Goldring N B, Orly J
J Biol Chem. 1985 Jan 25;260(2):913-21.
Ovaries of immature, intact rats were dispersed by collagenase-DNase treatment and cultured in serum-free medium (ovarian cell culture). The hormonal responsiveness of the ovarian cell was compared to that exhibited by pure granulosa cells isolated via needle puncturing. Surprisingly, despite the fact that the majority of the cultured cells should have been comprised of granulosa type, no follicle-stimulating hormone-inducible progesterone or 20 alpha-OH-progesterone (20 alpha-OH-P) could be detected by radioimmunoassay, as typically occurs in cultures of pure granulosa cells. Therefore, in order to unravel the cause for the different responsiveness between the granulosa and the ovarian cell, we applied thin-layer chromatography analysis to follow the metabolic fate of added radioactive pregnenolone to intact monolayers in culture. Such TLC analysis revealed that, after priming with follicle-stimulating hormone, added [3H]pregnenolone was converted to progesterone which was rapidly reduced and finally accumulated as 5 alpha-pregnane-3 alpha,20 alpha-diol(pregnanediol). In addition to pregnanediol, a second class of steroid hormones accumulated in the coculture medium, namely androsterone and 5 alpha-pregnane-3 alpha,17 alpha,20 alpha-triol (pregnanetriol). These latter two were specific products of the ovarian coculture, indicating the presence of theca-interstitial cells bearing 17 alpha-hydroxylase and 17,20-lyase activities. Pregnanediol, rather than progesterone, was found to be the progestin precursor for androgen formation. We thus conclude that due to exchange of steroid metabolites between the cocultured cell types, the final steroid products are different by far from the expected contributions of each individually cultured cell type. Moreover, these findings reveal an additional aspect of the "two-cell theory," suggesting a granulosa-thecal concerted metabolism of progestin steroids, rather than exchange of aromatizable androgens.
对未成熟、未阉割大鼠的卵巢进行胶原酶 - 脱氧核糖核酸酶处理以使其分散，并在无血清培养基中培养（卵巢细胞培养）。将卵巢细胞的激素反应性与通过针刺分离的纯颗粒细胞所表现出的反应性进行比较。令人惊讶的是，尽管培养的细胞大多数应该是颗粒细胞类型，但通过放射免疫测定法却检测不到促卵泡激素诱导的孕酮或20α - 羟基孕酮（20α - OH - P），而在纯颗粒细胞培养中通常会出现这种情况。因此，为了弄清楚颗粒细胞和卵巢细胞之间反应性不同的原因，我们应用薄层色谱分析来追踪添加到培养的完整单层细胞中的放射性孕烯醇酮的代谢命运。这种薄层色谱分析表明，在用促卵泡激素预处理后，添加的[3H]孕烯醇酮转化为孕酮，然后迅速还原，最终以5α - 孕烷 - 3α,20α - 二醇（孕烷二醇）的形式积累。除了孕烷二醇外，共培养培养基中还积累了第二类甾体激素，即雄酮和5α - 孕烷 - 3α,17α,20α - 三醇（孕烷三醇）。后两者是卵巢共培养的特异性产物，表明存在具有17α - 羟化酶和17,20 - 裂解酶活性的卵泡膜 - 间质细胞。发现孕烷二醇而非孕酮是雄激素形成的孕激素前体。因此我们得出结论，由于共培养细胞类型之间甾体代谢物的交换，最终的甾体产物与每种单独培养的细胞类型预期的贡献相差甚远。此外，这些发现揭示了“双细胞理论”的一个新方面，表明孕激素甾体存在颗粒细胞 - 卵泡膜协同代谢，而不是可芳香化雄激素的交换。
