EXPRESSION AND REGULATION OF TRANSFORMING GROWTH-FACTOR-BETA-1 MESSENGER-RIBONUCLEIC-ACID AND PROTEIN IN CULTURED PORCINE LEYDIG AND SERTOLI CELLS

In the present work, the expression and secretion of transforming grow th factor-beta 1 (TGF beta 1) by immature pig Leydig and Sertoli cells were investigated. Both cell types express two TGF beta 1 mRNA transc ripts of 2.5 and 3.5 kilobases, and the levels were 2.6-fold higher in Leydig than in Sertoli cells. In the latter cells, mRNA levels were e nhanced when cultured cells were stimulated by epidermal growth factor and phorbol ester (4 beta-phorbol 12-myristate 13-acetate) and signif icantly decreased by FSH and testosterone. Using a polyclonal antibody raised against a synthetic peptide that corresponded to the carboxyl- terminal region of TGF beta 1 and recognized this peptide, but not TGF beta 2 or TGF beta 3, specific immunostaining of both Leydig and Sert oli cells was demonstrated in situ, after cell isolation, and during c ulture. The immunostaining was more marked in Leydig cells than in Ser toli cells. Western blot analysis of Leydig or Sertoli cell-conditione d medium demonstrated a band of 25 kilodaltons, which was shifted to 1 2.5 kilodaltons under reducing conditions. Using the mink lung epithel ial cell bioassay for TGF beta 1, we could demonstrate the presence of TGF beta 1-like activity in Leydig and Sertoli cell-conditioned media after acid treatment, but not before activation. The inhibitory effec ts of both pure TGF beta 1 and acidified conditioned medium were almos t completely blunted by the TGF beta 1 antibody. The amounts of TGF be ta 1 secreted by Sertoli and Leydig cells were not significantly diffe rent and varied between 400-800 pg/48 h.10(6) cells. These studies dem onstrate for the first time that both pig Leydig and Sertoli cells exp ress TGF beta 1 mRNA, and the TGF beta 1-like activity secreted by the se cells corresponds to TGF beta 1. As TGF beta 1 has been demonstrate d to have strong effects on testicular cells, in particular on Leydig cell functions, it is suggested that local secreted TGF beta 1 may pla y a role in the autocrine/paracrine regulation of testicular functions .