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Theriogenology
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a b s t r a c t
Article history:
Received 7 November 2013
Received in revised form 13 June 2014
Accepted 18 June 2014
Vascular endothelial growth factor (VEGF) is known to induce endothelial cell proliferation, to promote cell migration, and to inhibit apoptosis, thus playing a central role in
angiogenesis and in the regulation of vasculogenesis. The expression of the VEGFligand
receptor system was studied in the placenta and uterus of the collared peccary in
nonpregnant females in the luteal phase and throughout pregnancy (>35, 75, 115, and
135 days). The material was examined by immunohistochemistry and by real-time reverse
transcription polymerase chain reaction. Intense positive immunolabeling was observed
for VEGF and its receptors in the uterine epithelium, uterine glands, and trophoblast. The
endothelial cells and smooth muscle cells in the maternal and fetal vessels, as well as the
connective tissue and mesenchyme, had weak immunoreactivity during all periods of
pregnancy. The regression analysis of the real-time polymerase chain reaction results
demonstrated cubic behavior, showing a specic time-dependent prole during pregnancy, which increased over the last gestational period to VEGF and VEGFR-1. The relative
expression of VEGFR-2 decreased in the middle-third of the pregnancy and increased in
late pregnancy. In the collared peccary, the expression of the VEGFligand receptor system
was similar to that in porcine and ruminant placentas, suggesting that an epitheliochorial
placenta has the same physiological and interhemal barrier during vascular gestational
development. The expression of VEGF among cells not related to the vascular system, such
as those of the uterine epithelium, trophoblast, and uterine glands, suggests a distinct
regulatory role for these cells in vasculogenesis and also a different role of VEGF pathway.
2014 Elsevier Inc. All rights reserved.
Keywords:
VEGF
Tayassu
Placenta
Epitheliochorial
Microcirculation
1. Introduction
The collared peccary (Pecari tajacu) is a mammal that
belongs to the same order as large ruminants and to the
same family as the domestic swine and has a diffuse, folded, epitheliochorial, chorioallantoic placenta with an
areolar gland complex [1]. The peccaries are polytocous
* Corresponding author. Tel.: 55 44 3011 4919; fax: 55 44 3011 4729.
E-mail address: tcsantos@uem.br (T.C. Santos).
0093-691X/$ see front matter 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2014.06.016
835
Multiplicao de Animais Silvestres da Universidade Federal Rural do Semi-rido [CEMAS]/Ufersa, RN, Brazilian
Institute of Environment and Renewable Natural Resources
{Instituto Brasileiro do Meio Ambiente e dos Recursos
Naturais Renovveis} [IBAMA] no. 1/24/92/0040-4). This
research was approved by the Bioethics Committee of the
School of Veterinary Medicine of the University of Sao
Paulo (protocol nos.17/2002 and 362/2003) and is registered in IBAMA (no. 2001.003237/05).
Twenty animals were used in this experiment, 16
pregnant and 4 nonpregnant. The details of their animal
breeders and tissue collection are available in the report of
Santos et al., [29]. The pregnant females (n 16) were
divided into four pregnant groups according to the gestation period: 35 days (<35 days), 75 days (7080 days),
115 days (90120 days), and 135 days (>125 days). The fth
group comprised nonpregnant females (n 4), which
included females with a developed CL in at least one ovary.
Tissue samples from the uterus (nonpregnant) and
uterus placenta (pregnant) were collected in lateral wall
of uterus around mesometrium insertion and umbilical
cord area and xed by immersion or perfusion in 4%
buffered formaldehyde in 0.1 M phosphate buffer, pH 7.3,
for 24 to 48 hours. Samples were processed by standard
histological procedures before being embedded in the
paraplast and were subsequently sectioned for immunohistochemistry. Parallel tissue samples were immediately
snap-frozen in liquid nitrogen and stored at 80 C to be
used for PCR.
