EXPRESSION OF PLACENTAL LACTOGEN AND PROLACTEIN RELATED PROTEIN-1 IN THE PLACENTAL TISSUES DURING GESTATION IN BLACK BENGAL GOAT

Abstract

Placental lactogen, PL and Prolactein Related Protein-1, PRP-1 are newly illustrated proteins synthesized from placenta in both bovine and caprine species having some major effect in gestation and fetal development. As Black Bengal goat (BBG) is the only recognized goat breed of Bangladesh, expression analysis of pregnancy specific genes in this breed is necessary to improve their reproductive efficiency. Aiming this fact, this study was designed to investigate the relative mRNA expression of this two genes (caprine Placental Lactogen, cPL and Prolactein Related Protein-1, PRP-1) in three different categories of caprine placental tissues (placentome, endometrium and fetal membrane) in three different stages of pregnancy (50, 60, 90 days) in Black Bengal goat. Same approach was performed in the same tissues using bovine placental lactogen, bPL which is a bovine specific protein. Analysis of relative mRNA expression of cPL and cPRP-1 in different placental tissue samples indicates their strong expression in placentome and endometrium as early as at 50 days of gestational age with their expression at also 60 or 90 days. Whereas, bPL was not properly expressed in any placental tissues during 50, 60 or 90 days of gestation as bPL is pregnancy specific gene especially in bovine species. This expression analysis indicates that both cPL and cPRP-1 has strong expression in Black Bengal does at as early as 50 days of gestation. So, cPL and cPRP-1 may be used as candidate gene in pregnancy diagnosis in Black Bengal goat.

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International Journal of Advances in Science Engineering and Technology, ISSN(p): 2321 –8991, ISSN(e): 2321 –9009
Volume-6, Issue-1, Jan.-2018, http://iraj.in
Expression of Placental Lactogen and Prolactein Related Protein-1 in the Placental Tissues during Gestation in Black Bengal Goat
1
EXPRESSION OF PLACENTAL LACTOGEN AND PROLACTEIN
RELATED PROTEIN-1 IN THE PLACENTAL TISSUES DURING
GESTATION IN BLACK BENGAL GOAT
1SHUVRA DEBNATH, 2M.A. M. YAHIA KHANDOKER, 3KEIICHIRO KIZAKI, 4JOBAIDA SHOVNA
KHANOM, 5MD. MAZHARUL ISLAM
1,2,4,5Department of Animal Breeding and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh,
3Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, 3-18-8
Ueda, Morioka, Iwate 020-8550, Japan
Email: 1to.shuvra@gmail.com, 2yahiakhabg@yahoo.com, 3kizaki@iwate-u.ac.jp, 4shovnajobaida@yahoo.com,
5mazharbau08@gmail.com
Abstract - Placental lactogen, PL and Prolactein Related Protein-1, PRP-1 are newly illustrated proteins synthesized from
placenta in both bovine and caprine species having some major effect in gestation and fetal development. As Black Bengal
goat (BBG) is the only recognized goat breed of Bangladesh, expression analysis of pregnancy specific genes in this breed is
necessary to improve their reproductive efficiency. Aiming this fact, this study was designed to investigate the relative
mRNA expression of this two genes (caprine Placental Lactogen, cPL and Prolactein Related Protein-1, PRP-1) in three
different categories of caprine placental tissues (placentome, endometrium and fetal membrane) in three different stages of
pregnancy (50, 60, 90 days) in Black Bengal goat. Same approach was performed in the same tissues using bovine placental
lactogen, bPL which is a bovine specific protein. Analysis of relative mRNA expression of cPL and cPRP-1 in different
placental tissue samples indicates their strong expression in placentome and endometrium as early as at 50 days of
gestational age with their expression at also 60 or 90 days. Whereas, bPL was not properly expressed in any placental tissues
during 50, 60 or 90 days of gestation as bPL is pregnancy specific gene especially in bovine species. This expression
analysis indicates that both cPL and cPRP-1 has strong expression in Black Bengal does at as early as 50 days of gestation.
