IDENTIFICATION AND PURIFICATION OF A HUMAN SERTOLI CELL-SECRETED PROTEIN (HSCSP-80) STIMULATING LEYDIG-CELL STEROID-BIOSYNTHESIS

Human Sertoli cells in vitro secrete a factor that stimulates steroid biosynthesis in purified human and rat Leydig cells as well as in the MA-10 mouse tumor Leydig cell line. MA-10 cells were used as a bioassay system to follow the characterization and purification of the active principle in the conditioned medium of human Sertoli cells. The Leydig cell stimulatory factor is a thermo-labile and trypsin-sensitive protein retained onto 10,000 mol wt (MW) cut-off filters. The following scheme was used to purify the active protein: concentration by ammonium sulfate (80%) precipitation, followed by dialysis using molecularporous membrane tubing of MW cut-off 12,000-14,000, heparin-agarose, Concanavalin-A-Sepharose, and immobilized reactive textile dye affinity chromatography. Yellow 86 and green 19 dyes immobilized on agarose matrix were used. This procedure resulted in the rapid (< 24-h) purification of a 79,000 +/- 6,082 (n = 3; under denaturating conditions) MW protein which stimulated Leydig cell steroid biosynthesis 25-fold at picomolar concentrations. The MW of the biologically active protein was further confirmed to be around 80,000 by gel filtration chromatography. This 80,000 MW human Sertoli cell-secreted protein (hSCSP-80) was shown to be different from human transferrin, human serum albumin, and rat testibumin. hSCSP-80, by modulating Leydig cell steroid biosynthesis, may play a significant role in the regulation of testicular function.