Elsevier

Theriogenology

Volume 84, Issue 6, 1 October 2015, Pages 969-982
Theriogenology

Research article
Prostacyclin receptor (PTGIR) in the porcine endometrium: Regulation of expression and role in luminal epithelial and stromal cells

https://doi.org/10.1016/j.theriogenology.2015.05.034Get rights and content

Abstract

The prostacyclin (PGI2) signaling pathway plays an important role during early pregnancy in rodents and ruminants. Abundant concentrations of PGI2 were also found in the endometrium and uterine lumen of gilts during the period of implantation. The present study was designed to examine (1) the expression of PGI2 receptor (PTGIR) messenger RNA (mRNA) and protein in the endometrium of cyclic and early-pregnant gilts; (2) possible regulation of endometrial PTGIR gene expression by conceptus products, estradiol and cytokines; (3) the effect of iloprost (a PGI2 analogue) on cAMP formation and the expression of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF; isoform 164) mRNA in luminal epithelial (LE) and stromal (ST) cells. Increased PTGIR mRNA expression in the endometrium was detected on Days 11 to 12 and 18 to 20 of pregnancy compared with respective days of the estrous cycle (P < 0.05). Moreover, greater PTGIR protein level was observed in pregnant than in cyclic gilts on Days 11 to 12 after estrus (P < 0.05). Gilts with unilateral pregnancy revealed abundant PTGIR expression in the endometrium collected from the gravid uterine horn of Day-11 pregnant gilts compared with the respective horn of cyclic animals (P < 0.01). Both LE and ST cells of the endometrium possess PTGIR protein. Moreover, IL1β, IFNγ, and conceptus-exposed medium, but not estradiol, stimulated PTGIR mRNA expression in LE and ST cells in vitro. Activation of PTGIR by incubation of LE and ST cells with iloprost resulted in greater cAMP generation (P < 0.01). Moreover, iloprost increased FGF-2 and VEGF164 mRNA expression in ST (P < 0.05), but not LE cells. In conclusion, this study revealed increased expression of PTGIR in the porcine endometrium during the peri-implantation period and reported a possible regulation of PTGIR abundance by conceptus-derived factors. Moreover, besides its important role in vascular system, PGI2 may promote the expression of proangiogenic genes in the uterine stroma.

Introduction

Successful implantation depends on the quality of embryos, a receptive endometrium, and the precise orchestration of embryo–maternal dialog. In ruminants and pigs, progesterone action is a prerequisite for the attainment of uterine receptivity and several progesterone-related genes have been identified in luminal epithelial (LE) and stromal (ST) endometrial cells. However, developing embryos further modulate the endometrial milieu, making it competent for implantation [1], [2], [3]. In pigs, pregnancy recognition is the result of estrogen synthesis and secretion by conceptuses between Days 10 and 15 of pregnancy [2], [4]. Effects of estrogens in the uterus include regulation of prostaglandin (PG) synthesis [5], [6], stimulation of expression of genes required for implantation [7], and increased uterine blood flow [8]. Additionally, uterine histotroph contains signaling molecules that support implantation and conceptus development [2].

Prostacyclin (prostaglandin I2; PGI2) is a member of the prostanoid family of lipid mediators, which are derivatives of arachidonic acid. The rate-limiting enzyme in prostanoid biosynthesis is prostaglandin endoperoxide synthase (PTGS2), also known as cyclooxygenase, that converts arachidonic acid into unstable intermediary PG, PGH2 [9]. Afterward, PGH2 is rapidly converted into PGI2 by the action of prostacyclin synthase (PGIS) [10]. Prostacyclin synthase is expressed mainly in endothelial cells and smooth muscle cells but is also present in several other cell types [11], [12]. In the cardiovascular system, PGI2 is a potent vasodilator and an inhibitor of platelet aggregation [10], [13]. Moreover, PGI2 inhibits the proliferation of vascular smooth muscle cells [14], reduces pulmonary blood pressure [15], and regulates renal blood flow [16]. This molecule is considered as a mediator of protective effects of vascular endothelial growth factor (VEGF) on vascular bed [17]. Moreover, PGI2 may also be involved in the establishment of pregnancy and implantation. Sustained overproduction of PGI2 is physiologically evident during pregnancy in women [18], mice [19], and domestic species [20], [21]. Prostaglandin I2 signaling pathway is critical for endometrial decidualization in mice because a PGI2 analogue restores implantation in Ptgs2-deficient females [19]. Moreover, PGI2 enhances blastocyst hatching and promotes live birth potential of mouse embryos [22], [23].

