Characterization of Latent Transforming Growth Factor‐β From Human Seminal Plasma

PROBLEM : Human seminal plasma is known to exhibit immunosuppressive activity. Transforming growth factor β (TGF‐β) has been identified as an immunosuppressive factor in human seminal plasma. Biologically active TGF‐β represents a family of 25‐kDa homodimeric proteins linked with disulfide bonds. TGF‐β associates with high molecular weight proteins noncovalently to form a type of latency that is biologically inactive. Quantitative distribution of active form of TGF‐β versus inactive latent form of TGF‐β, and mechanism of the TGF‐β activation in human seminal plasma remain to be elucidated. PURPOSE : To characterize seminal plasma latent form of TGF‐β, including its concentration, and the mechanism underlying the activation of TGF‐β. METHOD : Gel filtrations on ACA‐34 and Biogel P‐60 were used to fractionate seminal plasma. TGF‐β was measured by enzyme immunoassay using antibodies specific for TGF‐β 1 and TGF‐β 2 , respectively. Radioreceptor assay with recombinant human [ 125 I]‐TGF‐β 1 was applied to qualitatively identify TGF‐β 1 . Kinetic experiments with various pH, temperature and time, along with protease inhibitors, were performed to delineate the activation mechanism of latent TGF‐β. RESULTS : Human seminal plasma contained both TGF‐β 1 and TGF‐β 2 , predominantly in latent form. The total concentration of TGF‐β 1 averaged 238 ng/ml versus an average of 18 ng/ml for TGF‐β 2 . The in vitro activation or release of TGF‐β 1 , from latent TGF‐β 1 was achieved only at acidic pH of <4.0, and was time and temperature dependent. At pH 3.7 and 37°C, a significant activation of latent TGF‐β 1 was achieved after an incubation of only 15 min, reached the maximum at 120 min, and the activated TGF‐β 1 remained relatively stable for at least 24 h. The activation was not inhibitable by a series of protease inhibitors examined, alone or in combination (e.g., phenylmethylsulfonyl fluoride, E‐64, pepstatin, leupeptin, ethylenediamine tetraacetic acid). Competitive radioreceptor assay established the functional identity of TGF‐β 1 in human seminal plasma with recombinant human TGF‐β 1 . CONCLUSION : Human seminal plasma TGF‐β is biologically activated from high molecular weight latent TGF‐β by acid pH. The acidic environment of female lower genital tract could represent an in vivo physiological condition for activation of seminal plasma TGF‐β that may immunologically protect the integrity of sperm.