Methods of analysis of sperm antigens related to fertility

Abstract

Progress in the identification of novel sperm antigens related to fertility, particularly, immune infertility, has been made in recent years employing advanced proteomic analyses, mass spectrometry microsequencing and recombinant DNA technology. This chapter summarizes several approaches employed to identify and characterize previously unidentified sperm antigens and their encoding genes that are relevant to fertility and/or infertility, focusing on strategies that have the potential to yield candidate protein targets for immunocontraceptive or drug development programs. Methods aimed to enrich, isolate, and identify the relevant antigens include vectorial labeling, probing with antisperm antibodies (ASA) or monoclonal antibodies, subcellular fractionation, differential extraction, treatment of sperm to isolate and identify glycosylphosphatidylinositol (GPI)-anchored proteins, identification of glycoproteins by lectin blotting, cDNA library screening and PCR amplification. Several techniques employ two-dimensional (2D) gel electrophoresis to first separate and visualize immuno-reactive protein spots followed by mass spectrometry to sequence and identify the protein to obtain sequence information which can then be used to clone the corresponding gene. The recombinant proteins can be expressed in a variety of expression vectors and host systems, specific immunoreagents may be developed to the novel proteins and subsequent functional characterizations can lead to definition of the subcellular localization, definition of biological roles in various sperm functions, fertility, and validation of contraceptive potential. © 2009 Springer-Verlag Berlin Heidelberg.