Secretion of testicular transferrin by cultured Sertoli cells is regulated by hormones and retinoids

Abstract

We have previously reported that Sertoli cells in culture secrete a transferrin-like protein. The purpose of this study was to determine to what extent hormones such as follicle-stimulating hormone (FSH), insulin and testosterone, and vitamin A can regulate the amount of testicular transferrin secreted by cultured Sertoli cells. Sertoli cell culture medium was collected every 48 hr for the duration of culture and the amount of testicular transferrin in the medium was quantitated with a radioimmunoassay. The maximum amount of transferrin was detected in cell cultures which had been maintained in medium supplemented with FSH, insulin, retinol, and testosterone. During the first 48 h of culture these cells secreted 65±10 ng transferrin/105 cells compared to control cultures which secreted 56±8 ng transferrin/105 cells. In the 48 h collection ending on Day 6 of culture, the amount of transferrin secreted into the medium had risen to 164±16 ng transferrin/105 cells in treated cultures compared to 25±5 ng transferrin/105 cells in control cultures. It was found that the secretion of transferrin was regulated by the interaction of insulin with FSH, testosterone and retinol. When insulin was present in the medium, the cells, if untreated or treated with FSH or testosterone, secreted nearly twice as much transferrin as when the insulin was deleted. Both retinol and retinoic acid stimulated the secretion of transferrin by cultures maintained in medium which contained insulin. The secreted proteins were labeled with [35S]methionine and analyzed by two-dimensional gel electrophoresis and fluorography. Untreated Sertoli cell cultures secreted nearly undetectable amounts of [35S]methionine-labeled transferrin while cultures treated with FSH, insulin, testosterone and retinol secreted a greatly increased amount of radioactive transferrin. Transferrin secretion was also stimulated when calf serum was added to the medium. It was found that as the concentration of serum was increased, the relative response of the cells to FSH, insulin, testosterone, and retinol decreased. As the percentage of calf serum was increased to 10%, the amount of secreted transferrin in the medium approached a constant level which was equivalent to that amount of transferrin secreted by serum-free cultures which were maintained in FSH, insulin, testosterone and retinol. Sertoli cell cultures from 10, 20 and 60-day-old rats all secreted transferrin. No detectable transferrin was secreted by cultured peritubular fibroblasts. These results demonstrate that the secretion of transferrin by Sertoli cells in culture is regulated by a complex interaction of hormones, vitamin A and serum factors.