MODULATORY EFFECTS OF STEROID HORMONES, OXYTOCIN, ARACHIDONIC ACID, FORSKOLIN AND CYCLIC AMP ON THE EXPRESSION OF AQUAPORIN 1 AND AQUAPORIN 5 IN THE PORCINE UTERUS DURING PLACENTATION

Aquaporins are channel-forming trans-membrane proteins facilitating the water movement across the cellular plasma membrane (1). To date, there are 13 known mammalian aquaporins (AQP0-AQP12) that enable increased permeability of water as well as other small molecules such as glycerol and urea (2). Numerous studies have characterized the tissue-specific distribution of AQPs in rats, mice and humans (3). Furthermore, studies with animal knockout for genes encoding these proteins have proved the physiological significance of AQPs in the regulation of water transport in different organs, including those belonging to the reproductive system (4-7). Currently, AQPs are also considered as important players in processes associated with reproduction. Several AQP isoforms were found in the female reproductive tissues: ovary, uterus, placenta, amnion and chorion cytotrophoblast (8, 9). In the pig, the presence of AQP1 and AQP5 in uterus has been documented (10, 11). These AQPs are water selective and belong to the classical AQP family (2). AQP1 is a 28-kDa protein expressed in kidneys (12), pleural membranes, alveolar microvessels, gall bladder, fetal membranes, placenta, endothelia and different epithelia (13-15). AQP5 has mainly been localized in the apical plasma membranes of various secretory glands (16, 17) as well as in the apical membrane of the airway epithelium, corneal and pancreatic epithelium (18). In the pig uterus, AQP1 mostly appears in the capillary endothelium, but AQP5 in smooth muscles as well as in luminal and glandular epithelial cells (10, 11). Data accumulated so far, pertaining to the uterus, suggest that AQPs are involved in water movement between the intraluminal, interstitial and capillary compartments and their uterine expression is essential throughout the pregnancy, including its early stages (19, 15, 20) and parturition (21).

In the pig, the type of placenta is epitheliochorial, in which the chorion does not invade into the uterine endometrium, and the placental barrier includes both trophectoderm and uterine epithelium (22). The placentation in this species starts around Day 30 of pregnancy, when the free-floating embryos are already attached to the endometrium. This process relies on conceptus elongation to increase the available surface area for gas and nutrient exchange (23). Moreover, during early pregnancy, the intra-uterine microenvironment is enriched in a complex array of substances, mainly produced by the uterine glands, which play an important role in onset of pregnancy, i.e. conceptus development, implantation and placentation (24-28). Thus, we supposed that the uterine AQPs are implicated in water handling in connection with the high endometrial secretory activity. A participation of steroid hormones, OT, AA, cAMP and FSK in the regulation of AQP1 and AQP5 expression in the porcine uterus has been tested during the estrous cycle and implantation period (29, 30). In recently published papers, different aspects of placentation process in the pig were considered (31, 32), however data concerning the uterine expression of AQPs during this period are not available. Therefore, in this study, we evaluated the effects of chosen factors on AQP1 and 5 expressions in the pig uterus during placentation using the in vitro procedures. The study aimed to assess the influence of short (3 h) and long (24 h) in vitro incubation of the porcine uterine tissue, representing the placentation period, to P4, E2, OT, AA, cAMP and FSK on the expression of AQP1 and AQP5 mRNA and protein. Preliminary results of this research have been reported during national meeting of Polish Physiological Society (33).

MATERIALS AND METHODS

Animals and collection of uterine tissue

All experiments were performed in accordance with Animal Ethics Committee (AEC approval No. 66/2010 DTN), University of Warmia and Mazury in Olsztyn, Poland.

