Journal Pre-proof

The effect of oestradiol and progesterone on chemerin system expression in the

porcine endometrium during early pregnancy and the mid-luteal phase of the oestrous
cycle

Marlena Gudelska, Kamil Dobrzyn, Marta Kiezun, Katarzyna Kisielewska, Edyta

Rytelewska, Tadeusz Kaminski, Nina Smolinska
PII: S0093-691X(22)00467-8
DOI: https://doi.org/10.1016/j.theriogenology.2022.11.011
Reference: THE 16471

To appear in: Theriogenology

Received Date: 24 May 2022

Revised Date: 26 October 2022
Accepted Date: 5 November 2022

Please cite this article as: Gudelska M, Dobrzyn K, Kiezun M, Kisielewska K, Rytelewska E, Kaminski T,
Smolinska N, The effect of oestradiol and progesterone on chemerin system expression in the porcine
endometrium during early pregnancy and the mid-luteal phase of the oestrous cycle, Theriogenology
(2022), doi: https://doi.org/10.1016/j.theriogenology.2022.11.011.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. 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.

© 2022 Published by Elsevier Inc.

Marlena Gudelska: Conceptualization, Data curation, Formal analysis, Investigation,
Methodology, Validation, Resources, Visualization, Writing - original draft. Kamil Dobrzyn:
Supervision, Methodology, Validation, Resources. Marta Kiezun: Methodology, Validation,
Resources. Katarzyna Kisielewska: Methodology, Resources. Edyta Rytelewska:
Methodology, Resources. Tadeusz Kaminski: Writing - review & editing. Nina Smolinska:
Supervision, Conceptualization, Methodology, Funding acquisition, Project administration,
Writing - review & editing, Funding acquisition.

f
r oo
-p
re
lP
na
ur
Jo
 1 revised

2 THE EFFECT OF OESTRADIOL AND PROGESTERONE ON CHEMERIN SYSTEM

3 EXPRESSION IN THE PORCINE ENDOMETRIUM DURING EARLY PREGNANCY

4 AND THE MID-LUTEAL PHASE OF THE OESTROUS CYCLE

5 Marlena Gudelska1, Kamil Dobrzyn2, Marta Kiezun1, Katarzyna Kisielewska1, Edyta Rytelewska1,

6 Tadeusz Kaminski1, Nina Smolinska1*

of
7 *Corresponding author: Nina Smolinska, nina.smolinska@uwm.edu.pl, tel. +48 89 523 39 37, Fax

ro
8 +48 89 523 39 37

9 Marlena Gudelska: marlena.gudelska@uwm.edu.pl-p

re
lP

10 Kamil Dobrzyn: kamil.dobrzyn@uwm.edu.pl

na

11 Marta Kiezun: marta.kiezun@uwm.edu.pl

ur

12 Katarzyna Kisielewska: katarzyna.kisielewska@uwm.edu.pl

Jo

13 Edyta Rytelewska: edyta.rytelewska@uwm.edu.pl

14 Tadeusz Kaminski: tkam@uwm.edu.pl

1
15 Department of Animal Anatomy and Physiology, Faculty of Biology and Biotechnology,

16 University of Warmia and Mazury in Olsztyn, Poland

2
17 Department of Zoology, Faculty of Biology and Biotechnology, University of Warmia and

18 Mazury in Olsztyn, Poland

19

1
20 1. Abstract

21 Chemerin is an adipokine which is the product of the RARRES2 gene expressed mainly in

22 adipose tissue. This hormone exerts biological effects via three metabotropic receptors:

23 chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1), and C-C chemokine

24 receptor-like 2 (CCRL2). Chemerin exerts pleiotropic effects and participates in the regulation of

25 many processes, such as blood pressure control, immune system regulation, inflammation, and

26 angiogenesis. There is a growing body of evidence to indicate that chemerin is involved in the

of
27 regulation of reproductive system functions. However, little is known about the regulation of

ro
28 chemerin system (chemerin and its receptors) expression. The authors hypothesized that steroid

29
-p
hormones affect the expression of the chemerin system in the uterus of domestic pigs. Thus, the
re
30 aim of this study was to investigate the influence of steroids, oestradiol (E2), and progesterone (P4),
lP

