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      Comparative Analysis of Mafriwal ( Bos taurus × Bos indicus) and Kedah Kelantan ( Bos indicus) Sperm Proteome Identifies Sperm Proteins Potentially Responsible for Higher Fertility in a Tropical Climate

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          Abstract

          The fertility of zebu cattle ( Bos indicus) is higher than that of the European purebred ( Bos taurus) and crossbred ( Bos taurus × Bos indicus) cattle in tropical areas. To identify proteins related to the higher thermo-tolerance and fertility of Zebu cattle, this study was undertaken to identify differences in sperm proteome between the high fertile Malaysian indigenous zebu cattle (Kedah Kelantan) and the sub-fertile crossbred cattle (Mafriwal). Frozen semen from three high performance bulls from each breed were processed to obtain live and pure sperm. Sperm proteins were then extracted, and two-dimensional gel electrophoresis performed to compare proteome profiles. Gel image analysis identified protein spots of interest which were then identified by liquid chromatography mass spectrometry quadrupole time-of-flight (LC MS/MS Q-TOF). STRING network analysis predicted interactions between at least 20 of the identified proteins. Among the identified proteins, a number of motility and energy related proteins were present in greater abundance in Kedah Kelantan. Sperm motility evaluation by Computer Assisted Semen Analysis (CASA) confirmed significantly higher motility in Kedah Kelantan. While results from this study do identify proteins that may be responsible for the higher fertility of Kedah Kelantan, functional characterization of these proteins is warranted to reinforce our understanding of their roles in sperm fertility.

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          The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs.

          Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize each other, and fuse to form a new generation of life. The factors involved in this important membrane fusion event, fertilization, have been sought for a long time. Recently, CD9 on the egg membrane was found to be essential for fusion, but sperm-related fusion factors remain unknown. Here, by using a fusion-inhibiting monoclonal antibody and gene cloning, we identify a mouse sperm fusion-related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We have termed the gene Izumo and produced a gene-disrupted mouse line. Izumo-/- mice were healthy but males were sterile. They produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zona-free hamster eggs.
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            A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry.

            The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.
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              Identification of proteomic differences in asthenozoospermic sperm samples.

              Asthenozoospermia is one of the most common findings present in infertile males, but its aetiology remains unknown in most cases. Present proteomic tools now offer the opportunity to identify proteins which are differentially expressed in asthenozoospermic semen samples and potentially involved in infertility. We compared the expression of 101 sperm protein spots in 20 asthenozoospermic samples to that of 10 semen donor controls using two-dimensional proteomic analysis. Seventeen protein spots have been identified at different amounts in the asthenozoospermic samples compared with controls. These are cytoskeletal actin-B, annexin-A5, cytochrome C oxidase-6B, histone H2A, prolactin-inducible protein and precursor, calcium binding protein-S100A9 (2 spots), clusterin precursor, dihydrolipoamide dehydrogenase precursor, fumarate hydratase precursor, heat shock protein-HSPA2, inositol-1 monophosphatase, 3-mercapto-pyruvate sulfurtransferase/dienoyl-CoA isomerase precursor, proteasome subunit-PSMB3, semenogelin 1 precursor and testis expressed sequence 12. The detected amount of these proteins enabled the grouping of asthenozoospermic sperm samples in an unsupervised clustering analysis. We have identified several proteins present at different amount in asthenozoospermic sperm samples. These proteins could be candidates towards the development of diagnostic markers, and open up the opportunity to gain further insight into the pathogenic mechanisms involved in asthenozoospermia.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                Molecular Diversity Preservation International (MDPI)
                1422-0067
                August 2013
                30 July 2013
                : 14
                : 8
                : 15860-15877
                Affiliations
                [1 ]School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi UKM 43600, Selangor, Malaysia; E-Mails: ali_ash_3000@ 123456yahoo.com (A.A.); sheila@ 123456ukm.my (S.N.)
                [2 ]Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia
                [3 ]University of Malaya Centre for Proteomics Research, University of Malaya, Kuala Lumpur 50603, Malaysia
                Author notes
                [* ]Author to whom correspondence should be addressed; E-Mail: saiful72@ 123456um.edu.my ; Tel.: +603-7967-7139; Fax: +603-7967-4178.
                Article
                ijms-14-15860
                10.3390/ijms140815860
                3759890
                23903046
                82ecd3a0-bb65-4bfc-896e-e93034fe63a7
                © 2013 by the authors; licensee MDPI, Basel, Switzerland

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

                History
                : 08 May 2013
                : 24 June 2013
                : 22 July 2013
                Categories
                Article

                Molecular biology
                sperm,sperm proteins,fertility,motility,2d page
                Molecular biology
                sperm, sperm proteins, fertility, motility, 2d page

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