logging in or signing up spermatozoa proteomics and possible bull fertility biomarker utkarshtripathi2012 Download Post to : URL : Related Presentations : Let's Connect Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Copy embed code: Embed: Flash iPad Dynamic Copy Does not support media & animations Automatically changes to Flash or non-Flash embed WordPress Embed Customize Embed URL: Copy Thumbnail: Copy The presentation is successfully added In Your Favorites. Views: 246 Category: Education License: All Rights Reserved Like it (0) Dislike it (0) Added: April 27, 2012 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... Premium member Presentation Transcript PowerPoint Presentation: WELCOMEPowerPoint Presentation: ADDRESSING FERTILITY ISSUES IN BULL SEMEN USING PROTEOMIC APPROACHES Presented by - UTKARSH Kr. TRIPATHI M.V.Sc . 1st year (LPM)Introduction to Proteome & Proteomics: The word “proteome” is derived from PROTEins expressed by a genOME Coined by Marc Wilkins in 1994 Proteome is entire complement of proteins,including the modifications made to a particular set of proteins Proteomics is the large-scale study of proteins , particularly their structures and functions Introduction to Proteome & ProteomicsIntroduction to spermatozoa Proteomics: Spermatozoa proteomics is the identification and functional study of sperm proteins Separation of proteins suitable for mass spectrometry and subsequent protein identification Introduction to spermatozoa ProteomicsImportance of proteomics: Male infertility is a major problem for mammalian reproduction The nature of sub-fertility due to the male is as complex as that of the female ( Saacke &Dalton 2000) Spermatozoa transcriptionally inactive so only comprehensive method to understand the molecular functions in spermatozoa is via proteomics Importance of proteomicsImportance of spermatozoa proteomics: Herd reproductive management -primary focus Major fration of reproductive failure -attributable to semen quality In artificial insemination - important to assess the fertility level of the sire Importance of spermatozoa proteomicsPowerPoint Presentation: Proteomic analysis - compare the protein expression profiles from sperm of high- and low-fertility bulls Significant percentage of reproductive failure is attributable to semen quality and not to cow problems ( DeJarnette et al. 2004)Timeline of proteomics: 1975 – 1990 1994 1996 2002 Mass spectometry 2D – Gel Electrophoresis Nucleotide sequencing Proteomics Complex mixture analysis Genetic approaches Timeline of proteomics (The Open Reproductive Science Journal, 2011)High fertility spermatozoa differ from low fertility spermatozoa: There is higher expression of proteins involved in Energy metabolism Cell communication Spermatogenesis Sperm cell motility High fertility spermatozoa differ from low fertility spermatozoaPowerPoint Presentation: Cellular distribution GO annotations of reported proteins. Milardi . Seminal proteomics and male fertility. Fertil Steril 2012 Proteome Proportion In SpermatozoaMethods for proteomic analysis: Selection of high and low fertile bull Sample and Data Source Bull Fertility Prediction Isolation of pure sperm cells Protein extraction Proteomics Differential protein expression Methods for proteomic analysisPowerPoint Presentation: Semen collection Semen analysis Sample pre fraction The Basic Proteomic ProcessPowerPoint Presentation: Protein extraction Protein digestion Liquid chromatography Mass spectrography Protein analysis Protein sepration (Kashou et al., 2011)Global identification of protein: Protein solubilised from entire cell to produce whole cell lysates Sub cellular fractionation to get the protein of biological interest Global identification of proteinPowerPoint Presentation: Two-dimensional electrophoresis (2DE ) Individual protein is separated and removed as gel plug Trypsin digestion and resulting peptide are separated on the basis of charges & relative hydrophobicity by nano scale liquid chromatographyPowerPoint Presentation: Amino acid sequencing of these peptide determined by tandem mass spectrography (MS/MS) Matching of these existing database for protein identification ( Brewis & Gadella ,2010)PowerPoint Presentation: Outlay of proteomic analysis Spermatogenesis : SpermatogenesisCentral dogma in spermatogenesis: Central dogma in spermatogenesisSpermatozoal Proteins related to fertility: Spermatozoal Proteins related to fertility Positively correlated Negatively correlated Osteopontin PhospholipaseA2 P25b Acidic seminal fluid proteins a-L- fucosidase Cathepsin D Lipocalin -type prostaglandin D synthase Spermadhesin Z13 Clusterin Ubiquitin Kumar et al.,2012Factors affecting fertilizing ability of spermatozoa at fertilization: Capacitation Acrosome reaction Sperm- oocyte interaction Factors affecting fertilizing ability of spermatozoa at fertilizationComparison of proteins identified in high fertility and low fertility spermatozoa: Comparison of proteins identified in high fertility and low fertility spermatozoa 2051 1518 2281 325 1018 178 1520 312 219 325 1732 224 BMC Systems Biology .,2008Membrane and nuclear proteins: Sperm oocyte fusion is a key element for fertilization M ajority of membrane proteins involved Transport (33 %) Cell