Cell-specific expression and signal transduction of C-C motif chemokine ligand 2 and atypical chemokine receptors in the porcine endometrium during early pregnancy

Accepted Manuscript Cell-specific expression and signal transduction of C-C motif chemokine ligand 2 and atypical chemokine receptors in the porcine endometrium during early pregnancy Whasun Lim, Hyocheol Bae, Fuller W. Bazer, Gwonhwa Song PII:

S0145-305X(17)30401-9

DOI:

10.1016/j.dci.2017.12.020

Reference:

DCI 3061

To appear in:

Developmental and Comparative Immunology

Received Date: 25 July 2017 Revised Date:

21 December 2017

Accepted Date: 21 December 2017

Please cite this article as: Lim, W., Bae, H., Bazer, F.W., Song, G., Cell-specific expression and signal transduction of C-C motif chemokine ligand 2 and atypical chemokine receptors in the porcine endometrium during early pregnancy, Developmental and Comparative Immunology (2018), doi: 10.1016/j.dci.2017.12.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Cell-Specific Expression and Signal Transduction of C-C Motif Chemokine Ligand 2

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and Atypical Chemokine Receptors in the Porcine Endometrium during Early

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Pregnancy

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Whasun Lim1,†, Hyocheol Bae2,†, Fuller W. Bazer3 and Gwonhwa Song2, §

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Republic of Korea.

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6 Department of Biomedical Sciences, Catholic Kwandong University, Gangneung, 25601,

Institute of Animal Molecular Biotechnology and Department of Biotechnology, College of

Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea

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A&M University, College Station, 77843-2471, Texas, USA

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Center for Animal Biotechnology and Genomics and Department of Animal Science, Texas

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§

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Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea. Phone:

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+82-2-3290-3012; FAX: +82-2-3290-4994; E-mail: [email protected]

Correspondence: Gwonhwa Song, Ph.D., Department of Biotechnology, College of Life

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These authors contributed equally to this work.

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The authors have no conflicts of interest to declare.

21 Abstract

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Chemokines and atypical chemokine receptors (ACKRs; also known as chemokine decoy

24

receptors) play an important role in reproductive immunology by recruiting leukocytes during

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early pregnancy.

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chemokine ligand 2 (CCL2) and ACKRs in the endometrium during estrous cycle and early

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pregnancy, and to investigate the functional effects of CCL2 on porcine uterine luminal

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epithelial (pLE) cells.

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strongly detected in the glandular and luminal epithelium of the endometrium during early

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pregnancy compared to that in non-pregnant pigs.

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proliferation via activation of the PI3K and MAPK pathways and suppression of endoplasmic

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reticulum (ER) stress by reducing the expression of ER stress regulatory genes.

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these results provide novel insights into CCL2-mediated signaling mechanisms in the porcine

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endometrium at the maternal-fetal interface during early pregnancy.

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The aim of this study was to determine the expression of C-C motif

Our results indicated that CCL2, ACKR1, ACKR3, and ACKR4 were

Recombinant CCL2 improved pLE cell

Collectively,

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Key words: CCL2, ACKR, endometrium, proliferation, porcine, luminal epithelial cell

37 1. Introduction

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Successful implantation and establishment of pregnancy requires well-organized autocrine,

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paracrine, and endocrine interactions between sex steroid hormones, growth factors,

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cytokines, and chemokines, resulting in proliferation, migration, invasion, and differentiation

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of uterine and trophoblast cells (Bazer, 2013; Guzeloglu-Kayisli et al., 2009).

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unique immunological system that facilitates the attachment of a semiallogeneic conceptus to

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the endometrium is important for suppressing immune rejection response (Pijnenborg, 2002).

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For improving implantation, cytokines and chemokines participate in immune regulation at

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the maternal-fetal interface.

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and differentiation of trophoblast cells, expression of progesterone-activated genes in the

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endometrial luminal epithelium (LE), and migration and recruitment of uterine natural killer

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(uNK) cells, macrophages, and eosinophils to implantation sites in the uterus during early

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pregnancy in humans and mice (Kojima et al., 1995; Schofield and Kimber, 2005; Sherwin et

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al., 2004).

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(IL-6), IL-8, and tumor necrosis factor-alpha (TNF-α) by the endometrial cells and immune

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regulatory cells that are recruited to the site of implantation (Dekel et al., 2010; Mor et al.,

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2011).

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chemokine ligand 12 (CXCL12) are expressed significantly in the conceptuses and

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endometria of pregnant ewes for improvement of implantation and placentation compared to

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that in non-pregnant ewes (Ashley et al., 2011).

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endometrium regulates the recruitment of human uNK cells during early pregnancy (Starnes

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et al., 2006).

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In addition, a

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For instance, leukemia inhibitory factor stimulates the growth

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Moreover, early implantation is characterized by the secretion of interleukin 6

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Among the chemokines, C-X-C chemokine receptor 4 (CXCR4) and its ligand C-X-C

In addition, CXCL14 secreted by the

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In the chemokine family, C-C motif chemokine ligands (CCLs) play an important

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role at the maternal-conceptus interface during early pregnancy in mammals (Du et al., 2014).

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Their biological functions are commonly regulated by G protein-coupled chemokine

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receptors, but CCLs also bind to atypical chemokine receptors (ACKRs) (Nibbs and Graham,

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2013).

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chemokine receptor 3 (CCR3) by decidual stromal cells, the CCL24-CCL3 interaction has

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beneficial effects on decidualization during early pregnancy in humans (Li et al., 2013).

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CCL28 stimulates the proliferation and migration of porcine trophectoderm cells from day 12

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of pregnancy via CCR10, and both CCL28 and CCR10 are detected in the porcine

Following the secretion of CCL24 by trophoblasts, and its receptor C-C motif

ACCEPTED MANUSCRIPT endometrial tissues during the implantation period (Choi et al., 2016).

Moreover, ACKR2 is

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expressed in mouse extravillous trophoblast and apical side of syncytiotrophoblast, forming a

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barrier that regulates inflammatory chemokine expression at the maternal-fetal interface to

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reduce inflammation- or autoantibody-induced abortion (Martinez de la Torre et al., 2007).

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During early pregnancy, ACKR1 mRNA expression increases and CCL5 mRNA expression

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decreases significantly in the endometrium of pregnant pigs compared with that in non-

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pregnant pigs (Wessels et al., 2011).

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reduced from gestational day 20 to day 50 in the endometrium and trophoblast.

