Janszen F H, Cooke B A, van Driel M J, van der Molen H J
J Endocrinol. 1976 Sep;70(3):345-59. doi: 10.1677/joe.0.0700345.
An LH-responsive Leydig cell preparation (containing 6+/-2% Leydig cells) was obtained by collagenase treatment of rat testis. Centrifugation of this cell preparation through a 13% Ficoll solution for 10 min at 1500 g resulted in a four times purification of the Leydig cells, with a concomitant increases in steroidogenic activity. Addition of 0-2% albumin to the 13% Ficoll solution, adjusted to 280 mosmol/l, resulted in a further twofold purification of the Leydig cells paralleled by a twofold increase in steroidogenic activity. Centrifugation of these Ficoll-albumin-purified Leydig cells through a 6% dextran solution for 2 min at 100 g resulted in a further 1-7 times purification of the Leydig cells. A combination of the two centrifugation steps resulted in a 12-5 times purification of Leydig cells compared with the original crude cell suspension, while an increase in steroidogenic activity of 22-5 times was obtained. This final cell preparation contained 59 +/- 17% Leydig cells (mean +/- S.D., n = 6). The recovery of Leydig cells was 29%. Collagenase treatment of testes deficient in spermatogenesis resulted in a cell preparation with the same steroidogenic activity as Ficoll-purified cells from normal testes. Centrifugation of these cells through a 13% Ficoll solution gave only a limited increase in the steroidogenic activity. Isopycnic centrifugation of the crude cell preparation on a discontinous Ficoll metrizoate gradient resulted in two discrete peaks of Leydig cells, one peak at a density of 1-039-1-055 g/ml and one at a density of 1-068-1-088 g/ml. Both types of cells produced testosterone. In the presence of LH, cyclic AMP production in both types of Leydig cells increased, but testosterone production was only increased by LH in the "denser" Leydig cells and not in the "light" Leydig cells. No difference in sensitivity to LH could be observed between the Leydig cell preparations of different purity. Using a 60 min pre-incubation period the highest testosterone response was obtained with 100-1000 ng LH/ml. The same maximum testosterone response was obtained with 10-100 ng LH/ml when the pre-incubation period was omitted.
通过用胶原酶处理大鼠睾丸获得了对促黄体生成素（LH）有反应的睾丸间质细胞制剂（含6±2%的睾丸间质细胞）。将该细胞制剂通过13%的菲可溶液在1500 g下离心10分钟，使睾丸间质细胞得到了4倍的纯化，同时类固醇生成活性增加。向调整至280 mosmol/l的13%菲可溶液中添加0 - 2%的白蛋白，使睾丸间质细胞进一步纯化了2倍，同时类固醇生成活性增加了2倍。将这些经菲可 - 白蛋白纯化的睾丸间质细胞通过6%的葡聚糖溶液在100 g下离心2分钟，使睾丸间质细胞进一步纯化了1 - 7倍。这两个离心步骤相结合，与原始粗细胞悬液相比，睾丸间质细胞得到了12.5倍的纯化，同时类固醇生成活性增加了22.5倍。这种最终的细胞制剂含有59±17%的睾丸间质细胞（平均值±标准差，n = 6）。睾丸间质细胞的回收率为29%。对精子发生缺陷的睾丸进行胶原酶处理，得到的细胞制剂与来自正常睾丸经菲可纯化的细胞具有相同的类固醇生成活性。将这些细胞通过13%的菲可溶液离心，类固醇生成活性仅有限增加。将粗细胞制剂在不连续的菲可 - 泛影葡胺梯度上进行等密度离心，得到了两个离散的睾丸间质细胞峰，一个峰的密度为1.039 - 1.055 g/ml，另一个峰的密度为1.068 - 1.088 g/ml。这两种类型的细胞都能产生睾酮。在LH存在的情况下，两种类型的睾丸间质细胞中环状AMP的产生都增加了，但只有“密度较大”的睾丸间质细胞中的睾酮产生因LH而增加，“密度较小”的睾丸间质细胞中则没有增加。不同纯度的睾丸间质细胞制剂对LH的敏感性没有差异。使用60分钟的预温育期，当LH浓度为100 - 1000 ng/ml时可获得最高的睾酮反应。当省略预温育期时，LH浓度为10 - 100 ng/ml时可获得相同的最大睾酮反应。
