Hedger M P, Eddy E M
Gamete Biology Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Endocrinology. 1987 Nov;121(5):1824-38. doi: 10.1210/endo-121-5-1824.
Intertubular cells, isolated from adult rat testes by collagenase dispersal under conditions designed to minimize cell damage, were fractionated on Percoll density gradients. In the gradient fractions, there was a close cellular correlation between the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), determined by cytochemistry, and other Leydig cell markers (nonspecific esterase, autofluorescence, and an antigen defined by monoclonal antibody LC-1C6). As the reagents for 3 beta HSD cytochemistry are excluded by intact membranes, Leydig cells with damaged plasma membranes were identified by 3 beta HSD reactivity in suspended cell preparations, and the total number of 3 beta HSD-positive (3 beta HSD+) cells in the same preparations was determined after lysis of the cell membrane. Whole cells were differentiated from cytoplasmic fragments by counterstaining with the nuclear dye propidium iodide, and the number of intact Leydig cells in each preparation was determined subsequently by subtracting the number of damaged nucleated 3 beta HSD+ cells from the total number of nucleated 3 beta HSD+ cells. The majority of intact isolated Leydig cells were found in gradient fractions of 1.054-1.096 g/ml density. Acute (3-H) basal and hCG-stimulated testosterone production per intact Leydig cell were dependent upon the concentration of Leydig cells per assay well, indicating that there is cooperativity among Leydig cells in vitro. There was no difference in steroidogenic function among intact Leydig cells from different fractions of the above density gradient range at assay concentrations greater than 10,000 Leydig cells/well. At lower cell concentrations, Leydig cells from gradient fractions of lower density (1.054-1.064 g/ml) produced slightly less testosterone in response to hCG stimulation than Leydig cells from more dense fractions (1.070-1.096 g/ml). Prolonging the exposure of isolated cells to the dispersal conditions caused declines in the apparent buoyant density and basal testosterone and hCG-stimulated cAMP and testosterone production of all Leydig cells, without detectable changes in cell integrity. The data indicate that both the absolute steroidogenic function and the functional heterogeneity of isolated intact Leydig cells are, at least in part, dependent upon the procedures used for their isolation.
通过胶原酶分散法从成年大鼠睾丸中分离出曲细精管间细胞，分离条件旨在将细胞损伤降至最低，然后将这些细胞在Percoll密度梯度上进行分级分离。在梯度级分中，通过细胞化学测定的3β-羟基类固醇脱氢酶（3βHSD）的存在与其他睾丸间质细胞标志物（非特异性酯酶、自发荧光以及由单克隆抗体LC-1C6定义的一种抗原）之间存在密切的细胞相关性。由于完整的细胞膜会排斥3βHSD细胞化学试剂，因此在悬浮细胞制剂中，通过3βHSD反应性鉴定出具有受损质膜的睾丸间质细胞，并在细胞膜裂解后测定同一制剂中3βHSD阳性（3βHSD+）细胞的总数。通过用核染料碘化丙啶复染将完整细胞与细胞质片段区分开来，随后通过从有核3βHSD+细胞总数中减去受损有核3βHSD+细胞的数量来确定每个制剂中完整睾丸间质细胞的数量。大多数完整分离的睾丸间质细胞存在于密度为1.054 - 1.096 g/ml的梯度级分中。每个完整睾丸间质细胞的急性（3 - H）基础睾酮分泌以及人绒毛膜促性腺激素（hCG）刺激后的睾酮分泌取决于每个测定孔中睾丸间质细胞的浓度，这表明体外睾丸间质细胞之间存在协同作用。在测定浓度大于10,000个睾丸间质细胞/孔时，上述密度梯度范围内不同级分的完整睾丸间质细胞在类固醇生成功能上没有差异。在较低细胞浓度下，密度较低（1.054 - 1.064 g/ml）的梯度级分中的睾丸间质细胞对hCG刺激产生的睾酮略少于密度较高（1.070 - 1.096 g/ml）的级分中的睾丸间质细胞。延长分离细胞在分散条件下的暴露时间会导致所有睾丸间质细胞的表观浮力密度、基础睾酮分泌以及hCG刺激后的环磷酸腺苷（cAMP）和睾酮分泌下降，而细胞完整性没有可检测到的变化。数据表明，分离的完整睾丸间质细胞的绝对类固醇生成功能和功能异质性至少部分取决于用于分离它们的程序。
