Transforming growth factor-beta 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 grow th 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 r eceptors and to study their regulation by LH/hCG. TGF beta receptors have b een identified and characterized through three different approaches, includ ing cross-linking experiments and Western and Northern blotting analyses. I n cross-linking experiments, labeled TGF beta was shown to bind to three di fferent molecular species of 300, 80, and 53 kDa, which may correspond to t he protein betaglycan (also known as TGF beta type III receptor) and TGF be ta type II and I receptors (TGF beta RII and TGF beta RI), respectively. Th e presence of TGF beta RI and -RII was further demonstrated by Western blot ting 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 media tors of the TGF beta signal, Smads 1-7, were also detected in cultured Leyd ig 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-depende nt (maximal effect observed after 48 h of hCG treatment) manner. Furthermor e, to determine whether the stimulatory effect of LH/hCG was mediated by te stosterone, use was made of aminogluthetimide, an inhibitor of cytochrome P 450scc. The inhibition of testosterone formation did not affect the stimula tory effect of LH/hCG on TGF beta RI and -RII levels, suggesting that the g onadotropin action is not mediated by the steroid hormone. Together, the pr esent findings demonstrate that the TGF beta receptors are expressed and ar e under hormonal (gonadotropin) control in cultured porcine Leydig cells.