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IL-33-induced proliferation and invasion of human decidual stromal cells and stimulated

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CCL2 expression through activation of nuclear factor kappa B (NF-κB) and ERK1/2 signal

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transduction for successful establishment of pregnancy (Hu et al., 2014).

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activates ERK1/2 signaling to enhance integrin activity and chemotaxis for regulating

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adhesive molecules during the peri- and post-implantation periods (Jimenez-Sainz et al.,

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2003; Lin et al., 2013; Merviel et al., 2001).

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pathway during pregnancy has not yet been elucidated.

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Moreover,

Also, CCL2

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In addition, the expression of CCL2 and CCL4 is

However, their intracellular signal transduction

In the present study, we analyzed the sequence of porcine CCL2 compared to that of

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CCL2 from other species by multiple sequence alignment and generation of phylogenetic tree.

86

The main objectives of the present study were to: 1) compare the expression of CCL2 and its

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receptors (ACKRs) in the porcine endometrium during estrous cycle and early pregnancy; 2)

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investigate their expression in the endometrium between primiparous and multiparous sows;

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3) confirm the effects of CCL2 on the proliferation of porcine uterine luminal epithelial

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(pLE) cells; 4) demonstrate CCL2-mediated cell signal transduction in pLE cells; and 5)

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examine the inhibitory effects of CCL2 on tunicamycin-induced endoplasmic reticulum (ER)

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stress in pLE cells.

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receptivity at the maternal-fetal interface during early pregnancy in pigs, and CCL2 may play

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a key role in the recruitment of immune cells to implantation sites in the porcine

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endometrium.

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The results suggest that CCL2 and the ACKRs promote uterine

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2. Materials and Methods

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2.1. Sequence analysis

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For pair-wise comparisons and multiple sequence alignment, the amino acid sequences of

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CCL2 genes from each species were aligned using Geneious Pro version 10.2.2 software

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(Biomatters Ltd.) (Kearse et al., 2012), with default penalties for gap, and the protein weight

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matrix of Blocks Substitution Matrix (BLOSUM; Biomatters Ltd.).

A phylogenetic tree was

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constructed by the neighbor-joining method (Gascuel and Steel, 2006), using Geneious Pro

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version 10.2.2 software.

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phylogenetic tree, 1000 nonparametric bootstrap replications were used (Felsenstein, 1985).

To determine the confidence level for each internal node on the

106 2.2. Experimental animals and animal care

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Sexually mature gilts of similar age, weight, and genetic background were observed daily for

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estrus (day 0), and exhibited at least two estrous cycles of normal duration (18-21 days)

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before being used in this study.

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with the Guide for Care and Use of Agricultural Animals in Teaching and Research, and

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approved by the Institutional Animal Care and Use Committee of Texas A&M University.

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All experimental and surgical procedures were compliant

2.3. Experimental design and tissue collection

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Gilts were assigned randomly to either cyclic (day 9, 12, or 15 of the estrous cycle) or

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pregnant (day 9, 10, 12, 13, 14, 15, 20, or 30 of pregnancy) status.

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group were bred when detected to be in estrus, and 12 and 24 h later the gilts were

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ovariohysterectomized on either day 9, 12, or 15 of the estrous cycle or on day 9, 10, 12, 13,

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14, 15, 20, or 30 of pregnancy (n=3-4 pigs per day per status).

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groups at 1st, 3rd, and 6th parities were sacrificed on day of 30 pregnancy (n=3-4 pigs per

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group).

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was flushed with 20 mL of physiological saline and examined for the presence of

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morphologically normal conceptuses.

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to obtain several sections (~0.5 cm) from the entire uterine wall in the middle of each uterine

125

horn.

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(PBS; pH 7.2), and then embedded in Paraplast-Plus (Leica Microsystems, Wetzlar,

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Germany).

Those in the pregnant

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In addition, the pregnant

For confirmation of pregnancy prior to implantation, the lumen of each uterine horn

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Uteri from cyclic and pregnant gilts were processed

The tissue was fixed in fresh 4% paraformaldehyde in phosphate-buffered saline

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2.4. Cell culture

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An immortalized pLE cell line was first established by Wang and colleagues by stable

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transfection of primary pLE cells with a replication-defective retroviral (SV40) vector (Wang

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et al., 2000).

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have typical epithelial-like, cobblestone-shaped morphology, and show positive staining for

134

antibodies against epithelium-specific cytokeratin and negative staining for vimentin (Wang

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et al., 2000).

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present study were performed in pLE cells between passages 25-30.

That cell line was obtained and used in this in vitro study.

The pLE cells form a single monolayer at confluence.

The pLE cells

All analyses in the

Briefly, the monolayer

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cultures of pLE cells were grown to 80% confluence in Dulbecco’s modified Eagle’s medium

138

(DMEM)/F12 1:1 culture medium containing 20% fetal bovine serum in 100-mm tissue

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culture dishes.

140

subjected to various treatments.

For assays, in vitro-cultured pLE cells were serum-starved for 24 h, and then

141 2.5. RNA isolation

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Total cellular RNA was isolated from the endometrium of cyclic and pregnant gilts, using

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TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and purified using an RNeasy Mini Kit

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(Qiagen, Hilden, Germany) according to the manufacturer’s recommendations.

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quantity and quality of total RNA was determined by spectrometry and denaturing agarose

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gel electrophoresis, respectively.

The

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2.6. Quantitative polymerase chain reaction (PCR) analysis

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Specific primers were designed from sequences in the GenBank database using Primer 3 (ver.

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4.0.0) as illustrated in Table 2.

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of Korea).

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MO, USA) on a StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA,

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USA).

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64 °C for 40 sec, and 72 °C for 1 min, using a melting curve program (increasing the

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temperature from 55 to 95 °C at 0.5 °C per 10 sec) and continuous fluorescence

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measurements.

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which the CT value represented the number of cycles required for the fluorescent signal to

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exceed the background level.

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The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous

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control to standardize the amount of RNA in each reaction.

All primers were synthesized by Bioneer (Daejeon, Republic

Gene expression levels were measured using SYBR Green (Sigma, St. Louis,

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The PCR conditions were 95 °C for 3 min, followed by 40 cycles at 95 °C for 20 sec,

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Sequence-specific products were identified by generating a melting curve in

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Relative gene expression was quantified by the 2–∆∆CT method.

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2.7. Cloning of partial cDNA for porcine CCL2, ACKR1, ACKR3, and ACKR4

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cDNA was synthesized using AccuPower RT PreMix (Bioneer Inc.).

