The production of transforming growth factor-beta activity by rat granulosa cell cultures

We have examined whether granulosa cells (GC) secrete transforming growth factor-beta (TGF beta)-like activity using cell cultures prepared from diethylstilbestrol-primed female rats. Our results indicate that a significant level of active as well as latent TGF beta activity is found in defined GC culture medium as assessed by 1) potentiation of FSH-induced differentiation of rat GC, 2) neutralization of its activity by anti-TGF beta immunoglobulin, 3) inhibition of DNA synthesis in mink lung epithelial cells (CCl 64), and 4) activation of latent TGF beta activity by either acid or heat treatment. TGF beta production was more pronounced when the cells were seeded on fibronectin-coated plates. There was no difference in the level of TGF beta secretion by GC preparations derived from either diethylstilbestrol-primed immature or normal immature rats or adult rats. Furthermore, rat GC-conditioned medium contained much more TGF beta activity than medium from normal rat kidney cells (NRK 49-F), human prostatic adenocarcinoma cells (PC-3), or porcine GC. Rat thecal/interstitial cell culture medium contained activity comparable to that of GC medium. We conclude that rat GC preparations secrete a high level of TGF beta activity in vitro. Taken together with previous results, this indicates the possibility that TGF beta may be an autocrine regulator as well as a paracrine one within the ovarian follicle. Moreover, because of the high level of TGF beta activity produced, the rat GC culture system appears to be a useful experimental model for further exploring relationships between TGF beta production and its action.     

Regulation of human lung fibroblast C1q-receptors by transforming growth factor-beta and tumor necrosis factor-alpha

Transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) are two polypeptide mediators which are believed to play a role in the evolution of idiopathic pulmonary fibrosis (IPF). We have evaluated the effect of these two substances on the expression of receptors for collagen (cC1q-R) and globular (gC1q-R) domains of C1q and on type I collagen in human lung fibroblasts. Two fibroblast subpopulations differing in C1q receptor expression were obtained by culturing human lung explants in medium containing fresh human serum and heated plasma-derived serum and separating them based on C1q binding [Narayanan, Lurton and Raghu: Am J Resp Cell Mol Biol. 1998; 17:84]. The cells, referred to as HH and NL cells, respectively, were exposed to TGF-beta and TNF-alpha in serum-free conditions. The levels of mRNA were assessed by in situ hybridization and Northern analysis, and protein levels compared after SDS-polyacrylamide gel electrophoresis and Western blotting. NL cells exposed to TGF-beta and TNF-alpha contained 1.4 and 1.6 times as much cC1q-R mRNA, respectively, whereas in HH cells cC1q-R mRNA increased 2.0- and 2.4-fold. The gC1q-R mRNA levels increased to a lesser extent in both cells. These increases were not reflected in protein levels of CC1q-R and gC1q-R, which were similar to or less than controls. Both TGF-beta and TNF-alpha also increased procollagen [I] mRNA levels in both cells. Overall, TNF-alpha caused a greater increase and the degree of response by HH fibroblasts to both TGF-beta and TNF-alpha was higher than NL cells. These results indicated that TGF-beta and TNF-alpha upregulate the mRNA levels for cC1q-R and collagen and that they do not affect gC1q-R mRNA levels significantly. They also indicated different subsets of human lung fibroblasts respond differently to inflammatory mediators.     

Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 release IGF-binding protein-3 from human fibroblasts by different mechanisms.

