O-5: Identification of Novel ImmunodominantEpididymal Sperm Proteins Using CombinatorialApproach

Background: Alteration in the protein signatures of functionally immature testicular spermatozoa occurs during their journey through the epididymis. This leads to acquisition of sperm domain specific functions essential for successful fertilization. Epididymal sperm proteins are preferred targets for immunocontraception as well as in elucidating the causes of infertility. The Background of the study was to identify epididymis-specific proteins in different domains of the sperm which could be exploited as putative targets for the development of post testicular male contraceptives. Materials and Methods: We used a novel combinatorial approach comprising of modified subtractive immunization (SI), followed by proteomics. Neonatal mice were tolerized on day 0 and day 5 with testicular proteins and immunized in adulthood with proteins of the epididymal sperm domains, i.e. intact and soluble fractions of head and flagella (HI/ HS and FI/ FS). Post immune sera (PI) were characterized by ELISA, Indirect immunofluorescence (IIF), Immunohistochemistry and western blotting. Immunoreactive proteins were sequenced using mass spectrometry. Validation of novel proteins wasperformed by peptide ELISA. Results: ELISA indicates that the animals were tolerized to testicular proteins and upon challenge with the immunogen showed immune response specifically to epididymal proteins. IIF results indicate that post immune sera localized antigens specifically in the sperm regions with which they were immunized. Immunohistochemistry, using PI sera of all the four groups demonstrated intense reactivity starting from distal caput with three of the four sera (HI, HS and FI) and proximal caput with the serum of the fourth group (FS). Post immune sera were used for immunoproteomics which led to identification of 30 proteins, of which four proteins namely Sperm Head protein 1(SHP1) (Accession no. P85300), Sperm Flagella protein 2 (SFP2) (Accession no. P85301), Sperm Flagella protein 3 (SFP3) (Accession no. Q5BK63) and Sperm Flagella protein 4 (SFP4) (Accession no. Q06647) are being reported for the first time on sperm. Another group of four proteins namely Collagen alpha-2 (I) chain precursor, Homeodomain interacting protein kinase1, GTP binding protein Rab1 and Ubiquinol cytochrome c reductase core protein ΙΙ although reported earlier in testis are being reported for the first time in epididymal sperm. The possibility that the proteomics data represented abundant comigrating proteins instead of the immunoreactive group of proteins was ruled out by validation of the seven out of the eight novel proteins by peptide ELISA.Conclusion: The unique strategy helped in identifying number of new epididymal proteins on distinctly different domains of the sperm predominantly on the flagella. These proteins are immunogenic in nature, conserved in rodents. These data are a useful repository which could be exploited to develop targets for post testicular immunocontraception or biomarkers for infertility diagnosis and management.