Evidence that alpha 5 beta 1 integrins mediate Leydig cell binding to fibronectin and enhance Leydig cell proliferation stimulated by a Sertoli cell-secreted mitogenic factor in vitro

We reported previously that coculture of immature rat Sertoli cells with Leydig cells or the addition of a concentrate from Sertoli cell-conditioned medium (SCCM) stimulated Leydig cell [H-3]-thymidine incorporation, increased cell number, and altered Leydig cell morphology (Wu and Murono, 1994). In the present studies, the effect of various extracellular matrix proteins on immature Leydig cell binding, proliferation and response to SCCM concentrate was investigated. Pretreatment of culture wells with 50 mu g/mL collagen I or 10 mu g/mL laminin inhibited Leydig cell binding to culture wells about 95 and 89%, respectively; however, 5 mu g/mL fibronectin did not change the level of attachment. The binding of Leydig cells to fibronectin was reduced by antifibronectin or -beta 1 integrin antibodies (66 and 91%, respectively). Treatment of culture wells with five or 50 mu g/m L fibronectin alone increased [H-3]thymidine incorporation about twofold. When Leydig cells were cultured in wells precoated with increasing concentrations of fibronectin and then treated with SCCM concentrate for 2 d, [H-3]-thymidine incorporation increased progressively with the concentration of fibronectin, beyond the levels observed with SCCM concentrate alone. This response was associated with increases in both Leydig cell number and labeling indices. When Leydig cells were cultured on fibronectin, their numbers increased by 3.7- and 5.1-fold following treatment with SCCM concentrates or coculture for 6 d, respectively; whereas, they in creased 2.6- and 3.9-fold, respectively, when cultured on plastic. Labeling indices of Leydig cells cultured on plastic for 2 d and treated with SCCM or cocultured were 6.9 and 11.9%, respectively, while labeling indices of Leydig cells grown on fibronectin increased further to 17.6 and 26.3%, respectively. alpha 5 beta 1 integrin complexes and alpha 5 integrin mRNA were expressed in Leydig cells, suggesting that binding to fibronectin may be mediated by alpha 5 beta 1 integrins, a fibronectin receptor. These results suggest that Leydig cell proliferation stimulated by a Sertoli cell-secreted mitogenic factor(s) is enhanced by Leydig cell binding fibronectin, and that this binding may be mediated by alpha 5 beta 1 integrins.