Human neonatal fibroblasts in monolayer culture secrete insulin-like growth factor-binding proteins (IGFBPs), which may modulate IGF action. To examine whether an increase in extracellular concentrations of IGFBPs in response to IGF-I is due to the release of cell-associated IGFBPs, we measured secreted and cell-associated IGFBP-3 immunologically in fibroblast monolayers treated with IGF-I and IGF analogs with altered affinities for the IGF receptors and IGFBPs. IGFBP-3 in medium conditioned by fibroblasts treated with IGF-I was significantly increased (P < 0.05) compared with that in medium from untreated cultures; concomitantly, cell-associated IGFBP-3 was significantly decreased (P < 0.05). [Ser24]IGF-I (reduced affinity for IGF receptors) also increased secreted IGFBP-3 and decreased cell-associated IGFBP-3. In contrast, IGFBP-3 concentrations in medium conditioned by fibroblasts treated with B-chain IGF-I (reduced affinity for IGFBPs) were not significantly increased, and cell-associated IGFBP-3 was unchanged. Heparin, which releases proteins attached to cell surface proteoglycans, increased medium concentrations of IGFBP-3 and decreased IGFBP-3 binding to fibroblasts. An IGFBP of 29-31 kilodaltons (kDa) showed a pattern of regulation similar to that of IGFBP-3, while a third IGFBP, of 24 kDa, was decreased in IGF-I- and [Ser24]IGF-I-conditioned medium and unchanged by B-chain IGF-I and heparin. Preincubation with transforming growth factor-beta 1 (TGF beta 1), which stimulates fibroblast IGFBP-3 production, or human serum-derived IGFBP-3 did not increase cell-associated IGFBP-3. Analysis of total RNA isolated from fibroblasts revealed that IGFBP-3 mRNA was increased by TGF beta 1, but not by IGF-I. These data suggest that IGFs and TGF beta 1 release fibroblast IGFBPs by distinct mechanisms: IGFs by binding and subsequent release of cell-associated IGFBP-3 and 29- to 31-kDa IGFBP, and TGF beta 1 by increased de novo synthesis of IGFBP-3.     

Immunohistochemical localization of transforming growth factor-beta 1 and -beta 2 during follicular development in the adult rat ovary.

The transforming growth factors-beta (TGF-beta) affect the metabolic activities of each of the cell types in the ovary. In vitro studies using immature rat ovaries have shown the expression of TGF-beta 1 and/or TGF-beta 2 mRNA in thecal/interstitial cells and in granulosa cells (Mulheron and Schomberg, 1990; Mulheron et al., 1991). To obtain information on the localization of TGF-beta 1 and TGF-beta 2 in the rat ovary in vivo, we have examined the immunohistochemical staining using antibodies specific for either TGF-beta 1 or TGF-beta 2. In the adult ovary the immunostaining for TGF-beta 1 was intense, whereas the staining for TGF-beta 2 was faint. The pattern of immunostaining for TGF-beta 1 and TGF-beta 2 remained constant in the interstitial cell compartment and was not affected by the stage of the oestrous cycle. Since the interstitium surrounds follicles at all stages of development we conclude that TGF-beta is not actively involved in regulating the progression of follicles at discrete stages. At the time of antrum formation in the follicle, intense staining for TGF-beta 1 was observed in thecal cells. Around the preovulatory stage of development, TGF-beta 1 and TGF-beta 2 immunoreactivity was also found in the granulosa cells. In the corpus luteum, intense staining for TGF-beta 1 was found in some areas, whereas other areas were negative. Weak to moderate staining for TGF-beta 2 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of epidermal growth factor on insulin-like growth factor-I (IGF-I) and IGF-binding protein synthesis by adult rat hepatocytes.

Growth hormone has been established as a primary regulator of IGF-I gene expression in adults, not only in liver but also in many extrahepatic tissues. We considered the possibility that IGF-I production by adult rat liver could also be stimulated by epidermal growth factor (EGF), a peptide known to be involved in liver regeneration. Chromatographic analysis performed after acid treatment of conditioned media revealed the presence of both immunoreactive (IR) IGF-I and IGF binding protein (IGFBP). Both IR IGF-I and IGFBP were present in the conditioned medium of adult rat hepatocytes in basal conditions. The stimulation of IGF-I and IGFBP secretion by EGF appears to be dose-dependent with a significant increment already evident at 5 nM. That EGF stimulates secretion is supported by the finding that IGF-I and IGFBP-1 mRNA levels are increased after EGF supplementation. We conclude that adult rat hepatocytes spontaneously produce IGF-I and IGFBP, and that EGF is able to increase their synthesis and secretion. This non-growth hormone-dependent regulation of IGF-I and IGFBP-1 production by adult rat hepatocytes in culture indicates an important autocrine/paracrine role for IGF-I, particularly during liver regeneration after extensive organ mass loss.     

