Morphological and functional characterization of interstitial cells from mouse testes fractionated on Percoll density gradients.

Cell fractions were obtained by separation on Percoll density gradients after dissociation of mature mouse testes by mechanical or collagenase dispersion, and the ultrastructure, hCG receptor properties, and hCG-stimulated testosterone (T) production of these fractions were compared. Gradients were fractionated according to specific gravity, and all cell types were quantitated using morphometric techniques. Three peaks of specific [125I]iodo-hCG binding corresponding to densities of 1.0667 g/cm3 (fractions 2-3), 1.045 g/cm3 (fractions 6-7), and 1.0365-1.0215 g/cm3 (fraction 9) were obtained after collagenase dispersion, but the second peak of binding (fractions 6-7) was not observed after mechanical dispersion. Morphometric studies were performed by light microscopy on the cells present in the fractions corresponding to the first and second hCG binding peaks obtained by either mechanical or collagenase dispersion; the third representing germ cells and membranous debris was not studied further. Regardless of the method of preparation, morphologically intact Leydig cells represented 60-80% and 7-10% of the cells in fractions 2-3 and 6-7 that were associated with the first and second peaks of hCG binding Leydig cells obtained from fractions 6-7 contained greater numbers of lipid inclusions than Leydig cells from fractions 2-3. Mechanically dispersed Leydig cells exhibited similar numbers of hCG receptors in the dense and light Leydig cells, but hCG stimulated T production per Leydig cell was significantly greater in the dense Leydig cells containing few lipid inclusions. T production by dense and light collagenase-dispersed Leydig cells was not significantly different. The second hCG binding peak in collagenase-dispersed cell fractions 6-7 was associated with an increase in the number of an indeterminate connective cell type released from the testes by collagenase treatment, whose ultrastructure and limited hCG-binding capacity suggested that they may represent Leydig cell precursors. It is concluded that the identification and quantitation of different cell types in isolated testicular cell fractions is, therefore, of fundamental importance in the interpretation of receptor and secretory capacities of enriched preparations of Leydig cells.