Morphological and Functional Characterization of Interstitial Cells from Mouse Testes Fractionated on Percoll Density Gradients*

Abstract

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.

Differentiation of Leydig cells in the Mongolian gerbil

“…Mature ALC were found from the sixth week onward indicating that the differentiation of the gerbil ALC population is complete at that time. In conclusion, the differentiation of the gerbil ALC lineage began in a period similar (second week) to that in other laboratory rodents (Haider, 2004;Haider et al, 1995;Kerr et al, 1985;Mendis-Handagama and Ariyaratne, 2001;MendisHandagama and de Kretser, 1992;O'Shaughnessy et al, 2002;Wu et al, 2007), but the emergence of subsequent phases is delayed. On the other hand, the Leydig cell population in adult animals shows strikingly particular phenotypical features and functional heterogeneity.…”

“…The progressive stages of maturation of ALC were characterized based on criteria previously established for rats Benton et al, 1995;Haider, 2004;Hardy et al, 1989;Kerr and Knell, 1988;Mendis-Handagama and Ariyaratne, 2001;Mendis-Handagama et al, 1987;Wu et al, 2007) and mice (Baillie, 1964;Kerr et al, 1985;Wu et al, 2007). Some of these criteria are morphological, such as the distribution in the interstitial tissue, cellular shape and size, nuclear characteristics, abundance of cytoplasmic droplets whereas others concern functional activity such as the expression of AR and the enzymes 3b-HSD and 11b-HSD1.…”

“…Most information about postnatal differentiation of Leydig cells is derived from established models of laboratory rodents widely studied such as rats Benton et al, 1995;Haider, 2004;Hardy et al, 1989;Kerr and Knell, 1988;Mendis-Handagama and Ariyaratne, 2001;MendisHandagama et al, 1987;Wu et al, 2007) and mice (Baillie, 1964;Benton et al, 1995;Haider, 2004;Kerr et al, 1985;Mendis-Handagama and Ariyaratne, 2001;Mendis-Handagama and de Kretser, 1992;Wu et al, 2007). Two morphologically and functionally distinct populations of Leydig cells are involved in the abovementioned process-the fetal and adult ones, which arise respectively, in the prenatal and prepubertal period Haider, 2004;Mendis-Handagama and Ariyaratne, 2001;Roosen-Runge and Anderson, 1959).…”

Local Regulation of Macrophage Subsets in the Adult Rat Testis: Examination of the Roles of the Seminiferous Tubules, Testosterone, and Macrophage-Migration Inhibitory Factor1

“…Assay sensitivity was 0.09 ng/ml for LH and 1.5 ng/ml for FSH. Testicular IF and homogenates were assayed for testosterone without extraction using a direct 125 I-testosterone RIA [23], or after extraction with chloroform-hexane using a [ 3 H]testosterone RIA [24]. Extraction recoveries were 78-82%.…”

Leukocyte Populations of the Adult Rat Testis Following Removal of the Leydig Cells by Treatment With Ethane Dimethane Sulfonate and Subcutaneous Testosterone Implants1

“…Testosterone levels in testicular IF were measured by 25I-testosterone RIA, as previously described [44].…”