Gonadotropin regulation of Leydig cell DNA synthesis

Abstract

Adult male rats were injected s.c. with either saline, 100 IU hCG, 100 jug FSH, 50 μg LH, 100 μg PRL, 50 μg estradiol-17β, 500 μg or 10 mg testosterone; 50 μg estradiol-17β; animals were sacrificed at 12–120 h post-injection. Collagenase-dispersed interstitial cells (150–200 × 10 6 cells/2 ml) were incubated in vitro with 10 μCi [ 3H- methyl]thymidine for 1 h at 32°C. Centrifugation of the cells on discontinuous 11–27% metrizamide gradients revealed thymidine incorporation in the regions of population I and II Leydig cells. A significant increase in thymidine incorporation into DNA after treatment with either hCG or LH was first detectable at 48 h, was equivalent to control values at 72 h and was again significantly increased at 96 h in population I and at 120 h in population II cells. [ 3H]Thymidine incorporation at 48 h, expressed as dpm/10 6 cells, was 2205 ± 432 and 4119 ± 929 vs. 16473 ± 3795 and 11648 ± 3427 for control and hCG-treated population I and II cells, respectively. Addition of 20 mM hydroxyurea suppressed [ 3H]thymidine incorporation, 97% and 96% in hCG-treated population I and II cells, respectively. Autoradiographic analyses revealed that nuclei from control and 48 h hCG-treated population I and II cells exhibited 1.2% and 2.3% vs. 7% and 6.8% silver grains, respectively. PRL had no influence on LH/hCG-enhanced DNA synthesis; however, estradiol-17β administration for 48 h dramatically suppressed thymidine incorporation. Population I Leydig cells exhibited a higher level of LH/hCG-stimulated DNA synthesis compared to population II cells. It is concluded that replicative DNA synthesis is enhanced in Leydig cells by LH/hCG and occurs in waves at 48 h intervals.