Expression of adiponectin and adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) in the porcine uterus during the oestrous cycle

Animal Reproduction Science 146 (2014) 42–54

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Expression of adiponectin and adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) in the porcine uterus during the oestrous cycle Nina Smolinska ∗ , Kamil Dobrzyn, Anna Maleszka, Marta Kiezun, Karol Szeszko, Tadeusz Kaminski Department of Animal Physiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A, Kortowo, 10-719 Olsztyn, Poland

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Article history: Received 9 October 2013 Received in revised form 12 January 2014 Accepted 2 February 2014 Available online 15 February 2014

Keywords: Adiponectin Adiponectin receptors 1 and 2 Uterus Oestrous cycle Pig

a b s t r a c t Adiponectin is a hormone secreted primarily by white adipose tissue. Recent studies have shown that adiponectin and its receptors (AdipoR1 and AdipoR2) are expressed in different reproductive tissues, including the ovary and uterus. This newly discovered endocrine system plays an important role in the regulation of reproductive processes. The expression of the adiponectin system in the porcine uterus during the oestrous cycle has not been researched to date. The aim of the present study was to investigate the presence and changes in adiponectin system expression in the porcine uterus on days 2–3, 10–12, 14–16, and 17–19 of the oestrous cycle. The expression of the adiponectin gene was highest on days 14–16 and 2–3 in the endometrium and myometrium, respectively. In the endometrium, the content of AdipoR1 and AdipoR2 mRNAs was highest on days 10–12, whereas significantly higher expression levels of both genes were noted in the myometrium on days 17–19. The highest content of adiponectin and AdipoR1 protein in the endometrium was reported on days 2–3. In the myometrium, the expression levels of both receptor proteins were significantly higher on days 17–19. Adiponectin system proteins were localized in endometrial epithelial glandular cells, luminal epithelial cells and stromal cells as well as in longitudinal and circular muscles of the myometrium. This study demonstrated the presence of adiponectin, AdipoR1 and AdipoR2 genes and proteins in the porcine uterus and the effect of the stage of the oestrous cycle on the expression of the adiponectin system. Our results suggest that locally synthesized adiponectin directly affects uterine functions. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Adiponectin is a 30 kDa hormone secreted primarily by adipocytes (Hu et al., 1996; Scherer et al., 1995). In its full-length form of adiponectin consists of an N-terminal signal sequence, a collagenous domain and a C-terminal

∗ Corresponding author. Tel.: +48 89 523 42 26; fax: +48 89 523 39 37. E-mail address: [email protected] (N. Smolinska). http://dx.doi.org/10.1016/j.anireprosci.2014.02.001 0378-4320/© 2014 Elsevier B.V. All rights reserved.

globular region (Fruebis et al., 2001). Adiponectin levels in adipose tissue and blood plasma tend to be lower in obese subjects (Arita et al., 1999; Hu et al., 1996). Growing evidence suggests that adiponectin is an important regulator of energy metabolism and insulin sensitivity (Maeda et al., 1996; Yamauchi et al., 2001). Adiponectin has also been found to possess anti-diabetic, antiinflammatory, anti-atherosclerotic and anti-angiogenic properties (Brakenhielm et al., 2004; Goldstein and Scalia, 2004; Yokota et al., 2000).