The effect of transforming growth factor-beta 1 on glycosaminoglycan production by human marrow cultures

We have assayed the effect of transforming growth factor-beta 1 (TGF-beta 1), a potent modulator of hematopoiesis, on glycosaminoglycan production in human marrow cultures. Glycosaminoglycans are a component of the extracellular matrix known to affect cell growth and differentiation. TGF-beta 1 and [35S]sulfate were added simultaneously to hematopoietically active human marrow cultures, and radiolabeled glycosaminoglycan production was determined by cetylpyridinium chloride precipitation. TGF-beta 1 at 15 ng/ml for 72 h increased [35S]sulfate incorporation into media glycosaminoglycans to 190% of control levels but did not affect the [35S]sulfate incorporation into cell-associated glycosaminoglycans. Approximately 90% of the glycosaminoglycans in the media fraction and 85% of the glycosaminoglycans in the cell-associated fraction were susceptible to degradation by chondroitin ABCase in both treated and control cultures. Pulse-chase experiments suggested that the increase in glycosaminoglycan [35S]sulfate incorporation was not due to decreased glycosaminoglycan degradation. This concentration of TGF-beta 1 did not alter nonadherent granulocyte-macrophage colony-forming unit (CFU-GM) number per flask but significantly decreased the more primitive adherent CFU-GM number per flask (by 50%-70%). These data suggest that the ability of TGF-beta 1 to modulate hematopoiesis may be due, in part, to its effects on glycosaminoglycan production.     

Cytomegalovirus production by infected astrocytes correlates with transforming growth factor-beta release

Cytomegalovirus (CMV) encephalitis is well documented in immunosuppressed persons, but its pathogenesis has received little investigative attention. The examination of brain tissue from 2 patients with acquired immunodeficiency syndrome who had CMV encephalitis showed colocalization of CMV inclusions and transforming growth factor (TGF)-beta in cells that contained astrocyte-specific glial filaments. To investigate the relationship between CMV and TGF-beta in the brain, an ex vivo murine model of CMV-infected astrocytes was established. Cultures of primary murine (strain FVB/N) astrocytes inoculated with murine (Smith strain) CMV expressed, over time, increasing amounts of infectious CMV in parallel with increasing levels of TGF-beta mRNA and peptide. Astrocyte release of CMV declined in the presence of antibody to TGF-beta and increased substantially after the addition of exogenous TGF-beta. These findings suggest that CMV infection of astrocytes induces the production of TGF-beta, which in turn enhances productive CMV expression.     

Regulation of steroid production in cultured porcine thecal cells by transforming growth factor-beta.

Evidence that transforming growth factor-beta (TGF beta) is produced by porcine thecal cells and acts upon porcine granulosa cells suggests that this peptide may be a local regulator of follicular function in this species. The objective of the present study was to investigate the effects of TGF beta on steroidogenesis in thecal cells from 4-6 mm follicles of prepubertal gilts. In this culture system, cells undergo functional luteinization such that production of androstenedione, the major steroid product in 24 h incubations, declines, and in the presence of luteinizing hormone (LH) (250 ng/ml) and insulin (1 micrograms/ml), progesterone production increases over a 3-day culture period. TGF beta (0.1-10 ng/ml) had no effect on production of androstenedione from endogenous precursors in the presence or absence of LH, although there was a slight inhibition of androstenedione production in the presence of exogenous progesterone (up to 23%). As the cells luteinized in culture, the increase in progesterone production in response to LH increased (day 1, 4.4-fold; day 3, 13-fold). TGF beta at concentrations as low as 0.1 ng/ml caused marked (up to 90%) inhibition of LH-stimulated progesterone production in day 3 cultures. In the presence of TGF beta (10 ng/ml), the response to LH was completely abolished, and the response to dibutyryl cAMP was considerably attenuated (25% of controls). Since the primary site of action of TGF beta appeared to be distal to cAMP formation, the effect of TGF beta on conversion of exogenous 22-hydroxy-cholesterol and pregnenolone to progesterone was determined in day 3 cultures. 22-Hydroxycholesterol and pregnenolone restored progesterone production to at least 80% and 89% of controls, respectively. While the primary inhibitory action of TGF beta appears to be exerted distal to cAMP formation, neither cholesterol sidechain cleavage nor the 3 beta-hydroxysteroid dehydrogenase: delta 5-delta 4 isomerase reactions are primary targets of this factor. Together with evidence of thecal production of TGF beta, the results of this study indicate that this peptide may be an autocrine regulator of thecal steroidogenesis.     