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porcine CCL2, ACKR1, ACKR3, and ACKR4 mRNAs were amplified using specific primers

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based on data for porcine CCL2 (GenBank accession no. NM_214214.1; forward: 5′-ACT

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GCA GCC ACC TTC TGC-3′, reverse: 5′-CTT GTC CAG GTG GCT TAT GG-3′), porcine

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ACKR1 (GenBank accession no. NM_001244095.1; forward: 5′-CTT CAT CCT CGC CAG

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TAT CC-3′; reverse: 5′-CCA AAG TCC CAC AGT GAT CC-3′), porcine ACKR3 (GenBank

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accession no. XM_003133759.4; forward: 5′-TCA CCC ACC TCA TCT TCT CC-3′, reverse:

Partial cDNAs for

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5′-AGT AGA AGA CGG CGA TGA CC-3′), and porcine ACKR4 (GenBank accession no.

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NM_001097430.1; forward: 5′-CAG GAG TGG GAA AAC TGT GC-3′, reverse: 5′-AGG

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CAG CTG TGA AAG AGT GC-3′).

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ACKR4 were gel-extracted and cloned into the TOPO TA cloning vector (Invitrogen).

Partial cDNAs for CCL2, ACKR1, ACKR3, and

175 2.8. In situ hybridization analysis

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After verification of the sequences, the plasmids containing gene sequences were amplified

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with T7- and SP6-specific primers (T7: 5′-TGT AAT ACG ACT CAC TAT AGG G-3′; SP6:

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5′-CTA TTT AGG TGA CAC TAT AGA AT-3′), and then digoxigenin (DIG)-labeled RNA

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probes were transcribed using a DIG RNA labeling kit (Roche, Indianapolis, IN, USA).

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tissue sections were deparaffinized, rehydrated, treated with 1% Triton X-100 in PBS for 20

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min, and then washed twice in DEPC-treated PBS.

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paraformaldehyde, the sections were incubated in a prehybridization mixture containing 50%

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formamide and incubated in 4× standard saline citrate buffer for at least 10 min at room

185

temperature.

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with sheep anti-DIG antibody conjugated to alkaline phosphatase (Roche).

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visualized by exposure to a solution containing 0.4 mM 5-bromo-4-chloro-3-indolyl

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phosphate, 0.4 mM nitroblue tetrazolium, and 2 mM levamisole (Sigma-Aldrich).

The

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After hybridization and blocking steps, the sections were incubated overnight The signal was

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After post-fixation in 4%

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2.9. Reagents

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Recombinant human CCL2 (catalog number: 279-MC/CF) was purchased from R&D

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Systems (Minneapolis, MN, USA).

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recombinant porcine CCL2, recombinant human CCL2 was used in the present study.

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Tunicamycin from Streptomyces (catalog number: T7765) was purchased from Sigma.

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Antibodies against human phosphorylated (p)-AKT (Ser473, catalog number: 4060), human p-

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ERK1/2 (Thr202/Tyr204, catalog number: 9101), human p-JNK (Thr183/Tyr185, catalog number:

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4668), human p-P38 (Thr180/Tyr182, catalog number: 4511), human p-p70S6K (Thr421/Ser424,

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catalog number: 9204), human p-p90RSK (Thr573, catalog number: 9346), human p-S6

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(Ser235/236, catalog number: 2211), human p-cyclin D1 (Thr286, catalog number: 3300), human

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p-eIF2α (Ser51, catalog number: 3398), mouse total AKT (catalog number: 9272), rat ERK1/2

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(catalog number: 4695), human JNK (catalog number: 9252), human P38 (catalog number:

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9212), human p70S6K (catalog number: 9202), human p90RSK (catalog number: 9355),

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human S6 (catalog number: 2217), human cyclin D1 (catalog number: 2922), human eIF2α

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(catalog number: 5324), and human IRE1α (catalog number: 3294) were purchased from Cell

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Because there is no commercially available

ACCEPTED MANUSCRIPT Antibodies against human p-PERK (Thr981,

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Signaling Technology (Beverly, MA, USA).

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catalog number: sc-32577), human total PERK (catalog number: sc-13073), human ATF6α

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(catalog number: sc-166659), human BiP (catalog number: sc-13968), and mouse CHOP

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(catalog number: sc-7351) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,

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USA).

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number: EI305), and P38 (SB203580, catalog number: EI286) were obtained from Enzo Life

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Sciences (Farmingdale, NY, USA), and PI3K/AKT inhibitor (Wortmannin, catalog number:

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9951) was from Cell Signaling Technology.

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Inhibitors for ERK1/2 (U0126, catalog number: EI282), JNK (SP600125, catalog

213 2.10. Proliferation assay

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Proliferation assays were conducted using the Cell Proliferation ELISA, BrdU Kit (Cat No.

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11647229001, Roche) according to the manufacturer’s recommendations.

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were seeded in a 96-well plate, and then incubated for 24 h in serum-free DMEM/F12 1:1

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medium.

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final volume of 100 µL/well.

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culture and cells were incubated for an additional 2 h at 37 °C.

Briefly, pLE cells

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Cells were then treated with various concentrations of recombinant CCL2 in a After 48 h of incubation, 10 µM BrdU was added to the cell

221 2.11. Immunofluorescence analysis

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The effects of CCL2 on the expression of proliferating cell nuclear antigen (PCNA) were

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determined by immunofluorescence microscopy.

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seeded on confocal dishes (catalog number: 100350; SPL Life Sciences, Republic of Korea)

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and then incubated for 24 h in serum-free DMEM/F12 1:1 medium.

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protein, the serum-starved cells were treated with 20 ng/mL of recombinant CCL2 for 24 h,

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following which the cells were fixed using methanol and probed with mouse anti-human

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monoclonal PCNA antibody at a final dilution of 1:100.

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staining included substitution of the primary antibody with purified non-immune mouse IgG.

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Cells were then incubated with goat anti-mouse IgG Alexa 488 (catalog number: A11017,

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Invitrogen) at a 1:200 dilution for 1 h at room temperature.

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using 0.1% bovine serum albumin (BSA) in PBS and overlaid with DAPI.

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antibody, images were captured using a. LSM710 confocal microscope (Carl Zeiss,

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Thornwood, NY, USA).

pLE cells (3×104 cells per 300 µL) were

For detection of PCNA

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Negative controls for background

The pLE cells were then washed For each primary

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2.12. Western blot analysis

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The protein concentrations in whole-cell extracts were determined using the Bradford protein

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assay (Bio-Rad, Hercules, CA, USA) with BSA as the standard.

Proteins were denatured,

240

separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and

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then transferred to nitrocellulose membranes.