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Two adiponectin receptors (AdipoR1 and AdipoR2) have been recently identified. Both receptors contain seven transmembrane domains that are structurally and functionally distinct from G protein-coupled receptors (Yamauchi et al., 2003). The receptors differ in their binding preference for adiponectin isoforms, and both adiponectin receptors are differently distributed in tissue. AdipoR1 is abundantly expressed in skeletal muscles and has a high affinity for the globular form of adiponectin. AdipoR2 is found mainly in the liver and has a higher affinity for full-length adiponectin (Yamauchi et al., 2003). Emerging evidence shows that the cellular effects of adiponectin are mediated by the adenosine monophosphate-activated protein kinase (AMPK) pathway, the peroxisome proliferator-activated receptor ␣ (PPAR␣) pathway, or by activating p38 mitogen-activated protein kinase (MAPK) (Deepa and Dong, 2009). The relationship between nutritional status and reproductive success in animals has been studied for many years. Considerable evidence has been accumulated to implicate the existence of a common endocrine system that controls metabolism and the reproductive system. A growing body of evidence indicates that adiponectin belongs to a group of hormones that regulate metabolic status and reproduction. Adiponectin influences the reproductive system by exerting central effects on the hypothalamus–pituitary axis, inhibiting GnRH (Wen et al., 2008) and GnRH-induced LH secretion (Rodriguez-Pacheco et al., 2007; Lu et al., 2008). Both types of receptors are expressed in GT1-7 hypothalamic GnRH neuron cells (Wen et al., 2008) and in rat and porcine pituitary glands (Rodriguez-Pacheco et al., 2007; Kiezun et al., 2013). Several lines of evidence suggest that adiponectin also influences the reproductive system by exerting peripheral effects on the ovary and uterus. Adiponectin regulates periovulatory remodeling of the ovarian follicles and steroid synthesis as well as the inflammatory response of endometrial cells (Ledoux et al., 2006; Takemura et al., 2006; Chabrolle et al., 2007; Maleszka et al., 2014). Some studies suggest that adiponectin has biological implications for fertility (Mitchell et al., 2005). In particular, high circulating adiponectin levels have been described as being associated with IFV success (Bersinger et al., 2006), and plasma adiponectin is reduced in women with endometriosis (Takemura et al., 2005) and endometrial cancer (Dal Maso et al., 2004). Those findings suggest that adiponectin exerts certain effects on the endometrium. The observation that porcine plasma adiponetin levels change during the course of the oestrous cycle additionally implies that adiponectin secretion is regulated by gonadal steroids (Maleszka et al., 2014). The presence of the adiponectin system (adiponectin, adiponectin receptors) in the porcine endometrium and myometrium during the oestrous cycle and the possible effect of the phase of the cycle on adiponectin, AdipoR1 and AdipoR2 concentrations have not been investigated to date. For this reason, the aim of the current study was designed to compare the expression levels of: (1) adiponectin, AdipoR1 and AdipoR2 genes by quantitative real-time PCR and (2) adiponectin, AdipoR1 and AdipoR2 proteins by Western blotting and fluorescent immunohistochemistry in the endometrium and

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myometrium on days 2–3, 10–12, 14–16, and 17–19 of the oestrous cycle. 2. Materials and methods 2.1. 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. Twenty mature gilts (Large White × Polish Landrace; 7–8 months of age, body weight of 120–130 kg) descended from private breeding were used in the study. The gilts were divided into four experimental groups (n = 5 per group) as follows: 1/days 2–3 of the oestrous cycle: the early-luteal phase, development of new corpora lutea, 2/days 10–12: the midluteal phase, fully active corpora lutea), 3/days 14–16: the late-luteal phase, luteolysis, and 4/days 17–19: the follicular phase of the oestrous cycle. Females were monitored daily for oestrus behavior in the presence of an intact boar. The onset of the second oestrus was marked as day 0 of the oestrous cycle. The phase of the oestrous cycle was confirmed by the examination of ovarian morphology (Akins and Morrissette, 1968). Additionally, blood samples were collected into heparinized tubes, centrifuged (2500 × g, 15 min, 4 ◦ C) and the obtained plasma was stored at −80 ◦ C until progesterone measurements. The level of serum progesterone (P4) was determined to confirm the phase of the oestrous cycle (Nitkiewicz et al., 2010). Plasma P4 levels were 4 ± 2 ng/ml, 19 ± 3.4 ng/ml, 8 ± 2.2 ng/ml and 0.2 ± 0.03 ng/ml, respectively, on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle, and were consistent with previously published results (Henricks et al., 1972). The endometrium and myometrium was dissected out of the uterus within several minutes after slaughter. The tissue samples were frozen in liquid nitrogen and stored at −80 ◦ C for further analysis. 2.2. Total RNA isolation and cDNA synthesis Total RNA was extracted from all the tissue samples with the Absolutely RNA Miniprep Kit (Stratagene, USA). RNA concentration and quality were checked spectrophotometrically (NanoDrop ND-1000, NanoDrop Technologies Inc., USA). One microgram of RNA was reversely transcribed into cDNA in a total volume of 20 ␮l with 0.5 ␮g oligo(dT)15 primer (Roche, Germany) using the Omniscript RT Kit (Qiagen, USA) at 37 ◦ C for 1 h. The process was terminated by incubation at 93 ◦ C for 5 min. 2.3. Quantitative real-time PCR Specific primer pairs used to amplify parts of adiponectin, AdipoR1, AdipoR2, cyclophilin and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) are detailed in Table 1. Quantitative real-time PCR analysis was carried out using a PCR System 7300 (Applied Biosystems, USA). The PCR reaction included 20 ng cDNA, the appropriate forward and reverse primer at various concentrations (Table 1), 12.5 ␮l SYBR Green PCR Master Mix (Applied Biosystems, USA), and RNase free water in a final volume

