Testis‐Specific Antigen (TSA‐1) is Expressed in Murine Sperm and its Antibodies inhibit Fertilization

Abstract

We recently cloned and sequenced a sperm-specific antigen, designated as testis-specific antigen-1 (TSA-1), from human testis. The present study was conducted to examine its expression and function in murine sperm, in order to find out whether or not the mouse can provide a suitable model for examining its immunocontraceptive effects. The antibodies (Ab) were raised against purified human rTSA-1 in virgin female rabbits. The rTSA-1 was run in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the gel containing the approximately 18 kDa band was cut, minced and used for immunization to obtain the specific Ab. The immunoglobulins from preimmune bleed and from animals injected with adjuvant alone served as control. These Ab were analysed in enzyme-linked immunosorbent assay (ELISA), Western blot procedure, immunoprecipitation procedure, immunocytochemical technique (ICT), immunobead binding technique (IBT), acrosome reaction and sperm-zona binding assay. Active immunization of female rabbits with purified rTSA-1 protein of 18 kDa, produced high titer Ab against the recombinant antigen. These Ab to rTSA-1 were used in the present study. In Western blot procedure, rTSA-1 Ab recognized a specific protein band of approximately 24 +/- 3 kDa in murine sperm extract, the band similar to found in human sperm extract. In the immunoprecipitation procedure, rTSA-1 Ab immunoprecipitated the protein band of similar size from extracts of murine sperm and murine testis. The ICT and the IBT studies revealed the subcellular localization of TSA-1 on the surface of acrosome and tail regions of the non-capacitated and capacitated murine sperm cells. In functional bioassays, rTSA-1 Ab inhibited the acrosome reaction and sperm-egg binding in vitro. These data indicate that the TSA-1 is expressed in murine sperm and may have a biological role in sperm function and sperm-egg binding. In vitro inhibition of capacitation/acrosome reaction and sperm-zona binding suggests that the mouse can provide a suitable model to examine the immunocontraceptive effects of TSA-1 in actively immunized animals.