title: The electrophoretic separation of spermatozoa: an analysis of genotype, surface carbohydrate composition and potential for capacitation.
abstract: This study examines the properties of an electrophoretic device designed to effect the rapid isolation of spermatozoa for assisted conception purposes. In light of previous reports suggesting that X- and Y-bearing spermatozoa can be separated in an electric field, the first characteristic examined was the sex chromosome status of electrophoretically isolated spermatozoa. Exploiting sex chromosome-specific differences in the structure of the amelogenin gene, a quantitative PCR protocol was designed that allowed the rapid genotyping of isolated sperm suspensions. Reassuringly, application of this procedure demonstrated that the electrophoretic method did not result in a significant skewing of the ratio of X- and Y-bearing spermatozoa. Analysis of the molecular basis for electrophoretic sperm isolation demonstrated that sperm suspensions prepared in this manner were enriched in surface sialic acid residues that bound the Sambucus nigra agglutinin (SNA) lectin. Western blot analyses demonstrated the presence of four major SNA binding proteins, three of which were identified by MALDI-Tof mass spectrometry as aminopeptidase B, fucosyltransferase and prostatic acid phosphatase. The ability of neuraminidase to significantly suppress the electrophoretic isolation of spermatozoa emphasized the causative nature of this association between cell surface sialation and sperm behaviour in an electric field. Finally, seminal plasma proteins possessing decapacitation properties were shown to co-migrate with spermatozoa during their electrophoresis, necessitating their removal prior to in vitro fertilization. In terms of function, electrophoretically isolated cells were found to capacitate normally, exhibiting high levels of tyrosine phosphorylation and a capacity for extensive binding to homologous zonae pellucidae. We conclude that the electrophoretic procedure rapidly isolates functional spermatozoa via mechanisms that are independent of their genotype but reliant upon a net electronegative charge that is largely conferred by sperm surface glycoproteins.
authors.affiliation: Discipline of Biological Sciences and Priority Research Centre in Reproductive Science, Faculty of Science and IT, University of Newcastle, University Drive, Callaghan NSW, Australia.
authors.forename: C J
authors.initials: CJ
authors.lastname: Ainsworth
authors.forename: B
authors.initials: B
authors.lastname: Nixon
authors.forename: R J
authors.initials: RJ
authors.lastname: Aitken
mesh_terms: D002469:Cell Separation / Q000379:methods*; D004586:Electrophoresis / Q000379:methods; D005838:Genotype; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D037121:Plant Lectins / Q000378:metabolism; D011485:Protein Binding; D054788:Ribosome Inactivating Proteins / Q000378:metabolism; D013075:Sperm Capacitation / Q000502:physiology*; D013081:Sperm Motility; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000737:chemistry / Q000166:cytology*; D015044:Zona Pellucida / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D037121:Plant Lectins; C050600:Sambucus nigra lectins; D054788:Ribosome Inactivating Proteins; D019158:N-Acetylneuraminic Acid