Isolation and culture of Sertoli cells from the testes of adult Siberian hamsters: analysis of proteins synthesized and secreted by Sertoli cells cultured from hamsters raised in a long or a short photoperiod

Abstract

In the present study, we describe two procedures for isolation and culture of Sertoli cells from the testes of adult Siberian hamsters. In procedure I, collagenase and pancreatin were used for differential enzymatic digestion of the tissue at 32 degrees C, and effects of several attachment factors were examined. Sertoli cells isolated from adult hamsters were not affected by addition of Na selenite, epidermal growth factor, insulin, and transferrin, with or without 5% fetal bovine serum, to Dulbecco's modified Eagle's medium + Ham's F-12. Significant increase in the yield and plating efficiency of Sertoli cells was achieved by developing a novel procedure (procedure II) for Sertoli cell isolation. In this procedure, testicular tissue was exposed to only one enzyme (collagenase I) for two consecutive digestions at 37 degrees C, and the total period of enzymatic exposure was reduced relative to that of procedure I. Another objective of this study was to compare the functions of Sertoli cells isolated from spermatogenetically active and inactive adult testes. Isolated Sertoli cells from 60 +/- 5-day-old hamsters raised in long day conditions (LD, 16L:8D) or in short day conditions (SD, 6L:18D) were stimulated with FSH and testosterone in the presence of 35S-methionine for 24 h on Day 4 of culture. The medium was concentrated, and equal amounts of radioactive proteins from LD and SD hamster Sertoli cell cultures were analyzed by two-dimensional PAGE. Compared to Sertoli cells derived from SD hamsters, Sertoli cells from LD animals produced greater amounts of two secretory proteins and a smaller amount of one.