2.2. Immunohistochemistry
The paraplast blocks were sectioned (5 mm) using an
automatic microtome (Leica RM2155, Germany). Immunohistochemistry was performed for VEGF, VEGFR-1, and
VEGFR-2. The sections were rehydrated in an ethanol series, during the course of which they were submitted to
endogenous peroxidase blockage in 3% hydrogen peroxide
(vol/vol) in ethanol for 20 minutes. They were then placed
in 0.1 M citrate buffer at a pH of 6.0 and submitted to microwave irradiation at 700 MHz for 15 minutes. The sections were equilibrated in 0.1 M PBS at a pH of 7.4, and
nonspecic binding was blocked using Dako Protein Block
(DakoCytomation, Carpinteria, CA, USA) for 20 minutes.
Tissues were incubated with primary antibodies overnight
at 4 C in a humidied chamber. VEGF (anti-VEGFA (A-20):
sc-152), VEGFR-1 (anti-VEGFR-1 (C-17): sc-316), and
VEGFR-2 (anti-VEGFR-2 (C-20): sc-315) were detected by a
rabbit polyclonal antibody (diluted 1:300). All antibodies
were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA,
USA). The slices were then rinsed in PBS and incubated rst
with the biotinylated secondary antibody for 45 minutes
and then with streptavidinhorse radish peroxidase for
45 minutes (Universal Dako labelled streptavidin biotin
(LSAB) Kit, Peroxidase System-horse radish peroxidase,
DakoCytomation). After rinsing with PBS, binding was
visualized using diaminobenzidine as the chromogen. The
sections were counterstained with hematoxylin and
mounted. Negative controls were prepared using PBS
instead of primary antibody solution. The specicities of
the antibodies were determined by the incubation of
836
DNA polymerase; and 0.5 U AmpEraseR uracil-N-glycosylase (Applied Biosystems); 2.5 mL forward and 2.5 mL
reverse 900 mM primers (Invitrogen, Table 1); and 2.5 mL
water and 5 mL template (diluted 1:8 in nuclease-free
water) were added for a nal volume of 25 mL. All wells
were sealed with MicroAmp Optical Adhesive Covers
(Applied Biosystems), following the complete mixture of
all reagents. The amplication conditions were 2 minutes
at 50 C, 10 minutes at 95 C, 40 cycles of 15 seconds at
95 C (denaturation), and 1 minute at 60 C (annealing and
extension). For the dissociation curve, the samples ran for
15 seconds at 95 C, 1 minute at 60 C, and 15 seconds at
95 C.
The samples were analyzed in duplicate (one sample
and two PCR repeated on the same extracted sample), and
the expression of target genes was determined by relative
quantication with linear regression of the uorescence
data obtained by the real-time PCR. The relative expression
was determined by the ratio of the target gene and the
constitutive gene (GAPDH) by the following formula: relative expression N0 (target gene)/N0 (GAPDH). N0 values
were calculated in LinRegPCR 7.0 (linear regression PCR)
program [30].
We considered all of the samples that were obtained
with an accuracy of 1.8 and a correlation coefcient (R2)
between 0.9 and 1.0. The real-time PCR products were
transferred to an acrylamide gel (8%) and generated bands
consistent with the determined size of the amplicon
(Table 1).
2.5. Statistical analysis
The means obtained in each animal group were tested
for normality. The percentage of positive cells in the
immunohistochemical analysis were classied by score and
analyzed by the PROC GEN MOD of SAS [31] using Poisson
distribution. The difference between the percentage of cells
in each score (1 to 4) of each cell type (uterine epithelium,
uterine glands, and trophoblast) between the nonpregnant
and pregnant groups was analyzed by a contrast test for
each factor (VEGF, VEGFR-1, and VEGFR-2) and by t-test for
pregnant groups.
The results of the relative expression of the real-time
RT-PCR between pregnant and nonpregnant subjects
were compared using Dunnetts test. The relative means of
expression, assessed by real-time PCR, among the pregnant
groups were analyzed by regression analysis and the Tukey
test using PROC GLM of SAS.
