An interesting finding was that basal testosterone production increased significantly only in incubations with intact testes (Fig. 3), whereas no, or only a marginal, increase in basal testosterone was found during hours 1-5 of incubation with dispersed testicular cells (Fig. 4). We found that recombinant rat LH at a low concentration of 0.005 (jug/L (2.5- to 5-fold higher than in fetal plasma on days E17.5-19.5) was unable to stimulate testosterone production of E17.5 intact fetal testes, whereas at the higher concentration of 2.0 |xg/L, a clear response was observed (Fig. 5).
Stimulation of Dispersed Fetal, Neonatal, Immature, and Adult Rat Leydig Cells by Other Hormones and Growth Factors
We tested the acute (1-5 h) and chronic (12-72 h) effects on fetal testicular steroidogenesis of some putative paracrine factors, including activin, inhibin, epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), IGF II, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), leptin, VIP, and PACAP-27. Only the latter two significantly affected fetal testicular steroidogenesis. We found that both VIP and PACAP-27 stimulated cAMP and testosterone production by dispersed fetal Leydig cells significantly (p < 0.0001), with an effect equal to that attained with hCG (Fig. 6A). Interestingly, we found that this stimulatory effect of VIP and PACAP-27 on testosterone production was specific to fetal but not adult Leydig cells (Fig. 6B). We also found that VIP and PACAP-27 were unable to stimulate cAMP and testosterone production by mouse Leydig tumor cells (BLT-1; Fig. 6C). buy diabetes drugs
FIG. 5. The effect of recombinant rat LH on testosterone production by intact El 7.5 fetal testes. One testis from each fetus was incubated for 4 h in the absence and the other in the presence of 0.005 ^g/L or 2.0 jo-g/L of the hormone. Testosterone concentrations in the media were measured by RIA. Data points represent the mean ± SEM (n = 6). * Significant difference from the basal testosterone production < 0.0001).
FIG. 6. The effects of VIP, PACAP-27, and hCG on testosterone production by dispersed fetal rat (El 9.5) Leydig cells (A), purified adult rat Leydig cells (B), and murine Leydig tumor cells (BLT-1) (C). The different cells were prepared and incubated as described in Materials and Methods, in medium alone (C, black dotted bars), or in the presence of VIP, PACAP, or hCG. The contents of testosterone in the media were measured by RIA. Data points represent the mean ± SEM (n = 6-12). * Significant difference from the basal (C) testosterone production (***p < 0.001, ****p < 0.0001, NS = not significant).