31 on the gene (qPCR) and protein (Western Blot) expression of chemerin receptors, RARRES2 gene
na

32 expression as well as chemerin secretion (ELISA) by endometrial slices during early pregnancy
ur

33 (days 10 to 11, 12 to 13, 15 to 16, and 27 to 28) and the mid-luteal phase of the oestrous cycle
Jo

34 (days 10 to 11). Both steroid hormones modulated the expression of chemerin receptor genes and

35 proteins, as well as the abundance of RARRES2 mRNA transcripts. The study also demonstrated

36 that E2 increased chemerin protein secretion by endometrial cells during the maternal recognition

37 of pregnancy (days 12 to 13) and decreased its release during embryo implantation (days 15 to 16).

38 P4 had no significant effect on chemerin levels during the studied periods of gestation and the

39 oestrous cycle. These observations suggest that the effects of E2 and P4 on the chemerin system

40 are dependent on the period of gestation or the oestrous cycle. Our results support the hypothesis

41 that the hormonal environment could modulate the expression of the chemerin system in the

42 endometrium.

2
43 Key words: chemerin, chemerin receptors, steroid hormones, endometrium, early pregnancy,

44 pig

45 2. Introduction

46 The retinoic acid responder 2 gene (RARRES2), also known as tazarotene-induced gene 2

47 (TIG2), encodes the chemerin precursor pre-prochemerin [1,2]. Chemerin was first identified as a

48 ligand for chemokine like receptor 1 (CMKLR1), also known as Chemerin Receptor 23

of
49 (ChemR23), which is the best-known receptor for this hormone. The active form of adipokine

ro
50 generated after enzymatic cleavage can also be bound by two other metabotropic receptors: G

-p
51 protein-coupled receptor 1 (GPR1) and C-C chemokine receptor-like 2 (CCRL2) [2–4]. Chemerin
re
52 belongs to a group of adipokines which are secreted by white adipose tissue [5]. The hormone and
lP

53 its receptors (collectively referred to as the chemerin system) are present in various tissues of many

54 animal species, including humans, rodents, cattle, poultry, and pigs [6,7]. The RARRES2 gene was
na

55 found to be expressed in the mouse liver, white adipose tissue and placenta [8], human liver and
ur

56 pancreas [9], as well as in human [10] and rodent [8] ovaries. The expression of the CMKLR1 gene
Jo

57 was observed mainly in white adipose tissue, as well as in the lungs, heart, and placenta of mice

58 [8]. The second chemerin receptor, GPR1, is mostly present in the human central nervous system

59 [11], but it was also detected in human granulosa cells [10], and in human and mouse skin [12].

60 Moreover, our previous studies have shown that chemerin system genes and proteins are expressed

61 in the porcine hypothalamic-pituitary-ovarian axis [13–16].

62 Chemerin is a pleiotropic hormone that participates in the regulation of many processes, such

63 as blood pressure control, immune system regulation, inflammation, and angiogenesis [17]. It also

64 plays a key role in the regulation of metabolism [18]. Chemerin expression and secretion increase

65 during adipocyte differentiation [1,19]. Research has demonstrated that chemerin stimulates the

3
66 adhesion of macrophages to extracellular matrix proteins and other adhesion-related molecules to

67 sustain the recruitment, chemotaxis, and retention of macrophages at the inflammation site [20].

68 In addition, adipokine concentrations in human inflammatory fluids correlate with the

69 concentrations of tumour necrosis factor α (TNFα), interleukin 6 (IL6), and C-reactive protein

70 (CRP), which play a crucial role in the metabolic syndrome [21]. Inflammatory proteases,

71 including cathepsin G, tryptases, and elastases, are able to detach the C-terminus of pre-

72 prochemerin and activate the protein [22]. Chemerin is able to perform these roles by interacting

of
73 with its three receptors (CMKLR1, GPR1, and CCRL2). CMKLR1 relies on ERK 1/2 and Akt

ro
74 kinases for signal transduction [17]. Chemerin amplifies leukocyte chemotaxis by binding to

75
-p
CMKLR1 [23]. The expression of CMKLR1 in macrophages and immature dendritic cells also
re
76 confirms chemerin’s involvement in immune system regulation [1]. Despite its high structural
lP

77 similarity to CMKLR1, GPR1 performs somewhat different functions [17]. It is believed that
na

