spermatozoa proteomics and possible bull fertility biomarker

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WELCOME

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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 & Proteomics

Introduction 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 Proteomics

Importance 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 proteomics

Importance 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 proteomics

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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 spermatozoa

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Cellular distribution GO annotations of reported proteins. Milardi . Seminal proteomics and male fertility. Fertil Steril 2012 Proteome Proportion In Spermatozoa

Methods 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 analysis

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Semen collection Semen analysis Sample pre fraction The Basic Proteomic Process

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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 protein

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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 chromatography

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Amino acid sequencing of these peptide determined by tandem mass spectrography (MS/MS) Matching of these existing database for protein identification ( Brewis & Gadella ,2010)

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Outlay of proteomic analysis

Spermatogenesis :

Spermatogenesis

Central dogma in spermatogenesis:

Central dogma in spermatogenesis

Spermatozoal 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.,2012

Factors affecting fertilizing ability of spermatozoa at fertilization:

Capacitation Acrosome reaction Sperm- oocyte interaction Factors affecting fertilizing ability of spermatozoa at fertilization

Comparison 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 .,2008

Membrane 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 proteins

Capacitation 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 proteomics

Motility 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 capacitation

Bovine 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)

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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 .

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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 A2

Osteopontin:

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) Osteopontin

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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 Protein

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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) P25b

Spermadhesin 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 Z13

Sperm - 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 reaction

Cont....:

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....

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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 Conclusions

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