The biological actions of PGI2 are mediated by a PGI2-specific G-protein–coupled receptor, PTGIR (also known as IP), which has a typical seven transmembrane structure [24]. Binding of PGI2 to the Gs subunit of this receptor leads to adenylate cyclase activation and rapid cAMP formation. This classical PGI2 signaling is involved in the regulation of vascular function and smooth muscle relaxation [24], [25]. Ptgir-deficient mice show increased susceptibility to thrombosis and reduced inflammatory and pain responses [26]. An important role for PGI2 and PTGIR in menstruation was also suggested because the greatest expression of PTGIR in the human endometrium was observed during the menstrual stage compared with the proliferative and secretory stages of the cycle [27]. Moreover, PGI2 concentration in uterine venous blood is maximal during menstruation [28]. Additionally, PTGIR is involved in PGI2-stimulated expression of proangiogenic genes in the endometrium of women [29].

Among domestic species, the profiles of PTGIR expression have been investigated in the endometrium of cyclic and pregnant animals, as well as in conceptuses and embryos of cows and ewes [20], [30]. Recently, we reported differential expression of PTGIR in porcine peri-implantation conceptuses and found that PGI2 acting through PTGIR may promote the attachment and proliferation of trophoblast cells [31]. These data indicate that PGI2 may facilitate conceptus implantation in the pig. On the other hand, there are no data concerning PTGIR expression in the porcine endometrium and the role of PGI2 in this tissue during early pregnancy. Therefore, this study was designed to investigate (1) the profiles of PTGIR messenger RNA (mRNA) and protein expression in the endometrium of cyclic and early-pregnant gilts on Days 9 to 10, 11 to 12, 15 to 16, and 18 to 20 after estrus; (2) the effect of conceptus presence on PTGIR mRNA and protein expression in the porcine endometrium using an in vivo model; (3) the effect of estradiol (E2), conceptus-exposed medium (CEM), and cytokines on PTGIR mRNA expression in LE and ST cells of the porcine endometrium. Moreover, intracellular cAMP formation and the expression of fibroblast growth factor-2 (FGF-2) and VEGF164 mRNA were measured after in vitro stimulation of LE and ST cells with iloprost (a PGI2 analogue). Alternative splicing of VEGF mRNA from a single gene results in at least five different isoforms [17]. We analyzed VEGF164 mRNA expression because 164–amino acid isoform is the most abundant form in the porcine endometrium [32].

Section snippets

Animals and sample collection

All procedures involving the use of animals were conducted in accordance with the national guidelines for agricultural animal care and were approved by the Animal Ethics Committee, University of Warmia and Mazury, Olsztyn, Poland. In all experiments, a total of 73 crossbred gilts (Sus scrofa domesticus) of similar genetic background from one commercial herd were used.

To analyze endometrial PTGIR mRNA and protein expression, 36 pubertal gilts of similar age (8–8.5 months) and weight (140–150 kg)

PTGIR mRNA and protein expression in the endometrium of cyclic and early-pregnant gilts

PTGIR mRNA expression in the porcine endometrium was affected by the day (P < 0.001), reproductive status of animals (P = 0.009), and day-by-status interaction (P = 0.012; Fig. 1A). The relative level of PTGIR mRNA did not change during the studied period of the estrous cycle. During pregnancy, increased PTGIR mRNA expression was observed on Days 18 to 20 compared with Days 9 to 10 (P < 0.001), 11 to 12, and 15 to 16 (P < 0.01). Moreover, greater content of endometrial PTGIR transcripts was

Discussion

Abundant concentrations of PGI2 found in uteri of early-pregnant mice [19], rats [40], cows [20] and pigs [21] indicate that PGI2 is an important component of the proper embryo–maternal interactions during pregnancy establishment. As we have shown previously, increased expression of PGIS protein in the porcine endometrium on Days 12 and 16 of pregnancy was accompanied by greater concentrations of PGI2 in both the endometrial tissue and uterine lumen [21]. Recently, we found that PGI2 acting via

Acknowledgments

The authors are grateful to Ms Katarzyna Gromadzka-Hliwa and Mr Jan Klos for technical assistance in the laboratory and Mr Michal Blitek for help in care and handling of animals.

This work was supported by the National Science Centre (grant: 2011/01/B/NZ9/07069 to Agnieszka Blitek) and by basic grant of the Polish Academy of Sciences.

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