A testicular protein important for fertility has glutathione S-transferase activity and is localized extracellularly in the seminiferous tubules

Abstract

A 24-kDa protein isolated by preparative gel electrophoresis from rat testes was reported by us as an active immunogen in rats. Anti-24-kDa antibodies inhibited murine sperm-oocyte binding in vitro. Here, we show similarity at the NH2 terminus shared by this protein purified on Sephadex G-75 followed by anion exchange high performance liquid chromatography with glutathione S-transferase (GST)-μ subunits. This protein purified by glutathione affinity chromatography also demonstrated similarity to GST-μ NH2 terminus in a 30-amino-acid overlap. Both proteins showed activity toward the GST substrate 1-chloro-2,4-dinitrobenzene (Km of 33 μM and 50 μM) which was inhibited by 17β-estradiol 3-sulfate. Antisera against both proteins recognized liver GST-μ on Western blots and sperm acrosome of multiple species immunocytochemically. Both antisera significantly inhibited in vitro fertilization of goat oocytes by sperm preincubated with them while antiliver GST sera did not. GST activity was localized on rat sperm, seminiferous tubular fluid, and Sertoli cells. Seminiferous tubular fluid 24-kDa protein shared similarity to the NH2 terminus of GST-μ subunits in a 20-amino-acid overlap. Time-dependent accumulation of GST was detected in the spent culture medium of seminiferous tubules from rats of different ages suggesting secretion.