GROWTH-FACTOR REQUIREMENTS FOR DNA-SYNTHESIS BY LEYDIG-CELLS FROM THE IMMATURE RAT

Puberty in the male is dependent upon the elevated production of testosterone by the Leydig cells. LH affects this increase in testosterone output by increasing the total number of Leydig cells in the testis and by stimulating the steroidogenic pathway in these cells. Since Leydig cell proliferation is a prerequisite for the onset of puberty, we have examined the ability of LH and growth factors known to be present in the testis to promote DNA synthesis. Leydig cells were isolated from 21-day-old rats, cultured in serum-free medium for 48 h to become quiescent, and then treated with LH and growth factors for 18 h. [H-3]Thymidine incorporation into DNA was assessed over the subsequent 4-h incubation period. Cells in control cultures incorporated low levels of [H-3]thymidine into DNA and were stimulated after treatment with LH (100 ng/ml). Insulin/insulin-like growth factor-1 (IGF-1) and transforming growth factor-alpha (TGF-alpha), previously localized in Leydig cells by immunohistochemistry, also stimulated [H-2]thymidine incorporation into DNA. The responses of the Leydig cells to maximum levels of insulin and TGF-alpha were dependent on the cell density. Insulin and TGF-alpha alone and in combination increased the number of cells labeled with [H-3]thymidine, as assessed by autoradiography. TGF-beta, known to be secreted by Sertoli cells, also stimulated DNA synthesis under basal conditions, but the maximum response was significantly lower than that achieved in the presence of TGF-alpha. Pretreatment of the cells with a low level of LH (2 ng/ml) for 48 h caused a marked enhancement of the response of the cells to the subsequent 18 h of treatment with insulin plus TGF-alpha and insulin plus TGF-beta. In summary, whereas LH alone had little effect, it interacted synergistically with locally produced growth factors to promote DNA synthesis of immature Leydig cells. These interactions may determine the proliferative state of Leydig cells during development.