Hypoxia and transforming growth factor-beta 1 act independently to increase extracellular matrix production by placental fibroblasts

Villous fibrosis is associated with oxygen deprivation in placental pathology, but the signaling networks and growth factors involved in activating the relevant cellular repair mechanisms are largely unknown. TGF is a powerful enhancer of extracellular matrix (ECM) production and an important immune suppressor that has been linked with fibrosis in several tissues. Here, cell culture methods were used to investigate possible links between hypoxia, elevated TGF beta 1, and altered ECM production in placenta. Term placental fibroblasts were isolated and cultured under hypoxia (3% O(2)) or in the presence of TGF beta 1, and the expression of fibronectin, collagen I, and collagen IV was examined using immunohistochemistry, ELISA of cell monolayers with associated ECM, and real-time RT-PCR. The effect of hypoxia on endogenous production of TGF beta 1-3 was also examined. Both TGF beta 1 and hypoxia increased fibronectin, collagen I, and collagen IV protein and mRNA in placental fibroblasts. However, TGF beta 1-3 production was not increased by culturing the cells under hypoxic conditions for 5 d. Thus, increased ECM expression under hypoxia was not mediated directly by increased TGF beta. We conclude that ECM production can be stimulated independently by hypoxia and TGF beta 1.     

In situ expression of transforming growth factor-beta1-3, latent transforming growth factor-beta binding protein and tumor necrosis factor-alpha in liver tissue from patients with chronic hepatitis C

BACKGROUND: The mechanisms determining liver damage in chronic hepatitis C remain unclear. The aim was to evaluate the in situ expression of transforming growth factor beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha), two key cytokines implicated as important pathogenic mediators in the development of liver fibrosis. METHODS: In situ expression of TNF-alpha and of TGF-beta isoforms 1-3, and its transport protein latent TGF-beta binding protein (LTBP), was determined by immunohistochemistry in 9 untreated patients with chronic hepatitis C infection and in 6 controls without liver disease. In addition, TGF-beta1 expression was analyzed in 10 HCV patients before and after treatment with interferon-alpha alone, or in combination with ribavirin. RESULTS: Liver biopsies from HCV patients showed positive staining for TGF-beta1-3 isoforms and LTBP, and to a lesser degree for TNF-alpha, in areas with inflammation and fibrosis. Normal control liver showed no positive staining. TGF-beta1 expression before treatment, quantified by morphometric analysis, did not differ between non-responders and sustained responders. In patients responding to therapy, TGF-beta1 expression decreased in parallel with histological improvement, while no difference in TGF-beta1 expression was seen before and after treatment in non-responders. CONCLUSION: These results suggest that TNF-alpha and all three isoforms of TGF-beta are involved in the pathogenesis of HCV related liver disease, and that treatment leading to eradication of the virus affects the expression of TGF-beta1.     

Stimulation of collagen production by transforming growth factor-beta1 during differentiation of cardiac fibroblasts to myofibroblasts

The aim of the present study was to elucidate how transforming growth factor-beta(1) (TGF-beta(1)) can stimulate collagen deposition in cardiac tissue by interstitial cells via stimulation of fibroblasts, via myofibroblasts, or via differentiation of fibroblasts to myofibroblasts. The dose- and time-dependent stimulation of collagen production and of expression of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts, was studied in cultures of second-passage adult rat cardiac fibroblasts. The TGF-beta(1)-stimulated collagen production is positively correlated (r=0.68, P<0.001) with the appearance of alpha-SMA. Only at high concentrations (40 to 600 pmol/L) and after a long time (24 to 48 hours) of incubation, TGF-beta(1) increases the collagen production and stimulates the differentiation of fibroblasts to myofibroblasts. The maximal stimulation of the collagen production (2-fold, P<0.001) observed after incubation of cultures of fibroblasts with 600 pmol/L TGF-beta(1) for 48 hours is accompanied by a maximal stimulation of alpha-SMA expression (3.5-fold, P<0.001), when cultures consist mainly of myofibroblasts. The stimulation of collagen production cannot be reversed either after additional incubation of TGF-beta(1)-stimulated second-passage cultures for 2 days or in their offspring in the next third passage after incubation for 7 days without TGF-beta(1). The increased collagen production in these third-passage cultures cannot be further stimulated by TGF-beta(1). Our data suggest that TGF-beta(1)-stimulated collagen production in cultures of adult rat cardiac ventricular fibroblasts cannot be explained by a direct stimulation of the collagen production either in fibroblasts or in myofibroblasts. Instead, TGF-beta(1) induces the differentiation of fibroblasts to myofibroblasts, which have a higher activity for collagen production than fibroblasts.     

