Rat testicular germ cells and Sertoli cells release different types of bioactive transforming growth factor beta in vitro

Abstract

Several in vivo studies have reported the presence of immunoreactive transforming growth factor-beta's (TGF-beta's) in testicular cells at defined stages of their differentiation. The most pronounced changes in TGF-beta1 and TGF-beta2 immunoreactivity occurred during spermatogenesis. In the present study we have investigated whether germ cells and Sertoli cells are able to secrete bioactive TGF-beta's in vitro, using the CCl64 mink lung epithelial cell line as bioassay for the measurement of TGF-beta. In cellular lysates, TGF-beta bioactivity was only observed following heat-treatment, indicating that within these cells TGF-beta is present in a latent form. To our surprise, active TGF-beta could be detected in the culture supernatant of germ cells and Sertoli cells without prior heat-treatment. This suggests that these cells not only produce and release TGF-beta in a latent form, but that they also release a factor which can convert latent TGF-beta into its active form. Following heat-activation of these culture supernatant's, total TGF-beta bioactivity increased 6- to 9-fold. Spermatocytes are the cell type that releases most bioactive TGF-beta during a 24 h culture period, although round and elongated spermatids and Sertoli cells also secrete significant amounts of TGF-beta. The biological activity of TGF-beta could be inhibited by neutralizing antibodies against TGF-beta1 (spermatocytes and round spermatids) and TGF-beta2 (round and elongating spermatids). TGF-beta activity in the Sertoli cell culture supernatant was inhibited slightly by either the TGF-beta1 and TGF-beta2 neutralizing antibody. These in vitro data suggest that germ cells and Sertoli cells release latent TGF-beta's. Following secretion, the TGF-beta's are converted to a biological active form that can interact with specific TGF-beta receptors. These results strengthen the hypothesis that TGF-beta's may play a physiological role in germ cell proliferation/differentiation and Sertoli cell function.

Figure 1

Effect of anti-TGF-β₁ (filled bars) and anti-TGF-β₂ (hatched bars) antibodies on growth inhibition of mink lung CCl64 cells by heat treated culture supernatants of spermatocytes (sp.cytes), round spermatids (r. spert.), elongated spermatids (e.spert.) and Sertoli cells (S.c.). The dilutions of the culture supernatants that gave maximal growth inhibition were equivalent to 100% TGF-β activity (open bars). The final concentration of both antibodies used in this assay was 10 μg/ml. The values, which are expressed as percentage of residual TGF-β activity, represent means ± SD