Germ cell-conditioned medium contains multiple factors that modulate the secretion of testins, clusterin, and transferrin by Sertoli cells.

Abstract

In view of the evidence showing that germ cells regulate Sertoli cell (SC) function, the aim of this study was to examine if germ cell (GC)-conditioned media contained multiple biological factors that affect SC secretory functions. Total GC were isolated from adult rat testes. Pachytene spermatocytes (SPC) and early spermatids (SPT) were enriched to about 90% pure by centrifugal elutriation. GC, SPC, and SPT were cultured in serum-free medium for 20 hours with a viability greater than 95%. Conditioned media derived from these cells were fractionated by anion-exchange high-performance liquid chromatography (HPLC). An aliquot from each of these fractions was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were visualized by silver staining. The patterns of protein staining using media from GC, SPC, and SPT were similar. Bioassays of these column fractions on SC showed that the transferrin stimulatory, the testins inhibitory, and the clusterin inhibitory activities were eluted from the anion-exchange HPLC column in overlapping fractions. To determine whether these activities were confined to one or several molecules, further fractionations were performed. Eleven liters of GC-conditioned medium were fractionated by sequential HPLC using anion-exchange, gel permeation, and reversed-phase columns. Throughout the entire fractionation scheme, the HPLC fractions were bioassayed using primary SC-enriched cultures prepared from 20-day-old rats by incubating SC with aliquots of these fractions for 24 hours to monitor their effects on SC secretory function. The concentrations of transferrin, clusterin, and testins were quantified by specific radioimmunoassays. These studies showed that the transferrin stimulatory activity can be fractionated into four peaks (I, IIa, IIb, and IIc); clusterin inhibitory activity into three peaks (A, B, and C); and testins inhibitory activity into two peaks (1 and 2). Some of these bioactivities were eluted in overlapping fractions such as I and B, IIb and 1, and IIc and 2, whereas A, C, and IIa were not associated with any other assayed activities. In summary, additional GC modulators of SC function were identified for the first time, these include clusterin and testins inhibitors. The previously identified transferrin stimulatory activity was also resolved into multiple molecular forms.