Heterogeneity of Adult Mouse Leydig Cells with Different Buoyant Densities

Abstract

ABSTRACT: The authors investigated the morphologic characteristics and human chorionic gonadotrophin (hCG)‐stimulated testosterone production of adult mouse Leydig cells in vitro, which have different buoyant densities. Leydig cells from five testes of Swiss outbred male mice (15 weeks old) were isolated and purified by mechanical dispersion followed by density gradient centrifugation using Percoll. Two groups of Leydig cells were obtained with different buoyant densities: group 1 had densities of 1.0667 to 1.0515 g/cm3 and group 2 had densities of 1.0514 to 1.0366 g/cm3. In vitro testosterone production of these Leydig cells, in response to different doses of hCG (0, 5, 25,125, 625, and 3125 pg/mL), was determined by radioimmunoassay. Leydig cells were fixed and processed for electron microscopic stereology to quantify the organelles by volumes and surface area. In Leydig cells of Group 1, testosterone production per cell in vitro in response to 0 and 5 pg/mL hCG was not significantly different (P > 0.05). Increases in the dose up to 25 pg/mL produced a significant increase (P < 0.05) in testosterone production, although hCG doses of 125 and 625 pg/mL did not produce further increases in testosterone levels. However, 3125 pg/mL hCG further elevated the testosterone production by those Leydig cells with high buoyant density. In Leydig cells in group 2, the patterns of testosterone production in response to hCG doses of 0, 5, and 25 pg/mL were similar to those of Leydig cells in group 1. Those Leydig cells with low buoyant density, however, were unable to stimulate further testosterone production by an hCG dose of 3125 pg/mL. Comparison of testosterone production per Leydig cell between the two groups with different doses of hCG showed that Leydig cells in group 2 (lower buoyant density) showed a lower value than those in group 1 (higher buoyant density) for all hCG doses studied. Statistical significance (P < 0.05), however, was observed only with the hCG dose of 3125 pg/mL. These results demonstrated that Leydig cells with different buoyant densities were different in their sensitivity to hCG stimulation in vitro. Leydig cells in groups 1 and 2 were also different in their organelle composition, the most prominent feature being the differences in their lipid contents. Leydig cells with low buoyant density (group 2) were rich in lipid droplets, and Leydig cells with high buoyant density contained less lipid. Lipid‐rich Leydig cells with reduced testosterone production contained reduced surface area of smooth endoplasmic reticulum and membrane whorls, features characteristic to less differentiated Leydig cells in mice. Based on these results, the authors conclude that adult mouse Leydig cells exist as a heterogeneous population. Leydig cells with low buoyant density were less differentiated than those with high buoyant density with respect to their ultrastructure and their sensitivity to hCG stimulation in vitro.