This presentation summaries information regarding Pregnancy Associated Glyco-protein

1. Pregnancy Associated Biomolecules
(PABs) with reference to Pregnancy
Associated Glycoproteins (PAGs)

2.  Introduction
 Pregnancy Associated Biomolecules
 Interferon-τ (IFN-τ)
 Early Pregnancy Factor (EPF)
 Estrone Sulphate (ES)
 Chorionic Gonadotropins (CG)
 Placental Lactogens (PL)
 Pregnancy Associated Glycoproteins (PAG)
 PAG based diagnostic kits
 Conclusion

3. Biomolecules:
 Are the products of the harmonious and synchronous
process of the chemical reactions occurred in the
cell/body.
 All chemicals functional in living organism specially the
organic substances are known as biomolecules.
 Any molecule that is produced by a living organism
such as protein, carbohydrates, hormones, enzymes, etc.
(Michael et al., 2007)

4.  Some biomolecules are expressed or released into
maternal system at the initiation of pregnancy as well
as during pregnancy, which are commonly called as
‘Pregnancy Associated Biomolecules’.
 Pregnancy associated biomolecules play an important
role in the diagnosis of pregnancy and hence called
as ‘Pregnancy Biomarker’.
(Sousa et al., 2006)
Pregnancy Associated Biomolecules?

5. Maternal Recognition of Pregnancy
Conceptus
• Pregnancy is established and maintained by the two-
way communication between the conceptus and the
mother.
(Spencer et al., 2004)
Maternal System

6.  Conceptus secretes specific substances for its recognition by
mother which include steroid hormones, peptide hormones,
cytokines and growth factors etc.
(Szafranska et al., 2006)
 Conceptus-derived substances are considered to be precise
and reliable markers of pregnancy and fetal well-being.
(Sousa et al., 2006)
 These interactions between the conceptus and maternal
system emphasize the importance of both the components in
maternal recognition of pregnancy and embryonic
development.
(Roberts et al., 2008)

7. Pregnancy Associated Biomolecules
IFN-τ
PL
EPF
ES
CG
PAG
(Henderson et al., 1993)

8.  All these biomolecules secreted from the mono or
binucleate cells or both type of cells of the foetal
trophoblast.
 They enter in the blood of mother and also reach to
milk.
 Detected by RIA, ELISA, etc.
(Gajewski et al., 2008)

9. Interferon-τ
 Secreted by the trophectoderm of
the conceptus from day 10 to 24 of
gestation.
(Bazer et al., 1991)
 172 amino acid protein with
molecular weight of 20-24 kDa.
 Gives the first pregnancy signal
(Roberts et al., 1999)
 Peak level at day 17 in cattle and
day 16 in sheep.
(Roberts et al., 2007)

10. (Spencer et al., 2004)

11.  Interferon tau suppress estrogen receptor and
oxytocin receptor (OTR) expression at transcription
level .
(Fleming et al., 2001)
 Oxytocin induces uterine release of luteolytic
PGF2α, which cause structural and functional
regression of the ovarian CL.
(Fleming et al., 2001)
 Prevents endometrial luteolytic mechanism by
suppressing PGF2α secretion. Prevents recurrence of
estrus and ensures pregnancy to continue.
(Spencer et al., 2002)

13.  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 Early
Pregnancy Diagnosis (EPD).
(Bagnell, C. 2005)

14.  First described in mice and later
on to sheep, cattle and humans.
(Nancarrow et al., 1981)
 Characterized as glycoprotein or
polypeptide with a potent immuno
suppressive properties.
(Noonan et al., 1979)
Early Pregnancy Factor (EPF)

16.  Detected by Rosette Inhibition Test (RIT).
(Morton et al.,1984)
 Serum RIT values low on the day of AI, increased on
day 3 and remained high up to day 7 and subsequently
decreases.
(Sakonju et al.,1993)
 RIT assay is difficult and time consuming test.
 Indirect test, substances that have similar effects may
interfere with the results.
 It could not become popular test for routine pregnancy
diagnosis in farm animals.
(Threlfall, 1994)

17.  Major estrogen secreted from binucleate cells.
 Estimated in milk, plasma and urine.
(Henderson et al., 1993)
 Mean concentration in buffalo varies between 14.8 to
23.6 ng/ml from 2nd
to 9th
month of pregnancy.
(Prakash et al., 1990)
 RIA is used to detect estrone sulphate.
(Madan et al., 1993)
 Detects pregnancy from 2-4 months of gestation i.e.
too late to be considered as a nearly indicator of
pregnancy.

