Androgen production in primary culture of immature porcine leydig cells

Abstract

The present paper examines the steroidogenic responsiveness of immature porcine Leydig cells in primary culture. Both testosterone (T) and dehydroepiandrosterone sulfate (DHAS) secretion were measured under basal conditions and after stimulation with human chorionic gonadotropin (hCG) (25 ng/ml). In medium supplemented with insulin, transferrin, epidermal growth factor (3H) and 0.1% calf serum, cells survived 3–5 days in culture. The production of steroids (under hCG stimulation) is poor on day 0–1 of the culture. On day 2–4 basal T and DHAS levels are 1.9 and 17.0 ng/10 6 cells/24 h. The addition of hCG stimulated T and DHAS production 19- and 6-fold respectively and the average productions were 37 and 109 ng/10 6 cells/24 h. Increasing the serum to 0.5% did not change the viability of the cultures, but increased hCG stimulated T and DHAS production (183 and 188 ng/10 6 cells/24 h). The addition of α-tocopherol (vitamin E) to 0.1% calf serum led to a 4-fold increase in stimulated T production (142 ng/10 6 cells/24 h) and maintained full cell viability for more than 5 days. Measurement of 3β-ol steroid dehydrogenase activity indicates that the amount of enzyme is 4 times higher at day 2 than at day 0 and 1 (with or without hCG), suggesting a spontaneous maturation of the cells in culture. This might explain the increased T production with time in culture. In cumulative experiments (24 h) the cells do not seem to be desensitized to hCG stimulation following prolonged exposure to 25 ng hCG since the daily steroid production is increasing with time in culture. However, kinetic studies show that steroidogenesis is not linear over a 24 h period. In cumulative experiments the steroid production stops between 12 and 16 h following hCG exposure (5 and 100 ng/ml) and resumes following a medium change. These results suggest that some inhibitory compounds are accumulated in the medium and are controlling the Leydig cell function. Moreover high doses of hCG (100 ng/ml) result in a lower production of steroids and an earlier plateau in the case of DHAS. These results demonstrate that porcine Leydig cells can live and differentiate in hormone- and vitamin-supplemented medium and that auto-feedback mechanisms inhibiting steroid accumulation take place under in vitro conditions.