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chemiluminescence detection (SuperSignal West Pico, Pierce, Rockford, IL, USA) and

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quantified by measuring the intensity of light emitted from correctly sized bands under

244

ultraviolet light using a ChemiDoc EQ system and Quantity One software (Bio-Rad).

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Immunoreactive proteins were detected using goat anti-rabbit polyclonal antibodies against

246

phosphorylated- and total-proteins at a 1:1000 dilution, and separated by 10% SDS-PAGE.

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As a loading control, total protein and α-tubulin (TUBA) were used to normalize results for

248

detection of target proteins.

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linearity of chemiluminescent signals.

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Blots were developed using enhanced

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Multiple exposures of each western blot were used to ensure

250 2.13. Statistical analyses

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All quantitative data were subjected to least-squares analysis of variance (ANOVA) using the

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General Linear Model procedures of the Statistical Analysis System (SAS Institute Inc., Cary,

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NC, USA).

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protein or TUBA data as a covariate.

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appropriate error terms according to the expectation of the mean squares for error.

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value less than or equal to 0.05 was considered significant.

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square means (LSMs) with standard errors (SEs).

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Western blot data were corrected for differences in sample loading using total

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All tests of significance were performed using the

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A P-

Data are presented as least-

3. Results

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3.1. Multiple sequence analysis and phylogenetic analysis of CCL2 gene

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Porcine CCL2 cDNA is 757 bp in length with a 300 bp CDS that encodes a 99 amino acid

263

protein on chromosome 12.

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found to contain a critically conserved chemokine binding domain similar to that in other

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species (Figure 1A).

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sequences of mammalian and rodent CCL2 by the neighbor-joining method (Figure 1B).

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The phylogenetic tree was divided into three groups, including vertebrate, ruminant, and

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rodent clades.

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showed close genetic distance between horse, cow, and goat. However, rodent CCL2 was far

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from porcine CCL2.

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with CCL2 from horse, cow, and goat (Figure 1C and Table 1).

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80.6% sequence similarity with that of primates, including humans and chimpanzees,

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By multiple sequence alignment analysis, the CCL2 gene was

A phylogenetic analysis was performed with full-length amino acid

Porcine CCL2 was first clustered together with primates and dogs and

The full-length porcine CCL2 shared 83.8–86.9% sequence similarity Porcine CCL2 shared

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whereas the sequence similarity with rodent CCL2 (58.2–64.2% sequence similarity) was

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lower than that for other species.

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contains a conserved cytokine binding domain and that CCL2 protein has high sequence

276

similarity with the CCL2 protein of other species.

These results indicated that the porcine CCL2 gene

277 3.2. Spatiotemporal expression of CCL2 and ACKRs in uterine endometrium during

279

estrous cycle and early gestational period in pigs

280

To investigate the expression of porcine CCL2 and ACKRs mRNAs during estrous cycle (day

281

9, 12, or 15 of the estrous cycle) and early pregnancy (day 9, 10, 12, 13, 14, 15, 20, or 30 of

282

pregnancy), we performed quantitative RT-PCR analyses (Figure 2).

283

CCL2 mRNA in the porcine endometrium increased 2.1- (P < 0.01) and 1.5-fold (P < 0.05)

284

on day 12 and 15 of the estrous cycle, respectively, compared to that on day 9 of the estrous

285

cycle (Figure 2A).

286

0.01 and P < 0.001) in the porcine endometrium from day 13 to day 30 of pregnancy.

287

expression of ACKR1 decreased during the estrous cycle, but increased 1.5- (P < 0.05), 2.5-

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(P < 0.01), and 1.8-fold (P < 0.01) on day 14, 20, and 30 of pregnancy, respectively, in the

289

uteri of pigs (Figure 2B).

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gradually increased in the porcine uterus during early gestational period (Figure 2C).

291

Similarly, the expression of ACKR4 in the porcine endometrium was elevated approximately

292

2-fold (P < 0.05) on day 12 of the estrous cycle and 2.9- (P < 0.01), 6- (P < 0.01), and 6.2-

293

fold (P < 0.001) on day 14, 20, and 30 of pregnancy, respectively (Figure 2D).

294

ACKR2 expression was not detected in the endometrium of pigs (data not shown).

295

basis of these results, we determined the mRNA expression and localization of target genes in

296

the porcine endometrium by in situ hybridization analysis (Figure 3).

297

CCL2, ACKR1, ACKR3, and ACKR4 mRNA were strongly detected in the glandular

298

epithelium (GE) and LE of the porcine endometrium during early gestational period

299

compared to that in the estrous cycle.

300

and ACKR4 mRNAs in the porcine endometrium on day 30 of pregnancy to determine the

301

correlation with expression of their receptors and uterine receptivity due to parity of sows

302

(Figure 4).

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the porcine endometrium by multiparous pregnancy compared to that by primiparous

304

pregnancy, but ACKR3 was significantly decreased in the porcine endometrium at the 6th

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parity (Figures 4A–C).

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decreased specifically in GE and LE of the porcine endometrium (Figures 4D–F).

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The expression of

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However, CCL2 expression was highly increased (3.4 to 6.6-fold; P <

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ACKR3 mRNA expression was induced on cyclic day 12 and

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The

However, On the

Results indicated that

We then analyzed the expression of ACKR1, ACKR3,

Results showed that ACKR1 and ACKR4 were significantly downregulated in

With increased parity number the expression of all three receptors Taken

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together, these results indicated that expression of CCL2, ACKR1, ACKR3, and ACKR4

308

mRNAs increased in porcine endometria during early pregnancy and their expression may be

309

closely associated with uterine receptivity.

310 3.3. Stimulatory effects of CCL2 on proliferation of pLE cells

312

To demonstrate the effects of CCL2 on the proliferation of pLE cells, we performed cell

313

proliferation analysis using BrdU incorporated into cellular DNA in a dose-dependent manner

314

(0, 5, 10, and 20 ng).

315

cells by 114.6, 133.6 (P < 0.05), and 158.4% (P < 0.05) at 5, 10, and 20 ng/ml, respectively.

316

We then analyzed the expression of PCNA, a component of the replication and repair

317

machinery that enhances DNA replication, DNA repair, and cell cycle progression, by

318

immunofluorescence analysis (Figure 5B).

319

detected in non-treated pLE cells, PCNA was strongly expressed in the nuclei of CCL2-

320

treated pLE cells.

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pLE cells during the peri-implantation period.

