Background
Since the discovery of aquaporins (AQPs), water channel proteins, a high rate of transcellular water flow is believed to be mediated by these specialized protein transporters. Recently, AQPs have become considered to be important players in the field of reproduction, see reviews [
1,
2]. Several AQP isoforms are expressed in the female reproductive tissues: ovary, uterus, placenta, amnion and chorion cytotrophoblasts [
3‐
10]. Their specific expression pattern suggests that they participate in water movement between the intraluminal, interstitial and capillary compartments. Further studies have demonstrated that AQPs are also involved in endometrial development, cell migration and invasion [
11]. Aquaporin 1 and 5 are water selective and belong to the classical AQP family [
12]. AQP1 is a 28-kDa water channel protein expressed in the endothelial and epithelial cells of many tissues, increasing water permeability of the cell membrane. AQP5 has mainly been localized in apical plasma membranes of various secretory glands [
13]. The important role of AQP5 in water homeostasis is evidenced by AQP5-null mice which have reduced saliva secretion [
14].
The expression of AQPs in uterine tissue was first described by Li et al. [
15], who confirmed the presence of AQP1 transcript in the human uterus. Afterwards, Li et al. [
16] demonstrated that AQP1 mRNA expression in the rat uterus is up-regulated by estradiol. Accumulating evidence indicated that ovarian steroids can affect the expression of several AQPs in the reproductive system, including the uterus [
17,
18]. The presence of AQP1, 2 and 5 has also been studied throughout the estrous cycle in bitches [
19]. Very recently, Klein et al. [
20] showed uterine mRNA expression of 12 different AQPs subtypes in endometrium of cyclic and pregnant mares. Moreover, it was demonstrated that cAMP is involved in up-regulation of some AQPs in a variety of cell types [
9,
21‐
24]. The presence of AQP1 in human endometrial blood vessels indicates its involvement in the regulation of edema, and in the regulation of angiogenesis [
25] as well as pathological processes related to ovulatory uterine bleeding in women [
26].
Studies on pigs suggest a functional collaboration among diverse AQPs within the uterus during different phases of the estrous cycle and early pregnancy [
27]. It has been shown that AQP5 is localized in myometrial and epithelial cells of the uterus, but AQP1 in uterine endometrial and myometrial blood vessels [
7,
27]. Their expression at a protein level was also altered in distinct tissues depending on the phase of the estrous cycle and the stages of early pregnancy. However, the regulation of aquaporin genes and protein expression has not been examined in porcine uterine tissue. Therefore, we have designed an
in vitro experiment to explain whether steroid hormones, progesterone (P
4) and estradiol (E
2), and other factors: oxytocine (OT), arachidonic acid (AA; substrate for prostaglandins synthesis) as well as forskolin (FSK; adenylate cyclase activator) and cAMP (cyclic adenosine monophosphate; second messenger) may have impact on the AQPs expression. Consequently, the primary aim of this study was; (i) to examine the changes in AQP1 and AQP5 at mRNA and protein levels in porcine uterine explants in the presence of P
4, E
2, OT, AA, FSK and cAMP; and then (ii) to describe the effect of tissue exposition duration to the experimental factors on the AQPs expression; (iii) to compare their expression in the uterine explants, representing the mild-luteal phase of the estrous cycle and luteolysis; (iv) to determine the localization of AQP1 and 5 in uterine explants after the treatments. This study provided additional information concerning factors potentially responsible for water homeostasis in porcine uterus during the mid-luteal phase and luteolysis in the pig.
Discussion
In the present study, we have demonstrated that two isoforms of
AQPs (
1 and
5) mRNAs are expressed in the porcine uterine explants in both studied periods. In our previous study, performed with the use of immunohistochemistry and Western blot, AQP1 and 5 were clearly detected in untreated uterine tissue on Days 10–12 and 14–16 of the estrous cycle. In cyclic gilts, endometrial and myometrial expression of AQP1 did not change significantly, in turn endometrial AQP5 protein expression was significantly higher on Days 14–16 than on Days 10–12 of the cycle [
7]. In the present study, we aimed to further investigate, using an
in vitro system, whether these AQPs are regulated by steroid hormones, cAMP, forskolin, arachidonic acid or oxytocin in porcine uterine tissue at mRNA and protein levels.