78 chemerin regulates glucose homeostasis during obesity development by binding to GPR1 [24]. In

79 mice, chemerin/GPR1 signalling regulates the secretion of prostaglandin F2α (PGF2α)-induced

ur

80 progesterone (P4) by ovarian cells during follicular development, corpus luteum (CL) formation,
Jo

81 and luteolysis [25]. Rourke et al. [26] found that chemerin activates RhoA/RhoA kinase signalling

82 by binding to GPR1 in human or mouse cell-based assays. They concluded that similarly to

83 CMKLR1, GPR1 is coupled with Gαi/o and that GPR1 activates the MAPK/extracellular signal-

84 related kinase pathway [26]. The third receptor, CCRL2, has a completely different function. It

85 binds the N-terminus region of chemerin and does not induce signal transduction. CCRL2 is

86 responsible for presenting the C-terminal end of the hormone to neighbouring CMKLR1-expressed

87 cells [17].

4
 88 It is well known that adipose tissue-derived hormones, such as leptin and adiponectin, may act

89 as a link between metabolism and reproduction. This group of hormones includes chemerin which

90 appears to be involved in the crosstalk between metabolism and the reproductive system. In our

91 previous study, the chemerin system was identified in the uteri of non-pregnant and early pregnant

92 pigs [27]. The chemerin system is expressed in the porcine uterus, but the underlying regulatory

93 mechanisms remain unknown. The observed changes in chemerin system expression between the

94 phases of the oestrus cycle/early pregnancy suggest that steroids may be involved in the underlying

of
95 regulatory processes. This observation prompted the authors to hypothesize that steroid hormones

ro
96 influence the expression of the chemerin system in the uterus of domestic pigs. The research

97
-p
hypothesis was verified by analysing the effects of steroids, oestradiol (E2), and P4 on the gene and
re
98 protein expression of chemerin receptors, as well as RARRES2 gene expression and chemerin
lP

99 secretion by endometrial slices on days 10 to 11 (transuterine migration of embryos), 12 to 13

na

100 (maternal recognition of pregnancy), 15 to 16 (beginning of implantation), and 27 to 28 (end of

101 implantation) of early pregnancy, and the mid-luteal phase of the oestrous cycle (days 10 to 11 –
ur

102 mid-luteal phase of the oestrous cycle, when CL activity is similar to that observed during
Jo

103 pregnancy).

104

105 3. Material and methods

106 3.1. Experimental animals, tissue collection and in vitro cultures

107 All studies were executed according to Polish Act on the protection of animals used for

108 scientific or educational destination of the 15th of January 2015 (Polish Journal of Law of 2015

109 item 266) as well as directive 2010/63/EU of the European Parliament of the 22nd of September

110 2010 on the protection of animals used for a scientific destination. Uteri were collected as

5
111 described by Smolinska et al. [13] from 25 gilts destined for slaughter (7 to 8 months, 130 to 140

112 kg) and were divided into five groups (n=5 per group) as follows: days 10 to 11, 12 to 13, 15 to 16

113 and 27 to 28 of pregnancy, as well as on days 10 to 11 of the oestrous cycle. The day of the onset

114 of the second oestrus was designated as day 0 of the oestrous cycle. The phase of the cycle was

115 additionally evaluated by ovarian morphology [28]. Pigs were fertilized on days 1 to 2 of the cycle.

116 Immediately after slaughter, the uteri were transported to the laboratory in the ice-cold phosphate

117 buffered saline (PBS) supplemented with 1% antibiotic-antimycotic solution (Sigma-Aldrich,

of
118 USA). The day of pregnancy was confirmed by the presence and morphology of conceptuses

ro
119 isolated from the uterine horns [29]. Endometrial in vitro cultures were performed as described by

120
-p
Smolinska et al. [30]. Slices of the endometrial tissue isolated from the uterine horns (100 mg ±
re
121 10%) were washed three times in M199 medium (Sigma-Aldrich, USA). Subsequently, the tissue
lP

122 explants were individually placed into culture glass vials with 2 mL of phenol red-free M199
na

123 medium supplemented with 0.1% BSA (MP Biomedicals, USA), 5% new-born calf serum (FCS;

