A Partial Characterization of a Sertoli Cell-Secreted Protein Stimulating Leydig Cell Testosterone Production
1992 年 39 巻 2 号 p. 209-215
詳細
1992 年 39 巻 2 号 p. 209-215
To examine whether immature rat Sertoli cells in culture secrete a factor (s) which stimulates testosterone production by mature mouse Leydig cells, Sertoli cell-enriched cultures were prepared from 3-week-old male rats with trypsin and collagenase.
Sertoli cells were plated at an initial density of 3-5×106 cells/35mm well and cultured in 3ml serum free media supplemented with insulin (10μg/ml). Sertoli cell culture medium (SCCM) collected every 3rd day was added to Leydig cells (106 cells in 1ml of MEM with 2% steroid-free FCS) prepared from 10-week-old mice by mechanical separation and incubated for 3h at 34°C. Secreted testosterone was determined by RIA. SCCM 15 times concentrated by Amicon YM10 membrane demonstrated a dose-dependent stimulation of testosterone production, whereas there was no effect on testosterone secretion when Leydig cells were maximally stimulated by LH. Leydig cell stimulating activity was retained by both a dialysis membrane with a pore size of 24 Å and an ultrafiltration membrane with a molecular weight cut-off of 10kDa. However, activity was reduced by heating at 60°C for 30min and almost lost after incubation with 0.1% trypsin for 1 h at 37°C. This activity was not retained by means of a Con A-Agarose column and was demonstrated only in break-through fractions. HPLC gel filtration of a 15 times concentrated SCCM preparation on a TSK gel G3000SW revealed Leydig cell-stimulating activity at approximately 13kDa. In conclusion, immature rat Sertoli cells in culture secrete a factor (s) which stimulates testosterone production from adult mouse Leydig cells. This factor (s) is a heat-sensitive protein with a molecular size around 13kDa.
