animal
Animal (2015), 9:11, pp 1820–1831 © The Animal Consortium 2015
doi:10.1017/S1751731115001020
Expression of the orexin system in the porcine uterus, conceptus
and trophoblast during early pregnancy
N. Smolinska†, M. Kiezun, K. Dobrzyn, K. Szeszko, A. Maleszka and T. Kaminski
Department of Animal Physiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A, 10-719 Olsztyn-Kortowo, Poland
(Received 12 January 2015; Accepted 27 April 2015; First published online 2 July 2015)
Orexin A and B are hypothalamic peptides derived from the prepro-orexin (PPO) precursor. Orexins stimulate food intake
and arousal. Those peptides bind and activate two G protein-coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2
(OX2R). Numerous authors have suggested that orexins play an important role in the regulation of the reproductive functions.
The objective of the present study was to analyse the presence of and changes in the gene and protein expression pattern
of the orexin system in the porcine uterus, conceptus and trophoblast (chorioallantois) during early pregnancy. In the
endometrium, the highest PPO and OX1R gene expression was detected on days 15 to 16 of gestation. The OX2R mRNA
content in the endometrium was higher on days 10 to 11 and 15 to 16 than on days 12 to 13 and 27 to 28. In the trophoblasts,
PPO gene expression was higher on days 30 to 32 than on days 27 to 28. The highest PPO protein content in the endometrium
was noted on days 12 to 13. The highest OX1R protein content in the endometrium was detected on days 10 to 11, whereas
OX2R protein on days 15 to 16. In the trophoblasts, PPO and OX1R protein levels were more pronounced on days 27 to 28
than on days 30 to 32, but OX2R expression was higher on days 30 to 32. The expression of PPO, OX1R and OX2R was
different in the conceptuses and trophoblasts during early pregnancy. Local orexin production and the presence of the specific
orexin receptors suggest that the orexin system may participate in the control of porcine reproductive functions by exerting
endocrine and auto/paracrine effects on the uterus, conceptuses and trophoblasts during early pregnancy. This study provides
the first evidence for the presence of orexins and their receptors in the uteri, conceptuses and trophoblasts in pigs during early
pregnancy. The local orexin system is dependent on the stage of pregnancy.
Keywords: orexin A and B, orexin receptors 1 and 2, uterus, pregnancy, pig
Implications
Introduction
There is a growing body of evidence to implicate the
existence of a common endocrine system that controls
metabolism and the reproductive system. Orexin A and
orexin B, the hormones that control energy homoeostasis,
may be a part of the above system. This paper provides the
pioneering information about the presence of orexins and
orexin receptors in the porcine uterus, conceptus and
trophoblast during early pregnancy, the crucial period for
reproductive success. The expression levels of orexins and
their receptors were found to be correlated with the stages of
early pregnancy. A sound knowledge of mechanisms that
control energy homoeostasis and reproduction is needed in
animal breeding practice.
Orexin A (OXA) and B (OXB), also known as hypocretin 1
and 2, are hypothalamic neuropeptides isolated by two independent groups of investigators in 1998 (De Lecea et al., 1998;
Sakurai et al., 1998). Orexins are derived by proteolytic
cleavage from prepro-orexin (PPO), a common 130-amino acid
precursor that is synthesised mainly in neurons inside and in
the region of the lateral and posterior hypothalamus. OXA is a
33-amino-acid peptide of ~3.5 kDa with an N-terminal
pyroglutamyl residue and C-terminal amidation. OXB is a
28-amino-acid peptide of ~2.9 kDa with C-terminal amidation,
which is 46% identical in sequence to OXA. The biological
actions of those hormones are mediated by two closely related
G-protein-coupled receptors: orexin receptor type 1 (OX1R) and
orexin receptor type 2 (OX2R). OX1R is selective for OXA,
whereas OX2R binds both OXA and OXB with similar affinity
(De Lecea et al., 1998; Sakurai et al., 1998).
