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Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding

Significance statement

Mammalian sperm must undergo capacitation in order to fertilize an egg.  During capacitation, sperm gain a full ability to bind to the zona pellucida (ZP), the extracellular glycoprotein matrix surrounding the egg.  Capacitation occurs naturally when sperm swim through the female reproductive tract and can also be induced in vitro by incubating sperm in an albumin-containing medium.  This process involves modification and reorganization of sperm surface molecules, including proteins with inherent ZP affinity.  Recent studies suggest that many ZP-binding molecules likely act together as a complex during sperm-egg interaction. Some of these proteins are originally present on the sperm head plasma membrane of non-capacitated sperm. However, a number of them are known to localize to the acrosome – the membrane enveloped cap-like structure underneath the sperm anterior head plasma membrane (APM) where the initial sperm-ZP binding takes place.  How these proteins in different compartments work together during sperm-egg interactions still remains unclear.  The old dogma in the sperm-egg interaction field states that only capacitated sperm with intact acrosome bind to the ZP; acrosomal exocytosis then occurs on the ZP to release acrosomal proteins/enzymes essential for ZP interaction and digestion (to create paths of sperm to migrate further through the ZP layer).  However, a more recent result from the high performance videomicroscopy experiment1 indicates that acrosomal exocytosis already initiates during sperm migration through the cumulus cell layers that encompass the ZP.  Determining changes of protein profiles, especially those on the sperm anterior head plasma membrane, during capacitation would give a better understanding of how different sperm proteins act together in sperm-ZP interactions.  In this study, we used nitrogen cavitation (650 psi) to specifically isolate sperm anterior head plasma membrane as membrane vesicles from pig sperm before and after capacitation.  The sperm anterior head plasma membrane vesicle proteins in the high-molecular-weight (HMW) protein complexes that show ZP affinity were then further characterized by mass spectrometry.  Our proteomic characterization revealed that a number of acrosomal proteins, including zonadhesin and proacrosin/acrosin, were present with increased levels in HMW protein complexes of capacitated sperm sperm anterior head plasma membrane vesicles as compared with those of the non-capacitated sperm samples.  The trafficking of zonadhesin and other acrosomal proteins was further demonstrated at the intact sperm level by immunofluorescence and flow cytometry.  In summary, our results provided compelling evidence that trafficking of acrosomal proteins (especially those with ZP affinity) to the anterior head surface of sperm indeed occurs during capacitation and acrosomal proteins also play roles in the initial phase of sperm-ZP interaction.

1Jin, M., Fujiwara, E., Kakiuchi, Y., Okabe, M., Satouh, Y., Baba, S.A., Chiba, K., and Hirohashi, N. (2011). Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization. Proceedings of the National Academy of Sciences of the United States of America 108, 4892-4896.

Figure legend

 Immunofluorescence and flow cytometry showing an increasing number of sperm with zonadhesin on the surface of the sperm head anterior during the course of capacitation.  Merged fluorescent-phase contrast images (a-d) and flow cytometry histograms (e-h) showing anti-zonadhesin reactive signals of non-capacitated (Non-cap) sperm and sperm incubated for various times in capacitating medium (Cap-30 min, Cap-60 min, Cap-120 min).  Aldehyde-fixed sperm were used for anti-zonadhesin antibody and secondary antibody incubation.  Numbers in the flow cytometry histograms indicate percentages of sperm with positive staining signals of zonadhesin on the cell surface.  Flow cytometry histograms and micrographs shown were representative from 3 replicate experiments.  B) Proposed model for roles of acrosomal proteins in sperm-egg interactions.  During capacitation, a fraction of acrosomal proteins with ZP affinity (i.e., zonadhesin and proacrosin/acrosin) traffics to the sperm anterior head plasma membrane (APM) and exists as part of high-molecular-weight (HMW) protein complexes on the APM along with other ZP-binding proteins originally localized on the sperm head surface.  The direct affinity of HMW protein complexes for the ZP forms a basis of how capacitated sperm initially bind to the ZP.  With the continuation of acrosomal exocytosis, which leads to more exposure of the acrosomal content, the remainder of the same acrosomal proteins continues to play roles in the adhesion of sperm to the egg ZP.  Acrosomal exocytosis is completed when sperm have penetrated through the ZP layer and entered the perivitelline space; these “acrosome-reacted” sperm then bind to the egg plasma membrane but only one is involved in gamete plasma membrane fusion followed by its incorporation into the egg proper.

Proteomic Characterization Pig Sperm Anterior Head Plasma Membrane Reveals Roles Acrosomal Proteins in ZP3 Binding. Global Medical Discovery

 

 

 

 

 

 

 

 

Journal Reference

Kongmanas K, Kruevaisayawan H, Saewu A, Sugeng C, Fernandes J, Souda P, Angel JB, Faull KF, Aitken RJ, Whitelegge J, Hardy D, Berger T, Baker MA,Tanphaichitr N.

J Cell Physiol. Volume 230, Issue 2, pages 449–463,  2015.

Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Department of Biochemistry/Microbiology/ Immunology, University of Ottawa, Ontario, Canada.

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 sperm anterior head plasma membrane vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-bindingproteins 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 sperm anterior head plasma membrane 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 sperm anterior head plasma membrane 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 sperm anterior head plasma membrane HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with antizonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

© 2014 Wiley Periodicals, Inc.

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