Functional and Morphological Studies on Isolated Leydig Cells: Purification by Centrifugal Elutriation and Metrizamide Fractionation

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Abstract
Rat interstitial cells were fractionated by centrifugal elutriation to facilitate the purification of Leydig cells for analysis of mechanisms of gonadotropin action in vitro. By this procedure, 109 collagenase-dispersed interstitial cells from adult rat testes were separated into 12 fractions in about 1 h. Fractions 1-7 (sedimentation velocities, 1.9-12 mm/h per g) comprised 80-85% of the total cells applied, including erythrocytes, lymphocytes, germinal cells, macrophages, endothelial cells, damaged Leydig cells and contained less than 4% intact Leydig cells. Fractions 8-12 (sedimentation velocities, > 12 mm/h per g) comprised 15-20% of the original cells and contained 90-95% intact Leydig cells. Despite their different sedimentation velocities, the Leydig cell-rich fractions was similar in their LH [luteinizing hormone] receptor content (mean .+-. SD, 36,115 .+-. 4815 sites/cell) and showed similar 5-fold increases above the original cell preparation in testosterone and cAMP responses to hCG [human chorionic gonadotropin]. The pooled Leydig cell-rich fractions (8-12) were further resolved on 16-24% metrizamide gradients into 5 bands. Bands II-V (density range, 1.075-1.110 g/ml) contained pure Leydig cells, and band I (1.048 g/ml) contained pachytene spermatocytes, the contaminating cell type present in the Leydig cell-rich fractions obtained by elutriation. Each of the 4 Leydig cell-rich bands showed similar morphology and functional activity. Essentially similar results were observed using 14-32% metrizamide gradients. Leydig cells desensitized in vivo by hCG treatment and isolated by elutriation were also resolved by metrizamide gradients into 4 bands, but showed a redistribution in the gradient, due to the shift of about 50% of the cells originally present in the heaviest bands to lower density fraction. However, in spite of their changes in density, the Leydig cell bands still showed similar degrees of receptor down-regulation and impairment of the steroid responses to hCG in vitro. Centrifugal elutriation is a rapid and effective method for obtaining large quantities of purified (> 90%) and active Leydig cells. Further resolution of the Leydig cell-rich fractions in metrizamide gradients has allowed complete Leydig cell purification, which is not achieved by density gradient centrifugation alone. Since less responsive or inactive Leydig cells displayed various degrees of structural damage, such cells should not be considered as a population of physiological significance. Therefore, the morphologically intact Leydig cell population of the adult rat testis is composed of cells of different densities and a defined range of sedimentation velocities, but with similar morphology, biological responsiveness and susceptibility to desensitization by gonadotropins.