†
E-mail: [email protected]
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Expression of the orexins in the porcine uterus
The orexins were originally identified as regulators of food
intake and energy homoeostasis (Sakurai et al., 1998) and
later described as key modulators of the sleep–wake cycle
and arousal (Chemelli et al., 1999). Multiple lines of evidence
have also demonstrated that orexins participate in the control of different endocrine axes, including the hypothalamic–
pituitary–ovarian axis (Silveyra et al., 2007a and 2007b;
Kaminski et al., 2010a and 2010b; Nitkiewicz et al., 2010;
Maleszka et al., 2013; Smolinska et al., 2014b). Local orexin
production (Nitkiewicz et al., 2014) and the presence of
specific receptors in porcine and rat ovaries (Silveyra et al.,
2007a; Nitkiewicz et al., 2010) indicate that orexins could
also exert direct endocrine and auto/paracrine effects inside
glands. OXA was found in human and feline placenta, OX1R
protein was identified in feline placenta (in syncytiotrophoblast and decidual cells derived from the endometrium),
whereas the presence of OX2R protein was noted only in
cells forming the endometrial glands (Nakabayashi et al.,
2003; Dall'Aglio et al., 2012).
In view of our previous results, which demonstrated the
presence of orexins in the porcine uterus during the oestrous
cycle (Nitkiewicz et al., 2012) and the general scarcity of
research investigating the role of orexins in the uterus, the
aim of this study was to compare the expression levels of:
(1) PPO, OX1R and OX2R genes by quantitative real-time
PCR and (2) PPO, OX1R and OX2R proteins determined by
Western blotting in the endometrium and myometrium in the
mid-luteal phase of the oestrous cycle and during the early
stage of pregnancy from day 10 to 32 after insemination,
across implantation and placentation, and in the conceptuses and trophoblasts (chorioallantois) during early
pregnancy. Proteins of orexin system in the porcine
endometrium and myometrium were localised by fluorescent
immunohistochemistry.
Material and methods
Experimental animals
The experiments were carried out in accordance with the
ethical standards of the Animal Ethics Committee at the
University of Warmia and Mazury in Olsztyn. Thirty mature
gilts (Large White × Polish Landrace; 7 to 8 months of age,
BW of 120 to 130 kg) obtained from private breeding were
used in the study. Females were monitored daily for oestrus
behaviour in the presence of an intact boar. The day of onset
of the second oestrus was designated as day 0 of the
oestrous cycle. The phase of the oestrous cycle was also
confirmed on the basis of the morphology of the ovaries.
Insemination was performed on days 1 to 2 of the oestrous
cycle and the latter arbitrarily was designated as day 0 of
gestation. The gilts were assigned to one of six experimental
groups (n = 5 per group) as follows: days 10 to 11 of the
oestrous cycle (the mid-luteal phase connected with period
of fully active corpora lutea corresponding to the activity of
corpora lutea during pregnancy), days (after insemination)
10 to 11 (migration of the embryos to and within the uterus)
and 12 to 13 (just before implantation, the maternal
recognition of pregnancy), 15 to 16 (implantation), 27 to 28
(the end of implantation) and 30 to 32 (placentation) of
pregnancy. The endometrium (from implantation site) and
myometrium was collected from the uterus. Pregnancy was
confirmed by the presence of conceptuses. On days 10 to 11
and 12 to 13 of pregnancy, the uterine horns were flushed
with 20 ml of sterile phosphate buffered saline (PBS) to
recover conceptuses. On days 15 to 16 of pregnancy, whole
conceptus connected with fragments of trophoblast was
dissected from the endometrium. Dissection of trophoblast
tissues from conceptuses was done only on days 27 to 28
and 30 to 32 of pregnancy; therefore the expression of PPO
and its receptors genes and protein, and the levels of
OXA and OXB proteins were analysed in 15- to 16-day
conceptuses connected with fragments of trophoblast, in
27- to 32-day conceptuses and in 27- to 32-day trophoblast
tissues (chorioallantois). All samples harvested from the
endometrium, myometrium, conceptuses, trophoblast and
lateral hypothalamic area (LHA) were collected within several
minutes after slaughter and snap-frozen in liquid nitrogen
and stored at −80°C until further analysis.
