14 IGF-I AND IGF-II IMMUNOREACTIVITY IN CONDITIONED MEDIA FROM PRIMARYY CULTURES OF RAT SERTOLI CELIS

Abstract

IFG-I production by rat Sertoli cells has been reported, but evidences of IGF-II production are lacking. Trying to prove it, we measured IGF-II (as well as IGF-I) immunoreactivity in conditioned media from primary cultures of Sertoli cells from 8 day-old rats. After isolation the cells were cultured for 24 hrs in a 1: 1 mixture of mediums F12: DME with 10 ug/ml transferrin, 5 ug/ml insulin, 5 ug/ml vitamin E, 4 ug/ml hidrocortisone, 100 U/ml penicillin, 2.5 ug/ml amphotericin B. The cells were then cultured for 48 hs in medium without insulin and immediately afterwards the conditioned media and the cells were collected and analyzed separately. The RIAs employed human recombinant IGF-I and IGF-II for standard and tracer, a polyclonal anti IGF-I antibody and a monoclonal anti IGF-II antibody. To avoid interference by IGF binding proteins previous to RIAs the conditioned media were extracted with a mixture of formic acid-Tween 20-acetone. With the same purpose 8ng/tube IGF-I was added to the IGF-II RIA buffer. Recovery of added 1251 IGF-II after the extraction procedure was 101.2±3.9 % (X ± SD, n=4). Both IGFs were present in the conditioned media. Serial dilutions of the wxtracted media were parallel to the standard curves. IGF-I ommunoreactivity in the condiotioned media was 84±20 pg/ug DNA while IGF-II immunoreactivity was 802±209 pg/ug DNA(X-SEM, n=4). Our results tend to confirme IGF-II production by rat Sertoli cells.