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-β1 and TGF-β2, respectively. Radioreceptor assay with recombinant human [125I]-TGF-β1 was applied to qualitatively identify TGF-β1. 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-β1 and TGF-β2, predominantly in latent form. The total concentration of TGF-β1 averaged 238 ng/ml versus an average of 18 ng/ml for TGF-β2. The in vitro activation or release of TGF-β1, from latent TGF-β1 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-β1 was achieved after an incubation of only 15 min, reached the maximum at 120 min, and the activated TGF-β1 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-β1 in human seminal plasma with recombinant human TGF-β1.
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.