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Fig. 1. A comparison of adiponectin mRNA expression determined by quantitative real-time PCR in the endometrium (A) and the myometrium (B) between days 2 and 3, 10 and 12, 14 and 16 and 17 and 19 of the oestrous cycle, and (C) between the endometrium and the myometrium on days 2–3, 10–12, 14–16 and 17–19 of the cycle. 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.

Table 1 Characteristics of primers used in the study. Gene

Primer sequences

GenBank accession number

Complementary gene (nt)

Primer (nM)

Reference

Adiponectin

F: 5 -ATGATGTCACCACTGGCAAATTC-3 R: 5 -GACCGTGACGTGGAAGGAGA-3 F: 5 -GCCATGGAGAAGATGGAGGA-3 R: 5 -AGCACGTCGTACGGGATGA-3 F: 5 -TGTTCGCCACCCCTCAGTAT-3 R: 5 -AATGATTCCACTCAGGCCCA-3 F: 5 -GCACTGGTGGCAAGTCCAT-3 R: 5 -AGGACCCGTATGCTTCAGGA-3 F: 5 -CCTTCATTGACCTCCACTACATGGT 3 R: 5 -CCACAACATACG TAGCACCAGCATC-3

AY135647

514–536 565–584 148–168 204–222 322–341 373–392 219–237 269–299 61–85 219–243

900 300 300 50 50 50 300 300 500 500

Lord et al. (2005)

AdipoR1 AdipoR2 Cyclophilin GAPDH

AY452710 AY452711 AY266299 U48832

Lord et al. (2005) Lord et al. (2005) Lord et al. (2005) Bogacka et al. (2006)

AdipoR1: adiponectin receptor 1; AdipoR2: adiponectin receptor 2; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; F: forward; R: reverse.

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Fig. 2. A comparison of adiponectin receptor 1 (AdipoR1) mRNA expression determined by quantitative real-time PCR in the endometrium (A) and the myometrium (B) between days 2 and 3, 10 and 12, 14 and 16 and 17 and 19 of the oestrous cycle, and (C) between the endometrium and the myometrium on days 2–3, 10–12, 14–16 and 17–19 of the cycle. Results are means ± S.E.M. (n = 5). Bars with different superscripts are significantly different. Capital letters indicate p < 0.05; *** p < 0.001.

of 25 ␮l. 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 oestrous cycle. Real-time PCR cycling conditions were as follows: 50 ◦ C for 2 min, then enzyme activation and initial denaturation at 95 ◦ C for 10 min, followed by 40 cycles of denaturation at 95 ◦ C for 15 s, annealing at 60 ◦ C for 1 min. Negative controls were performed in which water was substituted for cDNA, 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 adiponectin, AdipoR1 and AdipoR2 genes was conducted based on the comparative cycle threshold method (CT ) (Livak and Schmittgen, 2001) and normalized using the geometrical means of reference gene expression levels: GAPDH and cyclophilin. Expression of adiponectin, AdipoR1 and AdipoR2 genes was calculated by the equation 2−CT , where CT was obtained by subtracting the corresponding geometrical means of reference genes (GAPDH and cyclophilin) CT value from the specific CT of the target (adiponectin, AdipoR1 or AdipoR2), and CT was determined by subtracting the CT of each experimental sample from CT of the reference sample, called the calibrator (the tissue with the lowest expression).