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As illustrated in Figure 5A, CCL2 increased the proliferation of pLE

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Although moderate expression of PCNA was

These results indicated that CCL2 is important for the proliferation of

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3.4. CCL2-regulated intracellular signaling pathways for proliferation of pLE cells

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To identify the CCL2-mediated intracellular signal transduction pathways involved in pLE

325

cell proliferation, we determined the phosphorylation of signaling molecules belonging to the

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PI3K/AKT and MAPK pathways, which are closely related to proliferation, by western blot

327

analysis in a time-dependent manner (0, 5, 15, 30, 60, and 120 min).

328

cells with 20 ng/mL of CCL2 gradually increased the phosphorylation of AKT by 4.6-fold (P

329

< 0.001) at 120 min post-treatment (Figure 6A).

330

the phosphorylation of p70S6K by 1.4-fold (P < 0.05) in pLE cells within 5 min post-

331

treatment (Figure 6B).

332

(P < 0.05) at 60 min post-treatment compared to that in non-treated pLE cells (Figure 6C).

333

In addition, phosphorylation of cyclin D1 was enhanced by approximately 2-fold (P < 0.01)

334

within 15 min post-treatment, and the increased phosphorylation was maintained until 60 min

335

post-treatment (Figure 6D).

336

phosphorylated ERK1/2 and P38 proteins by approximately 3.5- (P < 0.01) and 3.1-fold (P <

337

0.01), respectively, at 120 min post-treatment (Figures 6E and 6G).

338

phosphorylated JNK protein was rapidly stimulated 2-fold (P < 0.01) in pLE cells within 5

339

min post-treatment (Figure 6F).

340

of a downstream protein of MAPK (p90RSK) in pLE cells (Figure 6H).

Incubation of pLE

Treatment with CCL2 rapidly stimulated

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323

CCL2 also gradually activated the phosphorylation of S6 by 1.9-fold

In MAPK pathways, CCL2 significantly activated the

However, the

Furthermore, CCL2 significantly increased the expression To further

ACCEPTED MANUSCRIPT 341

determine the crosstalk between the CCL2-induced signal transduction pathways, pLE cells

342

were pre-incubated with pharmacological inhibitors, including wortmannin (a PI3K inhibitor,

343

1 µM), U0126 (an ERK1/2 inhibitor, 20 µM), SP600125 (a JNK inhibitor, 20 µM), and

344

SB203580 (a P38 inhibitor, 20 µM) prior to CCL2 treatment (Figure 7).

345

CCL2-activated AKT protein was completely blocked by wortmannin and was decreased by

346

U0126 and SB203580 in pLE cells (Figure 7A).

347

and S6 was almost inhibited by wortmannin and U0126 in pLE cells (Figures 7B and 7C).

348

The CCL2-induced increase in phosphorylation of cyclin D1 was decreased in response to

349

treatment with all inhibitors as compared to treatment with CCL2 alone (Figure 7D).

350

Activated ERK1/2 and p90RSK were completely blocked by U0126, but not by other

351

inhibitors (Figures 7E and 7H).

352

while CCL2-activated P38 was inhibited by all inhibitors in CCL2-treated pLE cells (Figures

353

7F and 7G).

354

pathways in pLE cells, and that there is cross-talk between the PI3K/AKT and ERK1/2 or

355

P38 MAPK pathways in response to CCL2 as illustrated in Figure 9.

The expression of

SC

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CCL2-induced phosphorylation of p70S6K

CCL2-activated JNK was suppressed only by SP600125,

M AN U

These results indicated that CCL2 activated the PI3K/AKT and MAPK

356

3.5. Effects of CCL2 on tunicamycin-induced ER stress in pLE cells

358

To investigate the effects of CCL2 on ER stress in pLE cells, we analyzed the expression of

359

ER stress regulatory proteins in the presence or absence of tunicamycin and CCL2 by western

360

blot analysis (Figure 8).

361

enhanced the expression of PKR-like ER resident kinase (PERK) and its downstream

362

signaling molecule, eukaryotic translation-initiation factor 2α (eIF2α) (Figures 8A and 8B);

363

however, this increase was suppressed by treatment with a combination of tunicamycin and

364

CCL2.

365

(IRE1α) and activating transcription factor 6α (ATF6α) were activated by treatment of pLE

366

cells with tunicamycin alone, but their expression decreased in response to additional

367

treatment with CCL2 (Figures 8C and 8D).

368

of binding immunoglobulin protein (BiP), which is a key regulator of ER stress that controls

369

PERK, IRE1α, and ATF6α signaling, was downregulated in pLE cells in response to

370

additional treatment with CCL2 (Figure 8E).

371

(CHOP), a transcription factor associated with ER stress-induced apoptosis, was activated in

372

tunicamycin-treated pLE cells, its expression was reduced in response to combined treatment

373

with tunicamycin and CCL2 (Figure 8F).

374

cleavage of caspase 3, which is an indicator of apoptosis in pLE cells, a combination of

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357

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Treatment of pLE cells with tunicamycin alone significantly

AC C

Furthermore, ER stress-associated molecules, such as inositol-requiring enzyme 1α

In addition, the tunicamycin-induced expression

Even though C/EBP homologous protein

Furthermore, while tunicamycin triggered the

ACCEPTED MANUSCRIPT 375

tunicamycin and CCL2 or CCL2 alone attenuated the cleavage of caspase 3 in pLE cells

376

(Figure 8G).

377

cells.

These results indicated that CCL2 suppressed ER stress-induced death in pLE

378 379

4. Discussion Results of the present study demonstrated the mRNA expression of CCL2, ACKR1,

381

ACKR3, and ACKR4 in the porcine endometrium during estrous cycle and early pregnancy.

382

In addition, it showed that the reduced expression of ACKRs in the porcine endometrium by

383

increasing parity might be associated with decreased uterine receptivity.

384

recombinant CCL2 increased the proliferation of pLE cells through activation of the PI3K

385

and MAPK pathways and suppression of ER stress.

386

that CCL2 improves uterine receptivity for implantation through receptor-mediated signaling

387

cascades in pigs, as illustrated in Figure 9.

RI PT

380

SC

Moreover,

M AN U

These results support our hypothesis

388

For establishment of pregnancy, immunological interactions between the conceptus

389

and the uterus/mother are required for survival of the semi-allogeneic conceptus (Mor et al.,

390

2011; Pijnenborg, 2002).

391

regulatory cells, including uNK cells, immature dendritic cells, T cells, and macrophages,

392

induce dynamic morphological changes in the uterine environment (PrabhuDas et al., 2015).