In the present study, steroid hormones (P4 and E2) differentially influenced the expression of AQP1 and 5 in porcine uterine tissue under in vitro conditions. AQP1 gene expression was down-regulated by E2 treatment for 3-h during the mid-luteal phase. Furthermore, P4 inhibited AQP1 mRNA gene expression during luteolysis, after short incubation (3 h). Steroid hormones (P4 and E2) increased AQP1 protein expression during both studied stages of the cycle and incubations. Moreover, E2 more effectively stimulated AQP1 protein expression on Days 14–16 than on Days 10–12 of the cycle. In turn, AQP5 gene expression was also down-regulated after treatments with E2 and P4 (3 h and 24 h) during the mid-luteal phase, but up-regulated by E2 during luteolysis (3 h). As in the case of AQP1, longer incubation with steroids down-regulated AQP5 mRNA expression during the mid-luteal phase. Interestingly, the basic (control) level of AQP1 transcript was higher (after 3- and 24-h incubations) in the mid-luteal phase (2.07 ± 0.61 and 1.03 ± 0.09, respectively) than during luteolysis, but in the case of AQP5 (after 3-h incubation) the relationship was inversed (1.59 ± 0.35 and 4.48 ± 0.71, respectively). At the protein level, the changes of AQP1 and 5 expression in response to E2 and P4, were not in full agreement with those noted for mRNAs.
Our findings concerning the E
2 effect on AQP expression are comparable to those reported by Li et al. [
16] and Kobayashi et al. [
31], who studied this steroid action on the uterine expression of AQP1 in rat and AQP5 in mouse, respectively. In addition, Kobayashi et al. [
31] revealed the presence of functional estrogen response element in AQP5 promoter regions which suggests the possibility of direct action of estrogens on AQP5 expression. Richard et al. [
5] found increased
AQP1 mRNA expression in mice myometrium in response to estrogen, but AQP5 expression was induced by estrogen only in progesterone-primed uteri. In several studies, the role of P
4 alone or in combination with estrogens in controlling AQP expression in uterus has been confirmed. Lindsay and Murphy [
6] reported increased expression of AQP5 in uterine epithelial cells by progesterone alone and in combination with estrogen. The same authors [
18] also noted progesterone-dependent expression of AQP5 and AQP1 in the rat uterus; AQP5 in glandular epithelium and AQP1 in the inner circular layer of myometrium. Aralla et al. [
19] demonstrated coincidently elevated expression of AQP5 in the apical plasma membrane of uterine epithelial cells with increased concentrations of P
4 in plasma. Furthermore, in the mouse uterus exogenous estrogen strongly up-regulated the expression of AQP2, without any effect on AQP5 [
17]. Very recently, the presence of mRNA for all
AQPs (
AQP0 to
12) has been shown in equine endometrium, while the Western blot analysis confirmed protein expression of AQP0, 2 and 5 [
20]. To the contrary, progesterone treatment of anoestrus mares did not enhance the expression of AQPs, indicating that factors other than progesterone or some factors in addition to progesterone are required for the up-regulation of certain AQP subtypes. The above observations are not entirely consistent, the discrepancies seem to result from different animal models and/or experimental protocols applied in the study. For example, Jablonski et al. [
17] used ovariectomized and hormonally treated mice, but in the present study porcine uterine explants were used. In turn, the lack of a full relationship – observed in the present study – between the concentration of gene transcripts and respective proteins may result from differentiated stability of mRNAs and/or proteins as well as from independent regulation of transcription, posttranscriptional processes or translation and functioning feedbacks,
i.e. high protein concentration may suppress mRNA synthesis. Moreover, steroids themselves may alter the stability of mRNA [
32]. It is also noteworthy that the processes of transcription and translation are not equally efficient.
The uterus is a target organ for ovarian steroid hormones and undergoes marked changes during the estrous cycle, including: tissue expansion (by 40-60%) and an increase in uterine gland activity during luteal phase [
33], hyperemia [
34] as well as increased capillary permeability [
33]. These changes require increased synthesis of AQPs. In the present study, as documented by Western blot analysis, P
4 and E
2 appeared to be effective in controlling AQP1 and AQP5 expression in pigs, suggesting their crucial role in this regulation during the mid-luteal phase of the estrous cycle and luteolysis.
Arachidonic acid is metabolized in the uterus to prostaglandins (PGE
2 and PGF
2alpha), which are involved in many reproductive activities including luteolysis, maternal recognition of pregnancy, endometrial gene expression and conceptus development [
35‐
37]. Prostaglandin synthesis is thus dependent upon accessibility of AA [
38] and the activity of enzymes involved in its metabolism [
39]. In our studies, its potential effect on AQP1 and 5 expression in uterine tissue of cyclic pigs was tested. The expression of
AQP1 and
5 mRNAs were similarly down-regulated by AA during the mid-luteal phase (after 3- and 24-h exposition). Nevertheless, the expression of both AQPs at the protein level was stimulated only during luteolysis in response to shorter exposition to AA. This well corresponds with physiological situation, since PGF
2alpha is released by endometrium (epithelial and stromal cells) [
40] in a pulsatile manner to cause corpus luteum regression [
41]. Thus, our data indirectly indicate that prostaglandins may exert regulatory effects on AQP1 and 5 in uterine tissue. Studies performed by Zelenina et al. [
42] have confirmed the interaction of PGE
2 with AVP in the regulation of AQP2 in the rat renal medulla
. Nevertheless, the potential involvement of prostaglandins in the regulation of AQPs in the pig uterus requires explanation in further experiments.