So, cPL and cPRP-1 may be used as candidate gene in pregnancy diagnosis in Black Bengal goat.
Key words - Black Bengal goat, Caprine Placental lactogen, cPL, Caprine Prolactein Related Protein-1, cPRP-1,
endometrium, fetal membrane (FM), placentome
I. INTRODUCTION
Placenta, a temporal organ plays a crucial role in
pregnancy by producing a number of proteins during
gestational period. Studies revealed that proteins like
Placental Lactogen, PL and Prolactein Related
Protein-1; PRP-1, both of which are member of
prolactin gene family has a major effect in gestation
which expressed early in the gestational period in
both bovine and caprine species. Placental lactogen
(bPL) and bovine prolactin-related protein-1 (bPRP1)
are members of the growth hormone (GH)/prolactin
(PRL) gene family, which are expressed
simultaneously in Bovine trophoblast binucleate cells
and are central to placentation and the progression of
pregnancy in cattle [1]. Firstly the Placental
Lactogen hormones of goats were detected by [2]
which are polypeptide hormones produced by
chorionic tissue in primates, rodents and ruminants
[3, 4]. The bPL mRNA is transcribed in
trophectoderm binucleate cells starting from Day 30
of pregnancy until the end of gestation which is
involved in the regulation of ovarian function,
mammogenesis, lactogenesis, and pregnancy stage-
dependent adaptation of nutrient supplies to the fetus
[5]. Placental lactogen (PL) [6, 7] specifically appear
in both caprine (goat) and bovine trophoblast whereas
in cattle prolactin-related protein (PRP) has been
known as a placental-specific molecule that
specifically expresses in trophoblastic binucleate
cells. The production of bPRP-I during early
implantation, and secretion into the uterine fluid and
serum of pregnant cows [8], indicate that it may be a
good candidate for pregnancy diagnosis
[9].According to [10], Prolactin-related protein I
(PRP-I) is a member of the nonclassical prolactin-
related family and PRP genes in caprine placenta
have coordination functions for gestation, as they do
in bovine.
Pregnancy diagnosis and determination of litter size
are of considerable value in improving efficiency of
reproduction in goats. Accurate pregnancy diagnosis
may provide essential information for effective herd
management practices [11]. As Black Bengal goat is
the most promising and valuable native goat breed of
Bangladesh, improved reproductive managemental
practices are major need for their economic
production. In this concept, early pregnancy diagnosis
is obviously one of the most important tools in this
recognized native breed of Bangladesh.
Pregnancy specific gene identification is of major
need in small ruminant like goat. Expression analysis
of these specific genes was considered to be essential
to evaluate their relative expression in different
placental tissues as there is very less information
related to this specific gene expression in this goat
breed. The present study was, therefore, undertaken
to determine the expression of this genes (placental

International Journal of Advances in Science Engineering and Technology, ISSN(p): 2321 –8991, ISSN(e): 2321 –9009
Volume-6, Issue-1, Jan.-2018, http://iraj.in
Expression of Placental Lactogen and Prolactein Related Protein-1 in the Placental Tissues during Gestation in Black Bengal Goat
2
lactogen and prolactin related protein) in placental
tissues in different days of pregnancy of Black
Bengal goat to assist in molecular factor
identification which will eventually be an aid for
early pregnancy detection of this goat breed.
II. METHODOLOGY
Tissue samples were collected in Artificial
Insemination Centre, Bangladesh Agricultural
University, Mymensingh, Bangladesh and further
study was conducted in reproductive Physiology
Laboratory, Iwate University, Morioka, Japan.
Caprine uterine tissues intended for total RNA
extraction, cDNA cloning and mRNA expression
were collected from 3 pregnant Black Bengal does of
three pregnancy stage (50, 60 and 90 days
respectively).
