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 (K_m of 33 μ M and 50 μ M) 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 anti-liver 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.