The functional activity of adult mouse Leydig cells in monolayer culture. Effect of lutropin and foetal calf serum.

Purified Leydig cells were obtained from adult mouse testes by mechanical dispersion followed by Percoll density-gradient centrifugation as described by Schumacher et al. (1978). The cells were then established in monolayer culture by maintaining them in medium and 10% serum at 32 degrees C in 95% O2, 5% CO2. The cells rapidly attached to the culture dishes, gradually flattened and became epitheloid in appearance. Testosterone production by the cells in response to maximum stimulating levels of LH (100 ng/ml) and dibutyryl cyclic AMP (1 mM) was maintained for at least 2 days (approximately 1 microgram/10(6) cells/2h) and then declined to lower levels by days 3-4. Cyclic AMP production in response to LH was higher on day 1 than day 0 and then declined to lower levels by days 3-4. Binding of [125I]hCG was similar on day 0 and day 1 (approximately 20 fmoles/10(6) cells) and then declined to lower levels by days 3-4. The functional activity of the cells cultured in 0, 1 and 10% foetal calf serum was also examined; no significant effect of the serum on LH-stimulated testosterone or cyclic AMP production was found; however, a decrease of up to 50% in the binding of [125I]hCG to the Leydig cells occurred in the presence of serum. These results demonstrate that the function of differentiated adult Leydig cells can be maintained for at least 2 days in culture.

High-pressure liquid chromatography of steroids secreted by human adrenal and testis cells in monolayer culture.

Reversed-phase high-pressure liquid chromatography with gradient elution on Zorbax-ODS columns has been used to separate, identify, and measure, spectrophotometrically, the steroids secreted by both human adrenal and testis cells in primary monolayer culture. Three related systems using exponential concave gradients have been developed with the specific objective of resolving the steroids produced by these two tissues. A methanol-water gradient has been used to separate most adrenal steroids, an acetonitrile-water gradient to separate testis steroids, and a dioxane-water gradient to separate polar steroids, including aldosterone. These three systems together permit the resolution of at least 43 naturally occurring steroids, plus four synthetic steroids with adrenocortical activity, with overall total elution times of 1 h or less for each system. Retention data for these steroids are given and the separation of steroids in the biological samples illustrated.

The heterogeneity of Leydig cells from mouse and rat testes--evidence for a Leydig cell cycle?

A method for purifying Leydig cells by centrifugation of testes cells on continuous density gradients of Percoll has been investigated. The distribution of Leydig cells in the separated bands of cells obtained and their receptor content and testosterone production after addition of lutropin (LH) has been measured. In agreement with previous work (Schumacher, Schäfer, Holstein & Hilz 1978) it was found that highly pure mouse Leydig cells (average density 1.070 g/ml) could be prepared by this method. These cells responded to LH and produced high amounts of testosterone (1 - 4 microgram/10(6) cells/2 h), and bound [125]hCG specifically (25 - 64 fmols hCG bound/10(6)). Similarly from rat testes, Leydig cells (average density 1.072 g/ml) were purified. These cells also responded to LH and produced 5 - 25 ng testosterone/10(6) cells/2 h and bound [125]hCG specifically (3 - 18 fmols hCG bound/10(6)cells). Two other bands of nucleated cells of lower density (approximately 1.045 and 1.052 g/ml) were formed on the gradients from both mouse and rat testes. Both these bands of cells were found to contain Leydig cells which bound [125]hCG specifically but little or not stimulation of testosterone production could be demonstrated. Fractionation of the gradients after separation of the cells into small aliquots demonstrated that fractions containing up to 100% Leydig cells could be isolated which were not stimulated to produce testosterone after addition of LH. It is concluded that in both the adult rat and mouse testes, Leydig cells of different densities and steroidogenic responsiveness to LH exist. The data obtained in this and other studies suggest that Leydig cells in the rat and mouse testes are not a homogeneous population and that they may be undergoing a continuous cycle of activity which involves changes in density and steroidogenic capacity.