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PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
PREGNANCY ASSOCIATED BIOMOLECULES
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PREGNANCY ASSOCIATED BIOMOLECULES

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PREGNANCY ASSOCIATED BIOMOLECULES

PREGNANCY ASSOCIATED BIOMOLECULES

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  • 1. PREGNANCY ASSOCIATED BIOMOLECULESJerome A,SK Singh & SK Agarwal (c) Jerome A
  • 2. PREGNANCY MATERNALCONCEPTUS SYSTEM(Spencer et al.,2004) (c) Jerome A
  • 3. IFN- τ PAG CG CONCEPTUS EPF PL ES(Aschheim & Zondek,1927;Kappes et al.,1990; Bartol et al.,1985; Morton et al., 1987; Zoli et al.,1991; Henderson et al., 1993) (c) Jerome A
  • 4. These signals-are potential markers ofpregnancy and embryonic viability as theyinfluence pregnancy recognition and successfulimplantation.Conceptus derived substances are considered tobe more precise and reliable markers ofpregnancy and fetal well being.Conceptus secretes specific substances for itsrecognition by mother. (Sousa et al.,2006;Szafranska et al.,2006) (c) Jerome A
  • 5. INTERFERON –τSecretory product of the conceptus, from day10 to 24 of gestation & peaks at day 17 in cattleand day 16 in sheep. (Bartol et al.,1985; Roberts et al.,2007)Prevents luteolysis by suppressing PGF2α secretion.Prevents recurrence of estrus and ensurespregnancy to continue.Up-regulates expression of no. of proteins andgenes.Provide suitable environment locally to theconceptus. (c) Jerome A
  • 6. Conti…Possessing a molecular weight of 20-24 kDawith 172 amino acids & considered as primesignal for MRP. (Knickerbockers et al.,1986)Remains within the uterine lumen and doesnot appear in measurable quantities inmaternal blood or other body fluids.Therefore, can not be used as a marker forEPD. (c) Jerome A
  • 7. EARLY PREGNANCY FACTOREarliest known signal claimed to be specific forfertilization.First described in mice and later on to sheep,cattleand humans. (Mortan et al.,1974;Nancarrow et al.,1981)Characterized as glycoprotein or polypeptide with apotent immunosuppressive properties. (Noonan et al.,1979)Detected by Rosette inhibition test. (Morton et al.,1984)Serum RIT values low on the day of AI, increased onday 3 and remained high until embryo recovery on day7 and subsequently decreased to a lower level by aweek after embryo recovery. (Sakonju et al.,1993) (c) Jerome A
  • 8. Conti…RIT assay isdifficult, tedious, cumbersome and timeconsuming test.Indirect test, substances that havesimilar effects may confound the results.Therefore, it could not become populartest for routine pregnancy diagnosis infarm animals.Attempt to isolate a protein responsiblefor EPF activity has produced variableresults without any convincingidentification or characterization.(Mehta et al., 1989; Klima et al., 1992;Threlfall, 1994) (c) Jerome A
  • 9. Conti…Many diagnostic kits – KEMS BioTest Ltd. (Littleton, CO), Early Conception Factor (ECF) test (Concepto Diagnostics, Knoxville, TN) & ECF dipstick test (Adams and Jordan, 1999; Concepto Diagnostics, 1999; Des coteaux et al.,2000)Detects pregnancy within 48 hrs afterconception.More false-positive & false-negative results.So ECF test in present form is not a reliablemethod for determining early pregnancy.Cordoba et al. (2001) (c) Jerome A
