Transforming growth factor-β receptor types I and II in cultured porcine Leydig cells:: Expression and hormonal regulation

The steroidogenic activity of testicular Leydig cells is controlled both by the pituitary hormone (LH) and by growth factors such as transforming growth factor-beta peptides (TGF beta 1, -2, and -3; inhibin/activin; and anti-Mullerian hormone). By using primary cultures of porcine Leydig cells as a model, the aim of the study was to identify and characterize the TGF beta receptors and to study their regulation by LH/hCG. TGF beta receptors have been identified and characterized through three different approaches, including cross-linking experiments and Western and Northern blotting analyses. In cross-linking experiments, labeled TGF beta was shown to bind to three different molecular species of 300, 80, and 53 kDa, which may correspond to the protein betaglycan (also known as TGF beta type III receptor) and TGF beta type II and I receptors (TGF beta RII and TGF beta RI), respectively. The presence of TGF beta RI and -RII was further demonstrated by Western blotting analysis using specific polyclonal antibodies. Finally, the expression of betaglycan, TGF beta RII, and TGF beta RI messenger RNAs, was confirmed by Northern blotting analysis, as shown by the presence of 6.4-, 4.6-, and 5.8-kb messenger RNhs, respectively. By using a RT-PCR approach, the mediators of the TGF beta signal, Smads 1-7, were also detected in cultured Leydig cells. TGF beta RI and TGF beta RII protein levels were enhanced by hCG/LH in a dose-dependent (maximal effect with 0.3 ng/ml hCG) and time-dependent (maximal effect observed after 48 h of hCG treatment) manner. Furthermore, to determine whether the stimulatory effect of LH/hCG was mediated by testosterone, use was made of aminogluthetimide, an inhibitor of cytochrome P450scc. The inhibition of testosterone formation did not affect the stimulatory effect of LH/hCG on TGF beta RI and -RII levels, suggesting that the gonadotropin action is not mediated by the steroid hormone. Together, the present findings demonstrate that the TGF beta receptors are expressed and are under hormonal (gonadotropin) control in cultured porcine Leydig cells.