RAT SPERM GALACTOSYL RECEPTOR - PURIFICATION AND IDENTIFICATION BY POLYCLONAL ANTIBODIES RAISED AGAINST MULTIPLE ANTIGEN PEPTIDES

We have previously reported the purification of rat testis galactosyl receptor, an equivalent to the Ca2+-dependent (C-type) minor variant o f rat hepatic lectin-2/3 (RHL-2/3). We now report the purification of galactosyl receptor from rat sperm and its immunolocalization in the i ntact rat testis and sperm by polyclonal antibodies prepared using mul tiple antigen peptides (MAP) as immunogens. Two MAP antigens (designat ed 27-mer and 28-mer), corresponding to amino acid sequences of the ca rbohydrate-recognition domain (galactose) and adjacent Ca2+-binding si tes of RHL-2/3, were used for immunization. Anti-RHL-2/3, anti-p27, an d anti-p28 sera crossreacted with rat hepatocyte RHL-2/3 and its rat t estis and sperm equivalent, galactosyl receptor, purified by chromatof ocusing followed by galactose-Hydropore-EP affinity chromatography. Ne ither anti-p27 nor anti-p28 sera crossreacted with the major hepatocyt e variant, RHL-1. A RHL-1-equivalent was not detected in rat testis an d sperm. Immunofluorescence studies demonstrated that anti-p27 and ant i-p28 sera recognize galactosyl receptor sites at the Sertoli cell-spe rmatogenic cell interface and on the dorsal surface of the sperm head, overlying the acrosome. The characteristic crescent-shaped immunoreac tive pattern in sperm was lost after induction of the acrosome reactio n. Further studies should determine whether antisera to MAP antigens 2 7-mer and 28-mer, corresponding to specific protein motifs, can serve as immunological probes for examining cell-cell interaction events dur ing spermatogenesis and at fertilization. (C) 1995 Wiley-Liss, Inc.