  • 10. ESTRONE SULPHATEMajor estrogen secreted from binucleate cells.Estimated in milk, plasma and urine. (Henderson et al.,1993)Mean conc. in buffalo varies between 14.8 to23.6 ng/ml from second to ninth month ofpregnancy. (Hung and Prakash,1990)RIA/EIA was used to detect estrone sulphate.(Henderson et al.,1993;Prakash & Madan,1993)Detects pregnancy at 31/2-4 months ofgestation i.e. too late to be considered as anearly indicator of pregnancy. (c) Jerome A
  • 11. Chorionic GonadotropinsIn 1927, Aschheim and Zondek first reported thepresence of a gonadotropic factor in the urine ofpregnant women.Known as human chorionic gonadotropin (hCG),glycoprotein secreted by the trophoblast.Based on this, an accurate method of EPD in human wasdeveloped.hCG is first detectable 8 to 11 days after conception.In 1930, Cole and Hart demonstrated PMSG, nowknown as equine chorionic gonadotropin (eCG) producedfrom the endometrial cups composed of trophoblasticcells.Detectable from day 35-40 days of pregnancy.Such substance does not exist in ruminants, cannot beused as indicator for EPD. (c) Jerome A
  • 12. PLACENTAL LACTOGENSynthesized by the binucleate cells & secretedthroughout gestation. (Kappes et al., 1990)MW – bovine- 32-34, ovine- 22 & caprine- 23kDa, (Byatt et al., 1986)Mammogenesis, regulation of fetal growth, ovariansteroidogenesis & maternal inter-mediary metabolism. (Byatt et al., 1992)Concentration remains low & varies from 1 to 2 ng/ml throughout gestation. (Beckers et al., 1982, Byatt et al.,1987)Since it is detected only from 3-4 month of gestationin cattle,it is not used as an indicator of earlypregnancy. (Byatt, 1987) (c) Jerome A
  • 13. PREGNANCY ASSOCIATEDGLYCOPROTEINS (c) Jerome A
  • 14. PREGNANCY ASSOCIATED GLYCOPROTEINSAcidic Glycoproteins belonging to AsparticProteinase family.Molecular structure – has more than 50 % AAsequence identity with Pepsin, Cathepsin D & E. (Xie et al.,1991,1994)Secreted by binucleate trophoblastic cells ofplacenta.Constitute a group of pregnancy–specific proteins.Nearly 22 different PAGs in ruminants has beenidentified at different stage of gestation.(Xie et al.,1991,1994,1997;Green et al., 2000; Garbayo et al., 2005) (c) Jerome A
  • 15. PAG1The first member of the family was isolated by Butleret al. (1982) from bovine placenta and was named aspregnancy specific protein B (PSPB).Subsequently, related proteins have been identified withthe different names such as PAG1 (Xie et al., 1991;Zoli et al., 1991), PSP-60 (Mialon et al., 1993), SBU3(Atkinson et al., 1993) in ruminant ungulates.These proteins are detectable in maternal blood andwere reported to be useful as prenatal markers forpregnancy diagnosis and embryonic viability in variousdomestic and wild ruminants.(Sasser et al.,1986; Zoli et al.,1992;Mialon et al., 1993;Perenyiet al., 2002; Szenci et al., 2003; Ayad et al., 2007, Panasiewiczet al., 2007) (c) Jerome A
  • 16. Conti… Conti…MW ranges- 37-78 kDa. (Butler et al.,1982; Sasser et al., 1989; Zoli et al., 1991)Moreover, PAG have also roles inplacentogenesis, detection of twins, sex ofthe foetus.(Patel et al.,1995,1997; Vasques et al., 1995; Willard etal., 1995;Green et al., 2005; Lopez-Gatius et al., 2007) (c) Jerome A