Effect of transforming growth factor-beta 1 on proliferation and death of rat prostatic cells

The ability of transforming growth factor B1 (TGF beta 1) to inhibit proliferation and activate death of rat ventral prostatic glandular cells was tested both in vivo and in vitro. In vivo administration of 50 ng TGF beta 1/day directly to the regressed ventral prostate of previously castrated male rats had no effect on the proliferative regrowth of the prostatic glandular cells induced by exogeneous androgen replacement. In addition, androgen-stimulated ventral prostatic cell proliferation in vitro in organ culture was not affected by exposure to 0.1-20 ng/ml TGF beta 1. In contrast in vivo administration of 50 ng TGF beta 1/day directly to the ventral prostate of intact noncastrated male rats resulted in the death of about 25% of the prostatic glandular cells within 7 days of treatment. Such TGF beta 1 treatment did not lower serum testosterone, nor did it affect the size or DNA content of the seminal vesicles, demonstrating the local nature of the response. Likewise, in androgen-maintained ventral prostate organ cultures in vitro, there was a dose-response relationship between glandular cell death and TGF beta 1 concentration in the medium. These results demonstrate that TGF beta 1 can induce the death of androgen-dependent prostatic glandular cells even when physiological levels of androgen are present. Previous studies have demonstrated that both the receptor and the mRNA for TGF beta 1 increase rapidly in the ventral prostate after castration. Taken with the present data, these results suggest that TGF beta 1 may be a physiological intermediate in the programmed cell death of rat prostatic glandular cells activated after androgen ablation.     

Postpartum thyroiditis is associated with fluctuations in transforming growth factor-beta1 serum levels

Postpartum thyroiditis (PPT) is characterized by a rapid evolution and recovery of euthyroidism. Therefore, it can represent a good model to study early cytokine fluctuations in autoimmune thyroid diseases. TGFbeta1 is an immunosuppressive cytokine, as it inhibits T and B cell proliferation, natural killer cell cytotoxic activity, and the generation of T cell cytotoxicity. The aim of this study was to assess serum concentrations of TGFbeta1 during pregnancy and to study possible serum fluctuations of this cytokine during the different phases of PPT. Thyroid biochemical pattern, antithyroid autoantibodies (ATA), and total and active TGFbeta1 (aTGFbeta1) serum concentrations were evaluated in 63 pregnant women. Thirty-four of them were ATA(+), and 29 were ATA(-). Twenty of the 34 ATA(+) women were followed in the postpartum year. Nine of these 20 women developed PPT; 11 remained euthyroid. All of the PPT women became euthyroid during the follow-up. Our results showed 1) detectable serum levels of aTGFbeta1 in 50% of ATA(+) pregnant women, suggesting that the presence of autoantibodies may characterize a favorable condition for TGFbeta1 activation; and 2) decreased total TGFbeta1 and increased aTGFbeta1 serum levels during the active phase of PPT in ATA(+) women. This seems to suggest that inflammation may be responsible for TGFbeta1 activation and autoantibody increase because of antigen release. Although further studies of women with persistent hypothyroidism after the postpartum year are needed, the possibility that the enhanced activation of TGFbeta1 may contribute to resolution of thyroid inflammation postpartum cannot be excluded.     

Early human thymocyte proliferation is regulated by an externally controlled autocrine transforming growth factor-beta 1 mechanism

Early thymocytes undergo extensive proliferation after their entry into the thymus, but cellular interactions and cytokines regulating this intrathymic step remain to be determined. We analyzed the effects of various T-cell growth factors and cellular interactions on in vitro proliferation of early CD2+CD3/TCR-CD4-CD8- (triple negative [TN]) human thymocytes. Freshly isolated TN cells were then assayed for their growth capacity after incubation with CD2I+III-monoclonal antibody (MoAb), recombinant human interleukin-2 (IL-2), IL-7, and/or IL-4. These cells displayed significant proliferative responses with IL-4, IL- 7, or CD2-MoAb+IL-2. The addition of recombinant transforming growth factor beta (TGF beta) or autologous irradiated CD3+CD8+CD4- cells to TN cell cultures dramatically decreased their growth responses to IL-2 and IL-7, whereas IL-4-induced proliferation was less sensitive to growth inhibition. We thus asked whether the CD8+ cell-derived inhibitory effect was due to TGF beta. The addition of neutralizing anti-TGF beta MoAb completely abolished CD8+ cell-derived inhibition of TN cell growth. Analysis of CD8+ cell-derived supernatants indicated that these cells had low TGF beta 1 production capacity, whereas TN cells secrete significantly high levels of TGF beta 1. Cell fixation studies showed that TN cells were the source of the TGF beta. TGF beta 1 released from TN cells was in the latent form that became the active inhibitory form through interaction of TN cells with CD8+ cells. Together, these data suggest a role for TGF beta 1 as an externally controlled, autocrine inhibitory factor for human early thymocytes, with a regulatory role in thymic T-cell output.     

