title: Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding.
abstract: The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.
authors: Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.|Department of Biochemistry/Microbiology/Immunology, University of Ottawa, Ontario, Canada.
authors: Kessiri
authors: K
authors: Kongmanas
authors: Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.|Department of Anatomy, Faculty of Medical Sciences, Naresuan University, Phitsanulok, Thailand.
authors: Hathairat
authors: H
authors: Kruevaisayawan
authors: Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.|Department of Biochemistry/Microbiology/Immunology, University of Ottawa, Ontario, Canada.
authors: Arpornrad
authors: A
authors: Saewu
authors: Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.|Department of Biochemistry/Microbiology/Immunology, University of Ottawa, Ontario, Canada.
authors: Clarissa
authors: C
authors: Sugeng
authors: Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.|Department of Biochemistry/Microbiology/Immunology, University of Ottawa, Ontario, Canada.
authors: Jason
authors: J
authors: Fernandes
authors: Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, California.
authors: Puneet
authors: P
authors: Souda
authors: Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.|Department of Biochemistry/Microbiology/Immunology, University of Ottawa, Ontario, Canada.|Division of Infectious Diseases, Ottawa Hospital-General Campus, Ottawa, Ontario, Canada.
authors: Jonathan B
authors: JB
authors: Angel
authors: Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, California.
authors: Kym F
authors: KF
authors: Faull
authors: The ARC Centre of Excellence in Biotechnology and Development, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia.
authors: R John
authors: RJ
authors: Aitken
authors: Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, California.
authors: Julian
authors: J
authors: Whitelegge
authors: Department of Cell Biology and Biochemistry, Health Sciences Center, Texas Tech University, Texas.
authors: Daniel
authors: D
authors: Hardy
authors: Department of Animal Science, University of California, Davis, California.
authors: Trish
authors: T
authors: Berger
authors: The ARC Centre of Excellence in Biotechnology and Development, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia.
authors: Mark A
authors: MA
authors: Baker
authors: Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.|Department of Biochemistry/Microbiology/Immunology, University of Ottawa, Ontario, Canada.|Department of Obstetrics and Gynaecology, University of Ottawa, Ontario, Canada.
authors: Nongnuj
authors: N
authors: Tanphaichitr
mesh_terms: D000177:Acrosome / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D047468:Immunoprecipitation / Q000379:methods; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D011956:Receptors, Cell Surface; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000378:metabolism*; D013552:Swine; D015044:Zona Pellucida / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface