Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
380
STUDY OF IMMUNOHISTOCHEMICAL TECHNIQUES FOR
PLACENTA IN PLACENTA
Ruah F. Al-mayahi*; Fawzi S. AL-Asadi**
* Department of Anatomy and Histology, College of Veterinary Medicine,University
of Basrah,Basrah,Iraq
Keywords: Placenta, Iummunohistochemical, sheep
ABSTRACT
The present study which include the demonstration of estrogen and progesterone
receptors and demonstration of CD34 protein in placenta of pregnant sheep. Twenty
one placenta from pregnant sheep were used in the present study. The
immunohistochemical study of CD34 protein as marker for vascularization appear at
day (27) few distribution in stroma of villi While at day (50) of gestation high density
of CD34 protein in And also showed at day (120) high density. The estrogen receptor
showed colour by kit at day (27) normal distribution in trophoblast cell of villus
epithelium and not present in binucleate cell While at day (50) high density of
estrogen receptor was showed ,And also at day (120) show high density of estrogen.
The immunohistochemical study of progesterone receptor as appear at day (27)
normal distribution in trophoblast cell of placentome and not present in binucleate cell
While at day (50) high density, and also at day (120) .
INTRODUCTION
The bovine placental has discrete regions where is co-development of
both maternal (caruncular) and fetal (cotyledonary) tissue . It is through these
regions, known as placentomes that the fetus obtains oxygen and nutrient and
excretes waste products, this
placentome mass to the development of the fetus has been demonstrated by
carunclectomy of sheep (1). (2) showed the placental growth in sheep by
measuring the diameter of cotyledons from early to mid-gestation. He reported
that nutrient restriction up to 90 days of gestation did not affect placental
growth , but that there was a pronounced effect on placental growth when
animals were nutrient – restricted later in gestation. Taken together, such data
show that the size of the placenta directly affects its capacity to transfer
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
381
nutrients which can influence the growth rate of the fetus and thus birth weight
(3).
The protective role of the placenta as a barrier and for removal of end products
of metabolism is vital as the fetal hepatic and renal systems have immature and
insufficient metabolic and excretory capacity (4). Pregnancy is characterized by
dynamic changes in multiple body systems resulting in increased basal oxygen
consumption and changes in energy substrate use by different organs including the
fetoplacental unit (5).
The shape of the placenta is variable due to the placental localization, so that
three shapes of placentomes in ruminants : convex, flat and concave. The convex
placentome is amushroom-shaped texture with a distinguish endometrial stalk, the flat
placentome has a flatter shaped less convex for both of these placentomes fetal
chorion coat the surface of the placentome. In contrast, in concave placentomes,
maternal tissue surrounds the fetal tissue (6). In sheep and goats, concave
placentomes general (7; 8),while in cattle convex placentomes general beside afew
number of flat ones(9). The production of estrogen is contrast to that of progesterone,
requires interaction between the fetus and the placenta ( 10). The formation of the
placenta is regulated by hormon (11) , cytokines, growth factors and substrate that are
present in the maternal and fetal circulation and bind to specific receptors on the
placental surface (12,13,14)
MATERIALS AND METHODS
Twenty one placenta from pregnant sheep were collected from the local
slaughterhouse of Basra city at ages of (27, 50, 120 ) day . Fine dissected to the
uterus of pregnant sheep were removed and open the uterus and remove the fetal ,
then the embryo age were determines by messurement the length of fetus by using an
electronic vernia , then removed number of placentomes at randomly by cutting from
its location and processing for the following morphological, histological,
Histochemical and immuonohistochemical studies. This work dwas done in laboratory
of veterinary medicine college in basra.
Histological study for preparation section :
For the preparation of histological sections, was processed according
to the following steps.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
382
Fixation, Dehydration, Clearing, Infiltration and Embedding, Trimming and
Sectioning at (4- 6) μm, Mounting: coated with mayers albumin, Staining.
Immunohistochemical study:
Estrogen receptor:
Demonstration of estrogen in placentome was done according to method (DAKO
company kit (2010)). The paraffin sections at 4um were cut(use chargeable slides ),
then de wax the slides in ovin at 56˚C for 30min , the slides were put in xyline two
jars 2min for each jar ,to Avoid slides dryness ,de hydration immediately with (100%
, 90% , 80 %, 70 %, 60 % ) ethanol alcohol then wash with (TBS) tris buffered saline ,
then the slides immerge in antigens retrieval solution target solution high PH from
DAKO for 20min at 121˚C under pressure and leave slides to cool at room
temperature with still in target solution , wash slides with buffered for 5min then
Incubate slides with hydrogen peroxide H2O2 for 5min and washing with buffered for
5min ,Incubate the slides with primary antibody dilution 1/60 for 30min from
(DAKO company ) then washing with buffered for 5min , Incubate slides with
secondary antibody one step (invasion secondary antibody ) from (DAKO company )
then wash with buffered for 5min, Then incubate slides with chromogen DAB (1-
5)min 1.5ml from dab buffered and 20um from chromogen dab , Wash slides with
buffered for 5min then stain slides with mayers haematoxylin for 5min and Wash with
buffered then distill water , covering via aqueous mounting medium and incubation
period 30min.