Tissue samples were recovered from mature cross-bred gilts (Large White × Polish Landrace) (n = 5) on Days 30 – 32 of gestation (placentation). Gilts were observed daily for estrus behavior (Day 0) and exhibited at least two estrous cycles of normal duration (Days 18 – 21). During third estrus they were naturally bred. The animals were slaughtered at a local abattoir on Days 30 – 32 of pregnancy. Pregnancy was additionally confirmed by the presence of morphologically normal conceptuses. Isolated uteri were placed immediately in ice-cold phosphate-buffered saline (PBS) supplemented with 100 IU/ml penicillin (Polfa, Poland) and 100 µg/ml streptomycin (Polfa, Poland) and transported to the laboratory on ice within 1 to 1.5 h for in vitro tissue culture.

Preparation and incubation of uterine slices

Sections of the middle part of uterine horn collected from pigs were opened longitudinally on the mesometrial surface. Uteri were washed three times with sterile PBS, cut into small pieces (400 mg weight) and washed three times in medium M199 (Sigma, USA). The uterine slices were placed in culture vials containing 2 ml Medium 199 supplemented with 0.1% BSA (Sigma), 20 µg nystatin (Sigma) and 20 µg gentamicin (Krka, Novo Mesto, Slovenia) and then preincubated in vitro under atmosphere of 95% O2 and 5% CO2 at 37°C for 18 h. After preincubation, the culture medium was replaced with fresh medium, and the explants were treated with vehicle (control) or P4 (10–5 M), E2 (10 nM), OT (10–7 M; Sigma), AA (10–5 M; Sigma), cpt-cAMP analogue (200 µM; Sigma) and FSK (10 ng/mL) and incubated for additional 3 or 24 h. Concentrations for the treatments were previously determined (29, 34, 35). All treatments were performed in duplicates. Furthermore, uterine tissue explants were snap-frozen in liquid nitrogen (for RNA and protein extraction) and stored at –80°C until further use.

Total RNA isolation, cDNA synthesis and quantitative real-time polymerase chain reaction analysis

Total RNA was extracted from the uterine explants collected after in vitro culture (n = 5 cultures), using fenozol (A&A Biotechnology, Gdansk Poland) in accordance with manufacturer`s instructions. RNA quality and quantity were determined with spectrophotometry (NanoDrop ND-1000, Thermo Scientific, Wilmington, DE, USA). Total RNA samples were transcribed to cDNA using an Enhanced Avian HS RT-PCR Kit (Sigma) and a mix of dNTPs and random hexamers as primers. Real-time PCR was performed in duplicate for each sample by 7300 Real-time PCR system and SYBR®Green PCR Master Mix (Life Technologies, Grand Island, NY, USA) using specific primers for AQP1 and AQP5 as described previously (29). Samples for the specificity control, non-template controls and dissociation curve analysis of the amplified products were used for each amplification. The specificity of amplifications was further validated with electrophoresis of obtained amplicons in a 2% agarose gel and, after extraction from gel, submitted to automated sequencing using 3730xl DNA Analyzer (Life Technologies). Levels of gene expression were calculated with the ΔΔCt method and normalized using the geometrical means for the expression of two reference genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S rRNA.

SDS-PAGE and Western blot

Following isolation, the tissues were immediately placed in ice-cold dissection buffer (0.3 M sucrose, 25 mM imidazol, 1 mM EDTA in ddH2O, pH 7.2) containing 8.4 µM leupeptin and 0.4 mM pefabloc. As described previously (29), the nitrocellulose membranes were incubated overnight at 5°C with anti-AQPs or β-actin antibodies, then with horseradish peroxidase-conjugated secondary antibody (P448, diluted 1:3,000, Dako A/S, Glostrup, Denmark) in PBS-T for 1 h. After washing with PBS-T, the sites of antibody-antigen reaction were visualized with an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Little Chalfont, UK) and exposed to photographic film (Hyperfilm ECL, RPN3103K, Amersham Pharmacia Biotech, Little Chalfont, UK). The products of Western blotting were quantified by densitometric scanning of immunoblots with GelScan for Windows version 1.45 software (Kucharczyk, Poland). Data were expressed as a ratio of AQP proteins relative to actin protein in OD units. In our previous study, we demonstrated that anti-AQP1 and anti-AQP5 antibodies preincubated with the respective immunizing peptides prevented labeling in the pig uterus (11).

