ISOLATION OF HUMAN LEYDIG-CELL MESENCHYMAL PRECURSORS FROM PATIENTS WITH THE ANDROGEN INSENSITIVITY SYNDROME - TESTOSTERONE PRODUCTION AND RESPONSE TO HUMAN CHORIONIC-GONADOTROPIN STIMULATION IN CULTURE

Mature Leydig cells, the main source of testicular testosterone in mammals, arise from immature mesenchymal precursors through an LH-dependent differentiation process. In order to study the steroidogenic potential of these precursors, undifferentiated mesenchymal cells were obtained from the testicular interstitium of two patients with androgen insensitivity syndrome. After double digestion with collagenase and separation of the suspensions in a Percoll density gradient, the cells were cultured in Ham's F12 medium: Dulbecco's Modified Eagle Medium (1:1) supplemented with antibiotics, transferrin, insulin, hydrocortisone, and vitamin E with or without 1 IU of hCG/ml. At 11 days in culture, samples were removed for morphological characterization and determination of 3-beta-hydroxysteroid dehydrogenase activity (3-beta-HSD). Testosterone concentration was determined by RIA in the culture medium at different intervals. Cultured cells were mesenchymal in appearance, elongated in shape, with numerous processes running in different directions. No mature Leydig cells were present. In basal conditions, the percentages of 3-beta-HSD-positive cells at 11 days on patients 1 and 2 were 33% and 28%. respectively, and the testosterone concentrations in the culture media were 4.8 and 8.4 ng . 10(6) cells . 24 h, respectively. In cultures stimulated with hCG, there was an increase of histochemical reactivity (47% and 42% in patients 1 and 2, respectively) and in the amount of testosterone secreted (10.2 and 12.0 ng . 10(6) cells, respectively). Electron microscopic studies of cultures grown in the absence of hCG demonstrated a homogenous population of poorly differentiated, fibroblastic-type mesenchymal cells. Cultures stimulated with hCG for 11 days showed a marked increase in organelles, particularly the number of mitochondria and smooth endoplasmic reticulum cisternae, which adopted a pattern resembling the one classically associated with steroid secretion. We report here a successful culture of human testicular mesenchymal cells, demonstrating that immature Leydig precursors are capable of testosterone synthesis and secretion and should, therefore, be included in the family of testosterone-producing cells. Addition of hCG increases testosterone secretion and promotes a trophic effect, triggering early cytologic differentiation toward mature Leydig cells.