Location And Function Of CCL2-CCR2Autocrine In The Porcine Endometrium During Implantation

In pig, litter size is one of the most important economic traits. Implantation plays a fatal role in litter size. It was reported that CCL2(CC Chemokine ligand2) was expressed in the human and sheep endometrium during implantation; however, regulatory machinism and biological function of CCL2remains understood. In this study, we aimed to investigate location and biological function of CCL2and its receptor CCR2(CC Chemokine receptor2) expressed in the porine endometrium.Firstly, we identified the location of both CCL2and CCR2in the porcine endometrium. Immunohistochemistry showed that both CCL2and CCR2were co-expressed in endometrial luminal epithelia. Uterine samples collected from sows which were slaughed on the pregnant Day13, Day18and Day24and estrous cyclic Day13and Day18. Secondly, porcine endometrial luminal epithelia was labeled respectively by same-condition immunohistochemistry using anti-CCL2antibody and anti-CCR2antibody. The results showed that expression of CCL2and CCR2during estrous cycle were stronger than their expression during implantation; during the estrous cycle, expression of CCL2and CCR2were weaker in endometrial luminal epithlia on the Day18comparing with their expression on the Day13; CCL2expressed in the implantation sites was higher than non-implantation sites; in the implantation sites, expression of CCL2reached the peak on the pregnant Day18; with implantation process, the expression of CCL2declined in the inter-sites. The expression of CCR2was declining during the implantation, and implantion sites were higher than inter-sites.This study founded the isolation, culture and identification of primary procine endometrial luminal epithelial cells. To explore the relationship between CCL2and CCR2, primary porcine endometrial luminal epitheliail cells were treated respectly with porcine CCL2recombine protein, anti-CCL2nutrual antibody, inhibitor of CCR2(RS102895), and RS102895+CCL2. Western blotting demonstrated that CCL2promoted significantly CCR2expression in the endometrial luminal epithelial cells; in contrast, CCL2could not effect the expression of CCL2, suggesting that porcine endometrial luminal epithelial cells formed a positive-feedback CCL2-CCR2autocrine. CCR2was increased rapidly in the endometrial luminal epithelial cells with RS102895treatment. Next, the study investigated the effect of progesterone on expression of CCL2and CCR2. In vitro, porcine endometrial luminal epithelial cells were treated with progesterone; In vivo, CCL2and CCR2in the endometrial luminal epithelia of ovariectomized gilts injected with progesterone were higher than control. These data demostreated that progesterone could increase the expression of CCL2and CCR2.To invistgate that function of CCL2-CCR2, porcine endometrial luminal epithelial cells were treated respectively recombine CCL2protein, anti-CCL2neutralizing antibody and PBS. RNA-Seq captured no differential gene between CCL2group and control. RNA-Seq captured48differential genes between anti-CCL2neutralizing antibody group and control, including11genes which are reported involve in implantation process such as VEGFA, CEBPB, MIF, CLDN3. To prove that effect of CCL2-CCR2on cell proliferation, cells were treated with recombinant CCL2protein, anti-CCL2antibody. Results showed that high concentration of CCL2inhibited cell proliferation. To prove effect of CCL2-CCR2on autophagy of procine endometrial luminal epithelial cell, the study assessed the effect of CCL2-CCR2on autophagy in procine endometrial epithelial cells. Results showed that CCL2-CCR2promoted cell autophagy to avoid death when these cells suffered with starving. Collectively, CCL2-CCR2with suitable concentration maintains procine endometrial luminal epithelial cells survivial for a successful implantation.