Relationship between hCG receptors disappearance and steroid accumulation during long term hCG stimulation in porcine Leydig cell cultures.

LH-hCG receptors and steroidogenesis can be maintained for more than a week in porcine Leydig cell culture (chemically defined medium). Free receptors and steroid accumulation were measured following long time exposure to hCG. As expected, the percent of receptor disappearance increases with the hCG concentration in the incubation medium. However, the steroidogenesis was also proportional to hCG concentration for 24 hours as well as 48 hours. A 50% receptor disappearance was obtained for 0.5 ng hCG/ml which led to a steroid accumulation of 50% of the maximal accumulation obtained with 100 ng/ml. A very significative positive correlation (rho) was observed between receptor occupancy (internalized and/or irreversibly occupied receptors) and steroid accumulation (T and DHAS) during a 24 or 48 hour period (rho = 0.97 and 0.98 for T, and rho = 0.96 and 0.94 for DHAS). Following 24 or 48 hour exposure to increasing hCG concentration, the cells were acutely stimulated by 25 ng hCG/ml for 4 hours. The steroidogenic response obtained to this acute stimulation was inversely proportional to the concentration of hCG during the previous period. A negative correlation was found between receptor occupancy and acute steroidogenic response (rho : -0.96 and -0.98 for T). These results indicate that during long term stimulation as well as during acute restimulation the absolute number of occupied receptors conditioned the steroidogenic response. These results do not fit into the concept of spare receptors and suggest that in these immature cells all receptors are potentially active in the regulation of steroidogenesis.

Hormonal regulation of pig Leydig cells in culture.

Using a primary culture of purified immature pig Leydig cells, the hormonal and non hormonal regulation of steroidogenic activity have been studied. The effects of LH/hCG, FSH, LHRH, o-prolactin, estradiol, epidermal growth factor (EGF), human low density lipoprotein (hLDL) and human high density lipoprotein (hHDL) on 125I-hCG binding and basal and hCG-stimulated cAMP and steroids (testosterone and dehydroepiandrosterone sulfate) production were evaluated. These data on the regulation of pig Leydig cell activity are compared with other published results, mainly in the rat model, obtained either in vivo and in vitro.

Androgen production in primary culture of immature porcine Leydig cells.

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 alpha-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 3 beta-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.

Changes in plasma gonadotropin after ovariectomy and estradiol supplementation at different stages at the end of the reproductive cycle in the rainbow trout (Salmo gairdneri R.).

To determine the effect of gonadal feedback on plasma GTH level, female rainbow trout were ovariectomized at three stages at the end of the reproductive cycle: at the end of vitellogenesis, during germinal vesicle migration and during the post-ovulatory period. A group of controls and one of castrates in each experiment were given an injection of physiological salt solution, and a third group of castrates was supplemented with estradiol-17 beta (E2) twice a week (200 micrograms/kg) from the day of surgery. The blood was sampled twice a week, and the GTH measured by RIA. At the end of vitellogenesis, castration induced a significant rise in the gonadotropic hormone level (P less than 0.001 from post-surgical day 5), and that response, unimpeded by E2, was homogeneous in all the fish. During germinal vesicle migration, the response to castration and to supplementary E2 varied with the individual. Ovariectomy induced a significant increase in GTH (P less than 0.005 from day 3), but that increase was immediate in 5 females and delayed in the other 4; E2 prevented GTH rise in only 6 females. At the post-ovulatory period we found no significant difference between the control fish and the castrates and E2, at least temporarily, prevented the post-ovulatory rise in GTH which is usually found in trout.