Brogesterone receptor:
Demonstration of estrogen in placentome was done according to method
(DAKO company,2010 ). The paraffin sections were cut at 4um(use chargeable slides
), then de wax the slides in oven at 56˚C for 30min , then put the slides in xyline two
jars 2min for each jar ,Avoid slides dryness …do hydration immediately with (100% ,
90% , 80 %, 70 %, 60 % ) ethanol alcohol then wash with (TBS) tries buffered saline ,
put the slides in antigens retrieval solution target solution high PH from DAKO for
20min at 121˚C under pressure and leave slides to cool at room temperature with still
in target solution , washing slides with buffered for 5min then Incubate slides with
hydrogen peroxide H2O2 for 5min then wash with buffered for 5min ,Incubate slides
with primary antibody dilution 1/50for 30min from (DAKO company) then wash with
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
383
buffered for 5min , Incubate slides with secondary antibody one step (invasion
secondary antibody) from (DAKO company) then wash with buffered for 5min, Then
incubate slides with chromogen DAB (1-5)min 1.5ml from dab buffered and 20um
from chromogen dab , Wash slides with buffered for 5min then stain slides with
mayers haematoxylin for 5min and Wash with buffered then distill water , covering
via aqueous mounting medium and incubation period 30min.
CD34 protein:
Demonstration of CD34 protein in placentome was done according to method
(DAKO company,2010 ), cut the paraffin sections at 4um(use chargeable slides), then
de wax the slides in ovin at 56˚Cfor 30min , the put the slides in xyline two jars 2min
for each jar ,Avoid slides dryness …do hydration immediately with (100% , 90% , 80
%, 70 %, 60 % ) ethanol alcohol then wash with (TBS) tries buffered saline , put the
slides in antigens retrieval solution target solution high PH from DAKO for 20min at
121˚C under pressure and leave slides to cool at room temperature with still in target
solution , wash slides with buffered for 5min then Incubate slides with hydrogen
peroxide H2O2 for 5min then wash with buffered for 5min ,Incubate slides with
primary antibody dilution 1/100 for 30min from (DAKO company ) then wash with
buffered for 5min , Incubate slides with secondary antibody one step (invasion
secondary antibody) from (DAKO company ) then wash with buffered for 5min, Then
incubate slides with chromogen DAB (1-5) min 1.5ml from dab buffered and 20um
from chromogen dab , Wash slides with buffered for 5min then stain slides with
mayers haematoxylin for 5min and Wash with buffered then distill water, covering
via aqueous mounting medium and incubation period 30min.
Result:
Estrogen Receptor:
The immunohistochemical study of estrogen receptor showed by brown colour
by kit (DAKO company ) at day (27) normal distribution in trophoblast cell of villus
epithelium and not present in binucleate cell (Figure.1).While showed at day (50) high
density of estrogen receptor in trophoblast cell of villus epithelium and not present in
binucleate cell compared to day (27) (Fig. 2) .And also at day (120) show high
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
384
density of estrogen receptor in trophoblast cell of villus epithelium and not present in
binucleate cell compared to day (50) (Fig .3).
Progesterone Receptor :
The immunohistochemical study of progesterone receptor as shown by brown
colour by kit (DAKO company ) appear at day (27) normal distribution in trophoblast
cell of placentome and not present in binucleate cell (Fig.4). While showed at day
(50) high density of progesterone receptor in trophoblast cell of placentomes and not
present in binucleate cell compared to day (27) (Fig. 5). And also at day (120)
showed high density of progesterone receptor in trophoblast cell and not present in
binucleate cell compared to day (50) (Fig. 6).
CD34 Antigen Receptor:
The immunohistochemical study of CD34 protein as marker for vascularization
showed by brown colour by kit (DAKO company ) appear at day (27) few distribution
in stroma of villi (Fig.7). While showed at day (50) of gestation high density of CD34
protein in stromal villi compared to day (27) ( Fig.8). And also showed at day (120)
high density of CD34 protein in stromal villi compared to day (50) ( Fig.9).
Fig.( 2): Cross section of sheep
placentom show: 1. Estrogen receptor (
)(brown color) at day 50 2.Binucleated
cell (A) (estrogen antibody 400x)
AA
Fig.(1): Cross section of sheep placentom
show: 1. Estrogen receptor ( )(brown
color) at day 27 2. Binucleated cell (A)
estrogen antibody 400x )
A
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
385
A
Fig.( 6): Cross section of sheep placentome
show:1. Progesterone receptor( )at day
120 2. Binucleate cell (A) (progesterone
antibody 400x).
