Implications
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.
Introduction
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., Reference De Lecea, Kilduff, Peyron, Gao, Foye, Danielson, Fukuhara, Battenberg, Gautvik, Bartlett II, Franlel, Van den Pol, Bloom, Gautvik and Sutcliffe1998; Sakurai et al., Reference Sakurai, Amemiya, Ishii, Matsuzaki, Chemelli, Tanaka, Williams, Richardson, Kozlowski, Wilson, Arch, Buckingham, Haynes, Carr, Annan, McNulty, Liu, Terret, Elshourbagy, Bergsma and Yanagisawa1998). 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., Reference De Lecea, Kilduff, Peyron, Gao, Foye, Danielson, Fukuhara, Battenberg, Gautvik, Bartlett II, Franlel, Van den Pol, Bloom, Gautvik and Sutcliffe1998; Sakurai et al., Reference Sakurai, Amemiya, Ishii, Matsuzaki, Chemelli, Tanaka, Williams, Richardson, Kozlowski, Wilson, Arch, Buckingham, Haynes, Carr, Annan, McNulty, Liu, Terret, Elshourbagy, Bergsma and Yanagisawa1998).
The orexins were originally identified as regulators of food intake and energy homoeostasis (Sakurai et al., Reference Sakurai, Amemiya, Ishii, Matsuzaki, Chemelli, Tanaka, Williams, Richardson, Kozlowski, Wilson, Arch, Buckingham, Haynes, Carr, Annan, McNulty, Liu, Terret, Elshourbagy, Bergsma and Yanagisawa1998) and later described as key modulators of the sleep–wake cycle and arousal (Chemelli et al., Reference Chemelli, Willie, Sinton, Elmquist, Scammell, Lee, Richardson, Williams, Xiong, Kisanuki, Fitch, Nakazato, Hammer, Saper and Yanagisawa1999). 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., Reference Silveyra, Lux-Lantos and Libertun2007a and Reference Silveyra, Catalano, Lux-Lantos and Libertun2007b; Kaminski et al., Reference Kaminski, Smolinska, Nitkiewicz and Przala2010a and Reference Kaminski, Smolinska, Nitkiewicz and Przala2010b; Nitkiewicz et al., Reference Nitkiewicz, Smolinska, Przala and Kaminski2010; Maleszka et al., Reference Maleszka, Smolinska, Nitkiewicz, Kiezun, Chojnowska, Dobrzyn, Jazowska and Kaminski2013; Smolinska et al., Reference Smolinska, Nitkiewicz, Maleszka, Kiezun, Dobrzyn, Czerwinska, Chojnowska and Kaminski2014b). Local orexin production (Nitkiewicz et al., Reference Nitkiewicz, Smolinska, Maleszka, Chojnowska and Kaminski2014) and the presence of specific receptors in porcine and rat ovaries (Silveyra et al., Reference Silveyra, Lux-Lantos and Libertun2007a; Nitkiewicz et al., Reference Nitkiewicz, Smolinska, Przala and Kaminski2010) 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., Reference Nakabayashi, Suzuki, Takahashi, Totsune, Muramatsu, Kaneko, Date, Takeyama, Darnel, Moriya and Sasano2003; Dall'Aglio et al., Reference Dall'Aglio, Pascucci, Mercati, Polisca, Ceccarelli and Boiti2012).
In view of our previous results, which demonstrated the presence of orexins in the porcine uterus during the oestrous cycle (Nitkiewicz et al., Reference Nitkiewicz, Smolinska, Maleszka, Kiezun and Kaminski2012) 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 real-time PCR
Total RNA isolation and cDNA synthesis was performed as described previously by Smolinska et al. (Reference Smolinska, Dobrzyn, Maleszka, Kiezun, Szeszko and Kaminski2014a). 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., Reference Smolinska, Dobrzyn, Maleszka, Kiezun, Szeszko and Kaminski2014a). 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. Real-time 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 (ΔΔC T ) and normalised using the geometrical means of reference gene expression levels: GAPDH and cyclophilin. 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.
Characteristics of primers used in the study
PPO=prepro-orexin; OX1R=orexin receptor 1; OX2R=orexin receptor 2; GAPDH=glyceraldehyde 3-phosphate dehydrogenase; F=forward; R=reverse.
Western blotting
Western blotting analysis was performed essentially as described by Smolinska et al. (Reference Smolinska, Kaminski, Siawrys and Przala2007). 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. (Reference Smolinska, Dobrzyn, Maleszka, Kiezun, Szeszko and Kaminski2014a). 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).
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).
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.
The content of OX1R mRNA in the endometrium was 2 to 12 times higher on days 15 to 16 of gestation than at all time-points 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).
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.
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).
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 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).
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.
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).
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.
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).
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.
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 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 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 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., Reference Gry, Rimini, Strömberg, Asplund, Pontén, Uhlén and Nilsson2009; Vogel and Marcotte, Reference Vogel and Marcotte2012).
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 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, Reference Bazer and Johnson2014). 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., Reference Waclawik, Jabbour, Blitek and Ziecik2009), boosts endometrial production of fibroblast growth factor 7 (FGF7), transforming growth factor beta (TGFβ), interleukin-1β, tumour necrosis factor alpha (TNFα), interferon delta (IFND) and interferon gamma (IFNG) (Harney and Bazer, Reference Harney and Bazer1989; Ka et al., Reference Ka, Jaeger, Johnson, Spencer and Bazer2001; Ross et al., Reference Ross, Ashworth, Hurst, Malayer and Geisert2003; White et al., Reference White, Kimball, Wyman, Stein, Ross, Ashworth and Geisert2009; Waclawik et al., Reference Waclawik, Blitek and Ziecik2010). The discussed growth factors and cytokines directly influence various genes whose products deliver luteoprotective effects and support conceptus development (Bazer and Johnson, Reference Bazer and Johnson2014). 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., Reference Nitkiewicz, Smolinska, Maleszka, Kiezun and Kaminski2012). 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., Reference Kaminski, Smolinska, Nitkiewicz and Przala2010a and Reference Kaminski, Smolinska, Nitkiewicz and Przala2010b; Nitkiewicz et al., Reference Nitkiewicz, Smolinska, Przala and Kaminski2010). 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., Reference Nakabayashi, Suzuki, Takahashi, Totsune, Muramatsu, Kaneko, Date, Takeyama, Darnel, Moriya and Sasano2003; Dall'Aglio et al., Reference Dall'Aglio, Pascucci, Mercati, Polisca, Ceccarelli and Boiti2012; Dehan et al., Reference Dehan, Canon, Trooskens, Rehli, Munaut, Van Criekinge and Delvenne2013), including our study of the porcine uterus (Nitkiewicz et al., Reference Nitkiewicz, Smolinska, Maleszka, Kiezun and Kaminski2012). 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., Reference Dehan, Canon, Trooskens, Rehli, Munaut, Van Criekinge and Delvenne2013). Our previous results suggest that orexins influence ovarian steroidogenesis in pigs (Nitkiewicz et al., Reference Nitkiewicz, Smolinska, Maleszka, Chojnowska and Kaminski2014). 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., Reference Steininger, Kilduff, Behan, Benca and Landry2004). 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., Reference Urbanska, Sokolowska, Woldan-Tambor, Bieganska, Brix, Jöhren, Namiecinska and Zawilska2012). Orexin neuropeptides were also localised in the chicken embryo brains (Godden et al., Reference Godden, Landry, Slepneva, Migues and Pompeiano2014). 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
