Nelson S E, McLean M P, Jayatilak P G, Gibori G
Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago 60612.
Endocrinology. 1992 Feb;130(2):954-66. doi: 10.1210/endo.130.2.1733737.
The aim of this investigation was to isolate, characterize, and culture the small and large luteal cell subpopulations forming the corpus luteum of the pregnant rat. Since the large luteal cells are extremely fragile and do not survive standard cell dispersion, a method which allows the survival and the long-term culture in serum-free media of small and large cells was developed. The two luteal cell populations differed not only by their size but also by their morphology in culture. The small luteal cells (12-20 mu in diameter) are characterized by a large oval nucleus, contain few lipid droplets and have a stellate shape. In contrast, the large luteal cells have a smaller spherical nucleus, high lipid content, and do not flatten out completely in culture, most probably due to the abundance of lipid droplets. Both luteal cell types express 3 beta HSD and the cytochrome P450 enzymes involved in steroidogenesis. However, it is the lipid filled large luteal cells that secrete the most progesterone, androgen, and estradiol; express greater amounts of P450scc and P450AROM; and possess more PRL and LH receptors. Despite the greater expression of LH receptor in the large luteal cells, small and large luteal cells responded to LH with equal increase in steroidogenic output. In serum free culture, luteal cells produced progesterone for up to 20 days; however, an exogenous source of cholesterol was a prerequisite for maximal progesterone secretion. The pattern of progesterone secretion by cultures of small and large luteal cells differed remarkably from that of mixed cell population. When nonsteroidogenic corpus luteum cells were cocultured with the large luteal cells, a severalfold increase in progesterone secretion was observed. This stimulation occurred even when cells were cocultured in the absence of exogenous source of cholesterol. In summary, a successful method was developed to disperse, isolate, and independently culture the two luteal cell populations forming the rat corpus lutem. The results indicate that the marked difference in the steroidogenic capacity of these two cell populations is due, in large part, to the difference in their size rather than to their origin in the follicle. In addition, the results have revealed an important effect of the nonsteroidogenic cells forming the corpus luteum on luteal cell steroidogenesis.
本研究的目的是分离、鉴定和培养构成妊娠大鼠黄体的小黄体细胞亚群和大黄体细胞亚群。由于大黄体细胞极其脆弱，在标准细胞分散过程中无法存活，因此开发了一种方法，使小细胞和大细胞能够在无血清培养基中存活并长期培养。两种黄体细胞群不仅大小不同，在培养中的形态也不同。小黄体细胞（直径12 - 20微米）的特征是有一个大的椭圆形细胞核，含少量脂滴，呈星状。相比之下，大黄体细胞有一个较小的球形细胞核，脂质含量高，在培养中不会完全扁平，很可能是由于脂滴丰富。两种黄体细胞类型均表达3β-羟类固醇脱氢酶（3βHSD）和参与类固醇生成的细胞色素P450酶。然而，富含脂质的大黄体细胞分泌的孕酮、雄激素和雌二醇最多；表达更多的胆固醇侧链裂解酶（P450scc）和芳香化酶（P450AROM）；并且拥有更多的催乳素（PRL）和促黄体生成素（LH）受体。尽管大黄体细胞中LH受体的表达更高，但小黄体细胞和大黄体细胞对LH的反应是类固醇生成量同等增加。在无血清培养中，黄体细胞可产生孕酮长达20天；然而，外源性胆固醇是最大量孕酮分泌的先决条件。小黄体细胞和大黄体细胞培养物中孕酮分泌模式与混合细胞群体的模式显著不同。当非类固醇生成的黄体细胞与大黄体细胞共培养时，观察到孕酮分泌增加了几倍。即使在没有外源性胆固醇的情况下细胞共培养，这种刺激也会发生。总之，开发了一种成功的方法来分散、分离和独立培养构成大鼠黄体的两种黄体细胞群。结果表明，这两种细胞群在类固醇生成能力上的显著差异在很大程度上是由于它们大小的差异，而不是它们在卵泡中的起源。此外，结果揭示了构成黄体的非类固醇生成细胞对黄体细胞类固醇生成的重要作用。
