Identification of a New, Testis-Specific Sperm Antigen Localized on the Principal Piece of the Spermatozoa Tail in the Fox (Vulpes vulpes)1

Abstract

Fox (Vulpes vulpes) sperm antigens were identified to assess them as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP13, a fox sperm protein of 97 kDa. The fSP13 protein was both auto- and iso-antigenic in foxes; it was recognized by sera of foxes immunized with fox sperm proteins and vasectomized foxes. The NH2-terminal sequence of fSP13 was determined, and a piece of cDNA was amplified from testicular RNA by reverse transcription polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 1662 base pairs was obtained, including a major open reading frame coding for 498 amino acid. Mass spectrometry analysis confirmed the position of the open reading frame and the presence of posttranscriptional modifications. Analysis of the predicted amino acid sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated the presence of four potential N-linked glycosylation sites. The fSP13 bears the closest amino acid similarity to two human sperm proteins: fibrousheathin 2 and testis-specific calcium binding protein 86-VII. The deduced 80 N-terminal amino acid sequence also presents similarity with the RIIalpha domain. By using a serum against fSP13, this antigen was localized on the principal piece of the fox spermatozoa. Northern blot analysis showed that fSP13 is specifically expressed in testis. The fSP13 is one of the first fox sperm antigens to be cloned and sequenced.