Reduced transforming growth factor-beta1 production by mononuclear cells from patients with active chronic idiopathic thrombocytopenic purpura

Chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder in which activated T-helper (Th) cells and different Th-cell cytokines might play an important role. We have recently reported that chronic ITP patients in remission had elevated plasma levels of the Th3 cytokine transforming growth factor-beta1 (TGF-beta1), possibly as a part of a bystander immune suppression. In the present study we found that, in ITP patients with active disease [platelet count (plc) < 50 x 10(9)/l], mitogen-stimulated peripheral blood mononuclear cells (PBMC) had a significantly reduced production of TGF-beta1 (444 +/- 178 pg/ml; n = 6) compared with patients with plc 50-150 x 10(9)/l (1293 +/- 374 pg/ml; n = 9; P < 0.05), patients with plc >150 x 10(9)/l (1894 +/- 244 pg/ml; n =12; P <0.005) and healthy controls (1698 +/- 241 pg/ml; n = 10; P < 0.01). Nineteen per cent of ITP patients expressed a platelet-induced PBMC proliferation. Surprisingly, 22% of the ITP patients had a PBMC proliferation below the normal range, i.e. a suppressed proliferation in the presence of platelets; five of these six patients had active disease. In summary, this study demonstrated that chronic ITP patients with active disease had reduced PBMC production of the Th3 cytokine TGF-beta1. This result gives further support to the theory that chronic ITP in active phase is associated with a downregulated Th3-response.     

Direct regulating effects of transforming growth factor-beta 1 on lactate production in cultured porcine Sertoli cells.

In the present study we have tested the direct effects of transforming growth factor-beta 1 (TGF beta 1) on lactate production by Sertoli cells isolated from immature porcine testes. In Sertoli cells cultured in a defined medium, TGF beta 1 was shown to stimulate lactate production in a time- and dose-dependent manner. The maximal and half-maximal effects of TGF beta 1 on lactate production were obtained in the picomolar concentration range, respectively 24 and 8 pM TGF beta 1. TGF beta 1 action was found closely related to that of insulin since 1) both TGF beta 1 (40 pM) and insulin (1 microgram/ml) induced the secretion of similar and nonadditive amounts of lactate; and 2) TGF beta 1 and insulin induced comparable increases in lactate production in FSH (1 microgram/ml)-treated Sertoli cells. Because lactate is derived from glucose, 2-deoxy-D-glucose (2-DOG) was used to investigate the hexose transport system of Sertoli cells after insulin, FSH, and TGF beta 1 treatments. Insulin (1 microgram/ml) and FSH (1 microgram/ml) were found to stimulate 2-DOG transport with a similar time course, with an effect detected up to 30 min and maximal at 150 min. In contrast, although TGF beta 1 also enhanced 2-DOG uptake by Sertoli cells, the increase in glucose transport was delayed, since the TGF beta 1 effect was first detected at 150 min and was maximal at 360 min. These effects of TGF beta 1 action on Sertoli cell activity are exerted through specific membrane TGF beta 1 receptors. Scatchard analysis of the binding of TGF beta 1 to cultured Sertoli cells revealed the presence of both a high affinity (Kd, approximately 180 pM) and a low affinity binding site systems for TGF beta 1. Affinity labeling of these receptors by covalent attachment to [125I] TGF beta 1 with disuccinimidyl suberate and subsequent electrophoretic analysis of the labeled complexes revealed the specific binding of [125I] TGF beta 1 to three predominant molecules of 260, 130, and 70 kDa. In conclusion, the present study demonstrates that testicular Sertoli cells are targets for TGF beta 1 action. In view of the importance of lactate as a substrate for germ cells, it is suggested that TGF beta 1 might also be involved in the development of normal germinal epithelium.