Fig.( 7): Cross section of sheep placentomes
show: Vascularization
( ) at day 27 ( CD34 400x) .
Fig.( 5): Cross section of sheep placentome
show:1. Progesterone receptor( )at day
50 2. Binucleate cell (A) (progesterone
antibody 400x).
A
Fig. (8): Cross section of sheep placentomes
show: Vascularization ( ) at day50
(CD34 400X).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
386
DISCUSSION
The present study showed that CD34 protein appeared as few distribution in
stroma of villi at day 27 in placenta while at day 50 of gestation showed high density
and also at day 120 higher density as compared to day 50 of gestation in sheep
placenta . CD34 is a marker that allow to appreciate the vascular density and it is
strongly correlated with angiogenesis (15) in human. The present study was agree
with results of (16) who noticed that embryonic capillaries in the chorionic villous
appeared between day 18 and 20 post- conception.
(17) found the immunostaining for CD34 in the endothelial cells in both cyto
and synsytiotrophoblast and all villous and also the fetal blood vessels were uniformly
distributed though out the chorionic villi in human (18)). (19) noticed that there were
interaction between trophoblast and vascular cells of the spiral arteries. The
angiogenesis is regulates by vascular endothelial growth factor, placental growth
factor and angiopoietins as well as protease such as the membrane –type matrix
metallo- proteinase (20) in human. The present study showed that estrogen receptor in
sheep placenta showed normal distribution at day 27 of gestation while at day 50 was
high density and also at day 120 showed higher density than at day 50 of gestation.
the present study show that progesterone receptor in sheep placenta showed
normal density at day 27 of gestation while increase in density in day 50 and day 120
of gestation. these result may be contributed to the lack of estrogen and progesterone
in maternal tissue. These result was agree with result of (21; 22,23)in rat,monkey and
human respectively .
(24), (25) noticed that bovine placentomes of progesterone localized into
nuclei of caruncular stromal cell and caruncular vascular pericytes suggesting that
these cells are rather under the control of placental than luteal progesterone. Also
(26) noticed that estrogen receptor blocker in pregnant cow had no effect on calving
process . Estrogen induce and maintain female secondary gland and stimulate the
Fig. (9): Cross section of sheep
placentomes show: Vascularization ( )
at day120 (CD34 400X).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
387
development of the endometrial lining (27) while (28) in mice noticed that estrogen
function was to stimulated proliferation luminal and glandular epithelium and increase
expression of progesterone receptor. The placental androgen are considered as
precursors for placental estrogen synthesis so that considerable levels of androgen
may also present in the uterine environment during pregnancy (29,30,31,32,33,) in
human, pig and rat respectively
دراسة الکیمیاء المناعیة النسیجیة للمشیمة فی الاغنام
فوزی صدام الاسدی رؤى فاضل المیاحی
فرع التشریح والانسجھ کلیة الطب البیطری، جامعة البصرة، البصره،العراق
الخلاصة
روتین تیرون و ب تروجین والبروجس تقبلات الاس ن مس ف ع ة الکش ة الحالی منت الدراس تض
ة ة المناعی رت الدراس ل. أظھ ام الحوام یمة للاغن رین مش د وعش ة واح ذه الدراس ی ھ تخدمت ف اس .CD3
ل ن الحم وم م ر ( 50 ) ی ا بعم وم بینم ر ( 27 ) ی ة بعم ب الزغاب ی ل ئیل ف انتشار ض CD النسیجیة لبروتین 34
کثافة عالیة وازداد بعمر ( 120 ) یوم .
ی أظھرت الدراسات الکیمیاء المناعیة النسیجیة لمستقبلات الاستروجین بعمر ( 27 ) یوم انتشار طبیعی ف
ا ی خلای ة ف ة عالی ت الکثاف وم کان ر( 50 ) ی خلایا التروفوبلاست وعدم وجودھا فی الخلایا ثنائیة النواة بینما بعم
ی ة ف ة عالی رت کثاف وم أظھ ر ( 120 ) ی ا بعم واة بینم التروفوبلاست وکذلک عدم وجودھا فی الخلایا الثنائیة الن
تقبلات یجیة لمس ة النس ات المناعی ا الدراس واة . أم ة الن ا الثنائی ی الخلای ا ف دم وجودھ ت وع ا التروفوبلاس خلای
ة ا ثنائی ی خلای ا ف دم وجودھ ت وع ا التروفوبلاس البروجسترون أظھرت بعمر ( 27 ) یوم توزیع طبیعی فی خلای
واة ة الن ا ثنائی ی الخلای النواة بنما بعمر ( 50 ) یوم کثافة عالیة من المستقبلات فی التروفوبلاست وعدم وجودھا ف
ی ا ف بینما بعمر ( 120 ) یوم أظھرت کثافة عالیة من البروجسترون فی خلایا التروفوبلاست وعدم وجودھا ایظ
خلایا ثنائیة النواة
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