Total RNA isolation, cDNA synthesis and quantitative realtime PCR
Total RNA isolation and cDNA synthesis was performed as
described previously by Smolinska et al. (2014a). Specific
primer pairs used to amplify parts of PPO, OX1R, OX2R,
cyclophilin and GAPDH (glyceraldehyde 3-phosphate
dehydrogenase) genes are detailed in Table 1. Quantitative
real-time PCR analysis was carried out using a PCR System
7300 (Applied Biosystems, Grand Island, NY, USA), as
described previously (Smolinska et al., 2014a). Briefly, the
PCR reaction included 20 ng cDNA, the appropriate forward
and reverse primer at various concentrations (Table 1),
12.5 μl SYBR Green PCR Master Mix (Applied Biosystems),
and RNase free water in a final volume of 25 μl. The details
concerning primers used in the study are described in
Table 1. The constitutively expressed genes, cyclophilin and
GAPDH, were used as the internal control to verify the
quantitative real-time PCR. During the preliminary experiments, it was found that expression of cyclophilin and
GAPDH was very similar in the endometrium and myometrium and was stable during the pregnancy and between
days 10 to 11 of the cycle and all days of pregnancy. Realtime PCR cycling conditions were as follows: initial denaturation and enzyme activation at 95°C for 10 min, followed
by 40 cycles of denaturation at 95°C for 15 s, annealing at
59°C for 1 min. Negative controls were performed in which
cDNA was substituted for water, or reverse transcription was
not performed prior to PCR. All samples were amplified in
duplicate. The specificity of amplification was tested at the
end of the PCR by melting-curve analysis. Product purity was
confirmed by electrophoresis. Calculation of the relative
expression level of PPO, OX1R and OX2R genes was conducted based on the comparative cycle threshold method
(ΔΔCT) and normalised using the geometrical means of
reference gene expression levels: GAPDH and cyclophilin.
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Smolinska, Kiezun, Dobrzyn, Szeszko, Maleszka and Kaminski
PCR-amplified DNA was electrophoresed on 1.5% agarose in
Tris-acetate buffer. After isolation from gel, DNA was
sequenced (ABI PrismTM BigDyeTM Terminator Cycle
Sequencing kit, ABI Prism 3777 DNA sequencer, Applied Biosystems) in both directions to confirm the accuracy of amplification. Comparison of the PCR-amplified DNA sequences to
those in the database indicated 100% (PPO) and 99% (OX1R,
OX2R) homology at the nucleotide level.
Western blotting
Western blotting analysis was performed essentially as
described by Smolinska et al. (2007). Briefly, equal amounts
of porcine uterine lysates (endometrium and myometrium
parts separately, 10 µg of total proteins) were resolved by
SDS–PAGE (12.5% gel) for separating PPO, OX1R, OX2R and
actin, and then transferred to nitrocellulose membranes
(Whatman, Piscataway, NJ, USA). Membranes were blocked
for 5 h at 4°C in Tris-buffered saline Tween-20 containing 5%
skimmed milk powder, then incubated overnight at 4°C with
rabbit polyclonal antibodies to PPO at a dilution of 1:250
(Merck Millipore, Billerica, MA, USA), sheep polyclonal
antibodies to OX1R at a dilution of 1 : 100 (Abcam,
Cambridge, MA, USA), mouse polyclonal antibodies to OX2R
at a dilution of 1 : 300 (Abcam), or rabbit polyclonal antibodies to actin diluted 1 : 200 (Sigma-Aldrich, Saint Louis,
MO, USA), which were used as a control for equal loading as
well as to quantify porcine PPO, OX1R and OX2R proteins. To
identify immunoreactive bands, membranes were incubated
for 1.5 h at room temperature (RT) with mouse anti-rabbit
IgG (Sigma-Aldrich; diluted 1 : 500 for PPO protein or
1 : 5000 for actin protein), rabbit anti-sheep IgG for OX1R or
goat anti-mouse IgG for OX2R (Santa Cruz Biotechnology,
Dallas, TX, USA; diluted 1 : 500) conjugated with alkaline
phosphatase. Nonspecific foetal calf serum (MP Biomedicals,
Santa Ana, CA, USA) was used instead of primary antibodies
to produce negative control blots. The immunocomplexes
were visualised using 4-nitroblue tetrazolium chloride (NBT)
and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), according
to the manufacture’s protocol (Promega, Madison, WI, USA).
The same procedures were used for preparing positive
control – LHA. The results of Western blotting were quantified
by densitometric scanning of immunoblots with GelScan for
Windows ver. 1.45 software (Kucharczyk, Warsaw, Poland).
Data were expressed as a ratio of PPO, OX1R or OX2R protein (relative abundance of the specific proteins) relative to
actin protein in arbitrary optical density units.
