Transforming Growth Factor- Receptor Types I and II in Cultured Porcine Leydig Cells: Expression and Hormonal Regulation

Abstract

The steroidogenic activity of testicular Leydig cells is controlled both by the pituitary hormone (LH) and by growth factors such as transforming growth factor-β peptides (TGFβ1, -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 TGFβ receptors and to study their regulation by LH/hCG. TGFβ 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 TGFβ 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 TGFβ type III receptor) and TGFβ type II and I receptors (TGFβRII and TGFβRI), respectively. The presence of TGFβRI and -RII was further demonstrated by Western blotting analysis using specific polyclonal antibodies. Finally, the expression of betaglycan, TGFβRII, and TGFβRI messenger RNAs, was confirmed by Northern blotting analysis, as shown by the presence of 6.4-, 4.6-, and 5.8-kb messenger RNAs, respectively. By using a RT-PCR approach, the mediators of the TGFβ signal, Smads 1–7, were also detected in cultured Leydig cells. TGFβRI and TGFβRII 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 TGFβRI and -RII levels, suggesting that the gonadotropin action is not mediated by the steroid hormone. Together, the present findings demonstrate that the TGFβ receptors are expressed and are under hormonal (gonadotropin) control in cultured porcine Leydig cells.