Immunohistochemistry

Paraffin embedded blocks of porcine uterus after culture were prepared as described in details elsewhere (11, 29). The tissue sections (4 µm) were exposed to the primary antibody overnight, then to a horseradish peroxidase conjugated goat anti-rabbit secondary antibody (P448; Dako). Nonspecific binding of IgG was eliminated by incubating the sections in 50 mM NH4Cl for 30 min, followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin and 0.2% gelatin. Labeling was visualized by 0.05% 3.3 diaminobenzidine tetrahydrochloride (DAB), and finally counterstained with Mayer`s haematoxylin. The microscopy was carried out using an Olympus light microscope (BX51, Japan). In addition, immunoglobulins from non-immunized rabbit were used as a negative control.

Statistical analysis

All numerical data were analyzed by one-way ANOVA and least significant difference (LSD) post hoc test and reported as the means (± S.E.M.) for five independent observations. Statistical analyses were performed using the Statistica program (StatSoft Inc., Tulsa, USA). Value of P < 0.05 was considered to be statistically significant.

RESULTS

AQP1 mRNA expression in the porcine uterine explants

The control abundance of AQP1 transcript in the uterine tissue harvested from gilts on Days 30 – 32 of pregnancy (the placentation period) did not significantly change during 3-h and 24-h incubations (Fig. 1A and 1B). The AQP1 mRNA expression in the uterine explants significantly (P < 0.05) decreased after 3-h treatments with P4, AA and FSK. Unlike, treatment with E2 for 24 h significantly (P < 0.05) increased AQP1 gene expression (Fig. 1A and 1B). Treatment with OT for 24 h significantly decreased AQP1 expression (P < 0.05). In turn, treatment with cAMP did not change AQP1 expression in the uterine explants (Fig. 1B).

Fig. 1. The expression of AQP1 mRNA in the uterine tissues harvested from pigs (n = 5) on Days 30 – 32 of pregnancy (the placentation period) after treatment with progesterone (P4; 10–5 M), estradiol (E2; 10–9 M), oxytocin (OT; 10–7 M), arachidonic acid (AA; 10–5 M), forskolin (FSK; 10 µg/mL) and cyclic-AMP (cAMP; 200 µM) for 3 (A) and 24 (B) h. The expression of studied gene, determined by real-time PCR, is presented as means ± S.E.M. in relation to the mean expression of reference genes, GAPDH and 18 s RNA. Different letters (a, b) indicate significant differences (P < 0.05) between each treatment and respective control values.

AQP5 mRNA expression in the uterine explants

The expression of AQP5 mRNA in the uterine explants on Days 30 – 32 of pregnancy is presented on Fig. 2A and 2B. The content of AQP5 transcript in the uterine explants following control incubations for 3 and 24 h did not significantly differ. The uterine explants incubated for 3 h with E2, OT, AA, FSK and cAMP exhibited significantly (P < 0.05) increased AQP5 mRNA expression. In contrast, P4 treatment for 3 and 24 h significantly (P < 0.05) reduced AQP5 mRNA expression (Fig. 2A and 2B). Similarly, AA (P < 0.05) and cAMP (P < 0.05) treatments for 24 h significantly decreased AQP5 mRNA expression (Fig. 2B).

Fig. 2. The expression of AQP5 mRNA in the uterine tissues harvested from pigs (n = 5) on Days 30 – 32 of pregnancy (the placentation period) after treatment with progesterone (P4; 10–5 M), estradiol (E2; 10–9 M), oxytocin (OT; 10–7 M), arachidonic acid (AA; 10–5 M), forskolin (FSK; 10 µg/mL) and cyclic-AMP (cAMP; 200 µM) for 3 (A) and 24 (B) h. The expression of studied gene, determined by real-time PCR, is presented as means ± S.E.M. in relation to the mean expression of reference genes, GAPDH and 18 s RNA. Different letters (a, b) indicate significant differences (P < 0.05) between each treatment and respective control values.