The tissues collected across specific stages of
gestation were designated as follows: (i) Day 50:
placentomal, intracaruncular endometrium and fetal
membrane tissue samples were collected from three
different cows on days 50 of gestation (n = 3 animals)
; (ii) Day 60: placentomal, intracaruncular
endometrium and fetal membrane tissue samples
were collected from three different cows on days 60
of gestation (n = 3 animals) ; (iii) Day 90: :
placentomal, intracaruncular endometrium and fetal
membrane tissue samples were collected from three
different cows on days 90 of gestation (n = 3
animals).After collecting these tissue samples they
were immediately stored at RNA Later solution and
preserved at 4°C. Pregnant placenta with fetus is
shown in figure 1.
(a) (b) (c)
Fig1: Pregnant placenta with fetus (a) 50 days pregnant
placenta with fetus (b) 60 days pregnant placenta with fetus (c)
90 days pregnant placenta with fetus
After that, total RNA isolation from the respective
tissue samples was done using RNeasy Minikit
(Qiagen, Japan) according to manufacturer’s
protocol. Total RNA was quantified and its integrity
was assesed using a spectrophotometer (by
Nanodrop® ND-1000, Agilent's 2100 Bioanalyzer).
Total RNA concentration was determined as the UV
absorption 260/280 nm OD ratio with a Nanodrop
ND-1000 spectrophotometer. Following that, reverse
transcription (RT) of total RNA to single-stranded
cDNA was done by using the High Capacity cDNA
Reverse Transcription Kits (Applied Biosystem).
PCR was performed using AmpliTaq Gold DNA
polymerase (Applied Biosystems, Foster City, CA,
USA). Caprine or bovine GAPDH was used as a
positive control for the PCR. Bovine cDNA was used
to see expression in bovine. The annealing
temperature was primer specific, and the PCR
involved 35 cycles. The PCR products were analyzed
by agarose gel electrophoresis and visualized by
ethidium bromide staining.
The respective primer sets for bPL, cPL, cPRP1, and
GAPDH are listed in Table 1. PCR condition is listed
in table 2 and 3.
Table 1: Primers used for RT-PCR
Table 2: Thermal cycling conditions for RT-PCR
Table 3: Annealing temperature and extension period for the
target genes
III. RESULTS AND DISCUSSION
Expression of GAPDH Gene around Day 50, 60
and 90 days of Gestation: RT-PCR analysis
confirmed GAPDH gene expression in Placental
tissues in goat (Black Bengal goat). The band image
(Figure2) shows strong single band for all the
placental tissues; placentome, endometrium, fetal
membrane (FT) of different gestational stage (50, 60
and 90 days respectively).
Expression of cPL Gene around Day 50, 60 and 90
days of Gestation: RT-PCR analysis confirmed cPL
gene expression in placental tissues of Black Bengal

International Journal of Advances in Science Engineering and Technology, ISSN(p): 2321 –8991, ISSN(e): 2321 –9009
Volume-6, Issue-1, Jan.-2018, http://iraj.in
Expression of Placental Lactogen and Prolactein Related Protein-1 in the Placental Tissues during Gestation in Black Bengal Goat
3
goat. The band image (Figure 2) shows double band
for all the placental tissues containing a strong and a
weak band. This represents that there may have some
related gene expression during gestation in Black
Bengal goat. Endometrial tissue around 90 days of
gestation showed weak band, which may be the result
of using low concentration RNA quality. The
expression of this gene was less in fetal membrane
during all days of gestation. . This result may indicate
that, caprine Placental lactogen expresses as early as
50 days of gestation in Black Bengal goat.
Consequently plasma concentration of cPL may be
high at 50 days of gestation in placentomes which
may assist in pregnancy diagnosis in Black Bengal
goat by possible radioimmuniassay. Along with that,
there is a good scope to measure the expression of
other related gene of Placental lactogen gene family
in this goat breed.
