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ORIGIN OF SEMINAL PLASMA PROTEIN Blood origin (Fowler and Meriono, 1983) Pre albumin Globulins Transferrins  Antitrypsin  Glycoprotein  liporpotein Immunoglobulins IgG, IgAand IgM Functions: 1. Osmotic pressure and P H 2. Transport of ions Reproductive tract origin Fertility associated proteins (Osteopontin and Prostaglandin D Synthase) Fertility associated antigen Heparin binding proteins Forward motility proteins IgG Fc binding proteins Clusterin Immobilin Inhibin Seminal Plasmin Heat shock protein (Hsp2A) BSP proteins (BSP- A 1, BSP- A 2, BSP- A 3 )

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REPRODUCTIVE TRACT ORIGIN Seminal Plasma Specific proteins from Testis Epididymis Vas deferens Prostate Seminal Vesicles Bulbourethral glands Control - Testosterone (Matousek, 1985 and Kulkarni et al., 1998)

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ISOLATION AND CHARACTERIZATION OF PROTEINS Ion Exchange chromatography Sephadex gel filteration SDS – PAGE 2 DHR- PAGE FPLC – Fast performance liquid chromatography

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Molecule Location Function Heparin binding proteins Accessory gland fluid and spermatozoa Increases affinity for heparin and enhances capacitation Fertility associated antigen (FAA) -do- Enhances capacitation Forward Motility Protein (FMP) Caput Epididymis Forward motility and Prevent head to head agglutination ATP Spermatozoa Enhances sperm motility Clusterin Testis and Epididymal fluid and sertoli cells Measures improper spermatogenesis and epididymal maturation Heat shock protein (Hsp2A) Spermatozoal tail membrane Measures Spermatozoal immaturity Spermatozoal antibodies Sperm head Blocks sperm oocyte binding Granulocyte elastase Seminal plasma Measure inflammation of genital tract Reactive Oxygen Species Spermatozoal membrane Damages spermatozoal membrane MARKERS OF SPERMATOZOAL FERTILITY (Braundmeier and Miller, 2001)

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HEPARIN BINDING PROTEINS (HBPs) Structure: 1. Major HBPs – 13,15 kDa 2. Minor HBPs – 16, 24, 30 kDa Synthesis : Seminal Vesicle, Prostate, Bulbourethral glands Functions: Binds to bull sperm membrane Acrosomal reaction and Capacitation Fertilisation and Sperm Concentration Increase in intensity of phosphorylation of 50 kDa sperm protein (Bellin, 1994) HBPs has high affinity for Heparin in Seminal Vesicular Fluid of Bull, Boar, Stallion and Man (Ronkko et al., 1994 and Krause et al., 2001

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FORWARD MOTILITY PROTEIN (FMP) Structure : 37 kDa Synthesis: Caput epididymis Purification: Gel chromatography, Affinity chromatography on Concavalin A agarose Functions: Develops forward motility in epididymal spermatozoa Prevent head to head agglutination of motile spermatozoa Stiffening of the flagella (Accott and Hoskins, 1983)

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SEMINAL PLASMIN (SPLN) Structure: 5.4 kDa 48 amino acids Basic Protein with P I 9.6-9.8 Synthesis: Seminal Vesicle, Prostate, Ampulla of the Vas deferens Functions: Transcript inhibitory Antimicrobial activity Gram positive and negative bacteria, Yeast and Retroviruses (Reddy et al., 1983 and Bhargava, 1985)

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GOSSACT Structure : Heat stable, Leucin rich, 19 kDa Isolated and Purified from Human Seminal plasma Function : Neutralise the inhibitory effect of Gossypol on LDH (Nakamura et al 1991)

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ANDROGEN BINDING PROTEIN (ABP) Structure: 70-90 kDa High affinity to testosterone and dihydro testosterone Synthesis: Sertoli cells, seminiferous tubule and epididymis (Larrea et al .,1981) Functions: Epididymal maturation of spermatozoa Acquisition of sperm motility and fertilizing ability Regulation of function of epididymis like synthesis and secretion of sperm maturation protein Mediate androgen action in epididymis by regulation of 5  reductase enzyme (Hinton et al., 2001)

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INHIBIN Structure: Glycoprotein hormone, 15-30 kDa, Two dissimilar disulfide linked sub units Two forms - Inhibin A (Long peptide chain,  and  A) - Inhibin B (Short peptide chain,  and  B) Synthesis: Sertoli cells Function: Suppresses FSH secretion and modulates reproductive function

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HEAT SHOCK PROTEINS (HspA2) Structure: 70 kDa It Indicates: Immature spermatozoa Incomplete plasma membrane remodelling (Huszar et al., 2000) ACROSIN Structure: Serine protease Synthesis: Stored in acrosome as a zymogen (Yanagimachi, 1994) It Indicates: High amount - high penetration rate Low amount - low penetration rate - Poor IVF Success Zona pellucida

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GRANULOCYTE ELASTASE Synthesis: Released from granulocytes during phagocytosis,degranulation and cell death It Indicates: Presence of WBCs in semen Inflammation of reproductive tract Reduces penetration in zona free hamster eggs (Maruyama et al., 1985)

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CLUSTERIN Structure: Recently discovered acidic glycoprotein, 75-85 kDa Synthesis: Testes and Epididymal fluid (Baily et al., 2000) Purification: SDS-PAGE, Western Blot and Flow cytometry. It indicates: Improper spermatogenesis and epididymal maturation Poor quality semen

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IgG Fc BINDING PROTEIN Structure: 94 kDa Functions: Regulating the female humoral and cellular responses of the inseminated spermatozoa by protecting them from immune destructions. IMMOBILIN Secreted by caput epididymus Inhibits sperm motility in cauda epididymis

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IMPLICATIONS Accurate Economically feasible Used as supplements to standard traits measured in semen analysis To identify semen with poor fertility and to suggest suitable therapeutic measures

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