18.  First detected in the urine of pregnant women.
(Aschheim and Zondek, 1927)
 It is known as human Chorionic Gonadotropin
(hCG), which is a glycoprotein secreted by the
trophoblast.
 hCG is first detectable from 8 to 11 days after
conception.

20.  First time Pregnant Mare Serum Gonadotropin
(PMSG) was detected
(Cole and Hart, 1930)
 It is now known as equine Chorionic Gonadotropin
(eCG) which is produced from trophoblastic cells.
 Detectable from 35-40 days of pregnancy.
 Such substance does not exist in ruminants, so it can
not be used as indicator for EPD.
(Jacob et al., 2007)

21.  Synthesized by the binucleate cells of trophoblast and
secreted throughout the gestation.
(Kappes et al., 1990)
 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)
 Detected only from 3-4 Month of gestation in cattle, it
is not used as an indicator of early pregnancy.
(Byatt et al., 1987)

22.  Glycoproteins belonging
to Aspartic Proteinase
family.
 Constitute a group of
pregnancy–specific
proteins.
 Secreted by mono and
binucleate trophoblastic
cells of placenta..
(Xie et al., 1994)

23. (Ushizawa et al.,2004)
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
PAGs Expression Profile

24.  PAGs have been discovered in the second half of the
20th century
(Zoli et al., 1991)
 The discovery of PAG brought a revolution in cattle’s
reproductive techniques.
(Mialon et al.,1993)
 Pregnancy-specific protein-B was the first pregnancy
specific protein identified in cattle
(Butler et al., 1982)
 Both pregnancy specific protein-B and PAG have
subsequently been reclassified as boPAG-1
(Green et al., 2005)

25.  PAGs are synthesized
in the outer epithelial
cell layer (chorion/
trophectoderm) of the
placenta
(Garbayo et al., 2000)
 Part of the molecules
traffics into maternal
blood and milk.
(Gajewski et al., 2008)

26.  PAG can be used as a reliable indicator of pregnancy
from 15 to 30 day of gestation.
(Humblot, 2001)
 PAG exist in milk throughout gestation.
 PAG level decline from 40 to 90 day of gestation,
but PAG levels steadily increase during mid and late
gestation.

27.  Placentogenesis
 Implantation
 Foetomaternal unit remodeling,
 Fetal number
 Detection of twins
 Sex of the foetus
 Neonatal birth weight in cattle
(Lopez-Gatius et al., 2007)

28.  The first discovered bovine PAG was boPAG-1, also
known as boPAG-67kDa
(Green et al., 1998)
 The rest of the molecules belonging to this family got
the following numbers as their names, which was
accepted in international nomenclature.
 Recent investigations have proved the existence and
sequences of 22 bovine PAGs, named appropriately
boPAG-1 – boPAG-22.
(Gajewski et al., 2009)

29. 1. The Pepsinogen-like PAG (boPAG-8) group
2. The Gonadotrophin-like PAG (boPAG-2) group
3. The major bovine PAG family (boPAG-1) group

30.  The boPAG-8 group is the family generally detected
among animals which belong to Placentalia. Those
specific peptides were also discovered in horses, cats,
zebras, mice and rats
(Green et al., 1998)
 In theory these glycoproteins are enzymatically active,
but so far this has not been scientifically confirmed
(Beckers et al., 1999)

31.  A break through discovery in cattle breeding.
 These molecules can stimulate the bovine granulosa
cell layer to produce progesterone
(Sousa et al., 2006)
 The gonadotrophin-like PAGs are built from a
polypeptide, which contains 372 amino acids
(Beckers et al., 1999)

