Identification of hormonally responsive proteins in primary Sertoli cell culture medium by anion-exchange high...

Twenty day-old rats were used to prepare Sertoli cell-enriched cultures which were grown for 4 days in serum-free medium supplemented with human transferrin, epidermal growth factor, and insulin. Proteins in the medium were first fractionated by anion-exchange HPLC. Forty-five to 50 fractions were collected, and each was examined by electrophoresis in sodium dodecyl sulfate-containing polyacrylamide gels. Using silver nitrate as a stain, more than 30 proteins were routinely identified. Rat androgen binding protein and rat transferrin were measured by immunoassays. When the cultures were supplemented with either testosterone, FSH, or both hormones and the media fractionated by HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these proteins could be tentatively categorized as follows: unresponsive to added hormones; responsive primarily to testosterone; responsive primarily to FSH; or responsive to testosterone or FSH but maximally responsive to both hormones. This report provides a method for identifying proteins in Sertoli cell-enriched cultures and has allowed us to show for the first time that many are responsive to hormones. The procedure takes advantage of the large loading capacity of the HPLC column to allow fractionation of liter quantities of medium. The high resolving power of the column permits preparation of fractions which are highly enriched with one or a limited number of proteins. The purification of many of these proteins is now feasible. The approach described is generally applicable for isolation of proteins from media from a variety of cells.