124 Sigma-Aldrich, USA) and 1% antibiotic-antimycotic solution (Sigma-Aldrich, USA). The tissue
ur

125 explants were preincubated in a shaking water bath for 2 h at 37°C in 95% O2 and 5% CO2
Jo

126 atmosphere. After preincubation, to examine the impact of E2 and P4 on the gene and protein

127 expression of chemerin system elements and the adipokine secretion by the porcine endometrium,

128 the explants were treated under the same conditions for 24 h with E2 (1, 10 and 100 nM; Sigma-

129 Aldrich, USA) or P4 (10, 100 and 1000 nM; Sigma-Aldrich, USA). The doses of steroid hormones

130 were established based on Blitek et al. [31] and Dobrzyn et al. [32]. Explants incubated under the

131 same conditions in the medium without any treatment were used as the controls. Our preliminary

132 studies have shown a negligible effect of E2 and P4 solvent (ethanol at the concentration of 0.01

133 M) on the gene/protein expression and chemerin secretion as well as on cell viability. All in vitro

6
134 cultures were performed in duplicates in five independent experiments (n=5). The vitality of tissue

135 explants was determined by measuring lactate dehydrogenase (LDH) activity in media at the end

136 of the 24-hour treatment. The LDH test was performed using a Liquick Cor-LDH kit (Cormay,

137 Poland) according to the manufacturer's instructions. The release of LDH by cultured explants was

138 compared to its activity in the medium obtained after homogenization of endometrial cells

139 (positive control indicating the total cell death and the maximal release of LDH). Mean activity of

140 LDH in the cultured slices after treatment period was 55.1 ± 4.5 U/L (1.8 % of maximal release of

of
141 LDH).

ro
142

143
-p
3.2. RNA isolation, cDNA synthesis and quantitative real-time PCR (qPCR)
re
144 Extraction of total RNA from endometrial explants was conducted using TRI Reagent® RNA
lP

145 Isolation Reagent (Sigma-Aldrich, USA) according to the manufacturer’s protocol. Purity and
na

146 quantity of RNA were measured spectrophotometrically (Infinite M200 PRO, Tecan,

147 Switzerland). To obtain cDNA, 1 μg of RNA was subjected to reverse transcription at the final
ur

148 volume of 20 μL with 0.5 μg oligo(dT)15 primers (Roche, Switzerland) using Omniscript RT Kit
Jo

149 (Qiagen, USA) for 1 h at 37°C. Reaction was terminated by the incubation at 93°C for 5 min.

150 Quantitative real-time PCR was carried out using AriaMX (Applied Biosystems, USA) as

151 described by Smolinska et al. [13]. In brief, the PCR reaction mixtures contained cDNA, primers,

152 12.5 µL Power SYBR Green PCR Master Mix (Applied Biosystems, USA) and

153 RNase-free water at the final volume of 25 µL. Primers used to amplify parts of RARRES2,

154 CMKLR1, GPR1, CCRL2 genes and reference genes: cyclophilin A (PPIA) and β-actin (ACTB),

155 as well as qPCR reaction conditions are detailed in Table 1. The qPCR no template controls (NTC)

156 included water instead of cDNA. All reactions were conducted in duplicates. The specificity of

7
157 amplification products was confirmed by the melting-curve analysis. Comparative cycle threshold

158 method (∆∆CT) was used to calculate the relative RARRES2, CMKLR1, GPR1 and CCRL2 gene

159 expression in comparison to the geometric mean of reference genes’ Ct values [33].

160

161 3.3. Protein isolation and Western blot

162 Total protein was extracted from tissue homogenates with the use of T-PER™ Tissue Protein

163 Extraction Reagent (Thermo Fisher Scientific, USA) with a mix of proteases inhibitors (Sigma-

of
164 Aldrich, USA). After isolation, the lysates were centrifuged twice at 10 000 X g for 5 min. The

ro
165 concentration of proteins in supernatants was measured using Bradford dye-binding procedure

166
-p
with BSA dilutions as the standards. Western blot analysis was carried out as described by
re
167 Smolinska et al. [13] with modifications. Briefly, 40 μg of total protein were separated by SDS-
lP

168 PAGE electrophoresis in a 12.5% polyacrylamide gels and transferred onto polyvinylidene
na

169 difluoride (PVDF) membranes (Merc Millipore, USA). Actin protein was used to quantify the

