Regulation of the synthesis and secretion of transferrin and cyclic protein-2/cathepsin L by mature rat Sertoli cells in culture

While numerous studies have examined the response of immature rat Sertoli cells to specific hormones and growth factors, the regulation of mature cells in vitro has not been well examined because highly purified cells have been difficult to isolate. We now describe a detailed method for isolating Sertoli cells from mature (> 60 days of age) rats and generating primary cultures of these cells greater than 90% in purity. We demonstrate that cell density, hormones, and growth factors regulate the synthesis or secretion of two Sertoli cell products, transferrin and Cyclic Protein-2 (CP-2)/cathepsin L. Cell density modulated the response of mature Sertoli cells to some hormones; insulin (at 10 μg/ml) and epidermal growth factor (EGF) acted synergistically to stimulate transferrin synthesis by 80% when cells were cultured at a density of 1.65 x 10⁵ cells/cm² but had no effect on transferrin synthesis by cells cultured at 1.46 x 10⁵ cells/cm². A mixture of FSH, retinol, and testosterone increased transferrin synthesis by 30% at both cell densities, and this stimulation was independent of the effect of EGF and insulin. CP-2/cathepsin L synthesis was significantly stimulated by increased cell density. FSH, retinol, and testosterone also stimulated CP- 2/cathepsin L synthesis by 30%; however, this stimulation just missed being statistically significant. Finally, we demonstrated that secretion of transferrin and CP-2 was reduced when cells were cultured in the presence of interleukin-1α, a cytokine synthesized by Sertoli cells. In summary, these data demonstrate differences in the regulation of the synthesis of transferrin and CP-2/cathepsin L by mature Sertoli cells; they also document that EGF plus insulin at high concentration substantially stimulated transferrin, but not CP-2 synthesis, and suggest that interleukin-1α may act in an autocrine manner to regulate Sertoli cell function.