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Fig. 3. A comparison of adiponectin receptor 2 (AdipoR2) mRNA expression determined by quantitative real-time PCR in the endometrium (A) and the myometrium (B) between days 2 and 3, 10 and 12, 14 and 16 and 17 and 19 of the oestrous cycle, and (C) between the endometrium and the myometrium on days 2–3, 10–12, 14–16 and 17–19 of the cycle. 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 < 0.001.

2.4. Fluorescent Immunohistochemistry Fluorescent immunohistochemistry was performed as described by Maleszka et al. (2013). The endometrium and myometrium were cut into 6 ␮m thick sections using the CM3050 cryostat (Leica, USA) and thaw-mounted onto poly-l-lysine-coated glass microscope slides (MenzelGlaser, Germany). Frozen tissue sections were fixed in 4% paraformaldehyde (Sigma-Aldrich, USA) after reaching RT. To decrease a nonspecific binding, sections were blocked in 10% normal goat serum (Sigma-Aldrich, USA) diluted in 0.01 M PBS with 0.1% bovine serum albumin (BSA; Sigma-Aldrich, USA) and 1% Triton X-100 (Sigma-Aldrich, USA) for 1 h RT. Tissue samples were left overnight in a moist chamber at 4 ◦ C with primary antibodies: rabbit

anti-adiponectin (Santa Cruz Biotechnology, USA), rabbit anti-AdipoR1 and anti-AdipoR2 (Phoenix Pharmaceuticals, Inc., USA) at 1:50 dilution. On the following day, the sections were incubated with biotinylated anti-rabbit IgG (Vector Laboratories, USA) at 1:100 dilution for 1 h at RT. The slides were incubated with the fluorescein (FITC)streptavidin complex (Vector Laboratories, USA) diluted 1:50 in 0.01 M PBS for 1 h at RT. The specimens were stained with propidium iodide to visualize cell nuclei. 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, Denmark). The slides were mounted in fluorescent medium (Sigma-Aldrich, USA). Fluorescence intensity was analyzed in each section. The labeled tissues were photographed with an fluorescence

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Fig. 4. Immunofluorescence localization of adiponectin (green) in endometrial and myometrial structures of the porcine uterus during the oestrous cycle. Cell nuclei stained with propidium iodide are shown in red. Right upper corners: negative controls. Scale bar = 100 ␮m; images are representative of n = 5. GC: glandular cells, LEC: luminal epithelial cells, SC: stromal cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

microscope, using a dual filter cube for FITC and TRITC (Olympus BX 51, Japan) attached to the digital camera, with a high-sensitivity charge coupled device (CCD) to provide a sufficient rate of the quantitative linearity (Olympus DP 72, Japan). 2.5. 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 ␮g of total proteins) were resolved by SDS-PAGE (12.5% gel) for separating adiponectin, AdipoR1, AdipoR2 and actin, and then transferred to nitrocellulose membranes (Whatman, 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 the rabbit polyclonal adiponectin antibodies at the dilution of 1:150 (Santa Cruz Biotechnology, USA), the rabbit polyclonal AdipoR1 antibodies at a dilution of 1:150 (Phoenix Pharmaceuticals, USA), rabbit polyclonal AdipoR2 antibodies

at a dilution of 1:200 (Phoenix Pharmaceuticals, USA), or rabbit polyclonal actin antibodies diluted 1:200 (Sigma, USA), which were used as a control for equal loading as well as to quantify porcine adiponectin, AdipoR1 and AdipoR2 proteins. To identify immunoreactive bands, membranes were incubated for 1.5 h at room temperature (RT) with mouse anti-rabbit IgG for adiponectin and AdipoR1 (Sigma, USA; diluted 1:2000), goat antirabbit IgG for AdipoR2 (diluted 1:500) or for actin (diluted 1:5000) conjugated with alkaline phosphatase (Santa Cruz Biotechnology, USA). Nonspecific foetal calf serum (MP Biomedicals, USA) was used instead of primary antibodies to produce negative control blots. The immunocomplexes were visualized using 4-nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), according to the manufacture’s protocol (Promega, USA). The same procedures were used for preparing positive controls—adipose tissue, skeletal muscles and liver (for adiponectin, AdipoR1 and AdipoR2, respectively). The results of Western blotting were quantified by densitometric scanning of immunoblots with GelScan for Windows ver. 1.45 software (Kucharczyk, Poland). Data