Fluorescent immunohistochemistry
Fluorescent immunohistochemistry was performed as
described by Smolinska et al. (2014a). The endometrium and
myometrium were cut into 6 µm thick sections using the
CM3050 cryostat (Leica, Wetzlar, Germany) and placed on
poly-L-lysine-coated glass microscope slides (Menzel-Glaser,
Braunschweig, Germany). Frozen tissue sections were fixed
in 4% paraformaldehyde (Sigma-Aldrich) after reaching RT.
To decrease a nonspecific binding, sections were blocked in
10% normal goat serum (Sigma-Aldrich) diluted in 0.01 M
PBS with 0.1% bovine serum albumin (BSA; Sigma-Aldrich)
and 1% Triton X-100 (Sigma-Aldrich) for 1 h at RT. Tissue
samples were left overnight in a moist chamber at 4ºC with
primary antibodies: rabbit to OXA (Phoenix Pharmaceuticals
Inc., Burlingame, CA, USA), rabbit to OXB (Phoenix
Pharmaceuticals Inc.), sheep to OX1R (Abcam) or mouse to
OX2R (Abcam) at 1 : 50 dilutions. On the following day, the
sections were incubated with biotinylated: anti-rabbit IgG,
anti-sheep IgG or anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) at 1 : 100 dilutions for 1 h at RT. The slides
were incubated with the fluorescein (FITC)-streptavidin
complex (Vector Laboratories) diluted 1 : 50 in 0.01 M PBS
for 1 h at RT. The specimens were stained with propidium
iodide to visualise cell nuclei. The same procedures were
used for preparing positive controls – LHA, data not shown.
For negative control, the primary antibody was omitted, and
tissues were incubated in 0.01 M PBS or in rabbit universal
negative control (Dako Cytomation, Glostrup, Denmark), or
alternatively, by pre-absorbing each primary antiserum with
an excess (100 µg/ml) of each antigen. The slides were
mounted in fluorescent medium (Sigma-Aldrich). The labelled tissues were photographed with a fluorescence
microscope, using a dual filter cube for FITC and TRITC
(Olympus BX 51, Tokyo, Japan) attached to the digital
camera (Olympus DP 72).
Table 1 Characteristics of primers used in the study
Gene
PPO
Primer sequences
F: 5’-AAGACGACACCCTTCCTGGAGAC-3’
R: 5’-TGATTGCCAGCGCCGTGTAGCA-3’
OX1R
F: 5’-ACCGCTGGTATGCCATCTACCAC-3’
R: 5’-ATAGAGGTCATCTGCCCAGCGTTCA-3’
OX2R
F: 5’-GACATCACGGAGACCTGGTTCTTC-3’
R: 5’-GATGTAGGAGACGATCCAGATGATGAC-3’
Cyclophilin F: 5’-GCACTGGTGGCAAGTCCAT-3’
R: 5’-AGGACCCGTATGCTTCAGGA-3’
GAPDH
F: 5’-CCTTCATTGACCTCCACTACATGGT 3’
R: 5’-CCACAACATACG TAGCACCAGCATC-3’
GenBank accession number
EF434655
AF097995
AF059740
AY266299
U48832
Complementary
gene, nt
41–63
240–261
27–49
208–232
30–53
207–233
219–237
269–299
61–85
219–243
Primer, nM
Reference
500
500
500
500
500
500
300
300
500
500
Smolinska et al. (2014a)
Kaminski et al. (2010a)
Kaminski et al. (2010a)
Smolinska et al. (2014b)
Smolinska et al. (2014a)
PPO = prepro-orexin; OX1R = orexin receptor 1; OX2R = orexin receptor 2; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; F = forward; R = reverse.
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Expression of the orexins in the porcine uterus
Statistical analysis
All data were analysed by one-way ANOVA followed by the
least significant difference post hoc test, and are reported as the
mean ± s.e.m. from five independent observations. Statistical
analysis was performed using the Statistica program (StatSoft
Inc., USA). Values for P < 0.05 were considered statistically
significant.