AQP1 protein content in the porcine uterine explants

The effects of P4, E2, OT, AA, FSK and cAMP on AQP1 protein expression in uterine explants harvested from pregnant gilts during the placentation period (Days 30 – 32) are shown in Fig. 3A and 3B. AQP1 protein expression in the uterine explants was significantly increased (P < 0.05) only by AA, after 3-h incubation (Fig. 3A). In turn, P4 and E2 significantly (P < 0.05) stimulated the expression of AQP1 protein (by ~3-fold) following 24-h incubation (Fig. 3B).

Fig. 3. Protein content of AQP1 determined by semi-quantitative Western blot analysis, in the uterine explants harvested from pigs (n = 5) on Days 30 – 32 of pregnancy (the placentation period) after treatment with progesterone (P4; 10–5 M), estradiol (E2; 10–9 M), oxytocin (OT; 10–7 M), arachidonic acid (AA; 10–5 M), forskolin (FSK; 10 µg/mL) and cyclic-AMP (cAMP; 200 µM) for 3 (A) and 24 (B) h. Data are presented as means ± S.E.M. in relation to β-actin tissue content. Different letters (a, b) indicate significant differences (P < 0.05) between each treatment and respective control values.

AQP5 protein content in the porcine uterine explants

The AQP5 expression in the uterine explants from gilts on Days 30 – 32 of pregnancy is depicted in Fig. 4A and 4B. The incubation of the explants with AA for 3 hours significantly (P < 0.05) increased in AQP5 protein expression (by ~2.7-fold), (Fig. 4A). Longer incubation (24 hours) with P4, E2, FSK and cAMP also significantly (P < 0.05) elevated AQP5 protein expression (by ~2.5 – 3.0 fold), Fig. 4B.

Fig. 4. Protein content of AQP5 determined by semi-quantitative Western blot analysis, in the uterine explants harvested from pigs (n = 5) on Days 30 – 32 of pregnancy (the placentation period) after treatment with progesterone (P4; 10–5 M), estradiol (E2; 10–9 M), oxytocin (OT; 10–7 M), arachidonic acid (AA; 10–5 M), forskolin (FSK; 10 µg/mL) and cyclic-AMP (cAMP; 200 µM) for 3 (A) and 24 (B) h. Data are presented as means ± S.E.M. in relation to β-actin tissue content. Different letters (a, b) indicate significant differences (P < 0.05) between each treatment and respective control values.

Immunohistochemical localization of AQP1 and AQP5 in the porcine uterine explants

In the uterine explants from gilts on Days 30 – 32 of pregnancy (the placentation period), immunoperoxidase labeling for AQP1 was associated with uterine endothelial cells under control conditions as well as following applied treatments. As exemplarily shown in Fig. 5, both the apical and basal cell membranes exhibited stable AQP1 labeling in control (Fig. 5A) and E2-treated uterine explants (Fig. 5B). In turn, AQP5 was associated with uterine epithelial cells (Fig. 5C-5H) and smooth muscle cells (Fig. 5I-5J). The prominent AQP5 labeling was seen in the apical (in control; Fig. 5C) and additionally in basolateral plasma membranes of the epithelial cells following treatments of the tissue explants with P4 (Fig. 5D), E2 (Fig. 5E), FSK (Fig. 5F) and cAMP (Fig. 5G) for 24 h, and with AA for 3 h (Fig. 5H). In the smooth muscle cells, in contrast to the epithelial cells, applied treatments did not cause any changes in AQP5 localization within the cell membranes.