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Fig. 5. Immunofluorescence localization of adiponectin receptor 1 (AdipoR1; green) in endometrial and myometrial structures of the porcine uterus during the oestrous cycle. Cell nuclei stained with propidium iodide are shown in red. Right upper corners: negative controls. Scale bar = 100 ␮m; images are representative of n = 5. GC: glandular cells, LEC: luminal epithelial cells, SC: stromal cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

were expressed as a ratio of adiponectin, AdipoR1 or AdipoR2 protein relative to actin protein in arbitrary optical density units. 2.6. Statistical analysis All data were analyzed by one-way ANOVA, followed by the least significant difference (LSD) 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. 3. Results

cycle, the expression of adiponectin mRNA was more pronounced in the myometrium than endometrium, whereas on days 10–12, 14–16 and 17–19, higher adiponectin gene expression was reported in the endometrium than in the myometrium (p < 0.05; Fig. 1C). The highest AdipoR1 and AdipoR2 genes expression was noted on days 10–12 (the endometrium) and 17–19 (the myometrium), and the lowest on days 2–3 and 17–19 of the cycle in the endometrium, and on days 10–12 and 14–16 of the cycle in the myometrium (p < 0.05; Fig. 2A and B and Fig. 3A and B). The AdipoR1 and AdipoR2 genes were significantly higher expressed in the endometrium than in the myometrium on days 2–3 (only AdipoR2), 10–12 and 14–16 of the cycle (p < 0.05). On days 17–19 differences between both tissues were negligible (Fig. 2C and Fig. 3C).

3.1. mRNA expression of adiponectin and its receptors The expression of adiponectin gene was highest on days 14–16 of the porcine oestrous cycle in the endometrium, and on days 2–3 of the cycle in the myometrium. The lowest adiponectin transcript content was found on days 2–3, 17–19 (the endometrium) and 14–16, 17–19 (the myometrium) (p < 0.05; Fig. 1A and B). On days 2–3 of the

3.2. Protein localization of adiponectin and its receptors in the porcine uterus Adiponectin (Fig. 4), AdipoR1 (Fig. 5) and AdipoR2 (Fig. 6) proteins were localized in the longitudinal and circular muscle layers of the porcine myometrium as well as endometrial epithelial glandular cells, luminal

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Fig. 6. Immunofluorescence localization of adiponectin receptor 2 (AdipoR2; green) in the endometrial and myometrial structures of the porcine uterus during the oestrous cycle. Cell nuclei stained with propidium iodide are shown in red. Right upper corners: negative controls. Scale bar = 100 ␮m; images are representative of n = 5. GC: glandular cells, LEC: luminal epithelial cells, SC: stromal cells.

epithelial cells and stromal cells on days 2–3, 10–12, 14–16 and 17–19 of the cycle. No staining was detected in the endometrium and myometrium when 0.01 M PBS or rabbit universal negative control was used instead of primary antibodies. 3.3. Protein expression of adiponectin and its receptors In the endometrium, adiponectin protein was observed in greater abundance on days 2–3 than on days 10–12 and 14–16 of the cycle. In the myometrium, adiponectin protein content was significantly higher on days 17–19 than in the remaining stages of the oestrous cycle. The lowest adiponectin protein was observed on days 10–12 in the endometrium and 2–3 in the myometrium (p < 0.05; Fig. 7A and B). Contrary to the adiponectin gene, on days 2–3 of the cycle, the expression of adiponectin protein was more pronounced in the endometrium than in the myometrium, whereas on days 10–12, 14–16 and 17–19, higher adiponectin protein expression was reported in the myometrium than in the endometrium (p < 0.05; Fig. 7C). The concentration of AdipoR1 protein was highest on days 2–3 of the porcine oestrous cycle in the endometrium, and on days 17–19 of the cycle in the myometrium. The