32.  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, it has been named as PAG1
(Zoli et al., 1991)
 This group also contains the proteins: boPAG-56kDa,
boPAG- 67kDa, boPAG-75kDa
(Klisch et al., 2006)

33.  The major bovine PAG family’s molecules are
expressed at the stage of early blastocyst to delivery
and even a few days after parturition
(Ushizawa et al., 2004)
 The synthesis in such early stage of pregnancy
suggests that PAGs are needed in the implantation
process and placentogenesis
(Wooding et al., 2005)

34.  Molecular weight - 67 kDa
 Half-life is approximately 7-9
days
(Kirakofe et al., 1993)
 Detected by RIA and ELISA
(Green et al., 2005
 Bovine and ovine PAG1
shows 73% amino acid
identity
(Sousa et al., 2006)
PAG1

35. PAG-1 is the
Best Pregnancy
Biomarker
Gajewski et al., 2009

36. 1. Rectal Palpation
 Difficult to diagnose pregnancy status
accurately earlier than 30 to 35 d after AI
(Youngquist et al., 2007)
2. Transrectal Ultrasonography (TU)
 Accuracy of pregnancy diagnosis by TU
decreases at a gestational age of less than 33 d.
(Romano et al., 2006)
PAG-1 v/s Traditional methods

37.  Early & easy diagnosis without disturbing dam
 Kits are chaeper and suitable for field condition, from
one kit > 72 animal diagnosis possible
 Detectable in the maternal blood soon after implantation
(Zoli et al., 1992)
 Present in measurable quantity from blood or milk
 It is detected from 15-30 days of pregnancy and upto
parturition
(Humblot, 2001)

38.  Recently, several immunological assays for the
quantification of PAG have been developed as accurate,
cost-effective and convenient alternatives
(Silva et al., 2009)
 ELISA and RIA are developed for the detection of
PAGs from milk, plasma, serum and blood samples of
pregnant cow.
 Pag-1 based diagnosis by RIA and ELISA are more
accurate, sensitive and specific rather then present
methods of pregnancy diagnosis

42. Species Mol. Wt.
(kDa)
Amino
Acids
pH
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 37,41 838 4.5
Zebra - 388 -
Deer 30,50,55 380 -
Pig 43 389 5.6-6.2

43.  RIA – 497 (PURIFIED) (Zoli et al.,1992)
Diagnosed
• pregnant - 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 et al., 2006)

44.  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 after AI RIA I
(497)
RIA II
(706)
RIA III
(708)
21 0.43 0.48 0.64
32 2.43 4.17 4.30
42 4.01 5.60 5.56

45. (Zoli et al., 1992)

46. (Silva et al., 2007)
Accuracy Rate of ELISA

47.  Reproductive performance in dairy herds depends greatly on
the accurate and early diagnosis of pregnancy (De Vries et al.,
2006)
 Traditionally used methods are found to be disadvantageous
for one or the other reasons.
 Pregnancy Associated Biomolecules (PAB) holds tremendous
potential for an accurate and early diagnosis of pregnancy in
case of farm animals.
 PABs are produced by the conceptus and enters into the
maternal system which play role in maintaining pregnancy.
 Amongst the PABs, PAGs has dragged the attention of the
researchers as well as the clinicians as a reliable tool for
pregnancy diagnosis, because of its early appearance in
maternal blood as well as in milk in measurable quantity as
compared to the other PABs.

48.  Many diagnostic kits have been developed based on ELISA and
RIA to detect PAG-1, which is now routinely used and found to be
accurate and cost effective.
 Further, PAG concentration may also give insight to embryonic or
foetal mortalities.
 Till date, PAGs have mostly been exploited as pregnancy biomarker
in case of large animals, particularly in cattle and buffalo.
 However, as report suggests that PAGs are present in all animal
species studied so far, hence in near future, there is every possibility
that PAG will replace all the traditional methods and will emerge as
the potential means of pregnancy diagnosis at an very early stage
not only in large animals but also in small animals like sheep and
goats.

49. Thank youThank you