Results
mRNA expression of PPO, OX1R and OX2R
In the endometrium, the expression of PPO gene was
about two times higher on days 15 to 16 of gestation relative
to days 10 to 11 of the porcine oestrous cycle and the
other studied stages of pregnancy (P < 0.01; Figure 1a). In
the myometrium, the highest PPO mRNA expression was
noted on days 15 to 16 of gestation, and the lowest on
days 27 to 28 and 30 to 32 of gestation (P < 0.05; Figure 1b).
On days 10 to 11 of the cycle and days 10 to 11 and 12 to
13 of gestation, the expression of PPO mRNA was more
pronounced in the myometrium than in the endometrium
(P < 0.01; Figure 1c). In the conceptuses, PPO gene expression was about two times higher on days 30 to 32 of
gestation relative to days 15 to 16 and 27 to 28 of gestation
(P < 0.05; Figure 1d). In the trophoblasts, PPO mRNA
expression was two times higher on days 30 to 32 than on
days 27 to 28 of gestation (P < 0.001; Figure 1e).
The content of OX1R mRNA in the endometrium was 2 to
12 times higher on days 15 to 16 of gestation than at all timepoints tested (P < 0.001; Figure 2a). In the myometrium, OX1R
gene expression on days 27 to 28 of gestation was similar to
days 10 to 11 of the oestrous cycle. There was about a two-fold
drop in the expression of this gene at all later time-points
(P < 0.05; Figure 2b). During all studied stages of pregnancy,
the expression of OX1R mRNA was more pronounced in the
endometrium than in the myometrium (P < 0.05; Figure 2c). In
the conceptuses, the content of OX1R mRNA was higher on
days 15 to 16 and 30 to 32 of gestation than on days 27 to
28 of gestation (P < 0.05; Figure 2d). In the trophoblasts, the
differences between days 27 to 28 and 30 to 32 of gestation
were insignificant (Figure 2e).
In the endometrium, the expression of OX2R gene was 3 to
15 times lower during all studied days of pregnancy relative
to days 10 to 11 of the cycle (P < 0.001; Figure 3a). Similarly
in the myometrium, the expression levels of OX2R mRNA
were significantly lower during all studied stages of gestation
than on days 10 to 11 of the cycle (P < 0.01; Figure 3b). On
days 10 to 11 of the cycle and days 10 to 11, 15 to 16 and 30
to 32 of gestation, the expression of OX2R mRNA was more
pronounced in the endometrium than in the myometrium,
whereas on days 12 to 13 and 27 to 28 of gestation, the
opposite pattern was observed (P < 0.05; Figure 3c). In the
conceptuses, the highest OX2R gene expression was noted
on days 27 to 28 and the lowest on days 15 to 16 of gestation (P < 0.001; Figure 3d). In the trophoblasts, OX2R mRNA
content was unchanged between days 27 to 28 and 30 to 32
of gestation (Figure 3e).
Protein expression of PPO, OX1R and OX2R
The content of PPO protein in the endometrium was higher on
days 10 to 11 and 12 to 13 of gestation, and it was lower on
days 15 to 16, 27 to 28 and 30 to 32 of gestation relative to
days 10 to 11 of the porcine oestrous cycle (P < 0.01; Figure 4a).
In the myometrium, PPO protein content was significantly
higher on days 30 to 32 of gestation, and it was lower on days
10 to 11, 15 to 16 and 27 to 28 of gestation than on days 10 to
11 of the oestrous cycle (P < 0.01; Figure 4b). At all time-points
tested (except for days 10 to 11 and 12 to 13 of gestation),
the content of PPO protein was more pronounced in the myometrium than in the endometrium (P < 0.05; Figure 4c). In the
conceptuses, the content of PPO protein was six to seven times
higher on days 15 to 16 and 27 to 28 of gestation than on days
30 to 32 of gestation (P < 0.001; Figure 4d). In the trophoblasts,
the expression of PPO protein was higher on days 27 to 28 than
on days 30 to 32 of gestation (P < 0.01; Figure 4e).
In the endometrium, OX1R protein content was higher on
days 10 to 11 of the cycle than during all studied days of
gestation (P < 0.001; Figure 5a). In contrast, the protein content
in the myometrium was more pronounced during all studied
stages of pregnancy, except for days 10 to 11 and
15 to 16 relative to days 10 to 11 of the cycle (P < 0.01;
Figure 5b). At all time-points tested (except for days 12 to 13 and
27 to 28 of gestation), the content of OX1R protein was higher in
the endometrium than in the myometrium (P < 0.01; Figure 5c).