lowest AdipoR1 protein content was found on days 17–19 (the endometrium) and 10–12 (the myometrium) (p < 0.05; Fig. 8A and B). The expression of the protein was higher in the endometrium than in the myometrium on days 2–3, 10–12 and 14–16 of the oestrous cycle (p < 0.05) and unchanged on days 17–19 (Fig. 8C). AdipoR2 protein in the myometrium was greatest on days 17–19, and lowest on days 10–12 of the cycle (p < 0.05). In the endometrium, the protein content was stable throughout the cycle (Fig. 9A and B). Expression of AdipoR2 protein was higher in the myometrium than in the endometrium on days 17–19 of the cycle (p < 0.05). During the remaining studied phases of the cycle differences in the protein expression were insignificant (Fig. 9C). 4. Discussion In this study, we have demonstrated that the adiponectin system (genes and proteins) is present in the porcine endometrium (epithelial glandular cells, luminal epithelial cells and stromal cells) and myometrium (longitudinal and circular muscle layers) on days 2–3, 10–12, 14–16 and 17–19 of the cycle. It is worth noting that the highest adiponectin protein contents in the endometrium

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Fig. 7. A comparison of adiponectin protein concentration determined by Western blotting analysis in the endometrium (A) and the myometrium (B) between days 2 and 3, 10 and 12, 14 and 16 and 17 and 19 of the oestrous cycle, and (C) between the endometrium and the myometrium on days 2–3, 10–12, 14–16 and 17–19 of the cycle. Upper panels: representative immunoblots (MM—molecular marker; adipose tissue—positive control); lower panels: densitometric analysis of adiponectin 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.

were observed on days 2–3 and 17–19 of the oestrous cycle, but adiponectin gene expression was lowest on the above days. Similarly to the endometrium, in the myometrium the highest adiponectin protein concentrations were noted on days 17–19 of the cycle, but the mRNA expression was lowest during the follicular phase. The reason for the differences in the expression patterns of adiponectin gene and protein remains unknown. The observed variations could be attributed to posttranscriptional processing of gene product and the presence of an intracellular regulatory mechanism of the gene expression. To date, the expression of adiponectin receptors (mRNA only) in the

porcine endometrium was analyzed by a single study (Lord et al., 2005), but unlike in our experiment, the authors did not observe the expression of adiponectin mRNA in an adult sow. This discrepancy could be attributed to methodological differences. In our experiment, we relied on real-time PCR, which is a more sensitive and reliable method than the PCR technique used by Lord et al. (2005). The results of several studies involving different species have revealed the expression of adiponectin mRNA in human endometrial epithelial and stromal cells (Takemura et al., 2006), the expression of adiponectin protein in endometrial stromal and epithelial cells and myometrial

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Fig. 8. A comparison of adiponectin receptor 1 (AdipoR1) protein concentration determined by Western blotting analysis in the endometrium (A) and the myometrium (B) between days 2 and 3, 10 and 12, 14 and 16 and 17 and 19 of the oestrous cycle, and (C) between the endometrium and the myometrium on days 2–3, 10–12, 14–16 and 17–19 of the cycle. Upper panels: representative immunoblots (MM—molecular marker; skeletal muscles—positive control); lower panels: densitometric analysis of adiponectin 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 < 0.001.

endothelial and smooth muscle cells in rabbits, and in epithelial cells of uterine glands in mice (Schmidt et al., 2008). The expression of adiponectin gene and protein was reported in the uteri of pregnant mice (Kim et al., 2011). The expression of both isoforms of the adiponectin receptors at mRNA and protein level was observed in the human (Takemura et al., 2006), rabbit and mouse endometrium (Schmidt et al., 2008). The expression of adiponectin receptors was also detected in the mouse uterus during pregnancy (Kim et al., 2011). Our findings and the result reported in studies of other species suggest that

the uterus is an important source of adiponectin and that the analyzed hormone could act as an autocrine/paracrine factor in the gland to directly modulate uterine functions. The physiological role of adiponectin in the regulation of reproductive processes is poorly understood. Adiponectin knockout mice are fertile (Kubota et al., 2002; Ma et al., 2002; Maeda et al., 2002), but transgenic female mice expressing adiponectin levels two- to three-fold higher than normal circulating levels are infertile (Combs et al., 2004), which underlines the role of adiponectin as an important factor regulating reproductive

