SEQUENCE AND LOCALIZATION OF THE MOUSE SPERM AUTOANTIGENIC PROTEIN, SP17

The present study characterizes the sperm protein Sp17 in the mouse. S p17 is a mammalian testis- and sperm-specific protein that has been is olated, sequenced, and characterized from rabbit testis and spermatozo a. In this study, a rabbit Sp17 cDNA probe representing the entire pro tein coding region was used to screen a mouse testis cDNA library to o btain the mouse Sp17 sequence. The mouse mRNA for Sp17 encodes a 149-a mino acid protein with a predicted molecular weight of 17 296. The mou se Sp17 (MSp17) cDNA sequence is 82% identical to, the rabbit Sp17 cDN A sequence while the MSp17 protein sequence is 74% identical to the ra bbit protein sequence. The presence of native Sp17 in mouse spermatozo a and testis was demonstrated by Western blot analysis, immunoprecipit ation, and immunolocalization. After SDS-PAGE, the native Sp17 has an apparent molecular mass of 24 kDa. The sequence of the native Sp17 was confirmed by Western blots of mouse testis and spermatozoa probed wit h two anti-peptide antibodies--one, anti-G22C, made against amino acid s 61-82 in the rabbit sequence (51-83 in the mouse), and a second, ant i-K18C, made against amino acids 120-136 in the C-terminal region in t he human sequence (118-134 in the mouse sequence). In the absence of p roteolytic inhibitors, part of the C-terminal of native MSp17 is cleav ed, giving rise to an 18-kDa band. Sp17 is present in spermatocytes an d spermatids in the testis. In spermatozoa, Sp17 is not available to b ind antibody on the surface of live, acrosome-intact spermatozoa, but it is present on the equatorial surface of live, acrosome-reacted sper matozoa. In fixed spermatozoa, staining is observed along the length o f the principal piece, weakly along the midpiece, and over the acrosom al region of the head. When the acrosome reaction begins, acrosomal st aining is seen throughout the equatorial region of the acrosome. Using mimotope analysis, this study has also demonstrated that native Sp17 is a sperm autoantigen and that recombinant mouse Sp17 is immunogenic in males with a highly restricted linear epitope.