Oxytocin is one of the key hormones implicated in controlling the uterine functions. Among others, it affects phosphoinositide hydrolysis [
43], expression of COX-2 in uterine tissue [
37] and regulates PGF
2alpha secretion [
43]. The main reason for investigating the effect of oxytocin on AQP1 and AQP5 expression was that pig endometrium secretes oxytocin [
44] and possesses its receptors [
45]. In the uterus, the concentration of OT receptor in the endometrium changes during the estrous cycle. It has been reported that in the middle luteal phase of the cycle, the density of OT receptor increases in the endometrium and myometrium [
46]. In the present experiments, OT decreased
AQP1 and
AQP5 mRNA expression (after 3- and 24-h incubations), without visible changes in protein content. In the pig during luteolytic period, oxytocin is responsible for pulsatile secretion of PGF
2alpha and contractions of myometrium [
47]. It might be hypothesized that under physiological conditions, inhibitory action of OT on
AQP1 and
5 uterine expression at a transcriptional level is connected with remodeling of endometrial tissues taking place at the end of the luteal phase [
33]. Very recently, Ducza et al. [
48] demonstrated increased
AQP2 mRNA, but reduced
AQP5 mRNA expression by OT in the rat uterus on Day 18 of pregnancy. Since the role of oxytocin, similar to PGs, in the regulation of AQPs expression and uterine fluid balance, so far is not sufficiently defined, therefore further work is needed to better delineate it.
Previous studies confirmed that intracellular signaling pathway consisting of adenylate cyclase (AC) and cAMP may be involved in the regulation of AQP expression [
21‐
23]. In the present study, the effects of cAMP and forskolin (AC activator) on AQP1 and 5 in the uterine tissue were tested. The treatment with cAMP did not cause striking changes in expression of studied
AQP mRNAs in the uterine tissue; only increased
AQP1 expression on Days 14–16 (after 24 h) and decreased
AQP5 on Days 10–12 (after 3 h) and Days 14–16 (after 24 h) of the estrous cycle. In a different experimental model, Wang et al. [
22] failed to see any changes in the expression of
AQP1, 8 or
9 genes in human amnion-derived WISH cells after
in vitro treatment with cAMP analog. In our studies, the opposite reaction of
AQP5 mRNA versus protein in response to cAMP is complicated. This discrepancy may result from the regulatory mechanism functioning at the transcription and translation levels, as discussed earlier and requires an explanation in further studies. The effects of forskolin on
AQP1 and
5 mRNAs were very similar to those observed in response to cAMP; i.e. stimulation of
AQP1 and mostly inhibition of
AQP5 (except stimulation on Day 14–16 after 24 h incubation). At the protein level, the expression of AQP1 and AQP5 was up-regulated by forskolin, as in response to cAMP. Studies performed with the use of different cells/tissues [
9,
22,
23,
49] or cell lines [
21,
23,
24] also confirmed the stimulatory effect of cAMP and/or forskolin on the expression of AQP1 [
24], AQP5 [
23] and AQP3, 8 and 9 [
9,
21,
23,
49]. Furthermore, AQP1 appeared to be up-regulated by arginine vasopressin and cAMP analogue in trophoblast cells [
24]. On the basis of our study, it might be concluded that the AC/cAMP pathway participates in the regulation of AQP5 expression in the uterine tissue. However, the engagement of the AC/cAMP pathway, particularly in the regulation of AQP1 expression in this tissue during the estrous cycle remains to be elucidated.
The changes in cellular localization of AQP1 and AQP5 in response to the studied factors, visualized by immunohistochemistry, are particularly interesting (Figure
5). In the uterine tissue, localization of AQP1 was predominantly associated with apical and basal membranes of endothelial cells, but AQP5 with apical membranes of epithelial cells. It is noteworthy that steroid hormones (P
4 and E
2), cAMP and forskolin caused an emergence of AQP5 in basolateral membrane of the epithelial cells during both studied periods. It might be thus hypothesized, that these changes are connected with potentially bidirectional transcellular water movement through uterine epithelial cells. Garcia et al. [
50] indicated that cAMP may induce insertion of AQP8 within intracellular vesicular structures and translocation to plasma membranes in the rat hepatocytes. Further experiments are necessary to further explain the role of AQPs in uterine water balance in cyclic gilts.
Authors’ contributions
MTS and AS conceived and designed the experiments. AS, PM and MTS performed the experiments. SN, BW, AS, MTS and SO contributed reagents/materials/analysis tools. AS wrote this manuscript. All authors read and approved the final manuscript.