A partial characterization of a Sertoli cell-secreted protein stimulating Leydig cell testosterone production.
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 x 10(6) cells/35 mm well and cultured in 3 ml serum free media supplemented with insulin (10 micrograms/ml). Sertoli cell culture medium (SCCM) collected every 3rd day was added to Leydig cells (10(6) cells in 1 ml of MEM with 2% steroid-free FCS) prepared from 10-week-old mice by mechanical separation and incubated for 3 h at 34 degrees 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 A and an ultrafiltration membrane with a molecular weight cut-off of 10 kDa. However, activity was reduced by heating at 60 degrees C for 30 min and almost lost after incubation with 0.1% trypsin for 1 h at 37 degrees 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 13 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
Murai
T
T
Department of Urology, Yokohama City University, School of Medicine, Japan.
Noguchi
K
K
Nagamoto
A
A
Hosaka
M
M
D000818:Animals; D001681:Biological Assay; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D007985:Leydig Cells / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000032:analysis; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012708:Sertoli Cells / Q000737:chemistry / Q000378:metabolism*; D013739:Testosterone / Q000096:biosynthesis*
D011506:Proteins; D013739:Testosterone; D007986:Luteinizing Hormone