  • 17. PAG geneThe PAG gene was first cloned in cattle &Sheep (Xie et al.,1991)Later on in many species- goat (Garbayo et al.,2000), pig (Szafranska et al.,1995) & other species (Xie et al.,1997).cDNA microarray analysis demonstratedexpression of several PAG molecules early asday 7 to 14 of pregnancy or even earlier incattle (Ushizawa et al.,2004). (c) Jerome A
  • 18. PAG 1 GENE (CATTLE)First gene to be cloned in cattle. 9 EXONS (99- 281 bp) 8 INTRONS (87-1800 bp) (Xie et al .,1995) Gen-Bank DATA ACC. NO gDNA IDENTIFIED SEQ.(bp) BY L27833 Exons 1-6 Xie et al., L22834 Exon 7-8 1995 L27832 Exon 9 L27833 1260 (Promoter) (c) Jerome A
  • 19. PAG 1 GENE PROFILE IN VARIOUS SPECIES SPECIES IDENTIFIED mRNA(bp) BY CATTLE Xie et al.,1991 1295 SHEEP -do- 1266 GOAT Garbayo et 1143 al.,2000 PIG Panasiewicz, 1353 2004 (c) Jerome A
  • 20. Conti…Recombinant protein technology hasprovided avenue for the generation of sufficient amount of specific proteins of interest from a single gene, used for the development of improved diagnostics for early pregnancy diagnosis & conceptus viability. (Patel et al.,2004) (c) Jerome A
  • 21. PAGs expression profile CELLS PAG profile BINUCLEATE CELLS 1,3,4,5,6,7,9 MONO- & 2,8,10,13 BINUCLEATE CELLS Days PAG profile 7 4,5,6 7 to 14 11,16,17 14 to 21 1,5-7,9-13,15- 17,19,21(Green et al.,2000; Ushizawa et al.,2004) (c) Jerome A
  • 22. PAG 1 Molecular weight - 67 kDa Various isoforms (PI:4.4, 4.6, 5.2 & 5.4) Amino acids -380 Bovine and ovine PAG1 shows 73% aa identity Proteolytically inactive Belong to boPAG1 group(Xie et al.,1991; Zoli et al.,1991;Guruprasad et al.,1996;Sousa et al.,2006) (c) Jerome A
  • 23. Conti...Half-life is approximately 7-9 days. (Kirakofe et al.,1993)Becomes detectable soon. After implantation isinitiated.Detected by RIA and ELISA (Zoli et al.,1992; Perenyi et al.,2002; Green et al.,2005) (c) Jerome A
  • 24. PAG 1 PROTEIN PROFILE IN VARIOUS SPECIES SPECIES MOL.WT AMINO pH (kDa) ACIDS CATTLE 37-78 380 4.0-4.4 SHEEP 47-90 382 4.0-6.8 GOAT 55,59,62 341 4.9-6.2 HORSE 37,41 388 4.5 ZEBRA - 388 - DEER 30,50,55 380 - PIG 43 389 5.6-6.2(Szafranska et al.,2006) (c) Jerome A
  • 25. DIAGNOSTICS DEVELOPED USING PAG 1 PROTEIN (CATTLE)RIA – 497 (PURIFIED) (Zoli et al.,1992) Diagnose pregnancy - 93% & non-pregnant - 97% Overall accuracy - 94%RIA – 706 & 708 (SP) (Perenyi et al.,2002)ELISA SYSTEM (SP) (Green et al.,2005) KIT DEVELOPED FROM PAG 1- BioPRYN(Sasser,2006) (c) Jerome A
  • 26. PAG1 concentration (ng/ml) in blood of cattleAble to detect PAG molecules in the maternal bloodas early as 21 days post AICompared three established RIA (Perenyi et al.,2002) Days RIA I RIA II RIA IIIafter AI 21 0.43 0.48 0.64 32 2.43 4.17 4.30 42 4.01 5.60 5.56 (c) Jerome A
  • 27. PAG 1 PROTEIN PROFILE IN VARIOUS SPECIES SPECIES MOL.WT AMINO pH (kDa) ACIDS CATTLE 37-78 380 4.0-4.4 SHEEP 47-90 382 4.0-6.8 GOAT 55,59,62 341 4.9-6.2 HORSE 37,41 388 4.5 ZEBRA - 388 - DEER 30,50,55 380 - PIG 43 389 5.6-6.2 (Szafranska et al.,2006) (c) Jerome A
  • 28. THANK YOU (c) Jerome A

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