Table 1
Oligonucleotide primers for swine VEGF, VEGFR-1, VEGFR-2, and GAPDH genes used in the real-time PCR assay in collared peccary.
Target genes
Oligonucleotide primers
Amplicon length
VEGF
50
50
50
50
50
50
50
50
71
75
75
82
VEGFR-1
VEGFR-2
GAPDH
TCGAGACCCTGGTGGACATC
CACACAGGACGGCTTGAAGA
TGAAGGAGGGCGTGAGGAT
GCCAACAGTCCAACATGATCTG
CGAGTGGAGGTGACAGATTGC
CCGATCACTTTTGGAATTGTGA
TCCCCACCCCCAACGT
TGTCATCATATTTTGCAGGTTTCTC
837
Table 2
Percentage of cells sorted by score in the immunolocalization of VEGF, VEGFR-1, and VEGFR-2 in the uterine epithelium, the glandular epithelium, and the
trophoblast of the placenta and uterus in collared peccary.
Scored
Group
VEGF
NP
35 d
75 d
115 d
135 d
VEGFR-1
NP
35 d
75 d
115 d
135 d
VEGFR-2
NP
35 d
75 d
115 d
135 d
% Uterine epithelium
% Uterine glands
% Trophoblast
1 ()
2 ()
3 ()
4 ()
1 ()
2 ()
3 ()
4 ()
1 ()
2 ()
3 ()
4 ()
05.83
12.44ea
00.22d
04.33eb
02.58ec
21.83
30.11ea
03.56ec
12.25eb
10.58eb
62.67
45.44ea
01.67ed
24.83ec
33.58eb
09.67
12.00c
94.56ea
58.58eb
53.25eb
00.00
00.22c
00.89ec
02.17eb
04.33ea
11.92
06.33ec
02.56ed
10.17b
13.17a
83.67
29.11ea
01.78ec
22.25eb
29.92ea
04.42
64.33eb
94.78ea
65.42eb
52.58ec
d
00.00
00.00
00.50
01.17
d
02.00c
00.56d
06.25a
04.17b
d
27.33b
00.00d
36.50a
14.75c
d
70.67c
99.44a
56.75d
79.92b
03.67
07.56ea
00.00e
01.83eb
01.42eb
14.42
26.33ea
01.33ec
08.42eb
09.25eb
76.75
52.89ea
05.67ec
17.67ec
19.83ec
05.17
13.22ec
93.00ea
72.08eb
69.50eb
00.25
00.78b
00.56b
03.67ea
03.00ea
03.42
04.11b
02.78b
12.75ea
11.08ea
88.33
30.33ea
03.89eb
26.08ea
32.92ea
08.00
64.78eb
92.78ea
57.50ec
53.00ec
d
00.83b
00.00c
02.25a
01.17b
d
06.92b
00.00c
11.17a
09.08ab
d
35.83c
66.67a
53.17b
53.25b
d
56.42a
33.33b
33.42b
36.50b
06.08
08.44ea
00.44ec
02.83eb
02.58eb
19.33
24.22ea
01.00ed
05.33ec
10.58eb
65.75
40.44ea
07.56ed
12.92ec
27.67eb
08.83
26.89ed
91.00ea
78.92eb
59.17ec
00.42
02.55eab
01.33eb
03.58ea
01.58eb
05.75
13.22ea
04.89b
06.58b
10.50ea
90.67
25.23ea
08.89eb
26.59ea
25.50ea
03.17
59.00eb
84.89ea
63.25eb
62.42eb
d
00.41
00.00
01.91
00.25
d
01.58b
00.00c
04.75a
03.50a
d
07.34c
01.00d
19.25a
12.83a
d
90.67ab
99.00a
74.09b
83.42b
a-c
Means in the same column with different superscripts differ by t test (P < 0.05).