Fig 2: Relative mRNA Expression of GAPDH, cPL, bPL, cPRP-1 for their gene specific primer in caprine placental tissues at three
gestational stages.
Expression of bPL Gene around Day 50, 60 and 90
days of Gestation: bPL is a bovine specific gene
expressed during gestation. According to Yamada et
al. 2002, the bPL mRNA is transcribed in trophoblast
binucleate cells in bovine spesies. In the maternal
circulation, bPL is detected in peripheral plasma
following day 60 of gestation [12].While using its
primer at caprine tissue DNA; it showed a relatively
weak double band. This may indicate its less
expression or expression of related gene of same gene
family in caprine tissues. This result support the
finding of [5] which states that the molecular
structure and plasma profiles of bovine placental
lactogen are very distinct from those described in the
sheep and goat. All the tissue samples of different
stages of gestation exhibit the same pattern at their
expression in band image (Figure 2) for bPL. This
may indicate that, bovine Placental lactogen
expression pattern may have some dissimilarity as in
caprine species.
Expression of cPRP-1 Gene around Day 50, 60
and 90 days of Gestation: RT-PCR analysis
confirmed cPRP-1 gene expression in placental
tissues in goat (Black Bengal goat). The band image
(Figure 2) shows strong single band for all the
placentomal tissues of different gestational stage (50,
60 and 90 days respectively). According to [10]
quantitative real-time RT-PCR analysis showed that
the cPRP1 expression intensities increased from Day
30 to Day 50 and then remained constant to Day 90
and thereafter decreased until Day 140 in placentome
which is similar to present study. Similar result was
found with bPRP-1 in bovine placental tissues in
gestation in the study of Yamada et al., 2002, which
stated that bovine prolactin-related protein I (bPRP-I)
is the only protein in the prolactin family other than
bovine placental lactogen (bPL) that has been shown
to be expressed in the bovine placenta.
On the other hand, band image shows relatively weak
band for endometrium and fetal membrane indicating
their relative low expression at different stage of
pregnancy (50, 60 and 90 days) in comparison to
placentome. The similar result was found in the
findings of [10] which states that in the caprine
intercotyledon (the area between the cotyledonary
villous and endometrium, the cPRP1 expression
intensity held steady from Day 30 to Day 90 and
thereafter decreased until Day 140, although the

International Journal of Advances in Science Engineering and Technology, ISSN(p): 2321 –8991, ISSN(e): 2321 –9009
Volume-6, Issue-1, Jan.-2018, http://iraj.in
Expression of Placental Lactogen and Prolactein Related Protein-1 in the Placental Tissues during Gestation in Black Bengal Goat
4
intensity was low compared to that in placentome.
This result indicates that, Prolactin Related Protein-1
expresses as early as 50 days of gestation in Black
Bengal goat. Consequently plasma concentration of
PRP-1 may be high at 50 days of gestation in
placentomes which may assist in pregnancy diagnosis
in BBG by possible radioimmuniassay.
CONCLUSION
The RT-PCR analysis revealed that, cPL and cPRP-
1was highly expressed in placentomes at all three
stages of pregnancy, while less expressed in
endometrium and fetal membrane which may be an
indicative of some other related gene of cPL
expression in the uterine wall and fetal membrane in
the pregnant Black Bengal does. On the other hand,
bPL gene was expressed at very low level and band
image indicated expression of some other related
gene in case of goat as bPL is a bovine specific gene
identified earlier. So it can be reported that caprine
placenta may have the expression of this two genes at
minimum 50 days of their gestational age. As a result,
cPL and cPRP-1 can be identified as candidate gene
for early pregnancy determination in Black Bengal
goat.
It may be noticed that this is the first study of gene
expression analysis in this goat breed in Bangladesh.
So, further studies are appreciated to identify more
candidate genes and molecular markers for pregnancy
detection with a view to establish an improved
method of early pregnancy diagnosis in Black Bengal
goat.
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