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Fig. 9. A comparison of adiponectin receptor 2 (AdipoR2) protein concentration determined by Western blotting analysis in the endometrium (A) and the myometrium (B) between days 2 and 3, 10 and 12, 14 and 16 and 17 and 19 of the oestrous cycle, and (C) between the endometrium and the myometrium on days 2–3, 10–12, 14–16 and 17–19 of the cycle. Upper panels: representative immunoblots (MM—molecular marker; liver—positive control); lower panels: densitometric analysis of adiponectin 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.

processes. AdipoR1 and AdipoR2 are highly expressed in the mid-secretory phase of the menstrual cycle (Takemura et al., 2006), which is equivalent to the implantation window in humans. Serum adiponectin levels decrease in response to selected disorders of the female reproductive system, including the polycystic ovarian syndrome (Ardawi and Rouzi, 2005), endometriosis (Takemura et al., 2005), endometrial cancer (Dal Maso et al., 2004) or preeclampsia (Ouyang et al., 2007), all of which are linked with implantation failure and pregnancy loss. The above findings suggest that the secretion of adiponectin from the endometrium may be timed to optimize both

uterine receptivity and blastocyst development. Kim et al. (2011) used a mouse model of delayed and activated implantation to demonstrate higher expression of adiponectin, AdipoR1 and AdipoR2 proteins in luminal epithelial cells upon the termination of delayed implantation, which suggests a putative role of adiponectin in uterine receptivity and implantation. The fact that adiponectin suppressed the proinflammatory cytokines interleukin 6, interleukin 8 and monocyte chemotactic protein 1 in endometrial epithelial and stromal cells points to the anti-inflamatory role of adiponectin in the endometrium (Takemura et al., 2006).

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In this study, the expression of both adiponectin receptors increased in the mid-luteal phase, which is when maternal recognition of pregnancy occurs in the pig, suggesting that adiponectin could also affect this process. Nevertheless, further studies are needed to determine the physiological role of the hormone in the reproductive system of mammals. It could be hypothesized that the adiponectin system is regulated by gonadal steroid hormones. The lowest concentrations of adiponectin, AdipoR1 and AdipoR2 proteins in the endometrium and myometrium during the mid-luteal and late-luteal phases suggest that progesterone suppresses the expression of adiponectin and both receptor proteins in the uterus. The highest contents of adiponectin and its receptor proteins were detected in the myometrium during the follicular phase, therefore, it cannot be ruled out that the expression of the adiponectin system in the myometrium was promoted by oestradiol. The possible impact of the oestrogens on the production of adiponectin in the body is controversial. According to Chu et al. (2006), the administration of exogenous oestradiol increases adiponectin levels in women. Other authors observed a drop in serum adiponectin levels under the influence of oestrogens (Im et al., 2006), whereas in some studies oestradiol produced no visible effects (Chalvatzas et al., 2009; Sumino et al., 2004). The results of our previous study on pigs (Maleszka et al., 2014) further contribute to the evidence of oestradiol’s inhibitory effect on plasma adiponectin concentrations. Similar adiponectin levels were observed during the early-luteal, mid-luteal and late-luteal phases of the oestrous cycle, and a significant decrease in adiponectin plasma levels was noted during the follicular phase. In healthy women, serum adiponectin levels remain stable throughout the menstrual cycle (Asimakopoulos et al., 2009; Dafopoulos et al., 2009; Hall et al., 2009; Kleiblova et al., 2006), which suggest that the relationship between circulating adiponectin levels and hormonal status linked to the phase cycle of reproductive cycle is species-dependent. Circulating levels of adiponectin are approximately two- to three-fold higher in females than in males, and it has been suggested that ovarian steroids could regulate adiponectin secretion (Bottner et al., 2004; Combs et al., 2003). 5. Conclusions This is the first ever study to demonstrate the presence of adiponectin and its receptors in the porcine uterus as well as changes in the expression of the adiponectin system during the course of the oestrous cycle. Our results suggest that locally synthesized adiponectin can control reproductive processes in pigs by directly influencing the uterus during the oestrous cycle. Further work is needed to determine the role of adiponectin in the regulation of the porcine uterus. Acknowledgment This research was supported by National Science Centre (projects no: 2011/03/B/NZ9/04187).

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