393

In addition, the accumulation of leukocytes in the endometrium is commonly upregulated by

394

chemokines and their receptors, and improves trophoblast invasion and trafficking and

395

uterine remodeling during the peri-implantation period of pregnancy (Dimitriadis et al., 2005).

396

As potent chemoattractants for uNK cells, CCL7, CCL8, CCL21, CCL22, and CX3CR1 are

397

secreted by decidualized stromal cells during pregnancy (Jones et al., 2004; Jones et al.,

398

1997).

399

the leukocyte trafficking by regulating the secretion of growth factors during embryogenesis

400

and placentation (Schanz et al., 2011; Tripathi et al., 2009).

401

CXCR4 system is abundant in the trophoblast and endometrium, and their interaction is

402

activated through the ERK1/2 and MAPK pathways during early pregnancy (Ashley et al.,

403

2011; Wu et al., 2004).

404

secreted by endometrial cells play crucial roles in the development of a receptive

405

endometrium at implantation sites (PrabhuDas et al., 2015).

406

with the recruitment of decidual immune cells, including uNK cells, dendritic cells, and

407

macrophages (Du et al., 2014).

408

roles of CCL2 and its ACKRs have not yet been well elucidated.

EP

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To maintain this unique early event in pregnancy, immune

AC C

ACKR3 (also known as CXCR7) expression increases the number of uNK cells and

In addition, the CXCL12-

Furthermore, high amounts of CCL2, CXCL8, and CXCL10

Moreover, CCL2 is associated

However, the spatiotemporal expression and functional Results of the present

ACCEPTED MANUSCRIPT 409

study indicated that CCL2 and ACKRs (ACKR1, ACKR3, and ACKR4) were predominantly

410

detected in the GE and LE of the porcine endometrium during early pregnancy as compared

411

to that in the non-pregnant state.

412

mRNAs was reduced in the endometria of multiparous pigs compared to primiparous sows.

413

Appropriate secretion of histotroph including nutrients, cytokines, hormones and growth

414

factors from the endometrial epithelial cells is important for maintenance of uterine

415

receptivity (Spencer et al., 2004).

416

placentation and a corresponding reduction in litter size.

417

dramatically reduced after the fifth parity due to changes in endometrial receptivity for

418

maintaining pregnancy in pigs (Fernandez-Rodriguez et al., 2011; Lawlor and Lynch, 2007;

419

Rodriguez-Zas et al., 2006).

420

implantation during early pregnancy in mammals and, in the present study, its expression

421

increased in the porcine endometrial tissues of sows at their 6th as compared to their 3rd

422

parities (Lim et al., 2017).

423

implantation during early pregnancy, decreased the expression of MUC1 mRNA in pLE cells.

424

Thus, there is a decrease in expression of ACKR1, ACKR3, and ACKR4 in endometria of

425

multiparous pigs compared to primiparous pigs that is closely related to porcine uterine

426

receptivity to implantation and placentation.

427

pLE cells isolated from day 12 of pregnancy.

428

may improve uterine receptivity and implantation rate by recruitment of leukocytes to

429

implantation sites in the porcine endometrium.

RI PT

Moreover, the expression of ACKR1, ACKR3, and ACKR4

Therefore, a deficiency in histotroph leads to abnormal

SC

In addition, fecundity was

M AN U

We reported that an anti-adhesive molecule, MUC1, inhibits

TE D

And, expression of FGF2, an important growth factor for

Moreover, CCL2 increased the proliferation of These results indicate that CCL2 and ACKRs

Consistent with results of previous studies,results of the present study showed that

431

CCL2-induced proliferation of pLE cells is regulated by activation of the PI3K and MAPK

432

pathways.

433

cells to decrease phosphorylation of AKT-p70S6K-S6 by the ERK1/2 inhibitor (U0126).

434

addition, studies with pLE involving growth factors, cytokines, and chemokines support the

435

role of these signaling pathways in cell proliferation (Jeong et al., 2017; Jeong et al., 2016;

436

Lim et al., 2017).

437

activities, such as reproduction.

438

proliferation and invasion in an autocrine manner, but aberrant levels of CCL2 affect eutopic

439

endometrial stromal cells from patients with endometriosis by activation of the AKT and

440

ERK1/2 pathways (Li et al., 2012).

441

phosphorylation of ERK1/2, JNK, and P38 MAPK in a dose- and time-dependent manner in

442

human umbilical vein endothelial cells (Werle et al., 2002).

EP

430

AC C

Moreover, crosstalk between ERK1/2 and AKT signaling was identified in pLE In

CCL2 regulates a variety of signaling pathways involved in biological In human endometrial stromal cells, CCL2 enhances

In addition, treatment with CCL2 activates the

Increased numbers of uNK cells

ACCEPTED MANUSCRIPT 443

isolated from human first trimester decidua in response to estrogen abundantly secrete CCL2

444

that increases angiogenesis in human endometrial endothelial cells, with the secretion of pro-

445

angiogenic factors during early pregnancy (Gibson et al., 2015).

446

proliferation and invasiveness stimulates CCL2 expression through activation of nuclear

447

factor kappa B (NF-κB) and ERK1/2 signal transduction for establishing successful

448

pregnancy (Hu et al., 2014).

449

protein kinase C (PKC), and PI3K is stimulated by CCL2 via integrin activation and

450

chemotaxis, which regulate expression of adhesive molecules for implantation (Jimenez-

451

Sainz et al., 2003; Lin et al., 2013; Merviel et al., 2001).

Moreover, IL-33-induced

RI PT

Furthermore, the phosphorylation of ERK1/2 mediated by Ras,

Results of the present study indicated that tunicamycin, which is an initiator of ER

453

stress, induced the expression of ER stress response signaling proteins, leading to pLE cell

454

death by cleavage of caspase 3.

455

expression of ER transmembrane signaling proteins such as PERK, eIF2α, IRE1α, ATF6α,

456

BiP and CHOP transcriptional factors.

457

pLE cells prevents ER stress that causes embryonic losses and severe pathological changes in

458

the placenta during pregnancy in pigs.

459

embryonic development and placental formation (Guzel et al., 2017; Yung et al., 2012).

460

ER, an essential organelle in eukaryotic cells, regulates biosynthesis of polypeptide hormones,

461

growth factors, and lipids, post-transcriptional modifications, and Ca2+ mobilization (Krebs et

462

al., 2015; Schwarz and Blower, 2016).

463

pathogenic stimuli results in the accumulation of unfolded and misfolded proteins in the

464

lumen, leading to ER stress (Guzel et al., 2017).

