Pachytene spermatocytes in co-culture inhibit rat Sertoli cell synthesis of inhibin βB-subunit and inhibin B but not the inhibin α-subunit

Abstract

This study investigates the effects of spermatogenic germ cells on inhibin α-subunit and βB-subunit expression, and inhibin α-subunit and inhibin B production by rat Sertoli cells in vitro. Sertoli cells isolated from 19-day-old rats were cultured for 48 h at 32 °C, in the presence or absence of FSH (2.3-2350 mIU/ml), and in the presence of pachytene spermatocytes, round spermatids or cytoplasts of elongated spermatids purified from adult rat testis by elutriation and density gradient separation. Sertoli cell secretion of inhibin α-subunit and inhibin B, as measured by immunoassay, was dose-dependently stimulated by FSH (maximal stimulation 13- and 2-fold, respectively). Round spermatids or cytoplasts co-cultured with Sertoli cells had no effect on basal or FSH-induced secretion of inhibin α-subunit or inhibin B. When Sertoli cells were co-cultured with pachytene spermatocytes, inhibin α-subunit secretion was unaltered, while inhibin B secretion was suppressed in a cell concentration-dependent manner to reach a maximal suppression of 45% compared with Sertoli cells alone (P<0.01). A similar suppression in inhibin B was still observed (64% of Sertoli cells alone) when the pachytene spermatocytes were separated from Sertoli cells by a 0.45 μm pore membrane barrier in bicameral chambers. Pachytene spermatocytes also suppressed FSH-induced inhibin B levels in Sertoli cell co-cultures and this suppression was attributed to a decrease in basal inhibin B production rather than a change in FSH responsiveness. Quantitation of Sertoli cell inhibin α- and βB-subunit mRNA by quantitative (real-time) PCR demonstrated that pachytene spermatocytes did not alter Sertoli cell α-subunit mRNA expression, but significantly (P<0.01) suppressed basal and FSH-induced βB-subunit mRNA expression to a similar degree to that seen with inhibin B protein levels. It is concluded that pachytene spermatocytes in vitro suppress Sertoli cell inhibin B secretion via factor-mediated suppression of inhibin βB-subunit expression. These findings support the hypothesis that specific germ cell types can influence inhibin B secretion by the testis independent of FSH regulation.