PREGNANCY ASSOCIATED BIOMOLECULES

1. PREGNANCY ASSOCIATED
BIOMOLECULES
Jerome A,SK Singh & SK Agarwal
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2. PREGNANCY
MATERNAL
CONCEPTUS SYSTEM
(Spencer et al.,2004)
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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)
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4. These signals-are potential markers of
pregnancy and embryonic viability as they
influence pregnancy recognition and successful
implantation.
Conceptus derived substances are considered to
be more precise and reliable markers of
pregnancy and fetal well being.
Conceptus secretes specific substances for its
recognition by mother.
(Sousa et al.,2006;Szafranska et al.,2006)
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5. INTERFERON –τ
Secretory product of the conceptus, from day
10 to 24 of gestation & peaks at day 17 in cattle
and day 16 in sheep.
(Bartol et al.,1985; Roberts et al.,2007)
Prevents luteolysis by suppressing PGF2α secretion.
Prevents recurrence of estrus and ensures
pregnancy to continue.
Up-regulates expression of no. of proteins and
genes.
Provide suitable environment locally to the
conceptus. (c) Jerome A

6. Conti…
Possessing a molecular weight of 20-24 kDa
with 172 amino acids & considered as prime
signal for MRP. (Knickerbockers et al.,1986)
Remains within the uterine lumen and does
not appear in measurable quantities in
maternal blood or other body fluids.
Therefore, can not be used as a marker for
EPD.
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7. EARLY PREGNANCY FACTOR
Earliest known signal claimed to be specific for
fertilization.
First described in mice and later on to sheep,cattle
and humans.
(Mortan et al.,1974;Nancarrow et al.,1981)
Characterized as glycoprotein or polypeptide with a
potent 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 on
day 3 and remained high until embryo recovery on day
7 and subsequently decreased to a lower level by a
week after embryo recovery.
(Sakonju et al.,1993)
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8. Conti…
RIT assay is
difficult, tedious, cumbersome and time
consuming test.
Indirect test, substances that have
similar effects may confound the results.
Therefore, it could not become popular
test for routine pregnancy diagnosis in
farm animals.
Attempt to isolate a protein responsible
for EPF activity has produced variable
results without any convincing
identification 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 after
conception.
More false-positive & false-negative results.
So ECF test in present form is not a reliable
method for determining early pregnancy.
Cordoba et al. (2001) (c) Jerome A

10. ESTRONE SULPHATE
Major estrogen secreted from binucleate cells.
Estimated in milk, plasma and urine.
(Henderson et al.,1993)
Mean conc. in buffalo varies between 14.8 to
23.6 ng/ml from second to ninth month of
pregnancy. (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 of
gestation i.e. too late to be considered as an
early indicator of pregnancy.
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11. Chorionic Gonadotropins
In 1927, Aschheim and Zondek first reported the
presence of a gonadotropic factor in the urine of
pregnant women.
Known as human chorionic gonadotropin (hCG),
glycoprotein secreted by the trophoblast.
Based on this, an accurate method of EPD in human was
developed.
hCG is first detectable 8 to 11 days after conception.
In 1930, Cole and Hart demonstrated PMSG, now
known as equine chorionic gonadotropin (eCG) produced
from the endometrial cups composed of trophoblastic
cells.
Detectable from day 35-40 days of pregnancy.
Such substance does not exist in ruminants, cannot be
used as indicator for EPD. (c) Jerome A

12. PLACENTAL LACTOGEN
Synthesized by the binucleate cells & secreted
throughout gestation.
(Kappes et al., 1990)
MW – bovine- 32-34, ovine- 22 & caprine- 23
kDa,
(Byatt et al., 1986)
Mammogenesis, regulation of fetal growth, ovarian
steroidogenesis & 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 gestation
in cattle,it is not used as an indicator of early
pregnancy.
(Byatt, 1987)
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13. PREGNANCY
ASSOCIATED
GLYCOPROTEINS
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14. PREGNANCY ASSOCIATED
GLYCOPROTEINS
Acidic Glycoproteins belonging to Aspartic
Proteinase family.
Molecular structure – has more than 50 % AA
sequence identity with Pepsin, Cathepsin D & E.
(Xie et al.,1991,1994)
Secreted by binucleate trophoblastic cells of
placenta.
Constitute a group of pregnancy–specific proteins.
Nearly 22 different PAGs in ruminants has been
identified at different stage of gestation.
(Xie et al.,1991,1994,1997;Green et al., 2000; Garbayo et al.,
2005) (c) Jerome A

15. PAG1
The first member of the family was isolated by Butler
et al. (1982) from bovine placenta and was named as
pregnancy specific protein B (PSPB).
Subsequently, related proteins have been identified with
the 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 and
were reported to be useful as prenatal markers for
pregnancy diagnosis and embryonic viability in various
domestic and wild ruminants.
(Sasser et al.,1986; Zoli et al.,1992;Mialon et al., 1993;Perenyi
et al., 2002; Szenci et al., 2003; Ayad et al., 2007, Panasiewicz
et al., 2007)
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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 in
placentogenesis, detection of twins, sex of
the foetus.
(Patel et al.,1995,1997; Vasques et al., 1995; Willard et
al., 1995;Green et al., 2005; Lopez-Gatius et al., 2007)
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17. PAG gene
The 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 demonstrated
expression of several PAG molecules early as
day 7 to 14 of pregnancy or even earlier in
cattle (Ushizawa et al.,2004).
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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)
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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
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20. Conti…
Recombinant protein technology has
provided
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)
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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)
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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)
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23. Conti...
Half-life is approximately 7-9 days.
(Kirakofe et al.,1993)
Becomes detectable soon. After implantation is
initiated.
Detected by RIA and ELISA
(Zoli et al.,1992; Perenyi et
al.,2002; Green et al.,2005)
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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)
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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 cattle
Able to detect PAG molecules in the maternal blood
as early as 21 days post AI
Compared three established RIA
(Perenyi et al.,2002)
Days RIA I RIA II RIA III
after AI
21 0.43 0.48 0.64
32 2.43 4.17 4.30
42 4.01 5.60 5.56
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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
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