Score: 1 negative, 2 positive weak, 3 positive medium, 4 positive strong; NP nonpregnant.
e
Mean difference compared with group NP by contrast test (P < 0.05).
d
3. Results
Collared peccaries females were studied in the luteal
phase and through pregnancy. VEGF and its receptors,
VEGFR-1 and VEGFR-2, were studied in the uterus and
placenta by immunohistochemistry (Table 2) and real-time
RT-PCR. Immunohistochemistry demonstrated that both
VEGF and its receptors showed a positive reaction in the
uterine and glandular epithelia and in the trophoblast of
both nonpregnant females and throughout pregnancy
(Figs. 14).
Fig. 1. Immunohistochemical localization of (A,D) VEGF, (B,E) VEGFR-1, and (C,F) VEGFR-2 in the uterus and placenta of (AC) nonpregnant and (DF) early
pregnant (35 days) collared peccaries. In the nonpregnant uterus, the uterine epithelium has cells with medium and strong staining. Approximately 35 days the
allantochorion is attached onto the uterine mucosa, and both the trophoblast (tro) and uterine epithelium (ep) are positive for VEGF and its two receptors.
Positive immunolabeling is also observed in uterine glandular cells (gla) and in the smooth muscle cells of vessels. Fetal vessels (fv). Bars: 40 mm.
838
Fig. 2. Immunohistochemical localization of (AB) VEGF, (CD) VEGFR-1, (EF) VEGFR-2, and the (G) negative control in placentas of a collared peccary (AD; G: 115 days; E
F: 75 days). Note positive immunostaining (brown) for the VEGF-ligand receptor system for trophoblast (tro) and uterine epithelium (ep). Mesenchymal cells (mes) and some
connective cells in maternal side were also positive. In the uterine glands (gla), strong positive immunostaining into cytoplasm is visible and also some negative cells (blue).
The vascular endothelium was positive in the fetal (**) and maternal (*) vessels (head arrow). (G) In the negative control primary antibody was omitted and replaced by PBS
and displayed no immunostaining. Bars: 40 mm. (For interpretation of the references to color in this gure, the reader is referred to the web version of this article.)
(P < 0.05) of VEGF for all periods and scores tested. However, the percentages of cells with a score of 4 in
nonpregnant females or in females after 35 days of pregnancy showed no differences.
In the uterine glands, the groups at 115 and 135 days of
pregnancy were negative or weakly positive to VEGFR-1 in
greater numbers than other groups, compared with
nonpregnant subjects.
In the trophoblast, it was noted that VEGF and VEGFR-2
were strongly positive throughout the pregnancy. VEGFR-1
was positive, having the highest percentage of cells with
score 4 in early pregnancy (56.42%), whereas during other
periods of pregnancy, approximately 60% of cells received a
score of 3 (moderate).
The VEGF system mRNA transcripts were quantied and
detected in all of the females studied, and the relative
expression results are shown in Table 3. The results showed
that the means for the relative expression of VEGF and
VEGFR-1 did not signicantly differ between the
nonpregnant females and those at 35, 75, and 115 days of
pregnancy; the expression levels only differed after
135 days of pregnancy (P < 0.05). With respect to VEGFR-2,
there were no signicant differences between the mean
relative expression of the nonpregnant group (in the luteal
phase) and the pregnant groups studied.
When the relative expression of VEGF and its receptors
was examined by regression analysis among the groups
during pregnancy, and the levels of mRNA for VEGF
839
Fig. 3. Immunohistochemical localization of (A) VEGF, (B) VEGFR-1, (C) VEGFR-2, and (D) the negative control in placentas of a collared peccary in late pregnancy
(135 days). The trophoblast (tro) and uterine epithelium (ep) are strongly positive for the VEGFligand receptor system. The capillary endothelium had strong
immunostaining in the maternal (mc) and fetal capillaries (fc). Bars: 40 mm.
4. Discussion
The relative expression of VEGF and its receptors have
here been studied in placentas throughout pregnancy in
collared peccaries as well as in nonpregnant animals.
