We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

Previously, we cloned and sequenced a sperm specific antigen, designated as HSS (EMBL nomenclature human sperm associated antigen 9: hSPAG9) from human testis (Shankar et al.: Biochem Biophys Res Commun 243:561-565, 1998). The present study was conducted to isolate baboon proteomic homologue in order to find out whether the baboon can provide a suitable model for examining its immunocontraception effects. Baboon SPAG9 (bSPAG9) was cloned and sequenced from the baboon testis cDNA library. The baboon cDNA contained open reading frame encoding 760 amino acids. A 90.6 and 96.8% homology between baboon and human SPAG9 was found at protein and DNA levels. Analysis for tissue specificity by Northern blot procedure using various baboon tissues indicated that bSPAG9 was specifically expressed only in the baboon testis. Further, cell type expression analysis by in situ hybridization in baboon testis demonstrated the expression of bSPAG9 mRNA transcript only in the round spermatid suggesting haploid germ cell expression. Anti-human SPAG9 antibodies recognized the acrosomal compartment region of baboon sperm in indirect immunofluorescence (IIF). Flow cytometry analysis showed surface localization of bSPAG9 in live baboon sperm. The amino acid sequence data for nonhuman primate SPAG9 suggest that antibodies generated by vaccinating baboon with hSPAG9 will recognize nonhuman primate SPAG9, supporting the testing of SPAG9 contraceptive vaccine based on hSPAG9 in the nonhuman primate model.

The present study was conducted to isolate macaque proteomic homologue of human SPAG9 (EMBL nomenclature human sperm associated antigen 9: hSPAG9; Shankar et al., 1998: Biochem Biophys Res Commun 243:561-565) in order to find out whether the macaque can provide a suitable model for examining its immunocontraception effects. Macaque SPAG9 was cloned and sequenced from the macaque testis cDNA library. The macaque cDNA contained open reading frame encoding 712 amino acids. A 84.9% and 94% homology between macaque and human SPAG9 was found at protein and DNA levels. Northern analysis and RNA in situ hybridization experiments revealed testis- and stage-specific expressions of macaque SPAG9 mRNA, mainly confined to round spermatid suggesting haploid germ cell expression. Anti-human SPAG9 antibodies recognized native SPAG9 in macaque sperm extract in Western blotting and the acrosomal compartment region of macaque sperm in indirect immunofluorescence. Flow cytometry analysis further revealed surface localization of macaque SPAG9 in live macaque sperm. The amino acid sequence data for nonhuman primate SPAG9 suggest that antibodies generated by vaccinating macaque with hSPAG9 will recognize nonhuman primate SPAG9, supporting the testing of SPAG9 contraceptive vaccine based on hSPAG9 in the nonhuman primate model.

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Problem:  It has been well documented that antisperm antibodies can be causative factors for infertility. In this report we have identified a protein on human sperm referred as human sperm‐associated protein (HSAP) using serum of an immunoinfertile woman; it is thus a sperm‐specific protein – a candidate molecule for control of fertility. Method of study:  An immunoinfertile woman serum showing head–head sperm agglutination and acrosomal localization, reacted with human sperm protein of apparent molecular weight of 48 kDa on Western blot. Anti‐48 kDa antiserum was raised in rabbit by eluting 48 kDa protein and was used to screen the human testis cDNA expression library. A putative positive hsap cDNA clone was obtained, sequenced and subjected to tissue specificities studies by Northern blotting. The cell type‐specific expression was done using in situ RNA hybridization studies. To obtain recombinant HSAP (r‐HSAP), hsap cDNA was cloned in pET 22b(+) expression vector. r‐HSAP was expressed as polyhistidine fusion protein in Escherichia coli and purified. Rabbits were immunized with the purified r‐HSAP, which led to generation of antibodies. In order to evaluate in vitro immunocontraceptive potential, the anti‐r‐HSAP antibodies were characterized by agglutination assay, zona‐free hamster egg penetration assay, indirect immunofluorescence (IIF) assay, and by flow cytometry analysis. Results:  We have cloned a human testis gene encoding a protein (HSAP) of 328 amino acids. Antibodies against the purified recombinant protein specifically recognized approximately 40 kDa r‐HSAP, and a cognate 48 kDa protein band in human sperm extract in Western blot procedure. The anti‐r‐HSAP antibodies localized acrosomal compartment, inhibited sperm binding/attachment in zona‐free hamster penetration assay and revealed surface binding with human live sperm by flow cytometry. The cDNA sequence has been submitted to EMBL and has been given the accession number Y16676. Conclusion:  This study has put in evidence that novel sperm‐specific r‐HSAP has role in sperm function and may have application in the development of a contraceptive vaccine. The availability of the recombinant protein will facilitate studies on the assessment of its potential as a contraceptive immunogen.