Rat sperm galactosyl receptor: Purification and identification by polyclonal antibodies raised against multiple antigen peptides

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Abstract
We have previously reported the purification of rat testis galactosyl receptor, an equivalent to the Ca2+-dependent (C-type) minor variant of rat hepatic lectin-2/3 (RHL-2/3). We now report the purification of galactosyl receptor from rat sperm and its immunolocalization in the intact rat testis and sperm by polyclonal antibodies prepared using multiple antigen peptides (MAP) as immunogens. Two MAP antigens (designated 27-mer and 28-mer), corresponding to amino acid sequences of the carbohydrate-recognition domain (galactose) and adjacent Ca2+-binding sites of RHL-2/3, were used for immunization. Anti-RHL-2/3, anti-p27, and anti-p28 sera crossreacted with rat hepatocyte RHL-2/3 and its rat testis and sperm equivalent, galactosyl receptor, purified by chromatofocusing followed by galactose-Hydropore-EP affinity chromatography. Neither anti-p27 nor anti-p28 sera crossreacted with the major hepatocyte variant, RHL-1. A RHL-1-equivalent was not detected in rat testis and sperm. Immunofluorescence studies demonstrated that anti-p27 and anti-p28 sera recognize galactosyl receptor sites at the Sertoli cell-spermatogenic cell interface and on the dorsal surfacae of the sperm head, overlying the acrosome. The characteristic crescent-shaped immunoreactive pattern in sperm was lost after induction of the acrosome reaction. Further studies should determine whether antisera to MAP antigens 27-mer and 28-mer, corresponding to specific protein motifs, can serve as immunological probes for examining cell-cell interaction events during spermatogenesis and at fertilization.

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