Progesterone, estradiol, arachidonic acid, oxytocin, forskolin and cAMP influence on aquaporin 1 and 5 expression in porcine uterine explants during the mid-luteal phase of the estrous cycle and luteolysis: an in vitro study

Abstract

BackgroundThe cell membrane water channel protein, aquaporins (AQPs), regulate cellular water transport and cell volume and play a key role in water homeostasis. Recently, AQPs are considered as important players in the field of reproduction. In previous studies, we have established the presence of AQP1 and 5 in porcine uterus. Their expression at protein level altered in distinct tissues of the female reproductive system depending on the phase of the estrous cycle. However, the regulation of aquaporin genes and proteins expression has not been examined in porcine uterine tissue. Therefore, we have designed an in vitro experiment to explain whether steroid hormones, progesterone (P4) and estradiol (E2), and other factors: oxytocine (OT), arachidonic acid (AA; substrate for prostaglandins synthesis) as well as forskolin (FSK; adenylate cyclase activator) and cAMP (second messenger, cyclic adenosine monophosphate) may impact AQPs expression.MethodsUterine tissues were collected on Days 10–12 and 14–16 of the estrous cycle representing the mid-luteal phase and luteolysis. Real-time PCR and Western blot analysis were performed to examine the expression of porcine AQP1 and AQP5. Their expression in the uterine explants was also evaluated by immunohistochemistry.ResultsThe results indicated that uterine expression of AQP1 and AQP5 potentially remains under control of steroid hormones and AA-derived compounds (e.g. prostaglandins). P4, E2, AA, FSK and cAMP cause translocation of AQP5 from apical to the basolateral plasma membrane of the epithelial cells, which might affect the transcellular water movement (through epithelial cells) between uterine lumen and blood vessels. The AC/cAMP pathway is involved in the intracellular signals transduction connected with the regulation of AQPs expression in the pig uterus.ConclusionsThis study documented specific patterns of AQP1 and AQP5 expression in response to P4, E2, AA, FSK and cAMP, thereby providing new indirect evidence of their role in maintaining the local fluid balance within the uterus during the mid-luteal phase of the estrous cycle and luteolysis in pigs.

Difference in expression between AQP1 and AQP5 in porcine endometrium and myometrium in response to steroid hormones, oxytocin, arachidonic acid, forskolin and cAMP during the mid-luteal phase of the estrous cycle and luteolysis

“…In turn, on Days 10–12 of the cycle, only the abundance of both AQPs proteins increased. In our recent in vitro studies [ 32 ], performed with the porcine uterine explants, consisting of endometrium and myometrium, P4 evidently stimulated the expression of both AQPs at the protein level, but mostly inhibited it at the transcriptional level. The data suggests that P4 is an important regulator of AQP1 and AQP5 expression in the porcine uterus, particularly in the endometrium.…”

“…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 using a 7300 Real-Time PCR system and SYBR®Green PCR Master Mix (Life Technologies, Grand Island, NY, USA) and specific primers for AQP1 and AQP5 as described previously [ 32 ]. Samples for the specificity control, non-template controls and dissociation curve analysis of the amplified products were used for each reaction.…”

“…Following isolation, the tissues were immediately placed in ice-cold dissection buffer (0.3 M sucrose, 25 mM imidazol, 1 mM EDTA in ddH 2 O, pH 7.2) containing 8.4 μM leupeptin and 0.4 mM pefabloc. As described previously [ 32 ], the membranes were incubated overnight at 5 °C with anti-AQPs or beta-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 antibody-antigen reaction sites 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 results of Western blotting were quantified by densitometric scanning of immunoblots with GelScan for Windows ver.…”

“…We also observed that these AQPs are differently localized and expressed in these structures during the estrous cycle and early pregnancy. Very recently, we described the response of AQPs (AQP1 and AQP5) to treatments with steroids, OT, arachidonic acid (AA), forskolin (FSK) and cAMP in the uterine explants from cyclic gilts during the mid-luteal phase (Days 10–12) and luteolysis (Days 14–16) [ 32 ]. However, to date, the potential of the porcine uterine tissues, endometrium and myometrium to express AQPs has not been studied separately.…”

The role of the aquaporin 5 in the function of late pregnant rat uterus: pharmacological studies

“…They observed the down-regulation of AQP5 gene expression after the progesterone and estrogen treatments during the mid-luteal phase, while in the time of luteolysis, it was increased by estrogen. (Skowronska et al, 2015) Ovariectomized rats were treated with testosterone, estrogen, or combination of them. It was determined that testosterone enhanced the expression of AQP5 in the uterus and this effect was abated by a consecutive estrogen treatment.…”