stimulating activity (MSA), compounds possessing insulin-like structure and activity, respectively, were incapable of replacing insulin as a requisite for FSH-mediated LH/hCG receptor induction or gonadotropinstimulated progesterone secretion.Our results indicate that insulin is critical for the maintenance of several functional properties of porcine granulosa cell monolayers, and thus should be considered for routine use in studies of the regulation of ovarian function in vitro.