Comparison and Characterization of Specific Fertilization Proteins in Human (Homo Sapiens) and Baboon (Papio Anubis) Spermatozoa

Abstract

Previous studies have shown presence of shared antigens on baboon and human spermatozoa and significant homology between some of the testicular and spermatozoa! antigens in the baboon and man. The baboon therefore, has been considered a possible non-human primate model for the study of human male reproduction. The present studies have investigated fertilisation proteins in the two species. These fertilisation proteins were localized on spermatozoa by immunofluorescence using a commercial monoclonal antibody (mAb) and monoclonal antibodies previously raised against specific antigens on the heads of rodent or human spermatozoa. Among the monoclonal antibodies used were mAb Ml (Noor and Moore, 1999), mAb 18.6, (Moore et al., 1990), mAb IAM-1 and mAb ES-1 (Al Eisa et al., 2001) and mAb 4G10 that was a commercial antibody. Biochemical characterisation was done using the same monoclonal antibodies and involved separation and identification of sperm proteins using one-dimensional gels and immunoblotting. The study then focused on characterisation of one of the fertilisation proteins, inner acrosomal membrane -1 (IAM-1). This entailed the use of two-dimensional gel electrophoresis, immunoblotting, end terminal sequencing, peptide mass spectrometry and partial amino acid sequencing to characterise the protein. This study identified shared determinants on baboon and human spermatozoa using mAbs 18.6, 4G10, LAM-1 and ES-1. These were VI immunolocalised to similar domains on spermatozoa in the two species. One dimensional immunoblots corroborated the immunolocalisation studies demonstrating similarity of determinants recognised by the same mAbs in the two species. Some determinants like Ml were found in rodents but not in the two primates. ES-1 was localized by immunofluorescence to the equatorial segment and tail in both human and baboon spermatozoa. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis of human sperm protein extracts and immunoblotting using mAb ES-1, revealed immunoreactive bands of apparent molecular weights, 24, 28, 40, 47 and 50 kDa, while in baboon sperm extracts immunoreactive bands were recognised at 34, 38, 47, and 50 kDa. Characterisation of IAM-1 using one dimensional immunoblots revealed it was a protein with four immunoreactive bands of apparent molecular weights 28, 32, 39 and 45 kDa in man while in the baboon, 3 immunoreactive bands of apparent molecular weights, 32, 38 and 44kDa were identified. Two- dimensional gel electrophoresis and immunoblotting revealed IAM-1 has native pi’s of between 3.86 - 4.0. Immunofluorescence localized this antigen to the anterior acrosome and equatorial region of both human and baboon spermatozoa. Treatment with different extraction solutions revealed that this protein was an integral membrane protein or a lipid-anchored protein intimately attached to the inner acrosomal membrane and equatorial segment of human spermatozoa. Mass spectrometry and amino acid sequencing isolated several peptides and the overall picture obtained suggested IAM-1 was end-terminally blocked, non- VII glycosylated and a trypsin-like protease. Taken together, immunological and biochemical data obtained suggested that the protein IAM-1 could be a membrane anchored trypsin-like protease most likely involved in sperm-zona penetration The ultimate goal of this study was identification of suitable gamete- specific proteins integrally involved in the fertilisation process, which can be used for immunocontraception. Further work remains to be done to confirm the suitability of the IAM-1 as a possible candidate protein for testing. This work provides additional evidence for use of the baboon as a model for study of human reproduction.