465

including PERK, IRE1α, and ATF6α in combination with BiP are induced by ER stress (Hetz,

466

2012).

467

stimuli, it activates the ER stress response proteins located in the ER transmembrane.

468

response to ER stress, the activation of PERK-eIF2α and IRE1α promotes NF-κB-mediated

469

inflammation with increased expression of pro-inflammatory cytokines (Deng et al., 2004;

470

Kaneko et al., 2003).

471

phosphorylation of AKT (Yamazaki et al., 2009).

472

response proteins, including IRE1α, eIF2α, and CHOP, leads to pathophysiological changes

473

in the placenta, including pre-eclampsia and intra-uterine growth restriction (Burton et al.,

474

2009; Iwawaki et al., 2009; Yung et al., 2008).

M AN U

However, additional treatment with CCL2 reduced the

These results indicate that administration of CCL2 to

TE D

ER stress in pregnancy exerts negative effects on The

However, disruption of ER homeostasis through

EP

Three ER transmembrane proteins,

AC C

Even though BiP interacts with the three proteins in an inactive state without stress

475 476

SC

452

5. Conclusions

In

ATF6α also induces NF-κB-stimulated inflammation by In pregnancy, up-regulation of ER stress

ACCEPTED MANUSCRIPT 477

In summary, CCL2 and ACKRs (ACKR1, ACKR3, and ACKR4) are abundantly present in

478

the GE and LE of the endometrium during early pregnancy compared to that in the non-

479

pregnant state.

480

endometrium by increasing the number of parity compared to that in primiparous pigs.

481

Administration of CCL2 to pLE cells induces proliferation through activation of PCNA

482

expression and PI3K/AKT and MAPK pathways during early pregnancy. A cross-talk

483

between the PI3K and MAPK signaling pathways was identified in pLE cells in response to

484

CCL2. Moreover, CCL2 suppressed tunicamycin-induced ER stress on pLE cells by reducing

485

the expression of UPR signaling molecules. Taken together, CCL2 may play an important

486

role in maternal-fetal interaction by improving uterine receptivity by recruitment of immune

487

cells during early gestational period in pigs.

488

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RI PT

The expression of these three receptors is downregulated in the

Acknowledgements

490

This research was supported by a grant of the Korea Health Technology R&D Project through

491

the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health

492

& Welfare, Republic of Korea (grant number : HI15C0810).

493

supported by a Korea University Grant

This research was also

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ds at the porcine maternal-fetal interface. Immunol Cell Biol 89, 304-313.

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Yamazaki, H., Hiramatsu, N., Hayakawa, K., Tagawa, Y., Okamura, M., Ogata, R., Hu

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ental morphogenesis: implications for human intrauterine growth restriction. J Pathol 228, 554-564.

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Figure legends

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Figure 1.

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[A] Multiple alignment of DNA sequences of pig (Sus scrofa), human (Homo sapiens),

663

chimpanzee (Pan troglodytes), horse (Equus caballus), sheep (Ovis aries), goat (Capra

664

hircus), cow (Bos taurus), dog (Canis lupus familaris), rat (Rattus norvegicus), and mouse

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(Mus musculus) CCL2 was analyzed using Geneious Pro Version 10.2.2.

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indicates a conserved domain known as the chemokine binding domain found in Pfam, a

667

family matrix and NCBI conserved domain database.

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relationship between the pig CCL2 nucleic acid sequence and the CCL2 nucleic acid

669

sequences of other species.

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Geneious software, and the bar shows the genetic distance between the species.

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Relative sequence homology with CCL2 protein in pigs.

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The blue box

[B] The phylogenetic tree shows the

It was generated by the neighbor-joining method using

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Multiple sequence alignment and phylogenetic tree analysis of CCL2 in pigs.

[C]

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Figure 2.

Relative mRNA expression of CCL2 and ACKRs in the porcine endometrium

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during estrous cycle and early pregnancy period.

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[A], ACKR1 [B], ACKR3 [C], and ACKR4 [D] mRNAs in the porcine endometrium was

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analyzed by quantitative RT-PCR.

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ACKR4 were normalized based on that of GAPDH gene [mean ± standard error of mean

678

(SEM), n=3].

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pigs (***P < 0.001, **P < 0.01, and *P < 0.05).

[A–D] Differential expression of CCL2

Messenger RNA levels of CCL2, ACKR1, ACKR3, and

The asterisks reveal significant differences compared to day 9 estrous cycle in

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Figure 3.

Spatiotemporal expression of CCL2, ACKR1, ACKR3, and ACKR4 mRNA in the

682

porcine endometrium during estrous cycle and early gestational period.

683

localization of CCL2 [A], ACKR1 [B], ACKR3 [C], and ACKR4 [D] mRNA was determined

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by in situ hybridization analysis.

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hybridized with anti-sense and sense CCL2, ACKR1, ACKR3, and ACKR4 cRNA probes.

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GE, glandular epithelium; LE, luminal epithelium.

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panels and sense) and 20 µm (the second horizontal panels).

Cell-specific

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Cross-sections of porcine endometrial tissues were

Scale bar: 50 µm (the first horizontal

688 Figure 4.

690

endometrium by increasing parity.

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[B], and ACKR4 [C] mRNAs in the porcine endometrium on day 30 of pregnancy was

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analyzed by quantitative RT-PCR.

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were normalized based on that of GAPDH gene (mean ± SEM, n=3).

The asterisks reveal

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significant differences compared to primiparous pigs (**P < 0.01).

[D–F] Cell-specific

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localization of ACKR1 [D], ACKR3 [E], and ACKR4 [F] mRNA was determined by in situ

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hybridization analysis.

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anti-sense and sense ACKR1, ACKR3, and ACKR4 cRNA probes.

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LE, luminal epithelium.

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(the second horizontal panels).

[A–C] Differential expression of ACKR1 [A], ACKR3

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Messenger RNA levels of ACKR1, ACKR3, and ACKR4

Cross-sections of porcine endometrial tissues were hybridized with

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Changes in mRNA expression of ACKR1, ACKR3, and ACKR4 in the porcine

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GE, glandular epithelium;

Scale bar: 50 µm (the first horizontal panels and sense) and 20 µm

Figure 5.

Effects of CCL2 on the proliferation of pLE cells.

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assay was performed to determine the stimulatory effects of CCL2 on pLE cells in a dose-

703

dependent manner (0, 5, 10, and 20 ng/mL).

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non-treated control cells (100%).

The asterisks reveal significant differences compared to

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non-treated pLE cells (*P < 0.05).