In the conceptuses, OX1R protein expression was four times
higher on days 15 to 16 and 27 to 28 of gestation than on days
30 to 32 of gestation (P < 0.001; Figure 5d). In the trophoblasts,
the content of OX1R protein was higher on days 27 to 28 than
on days 30 to 32 of gestation (P < 0.05; Figure 5e).
The content of OX2R protein in the endometrium was higher
on days 15 to 16 of gestation and it was lower on days 10 to 11,
27 to 28 and 30 to 32 of gestation than on days 10 to 11 of the
cycle (P < 0.05; Figure 6a). In the myometrium, the expression
of OX2R protein on days 10 to 11 and 15 to 16 of gestation was
similar to days 10 to 11 of the oestrous cycle. There was an
increase in the expression of OX2R protein on days 12 to 13 and
27 to 28 of gestation, and a two-fold drop on days 30 to 32 of
gestation in relation to earlier mentioned period of the cycle
(P < 0.05; Figure 6b). On days 15 to 16 and 30 to 32 of
gestation, the content of OX2R protein was higher in the
endometrium than in the myometrium, whereas on days 10 to
11 of the cycle, higher OX2R protein expression was found in
the myometrium than in the endometrium (P < 0.05; Figure 6c).
In the conceptuses, the content of OX2R protein was about
eight times higher on days 15 to 16 than days 27 to 28 and 30
to 32 of gestation (P < 0.001; Figure 6d). In the trophoblasts,
OX2R protein expression was higher on days 30 to 32 than on
days 27 to 28 of gestation (P < 0.001; Figure 6e).
Protein localisation of OXA, OXB, OX1R and OX2R in the
porcine uterus
OXA (Supplementary Figure S1), OXB (Supplementary
Figure S2), OX1R (Supplementary Figure S3) and OX2R
(Supplementary Figure S4) proteins were localised in the
longitudinal and circular muscle layers of the porcine
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Smolinska, Kiezun, Dobrzyn, Szeszko, Maleszka and Kaminski
Figure 1 Prepro-orexin (PPO) mRNA expression in the porcine uterus, conceptus and trophoblast during early pregnancy and the oestrous cycle.
A comparison of PPO mRNA expression determined by quantitative real-time PCR in the endometrium (a) and myometrium (b) between days 10 to 11 of
the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy and (c) between the endometrium and myometrium on days
10 to 11 of the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy, (d) between the conceptuses on days 15 to 16,
27 to 28 and 30 to 32 of pregnancy, (e) between the trophoblasts on days 27 to 28 and 30 to 32 of pregnancy. Results are means ± s.e.m. (n = 5). Bars
with different superscripts are significantly different. Capital letters indicate P < 0.05; **P < 0.01; ***P < 0.001.
myometrium as well as endometrial epithelial glandular cells,
luminal epithelial cells and stromal cells on days 10 to 11 of
the cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and
30 to 32 of gestation. No immunostaining was observed in
the negative controls.
Discussion
In this study, the expression of PPO, OX1R and OX2R genes
and proteins was evaluated in the endometrium and myometrium of cyclic and early-pregnant gilts and in porcine
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Expression of the orexins in the porcine uterus
Figure 2 Orexin receptor 1 (OX1R) mRNA expression in the porcine uterus, conceptus and trophoblast during early pregnancy and the oestrous cycle.
A comparison of OX1R mRNA expression determined by quantitative real-time PCR in the endometrium (a) and myometrium (b) between days 10 to 11 of
the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy and (c) between the endometrium and myometrium on days
10 to 11 of the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy, (d) between the conceptuses on days 15 to 16,
27 to 28 and 30 to 32 of pregnancy, (e) between the trophoblasts on days 27 to 28 and 30 to 32 of pregnancy. Results are means ± s.e.m. (n = 5). Bars
with different superscripts are significantly different. Capital letters indicate P < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.
conceptuses and trophoblasts. The presence of OXA, OXB,
OX1R and OX2R was determined in porcine uterine tissues.
