Elsevier

Theriogenology

Volume 78, Issue 5, 15 September 2012, Pages 951-964
Theriogenology

Research article
Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs

https://doi.org/10.1016/j.theriogenology.2012.05.016Get rights and content

Abstract

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.

Introduction

Successful embryo implantation and establishment of pregnancy depends on two major factors: proper development of embryos capable of implanting and, at the same time, a receptive endometrium. Under the influence of ovarian steroids, locally acting soluble factors synthesized by the endometrium can affect conceptus development and implantation. Developing embryos, in turn, synthesize and secrete molecules that can act in an autocrine or paracrine manner to improve conceptus survival in the uterus. Among the factors involved in embryo-maternal interactions are prostaglandins, growth factors and cytokines and their receptors [for review see references: [1], [2], [3], [4]]. Leukemia inhibitory factor (LIF) and interleukin (IL)-6 are present within the uterine luminal microenvironment during early pregnancy, and seem to be good candidates for involvement in conceptus-endometrial signaling during the peri-implantation period [1], [5], [6].

LIF is a pleiotropic cytokine that was first described as a stimulator of differentiation of the myeloid leukemia cell line M1 [7]. This cytokine exhibits a variety of functions, including cell proliferation, differentiation, and survival that are important for embryo development and implantation [8]. Thus, LIF and LIF receptor (LIFR) expression in conceptus and/or endometrial tissue and LIF content in the uterine lumen during pregnancy have been studied in many species, especially in humans and mice [5], [8], [9]. Female mice lacking functional LIF are unable to support implantation, even if exogenous LIF was infused into the uterine lumen. On the other hand, blastocysts collected from LIF-deficient mice can implant after transfer to wild type recipients [10]. These data indicate that maternal expression of LIF is essential for implantation in mice. In the human endometrium, LIF mRNA and protein content are low or undetectable during the proliferative phase but increase significantly during mid- and late secretory phases [9], [11]. LIF protein has been also detected in human uterine flushings during the expected time of implantation in fertile women. Interestingly, the concentration of LIF in the uterine lumen of infertile women is significantly lower than in fertile women during the same phase of the menstrual cycle [12]. In contrast to the endometrium, no LIF mRNA was observed in preimplantation human embryos [13]. In humans and mice, LIFR is expressed both in embryos and in the luminal epithelium of the endometrium [11], [13].

IL-6 is a multifunctional cytokine whose role in embryo implantation has been studied. IL6-deficient mice exhibit reduced fertility and decreased numbers of implantation sites with viable embryos [14]. Moreover, in the human endometrium, IL-6 protein is weakly expressed during the proliferative phase, but immunoreactivity to IL-6 increases during the midsecretory phase, corresponding to the implantation window [15]. Reduced endometrial IL6 mRNA expression was demonstrated in women with unexplained recurrent miscarriages compared with fertile women [16]. IL-6 receptors are localized in preimplantation embryos [13], and also in glandular epithelium and stromal cells of the human endometrium [15]. Because IL-6 is an important regulator of expression of other genes crucial for embryo development [17], a role in implantation is also postulated for this cytokine.

In the pig, the expression of LIF, IL-6, and their receptors has been studied in the endometrium and/or conceptuses [18], [19], [20], [21], [22]. It was demonstrated that LIF [18], [19], [21] and IL6 [19], [20] mRNA is expressed in the endometrium of pregnant gilts. Both IL-6 and LIF are detected in uterine flushings of early pregnant pigs [18]. Moreover, LIFR mRNA is expressed in porcine conceptuses and endometrial cells during early pregnancy [19]. Expression of IL6R mRNA in the pig endometrium was also demonstrated [22]. These data indicate that LIF and IL-6 may be important for embryo-maternal interactions in pigs, but no study has been performed on the regulation of expression of these cytokines in the endometrium or on the role of LIF and IL-6 during implantation in this species. Additionally, there are no data concerning the presence of IL6R in porcine conceptuses. Therefore, the current study was conducted to examine: (1) the profiles of LIF and IL6 mRNA expression and protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus secretions on LIF and IL-6 synthesis in the pig endometrium during the peri-implantation period using an in vitro model; (3) profiles of IL6R and LIFR mRNA expression in porcine conceptuses on Days 10 to 18 of gestation; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells in vitro.

Section snippets

Animals

All procedures involving the use of animals were approved by the Animal Ethics Committee, University of Warmia and Mazury in Olsztyn, Poland, and were conducted in accordance with the national guidelines for agricultural animal care. In all experiments, 58 pubertal, crossbred gilts (Sus scrofa domesticus) of similar age (8 to 8.5 mo), weight (140 to 150 kg) and genetic background from one commercial herd were used.

To analyze IL6 and LIF mRNA expression, IL-6 protein content in the endometrium,

LIF and IL6 mRNA expression in the endometrium during the estrous cycle and early pregnancy

The expression of LIF mRNA was affected by day after estrus (P < 0.001) and by reproductive status of animals (P < 0.05; Fig. 1A). The lowest LIF mRNA content was detected on Day 10 of the estrous cycle and pregnancy, when compared with all other days studied (P < 0.05). After an initial sharp increase between Day 10 and 12 in both cyclic and pregnant animals (P < 0.001), the expression of LIF mRNA in the endometrium of cyclic gilts did not vary significantly on Days 12 to 18. In the pregnant

Discussion

Implantation is a critical step for the establishment of pregnancy that requires coordination of molecular and cellular events resulting in uterine growth and differentiation, conceptus development and implantation, and placental formation. Among factors involved in embryo-uterine cross-talk during the peri-implantation period are members of the IL-6 family of cytokines, IL-6 and LIF, and their receptors [2], [5]. Intensive studies on the role of LIF and its receptors during implantation began

Acknowledgments

This research was supported by grant 717/N-COST/2010/0 from Ministry of Science and Higher Education in Poland.

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