170 CMKLR1, GPR1 and CCRL2 proteins expression on separate blots. Membranes were blocked for
ur

171 1 h at room temperature in Tris-buffered saline with Tween-20, containing 5% skimmed milk
Jo

172 powder. Subsequently, the membranes were incubated overnight at 4°C with specific primary

173 antibodies: mouse polyclonal antibodies to GPR1 (1:500; ab169331; Abcam, UK), rabbit

174 polyclonal antibodies to CMKLR1 (1:1000; ab230442; Abcam, UK), CCRL2 (1:600; ab85224;

175 Abcam, UK) and actin (1:200; A2066; Sigma-Aldrich, USA). To identify immunoreactive

176 products, membranes were incubated for 1.5 h at room temperature with secondary antibodies

177 conjugated with horseradish peroxidase (HRP): goat anti-mouse IgG for GPR1 (1:2500; 115-035-

178 003; Jackson ImmunoResearch Laboratories, USA) and goat anti-rabbit IgG for CMKLR1,

179 CCRL2 and actin (1:5000; sc-2054; Santa Cruz, USA). Visualisation of immunocomplexes was

8
180 conducted using chemiluminescence HRP substrate (Merck Millipore, USA) as described by the

181 manufacturer. Next, the membranes were analysed and archived using G:Box EF with

182 chemiluminescent imaging software (Syngene, USA). The results were quantified by the optical

183 density (OD) analysis with Image Studio Lite v.5.2 (LI-COR, USA). Data were presented in the

184 arbitrary units as a ratio of OD of examined protein relative to OD of actin protein.

185

186 3.4. Enzyme-linked immunosorbent assay (ELISA)

of
187 The concentrations of chemerin protein in the culture media were determined using

ro
188 commercial ELISA kit (FineTest, China) as described by the manufacturer. The range of standard

189
-p
curve was 0.156-10 ng/mL. Sensitivity of the assay was defined as the lowest concentration that
re
190 could be distinguished from the zero samples (0.001 ng/mL). According to the manufacturer, no
lP

191 significant cross-reactivity or interference between chemerin and any homologous proteins
na

192 assayed have been noted. Absorbances were measured using Infinite M200 PRO reader with Tecan

193 I-control software (Tecan, Switzerland) at 450 nm. The results were linearized by plotting the log
ur

194 of chemerin concentrations in comparison with the log of the optical density. Regression analysis
Jo

195 was used to define the best fit line. Intra-assay variability was 3.22% ± 0.39, whereas inter-assay

196 was 8.1%.

197

198 3.5. Statistical analysis

199 Statistical analysis was conducted using Statistica software (StatSoft Inc., USA). Differences

200 between groups were affirmed using one-way ANOVA followed by the Duncan's post-hoc test

201 and were reported as mean ± standard error of the mean (SEM) from five independent

202 observations. Values for P<0.05 were considered as statistically significant.

9
203 4. Results

204 4.1. The effect of E2 on RARRES2 gene expression and chemerin secretion

205 On days 10 to 11 of pregnancy, RARRES2 gene expression in the endometrial slices was

206 enhanced by E2 at the doses of 1 and 10 nM (Fig. 1A), however the protein release was unchanged

207 (Fig. 1B). On days 12 to 13 of pregnancy, chemerin gene expression was increased under the

208 influence of E2 at the doses of 1 and 10 nM (Fig. 1C), whereas chemerin protein release was

209 enhanced by E2 only at the dose of 1 nM (Fig. 1D). On days 15 to 16 of gestation, E2 had no effect

of
210 on RARRES2 mRNA content (Fig. 1E), while protein concentrations were reduced by the steroid

ro
211 at the doses of 10 and 100 nM (Fig. 1F). On days 27 to 28 of gestation, chemerin gene expression

212
-p
was decreased by E2 at all tested doses (1–100 nM; Fig. 1G), whereas the steroid had no effect on
re
213 the protein concentration (Fig. 1H). On days 10 to 11 of the oestrous cycle, both the gene
lP

214 expression in the endometrium and protein concentration in culture media were not affected by E2
na

215 (Fig. 1I, J).

