EFFECT OF LUTEINIZING-HORMONE DEPRIVATION INSITU ON STEROIDOGENESIS OF RAT LEYDIG-CELLS PURIFIED BY A MULTISTEP PROCEDURE
EFFECT OF LUTEINIZING-HORMONE DEPRIVATION INSITU ON STEROIDOGENESIS OF RAT LEYDIG-CELLS PURIFIED BY A MULTISTEP PROCEDURE
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DOI：
10.1095/biolreprod36.3.769
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发表时间：
1987-04-01
影响因子：
3.6
通讯作者：
EWING, LL
中科院分区：
文献类型：
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作者：
KLINEFELTER, GR;HALL, PF;EWING, LL
剥夺大鼠的促黄体激素（LH）导致Leydig细胞失去平滑内质网并降低其P450 C17-羟化酶/C17，20-裂解酶活性（Wing等人，1984年）。垂体切除大鼠给予LH可防止Leydig细胞结构和功能的这些变化（Ewing和Zirkin，1983）。我们采用多步骤分离大鼠Leydig细胞的方法，研究LH对Leydig细胞类固醇合成的营养作用。我们的方法采用血管灌注，酶解，离心淘洗，和Percoll梯度离心。Percoll密度梯度离心后获得的纯化Leydig细胞级分含有95%保存良好的3 β-羟基类固醇脱氢酶（3 β- HSD）染色的细胞具有间质细胞的超微结构特征。这些Leydig细胞产生248和29 ng的睾酮/106 Leydig细胞孵育3小时时，有和没有最大刺激浓度的绵羊LH。将从对照大鼠和用睾酮-雌二醇（T-E）植入物处理4天以抑制LH产生的大鼠获得的纯化Leydig细胞与饱和浓度（2 μ m）的去甲烯醇酮一起孵育。来自对照和T-E-植入大鼠的Leydig细胞分别产生537和200 ng睾酮/106 Leydig细胞·3 h，表明在来自T-E-植入大鼠的Leydig细胞中将双烯醇酮转化为睾酮的类固醇生成反应中存在缺陷。通过使用兔抗P450 C17-羟化酶/C17，20-裂解酶猪微粒体酶的抗体，对对照组和T-E植入4天和12天的大鼠的Leydig细胞微粒体蛋白进行一维十二烷基硫酸钠聚丙烯酰胺凝胶免疫印迹，结果显示随着LH停药期的继续，酶持续丢失。这些结果表明，剥夺LH的动物的Leydig细胞将阿替诺龙转化为睾酮的能力降低，P450 C17-羟化酶/C17，20-裂解酶含量降低。
Depriving rats of luteinizing hormone (LH) causes Leydig cells to lose smooth endoplasmic reticulum and diminishes their P450 C17-hydroxylase/C17,20-lyase activity (Wing et al., 1984). LH administration to hypophysectomized rats prevents these changes in Leydig cell structure and function (Ewing and Zirkin, 1983). We adopted a multistep procedure of rat Leydig cell isolation to study the trophic effects of LH on steroidogenesis in the Leydig cell. Our method employs vascular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll gradient centrifugation. The purified Leydig cell fraction obbtained after Percoll density-gradient centrifugation contains 95% well-preserved 3.beta.-hydroxysteroid dehydrogenase (3.beta.-HSD)-staining cells with ultrastructural characteristics of Leydig cells. These Leydig cells produced 248 and 29 ng of testosterone/106 Leydig cells when incubated for 3 h with and without a maximally stimulating concentration of ovine LH. Purified Leydig cells obtained from control rats and rats treated with testosterone-estradiol (T-E) implants for 4 days to inhibit LH production were incubated with a saturating concentration (2 .mu.m) of pregnenolone. Leydig cells from control and T-E-implanted rats produced 537 and 200 ng of testosterone/106 Leydig cells.cntdot.3 h, respectively, suggesting a defect in the steroidogenic reactions converting pregnenolone to testosterone in Leydig cells from T-E-implanted rats. By usingg rabbit antibodies to the P450 C17-hydroxylase/C17,20-lyase pig microsomal enzyme, immunoblots of one-dimensional sodium dodecyl sulfate polyacrylamide gels of Leydig cell microsomal protein from control and 4- and 12-day T-E implanted rats revealed a continued loss of enzyme as the period of LH withdrawal continues. These results show that Leydig cells from animals deprived of LH had diminished capacity to convert pregnenolone to testosterone and reduced P450 C17-hydroxylase/C17,20-lyase content.