communication (18%) Metabolism (17 %) (Bhattacharyya & Kanjilal,2003) Membrane and nuclear proteinsCapacitation proteomics: Capacitation is coupled to a specific type of metabolism, that is glycolysis or oxidative respiration In glycolysis , expression of pyruvate kinase (PKM2) was higher in high fertility spermatozoa Capacitation proteomicsMotility of sperm and capacitation: COX 3 and ATP5B higher in high fertility spermatozoa compared to low fertility spermatozoa (Chapman et al., 2006) COX 3 is responsible for Coupling of electron transport to oxidative respiration ATP5B catalyzes the production of ATP ( Wright et al.,2009) M otility of sperm and capacitationBovine seminal plasma proteins (BSPs): Bovine seminal plasma proteins (BSPs) secreted by the seminal vesicles Family of heparin-binding proteins Represent approximately 70% of the total protein content of bovine seminal plasma ( Manjunath et al ., 2000 ) Bovine seminal plasma proteins (BSPs)PowerPoint Presentation: Bovine seminal plasma protein are of 3 types. BSP1 , BSP2 , BSP5 , respectively and BSPs are collectively called as binder of sperm . ( Manjunath et al .,2000) BSPs also bind to choline phospholipids of sperm plasma membrane ( Desnoyers & Manjunath.,1992 ) This prevents the free movement of phospholipids and stabilizes the sperm membrane .PowerPoint Presentation: BSPs responsible for capacitation by stimulating cholesterol and phospholipids efflux from the sperm membrane with capacitating agents such as heparin or HDL ( Sutovsky et al.,2003 ) BSP bind to the low-density fraction (LDF) of a lipoprotein of a lipoprotein component of the egg yolk (EY) extender ( Therien & Moreau.,1998) This binding of BSPs to LDF prevent detrimental effect on sperm membrane - crucial for sperm storage (Jenkins et al., 2003 )Phospholipase A2: Role in the late maturation of spermatozoa , the acrosomal reaction and sperm egg fusion ( Taketomi et al.,2010) The PLA2 attached to sperm membranes , synthesizes arachidonic acid, which is converted to PGE2 , leading to acrosome reaction ( Breitbart & Spungin .,1997) PLA2 stimulates immune cells (Graham et al.,1980 ) Antimicrobial activity in the seminal plasma Phospholipase A2Osteopontin: OPN , highly acidic glycoprotein , initially isolated from the mineralized matrix of bovine diaphysial bone Associated with cell adhesion, immune cell stimulation, intracellular signalling (Souza et al.,2008) OsteopontinPowerPoint Presentation: OPN promotes capacitation of sperm ( Moura et al.,2007 ) I ncrease sperm viability possibly by blocking apoptotic pathways ( Erikson et al.,2007) OPN bind to sperm at ejaculation , possibly through integrin and/or CD44 receptors ( Killian et al.,2008) T rigger intracellular signalling , affecting post-fertilization events ( Goncalves et al.,2007)Acidic Seminal Fluid Protein: A spermadhesin protein R eversible inhibition of spermatozoal motility may be by interaction between aSFP and spermatozoa H igh aSFP levels restrict motility ( Schoneck et al.,1996) H igh aSFP in ampulla prevent spermatozoa from wasting energy on ejaculation ( Dostilovi et al.,1994) But it could stimulate their activity after dilution in the female genital tract ( Einspanie.,et al 1996) Acidic Seminal Fluid ProteinPowerPoint Presentation: aSFP protect spermatozoa from oxidative damage by diminishing lipid peroxidation at its physiological concentration ( Einspanier et al.,1994) Higher concentration of aSFP - high freezability of bull semen ( Jobim et al.,2004)P25b: I nvolved in the binding the spermatozoa to the surface of the egg and acquisition of sperm fertilizing ability (Sullivan et al ., 2003) C onsidered as a marker of epididymal maturation of male gametes associated with freezing-thawing procedures ( Lessard et al.,2004) P25bSpermadhesin Z13: Spermadhesin - abundant in seminal plasma of bulls of low fertility and named it anti-fertility factor (Killian et al .,1997) Lower fertility of bulls with high levels of spermadhesin Z13 due to adverse effect of this protein on sperm motility ( Moura et al .,1) Spermadhesin Z13Sperm - oocyte reaction: EGF and PDGF signaling most prominent pathway specific to high fertility sperm ( Braundmeier et al,2002 ) EGF signaling has an important role in sperm capacitation ( Saacke et al,1999) EGF stimulate phospholipase C (PLC) ( Dikic et al,2003) Sperm - oocyte reactionCont....: PLC is important for the acrosome reaction fertilization and embryo development ( DeJarnette et al2003) PLC catalyzes the production of inositol 1, 4, 5 triphosphate (IP3) from phosphatidylinositol 4, 5-biphosphate IP3 generated by PLC activates the extra cellular calcium influx required for the AR ( Karabinus & Whitman,1998) Cont....PowerPoint Presentation: Comparative proteomic analysis showed that the expression levels of several proteins are related to high and low fertility Expression levels of these proteins explain spermatozoa functions & sperm- oocyte communication Proteomics analysis - a valuable tool to identify protein changes related to fertility Expression profiles could help identify protein biomarkers of bull fertility ConclusionsPowerPoint Presentation: THANK YOU You do not have the permission to view this presentation. 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