To our knowledge, this is the first study demonstrating the
presence of the orexin system in the uterus of pregnant
mammals. Interestingly, significant discrepancies or even
opposite values of the gene expression and protein concentration were noted. The absence of correlations between
the protein concentration and gene transcript could be
attributed to transcriptional and post-transcriptional regulation (RNA processing and stability), differences in mRNA and
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Smolinska, Kiezun, Dobrzyn, Szeszko, Maleszka and Kaminski
Figure 3 Orexin receptor 2 (OX2R) mRNA expression in the porcine uterus, conceptus and trophoblast during early pregnancy and the oestrous cycle.
A comparison of OX2R mRNA expression determined by quantitative real-time PCR in the endometrium (a) and myometrium (b) between days 10 to 11 of
the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy and (c) between the endometrium and myometrium on days
10 to 11 of the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy, (d) between the conceptuses on days 15 to 16,
27 to 28 and 30 to 32 of pregnancy, (e) between the trophoblasts on days 27 to 28 and 30 to 32 of pregnancy. Results are means ± s.e.m. (n = 5). Bars
with different superscripts are significantly different. Capital letters indicate P < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.
protein stability and functioning feedbacks that suppress
mRNA expression through high protein concentrations and
attenuate post-transcriptional processes through high levels
of gene expression (Gry et al., 2009; Vogel and Marcotte,
2012).
Gene and protein expression levels differed in various
stages of pregnancy. It seems that all components of the
orexin system in the porcine uterus and conceptuses may be
regulated by the local hormonal environment. Significant
changes in the hormonal milieu of the uterus are probably
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Expression of the orexins in the porcine uterus
Figure 4 Prepro-orexin (PPO) protein expression in the porcine uterus, conceptus and trophoblast during early pregnancy and the oestrous cycle.
A comparison of PPO protein concentration determined by Western blotting analysis in the endometrium (a) and myometrium (b) between days 10 to 11
of the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy and (c) between the endometrium and myometrium on
days 10 to 11 of the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy, (d) between the conceptuses on days 15
to 16, 27 to 28 and 30 to 32 of pregnancy, (e) between the trophoblasts on days 27 to 28 and 30 to 32 of pregnancy. Upper panels: representative
immunoblots (MM = molecular marker; LHA = lateral hypothalamic area – positive control); lower panels: densitometric analysis of PPO protein relative
to actin protein. Values are expressed as means ± s.e.m. in arbitrary optical density units (n = 5). Bars with different superscripts are significantly different.
Capital letters indicate P < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.
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Smolinska, Kiezun, Dobrzyn, Szeszko, Maleszka and Kaminski
Figure 5 Orexin receptor 1 (OX1R) protein expression in the porcine uterus, conceptus and trophoblast during early pregnancy and the oestrous cycle.
A comparison of OX1R protein concentration determined by Western blotting analysis in the endometrium (a) and myometrium (b) between days 10 to 11
of the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy and (c) between the endometrium and myometrium on
days 10 to 11 of the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy, (d) between the conceptuses on days 15
to 16, 27 to 28 and 30 to 32 of pregnancy, (e) between the trophoblasts on days 27 to 28 and 30 to 32 of pregnancy. Upper panels: representative
immunoblots (MM = molecular marker; LHA = lateral hypothalamic area – positive control); lower panels: densitometric analysis of OX1R protein relative
to actin protein. Values are expressed as means ± s.e.m. in arbitrary optical density units (n = 5). Bars with different superscripts are significantly different.
Capital letters indicate P < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.
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Expression of the orexins in the porcine uterus
Figure 6 Orexin receptor 2 (OX2R) protein expression in the porcine uterus, conceptus and trophoblast during early pregnancy and the oestrous cycle.
A comparison of OX2R protein concentration determined by Western blotting analysis in the endometrium (a) and myometrium (b) between days 10 to 11
of the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy and (c) between the endometrium and myometrium on
days 10 to 11 of the oestrous cycle and days 10 to 11, 12 to 13, 15 to 16, 27 to 28 and 30 to 32 of pregnancy, (d) between the conceptuses on days 15
to 16, 27 to 28 and 30 to 32 of pregnancy, (e) between the trophoblasts on days 27 to 28 and 30 to 32 of pregnancy. Upper panels: representative
immunoblots (MM = molecular marker; LHA = lateral hypothalamic area – positive control); lower panels: densitometric analysis of OX2R protein relative
to actin protein. Values are expressed as means ± s.e.m. in arbitrary optical density units (n = 5). Bars with different superscripts are significantly different.