[B] Immunofluorescence analysis showed the expression

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of a proliferation marker, PCNA, in response to CCL2 treatment (20 ng/mL) of pLE cells.

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Green fluorescence indicated that PCNA expression was stronger in the nuclei of CCL2-

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treated pLE cells than that of non-treated pLE cells.

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with DAPI (blue fluorescence).

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µm (the second and fourth vertical panels).

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[A] BrdU cell proliferation

Data are expressed as percentage relative to

The cell nuclei were counterstained

Scale bar: 40 µm (the first and third vertical panels) and 20

711 712

Figure 6.

CCL2 activates PI3K/AKT and MAPK signaling in pLE cells.

[A–H] Western

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blot analysis of phosphorylated AKT [A], p70S6K [B], S6 [C], cyclin D1 [D], ERK1/2 [E],

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JNK [F], P38 [G], and p90RSK [H] after treatment of pLE cells with CCL2 (20 ng/mL) for

715

the indicated time periods.

716

which are presented as value of phosphorylated proteins relative to total proteins.

717

bars represent SEM of representative experiments performed in triplicate.

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reveal significant differences compared to non-treated pLE cells (***P < 0.001, **P < 0.01,

719

and *P < 0.05).

Immunoblots were detected to calculate the normalized values, All error

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The asterisks

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Figure 7.

Blockage of the PI3K and MAPK pathways influences the phosphorylation of

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their downstream signaling molecules.

723

[A], p70S6K [B], S6 [C], cyclin D1 [D], ERK1/2 [E], JNK [F], P38 [G], and p90RSK [H]

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was regulated by pre-incubation with pharmacological inhibitors to inhibit PI3K

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(wortmannin), ERK1/2 (U0126), JNK (SP600125), and P38 (SB203580) prior to CCL2

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treatment of pLE cells.

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which are presented as value of phosphorylated proteins relative to total proteins.

728

bars represent SEM of representative experiments performed in triplicate. The asterisks

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reveal significant differences compared to non-treated pLE cells (***P < 0.001, **P < 0.01,

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and *P < 0.05).

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compared to CCL2 treatment alone.

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[A–H] The CCL2-activated phosphorylation of AKT

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Immunoblots were detected to calculate the normalized values, All error

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Lower case letter (a) indicates statistically significant differences (P < 0.05)

732 Figure 8.

CCL2 suppresses tunicamycin-induced ER stress in pLE cells.

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blot analysis of p-PERK [A], p-eIF2α [B], IRE1α [C], ATF6α [D], BiP [E], CHOP [F], and

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cleavage caspase 3 [G] showed the inhibitory effects of CCL2 on tunicamycin-induced ER

736

stress in pLE cells.

737

are presented as value of target proteins relative to total protein or TUBA.

738

represent SEM of representative experiments performed in triplicate.

739

significant differences compared to non-treated pLE cells (***P < 0.001, **P < 0.01, and *P

740

< 0.05).

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compared to CCL2 treatment alone.

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[A–G] Western

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Immunoblots were detected to calculate the normalized values, which All error bars

The asterisks reveal

Lower case letter (a) indicates statistically significant differences (P < 0.05)

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Figure 9.

Hypothetical illustration of the present study demonstrating the effects of CCL2

744

on the porcine endometrium.

745

in the porcine endometrium during early pregnancy, CCL2 participates in the proliferation of

746

pLE cell isolated from day 12 of pregnancy through activation of AKT-p70S6K-S6 axis and

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ERK1/2-JNK-P38 MAPK signaling cascade with abundant expression of PCNA.

In the presence of CCL2 and ACKR1, ACKR3, and ACKR4

Moreover,

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tunicamycin-activated ER transmembrane proteins, including PERK, IRE1α, and ATF6α are

749

suppressed in response to additional treatment of pLE cells with CCL2.

750

CCL2 regulates pLE cell proliferation, and may improve porcine endometrial development

751

and uterine receptivity during early pregnancy.

Taken together,

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ACCEPTED MANUSCRIPT Table 1. Pairwise comparison of CCL2 between pig and other species. Symbol

Pig (Sus scrofa)

CCL2

vs. Horse (Equus caballus)

CCL2

86.9

vs. Cow (Bos taurus)

CCL2

86.9

vs. Goat (Capra hircus)

CCL2

83.8

vs. Dog (Canis lupus familaris)

CCL2

vs. Chimpanzee (Pan troglodytes)

CCL2

vs. Human (Homo sapiens)

CCL2

vs. Sheep (Ovis aries)

CCL2

vs. Mouse (Mus musculus)

Ccl2

vs. Rat (Rattus norvegicus)

Ccl2

755 756

80.6 74.7

64.2

58.2

Table 2. Primer information for quantitative RT-PCR analysis

CCL2

ACKR1

ACKR2

Forward: 5′-GTC ACC AGC AGC AAG TGT CC-3′ Reverse: 5′-CTT GTC CAG GTG GCT TAT GG-3′ Forward: 5′-GGA TCA CTG TGG GAC TTT GG-3′

Reverse: 5′-GGG CAA CAA GAC AAA GAT GG-3′ Forward: 5′-GAT GAG GTG CTG TCC TTT GG-3′ Reverse: 5′-GCA AGA CCA CGA GAA GAA GG-3′ Forward: 5′-GCT GGA CAT CTT CTC CAT CC-3′ Reverse: 5′-GGC TTT CAT CAG CTC GTA CC-3′

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ACKR3

Sequence (5′ → 3′)

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Gene

ACKR4

GAPDH

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80.6

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83.3

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Identity (%)

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Species

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Forward: 5′-GGC AGT GGC AGA TTT ACT CC-3′

Reverse: 5′-CAA GCC AAA AAC TGC ATT CC-3′ Forward: 5′-CAA TGA CCC CTT CAT TGA CC-3′ Reverse: 5′-TAC GTA GCA CCA GCA TCA CC-3′

Accession No.

Size (bp)

NM_214214.1

117

NM_001244095.1

164

NM_001256773.2

100

XM_003133759.4

169

NM_001097430.1

143

NM_001206359.1

183

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ACCEPTED MANUSCRIPT Highlights · CCL2-ACKR system is regulated in the porcine uterine endometrium · CCL2 induces the proliferation and cell cycle progression of porcine uterine cells

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· CCL2 activates PI3K/AKT and MAPK pathways for cellular proliferation in the uterus · CCL2 reduces ER stress regulatory gene expression in porcine luminal epithelia cells

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· CCL2 may play an important role in maternal-fetal interaction during early pregnancy