Fig. 5. Immunohistochemical localization of AQP1 and AQP5 in the uterine explants from pigs on Days 30 – 32 of pregnancy (the placentation period) after treatment with progesterone (P4; 10–5 M), estradiol (E2; 10–9 M), oxytocin (OT; 10–7 M), arachidonic acid (AA; 10–5 M), forskolin (FSK; 10 µg/mL) and cyclic-AMP (cAMP; 200 µM) for 3 or 24 h. Anti-AQP1 antibody labeling of endothelial cells (arrows): (A) control and (B) E2-treated uterine explants (for 3 h) - both the apical and basal plasma membranes exhibited stable AQP1 staining. Anti-AQP5 antibody labeling of epithelial cells (arrows): (C) control - staining is seen only in the apical plasma membranes; (D) P4-treated; (E) E2-treated; (F) FSK-treated and (G) cAMP-treated tissue explants (for 24 h) and (H) AA-treated explants (for 3 h) - prominent AQP5 staining is seen in both the apical and basolateral plasma membranes. AQP5 antibody also stained smooth muscle cells (arrows): (I) control and (J) E2-treated uterine explants (for 24 h). Negative controls for AQP1 and AQP5 were performed by using NRS (normal rabbit serum) - no specific immunostaining is observed ((K) and (L), respectively). The pig kidney cortex was used as a positive control for AQP1 (M) - specific immunostaining is seen in the apical and basolateral cellular membranes in proximal tubules. The pig lung was used as a positive control for AQP5 (N) - specific immunostaining is seen in the apical membranes of type I pulmonary epithelial cells. L, lumen. Bar = 50 µm.

Fig. 6. Hypothetical mechanism of transcellular water movement between the uterine lumen and blood vessels in pigs. Distribution of AQP1 and AQP5 in the uterine explants from pigs on Days 30 – 32 of pregnancy (the placentation period): (A) under control condition - AQP1 was associated with the apical and basal membranes of endothelial cells, but AQP5 was mainly localized in the apical membranes of uterine epithelial cells; (B) after treatment with P4, E2, AA, cAMP or FSK - the AQP1 localization did not change, but AQP5 labeling additionally appeared in the basolateral membranes of epithelial cells (indicated by arrow).

DISCUSSION

In this in vitro study, performed with the uterine explants derived from pregnant gilts during placentation period (Days 30 – 32), we observed the following effects of tested factors; 1) stimulatory of E2 on AQP1 and 5 mRNA expression, 2) predominantly inhibitory of progesterone on AQP1 and 5 mRNA levels, 3) stimulatory of P4, E2 and AA on both AQPs, but cAMP and FSK only on AQP5 protein content, and 4) prominent AQP5 labeling in both the apical and basolateral plasma membranes of the glandular epithelial cells in response to 24-h exposure to steroid hormones, AA, cAMP and FSK.

In previous study, we have described the effects of steroid hormones on the expression of AQP1 and 5 mRNAs on Days 14 – 16 of pregnancy when the implantation occurs (30). In that study, E2 decreased the expression of both AQPs, but P4 affected neither AQP1 nor AQP5 mRNA expression. In the present study, both steroids influenced the expression of studied AQPs during placentation. Namely, E2 up-regulated, but P4 down-regulated AQP1 and 5 expressions. Notably, the response of AQP mRNAs to estradiol required longer (24 h) exposition to the treatment. This comparison implies the existence of distinct expression patterns of genes encoding these AQPs in response to P4 and E2 during implantation and placentation. The change of estradiol action on the expression of AQPs from inhibitory to stimulatory with progressing gestation seems to be the most intriguing observation. It was already found that uterine tissues in pigs produce considerable amounts of steroids (36, 25), including estradiol, of which daily uterine production in this species during early pregnancy reaches about 1 ug (25). It is also likely, that estrogens secreted by developing placenta may additionally modify the uterine AQP expression (37). In previous studies, stimulatory effect of E2 on the uterine expression of AQP1 in rat and AQP5 in mouse were documented by Li et al. (38) and Kobayashi et al. (39), respectively. In addition, Kobayashi et al. (39) have revealed the presence of functional estrogen response element in AQP5 promoter what suggests a possibility of direct action of estrogens on this AQP gene expression. Moreover, Richard et al. (15) found increased AQP1 mRNA expression in mice myometrium in response to estrogens. In contrast to the opposite AQP mRNAs response to E2 and P4 found in the present experiment, the expression of studied AQPs at the protein level was stimulated by both steroids. In many other studies, the lack of correlation between mRNA and protein expression has been already reported (40-44). Our data suggest, that progesterone is engaged in stimulation of AQP protein synthesis in the uterine tissue during placentation period, despite the lack of evident increase in AQP transcript content. Possibly, this effect was induced at the posttranscriptional level.