Capital letters indicate P < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.
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Smolinska, Kiezun, Dobrzyn, Szeszko, Maleszka and Kaminski
triggered by oestrogens, which are produced in conceptuses
as signals of maternal recognition of pregnancy. Oestrogens
and ovarian progesterone make up the specific hormonal
microenvironment (Bazer and Johnson, 2014). The first oestrogens peak (days 10 to 12 of gestation) stimulates prostaglandin E2 (PGE2) synthesis and increases the PGE2/
prostaglandin F2α (PGF2α) ratio (Waclawik et al., 2009),
boosts endometrial production of fibroblast growth factor 7
(FGF7), transforming growth factor beta (TGFβ), interleukin1β, tumour necrosis factor alpha (TNFα), interferon delta
(IFND) and interferon gamma (IFNG) (Harney and Bazer,
1989; Ka et al., 2001; Ross et al., 2003; White et al., 2009;
Waclawik et al., 2010). The discussed growth factors and
cytokines directly influence various genes whose products
deliver luteoprotective effects and support conceptus development (Bazer and Johnson, 2014). The variations in the
expression of PPO and orexin receptors observed in this study
are temporally associated with the above changes in the
local hormonal environment. The postulated involvement of
the hormonal milieu, in particular steroids, in the regulation
of orexin expression is supported by our earlier observation
that OXA and OXB production in the uterus of cyclic pigs is
dependent on the hormonal status of animals. The highest
expression of PPO mRNA was noted in the endometrium and
myometrium on days 14 to 16 of the oestrous cycle, that is,
during luteolysis (Nitkiewicz et al., 2012). Therefore, it cannot be ruled out that the orexin system in the porcine uterus
and placenta is controlled by selected steroids, prostaglandins, growth factors and cytokines. Further studies are
needed to validate this hypothesis and supply detailed data.
Hormone-dependent changes in expression levels of both
orexin receptors were also noted in all branches of the
porcine hypothalamic–pituitary–ovarian axis (Kaminski et al.,
2010a and 2010b; Nitkiewicz et al., 2010). In the present study,
the expression of orexin receptors genes and proteins was also
observed in the porcine uterus and trophoblast, which could
point to the auto/paracrine action of orexins in those structures.
The role of those peptides in uterine/placental physiology
remains unknown. To our best knowledge, the presence of the
orexin system components in human and feline placenta and
uteri has been demonstrated by very few studies (Nakabayashi
et al., 2003; Dall'Aglio et al., 2012; Dehan et al., 2013),
including our study of the porcine uterus (Nitkiewicz
et al., 2012). The involvement of orexins in the control
of endometrial epithelial cell proliferation through type 2
receptors has been suggested in only one study (Dehan et al.,
2013). Our previous results suggest that orexins influence
ovarian steroidogenesis in pigs (Nitkiewicz et al., 2014). Further
work is needed to explore the role of orexin peptides in the
control of uterine/placental functions.
Here we demonstrated, for the first time, the presence of
the complete orexin system (gene and protein) in the porcine
conceptuses. Interestingly, the content of PPO and OX1R
proteins was greater on days 15 to 16 and 27 to 28 of
gestation than on days 30 to 32 of gestation. The highest
expression of OX2R protein was determined during implantation. Past studies have found the localisation of orexin
mRNA and protein in the rat embryo brains on days
18 of gestation (Steininger et al., 2004). The expression of
both types of orexin receptors genes was determined in the
primary neuronal cell cultures derived from rat embryos on
day 16 of gestation (Urbanska et al., 2012). Orexin neuropeptides were also localised in the chicken embryo brains
(Godden et al., 2014). The role of orexin system in controlling
the physiology of the embryo remains completely unknown
and further studies are needed.
In this study, the expression of PPO, OXA and OXB, OX1R
and OX2R was observed in the trophoblasts, conceptuses
and uterine structures of pregnant sows, and orexin expression levels were found to be correlated with the stages of
early pregnancy. Our findings suggest that orexins are
involved in the control of uterine and trophoblasts functions,
but further work is needed to validate this hypothesis.
Acknowledgement
This research was supported by National Science Centre (projects no: 2011/03/B/NZ9/04187).
Supplementary material
To view supplementary material for this article, please visit
http://dx.doi.org/10.1017/S1751731115001020
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