Metabolic demand is large in the placenta during pregnancy. The transfer of nutrients across the placenta can
occur by simple diffusion, facilitated diffusion, active
transport, and receptor-mediated endocytosis [32]. All of
these pathways are inuenced by the surface area available
for exchange [33].
From the start of the apposition of the trophoblast onto
the uterine epithelium, proliferation factors are involved.
The placenta of the peccaries, similar to that of other suiform, is epitheliochorial and lacks trophoblast invasion at
the uterine tissue. In the collared peccary, both the uterine
mucosa and the allantochorionic membrane form complementary folds, which increase throughout pregnancy in
height and complexity to provide an increase in the contact
area for exchange between maternal and fetal tissues
840
Fig. 4. Immunohistochemical localization of (A) VEGF, (B) VEGFR-1, and (C) VEGFR-2 of the uteri of the collared peccary in late pregnancy (135 days). Strongly
positive glandular cells (gla) are evident with some negative cells (*). The smooth muscle cells (arrows) and endothelial cells (head arrows) of vessels had strong
positive immunostaining of the VEGFligand receptor system. Bars: 40 mm.
vascular development is critical to the growth and maintenance of the placenta. In pigs, from Day 1 to Day 12 of
pregnancy, there is an increase in the mRNA for VEGF and
its receptors [37], which remains high throughout the
pregnancy [18]. In the present study, the VEGFligand receptor system of the collared peccary showed immunoreactivity at Day 35 in the uterine and placental cells and
were expressed in placental tissues, as assessed by real
time RT-PCR. The peccaries have longer pregnancies than
pigs, requiring about 146 days to produce two offspring
[2,38]. Despite differences, the morphological aspects of
the placenta are very similar between species and a proportional analysis throughout pregnancy reduces them.
Immunohistochemistry in this study demonstrated an
expression in maternal epithelial cells, trophoblastic cells,
and the uterine glands for VEGF, VEGFR-1, and VEGFR-2.
This result has been observed in porcine placentas [16,18].
Charnock-Jones et al. [17] also identied VEGF in these cells
by immunohistochemistry; however, when tested by in situ
hybridization, the uterine glands showed a weaker reaction
compared with immunohistochemical reactivity.
In our study, there was a pronounced reduction in the
relative expression of VEGFR-2 around Days 75 and 115 of
pregnancy, which was much lower compared with other
Table 3
Relative expression of the VEGF, VEGFR-1, and VEGFR-2 (means SE) observed after analysis in LinRegPCR 7.0 program (Linear regression PCR).
Group
Relative expression
No pregnant
35 d
75 d
115 d
135 d
Regression
P value
0.0114
0.0574
0.0179
0.0194
0.3635
Cubicd
0.005
VEGF
a-b
VEGFR-1
0.030
0.013b
0.005b
0.014b
0.076ac
0.0039
0.0127
0.0082
0.0057
0.0375
Cubice
0.014
VEGFR-2
0.0013
0.0083b
0.0021b
0.0015b
0.0068ac
0.002028
0.002718
0.000722
0.000042
0.001863
Quadraticf
0.003
0.0009
0.00052a
0.00006b
0.00001b
0.00086a
841
Fig. 5. Regression analysis of the relative mRNA expressions of VEGF, VEGFR-1, and VEGFR-2 (/GAPDH)) in collared peccary placenta tissue during pregnancy.
VEGF 0.6998 0.0394x 0.0006x2 0.000003x3 (R2: 0.77); VEGFR-1 0.0631 0.004x 0.00006x2 0.00000027x3 (R2: 0.63); and VEGFR-2 0.0072
0.00016x 0.0000008x2 (R2: 0.53).
842
Acknowledgments
The authors thank the Wild Animal Multiplication
Center from UFERSA for the animals. This research was
supported by grants from the Fundao de Amparo a Pesquisa do Estado de So Paulo (FAPESP) (Processo n 02/
13946-5).
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