Oxytocin is an important factor that stimulates contraction of uterus (45), as well as affects phosphoinositide hydrolysis (46), expression of COX-2 in pig uterine tissue (47) and PGF2α secretion (46). It was also evidenced that endometrially secreted OT may play a role in the CL maintenance during early pregnancy. In the porcine uterine lumen, significantly higher concentration of OT is observed on Days 11 – 14 of pregnancy than during corresponding days of the estrous cycle (48). In the present studies, we observed the up-regulation of uterine AQP5 gene expression after 3-h incubation with OT. In turn, OT treatment decreased AQP1 mRNA expression after longer incubation. However, similarly to implantation period (30), OT affected neither AQP1 nor AQP5 protein expression during placentation. Very recently, Ducza and coworkers (49) proposed that OT selectively influences the expression of AQP5 at the end of pregnancy and may serve as a marker that indicates the initiation of delivery in rats.

Prostaglandins (PGs), especially PGE2 and PGF2α, are implicated in the regulation of reproductive processes at the utero-ovarian level, including the crucial role of PGE2 in maintenance of pregnancy and conceptus development (50). Both prostaglandins are produced in the porcine uterus, mainly by endometrial tissue (47), but also by myometrium (34). It is well known that prostaglandin synthesis depends on accessibility of arachidonic acid (51) and the activity of enzymes involved in its metabolism, such as COX-1 and COX-2 (52). Previous studies have suggested that prostaglandins may affect the expression of AQPs. Namely, it was found that the distribution of AQP2 in rat inner medulla is regulated by a balance between AVP and PGE2 (53), and that mice lacking the initial enzyme in prostaglandin synthesis, cPLA2, have abnormal AQP1 expression in the kidney (54). In our recent in vitro study performed with uterine explants derived from gilts on Days 10 – 12 and 14 – 16 of the estrous cycle, the expression of AQP1 and 5 was stimulated at the protein level by AA only during luteolysis (29). During the placentation, the AQP1 expression at the mRNA level was significantly reduced, but at the protein level was stimulated by AA. In turn, the expression of AQP5 was stimulated at the mRNA and protein levels by AA during 3-h exposition. Interestingly, during implantation the inverse relation was found in case of AQP5; AA decreased its mRNA, but increased its protein (30). Collectively, these data indirectly suggest that during placentation the AQP1 and 5 expression may be specifically affected by prostaglandins. Nevertheless, an examination of colocalization of these AQPs with major enzymes of prostaglandin pathway, COX-1 or COX-2, would provide deeper insight into the role of prostaglandins in the uterine AQPs expression during early pregnancy.

In the present study, mainly AQP5 was submitted to regulatory effect of cAMP and forskolin, since these compounds markedly increased AQP5 mRNA during 3-h exposure and protein abundance after 24-h incubation. This indicates that elevated AQP protein abundance most likely resulted from increased number of its transcripts, which was accompanied by subsequent decrease in AQP5 mRNA level (24 h). Similar relationship was found by Yang et al. (35) in studies concerning mouse lung tissue. Moreover, Sidhaye et al. (55) observed that the exposure of lung epithelial cells for several hours to cAMP or forskolin increased AQP5 protein abundance. In the present study, only forskolin decreased AQP1 mRNA after 3-h incubation, whereas other treatments of the uterine tissue with cAMP or FSK were ineffective. This notation is in agreement with the results published by Wang et al. (56), who failed to see any effects of cAMP analogue on the expression of AQP1 gene in vitro in human amnion epithelia, although AQP1 mRNA was increased by FSK. In contrast, during implantation, cAMP and FSK increased both AQP1 mRNA and protein expression (30). Altogether, the previous (30) and present our studies indicate that participation of cAMP in the regulation of AQP1 expression in the uterine tissues is modified depending on the stage of gestation.

In the present study, we also established the uterine localization of AQP1 and 5 by immunohistochemistry in pigs during placentation. Previous studies have documented predominant localization of AQP1 in the apical and basal membranes of uterine endothelial cells and AQP5 in the apical membranes of epithelial cells (10, 11). Such distribution of AQP5 protein in the uterine glands is in line with its epithelial localization in type I and type II pulmonary alveolar cells as well as in the yolk sac and salivary glands (35, 57-59). In the present study, in vitro treatment of the uterine explants with steroid hormones (P4 and E2) has caused an intense accumulation of AQP5 signal additionally in the basolateral membrane of the epithelial cells. The effects of steroid hormones on AQPs expression were also studied in rodents mainly during implantation period (15, 19, 20, 60). Lindsay and Murphy (20, 60) reported increased expression of AQP5 in uterine epithelial cells by progesterone alone and in combination with estrogens. They also noted progesterone-dependent expression of AQPs in the rat uterus; AQP5 in glandular epithelium and AQP1 in the inner circular layer of myometrium. Moreover, in our study, AA, cAMP and FSK have caused - in contrast to control - a prominent accumulation of AQP5 in the basolateral membrane of the uterine epithelial cells. Similar effects of studied factors on localization of AQP5 were noted in uterine tissue representing the implantation period (30). The above observations, concerning AQP5 distribution in the uterine tissue, are summarize in Fig. 6. The appearance of AQP5 on surface of the basolateral membrane following treatment with examined substances indicates that this aquaporin is markedly engaged in water efflux and influx within uterus. These changes seem to be connected with high secretory activity of endometrial glands during early pregnancy, which release multiple substances that promote uterine receptivity and embryonic development (61, 25).

In summary, this research has demonstrated the uterine expression of mRNAs and proteins for AQP1 and AQP5 in pregnant gilts during placentation as well as the effects of studied factors (P4, E2, OT, AA, cAMP and FSK) on this expression. Estradiol stimulated AQP1 and 5 mRNA expression, but progesterone predominantly inhibited AQP1 and 5 mRNA levels. In turn, at the protein level, steroid hormones and AA stimulated the expression of both AQPs, but cAMP and FSK stimulated only AQP5. Interestingly, the basal uterine expression of AQP1 during placentation appeared to be markedly lower than during implantation in previous study. Moreover, studied factors (P4, E2, AA, cAMP or FSK) have caused appearance of AQP5 in the basolateral membranes of epithelial cells, in addition to its presence in the apical membranes of these cells.

Based on our previous and present studies, it might be concluded that AQP1 and AQP5 play an important physiological role in maintaining water homeostasis and transcellular water movement within endometrial and endothelial cells in the pig uterus during early pregnancy. The present study also indicates that uterine expression of AQP1 and AQP5 remains under control of steroid hormones (P4 and E2), AA derivatives and cAMP, which contribute to embryo placentation in pigs.

Author contributions: conception and design of research: MTS and AS, the protein analysis: MTS and AS, the gene expression analysis: PM and AS, contribution to the analysis and interpretation of data: AS, the manuscript was written by AS, revised by: SO, SN and MTS, prepared figures: MTS and AS.

Acknowledgements: This work was support by Grant Number N N308 5848 40 awarded to MTS from the Polish Ministry of Science and Higher Education.

Conflict of interests: None declared.

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