216
ur

217 4.2. The effect of E2 on CMKLR1 gene and protein expression

Jo

218 On days 10 to 11 of pregnancy, E2 at the dose of 1 nM enhanced the expression of CMKLR1

219 gene (Fig. 2A) and reduced the receptor protein concentration in the endometrial explants (Fig.

220 2B). On days 12 to 13 of gestation, E2 (1–100 nM) increased the CMKLR1 mRNA contents (Fig.

221 2C). The opposite situation was observed in the case of the receptor protein (Fig. 2D). On days 15

222 to 16 of pregnancy, E2 at the dose of 1 nM increased whereas at the dose of 100 nM decreased

223 CMKLR1 gene expression (Fig. 2E). During these days, endometrial expression of CMKLR1

224 protein was reduced by E2 at the dose of 10 nM (Fig. 2F). In the endometrium collected from pigs

225 on days 27 to 28 of pregnancy, the contents of CMKLR1 transcripts were decreased by E2 at the

226 doses of 1 and 100 nM (Fig. 2G). Receptor protein concentration was increased by the presence of
10
227 E2 at the dose of 100 nM (Fig. 2H). During the mid-luteal phase of the cycle, E2 (1–100 nM) caused

228 an increase in the receptor gene expression (Fig. 2I) but decreased the receptor protein contents

229 (Fig. 2J).

230

231 4.3. The effect of E2 on GPR1 gene and protein expression

232 On days 10 to 11 of gestation, GPR1 gene expression was enhanced by E2 at the dose of 1 nM

233 (Fig. 3A). On the contrary, E2 at the same dose caused a decrease in the receptor protein content

of
234 (Fig. 3B). On days 12 to 13 of gestation, GPR1 gene expression was reduced under the influence

ro
235 of E2 at the doses of 1 and 100 nM (Fig. 3C). During these days, GPR1 protein content was

236
-p
increased under the influence of E2 at the dose of 100 nM (Fig. 3D). On days 15 to 16 of pregnancy,
re
237 enhanced GPR1 gene expression was observed under the influence of E2 at the dose of 10 nM (Fig.
lP

238 3E), whereas the protein concentrations were increased by E2 at the doses of 1 and 100 nM (Fig.
na

239 3F). On days 27 to 28 of pregnancy, E2 reduced the expression of GPR1 gene (1-100 nM; Fig.

240 3G), and protein (10 nM; Fig. 3H). On days 10 to 11 of the oestrous cycle, E2 had no effect on
ur

241 GPR1 gene expression (Fig. 3I), whereas the protein content was enhanced by the steroid at the
Jo

242 dose of 100 nM (Fig. 3J).

243

244 4.4. The effect of E2 on CCRL2 gene and protein expression

245 On days 10 to 11 of pregnancy, CCRL2 gene expression was decreased by E2 at all tested

246 doses (Fig. 4A), whereas the protein content was reduced only by E2 at the dose of 1 nM (Fig. 4B).

247 On days 12 to 13 of pregnancy, the receptor gene expression was down-regulated by E2 (1–100

248 nM; Fig. 4C). The content of the receptor protein was increased by E2 at the dose 10 nM (Fig. 4D).

249 On days 15 to 16 of pregnancy, the expression of CCRL2 gene was decreased by E2 (1-100 nM;

11
250 Fig. 4E), whereas the protein concentrations were increased by the steroid at the doses of 10 and

251 100 nM (Fig. 4F). On days 27 to 28 of gestation, E2 at the doses of 10 and 100 nM caused a

252 reduction in CCRL2 gene expression (Fig. 4G), and an enhancement in the receptor protein

253 contents (Fig. 4H). On days 10 to 11 of the cycle, E2 (100 nM) enhanced the gene expression (Fig.

254 4I), whereas the steroid (1 and 10 nM) caused a drop in the protein contents (Fig. 4J).

255

256 4.5. The effect of P4 on RARRES2 gene expression and chemerin secretion

of
257 In the endometrial tissue slices on days 10 to 11 (Fig. 5A) and 12 to 13 (Fig. 5C) of pregnancy,

ro
258 P4 (10 and 1000 nM) caused an increase in RARRES2 gene expression. On days 15 to 16 of

259
-p
gestation, higher chemerin mRNA content was observed under the influence of P4 at the dose of
re
260 1000 nM (Fig. 5E). On days 27 to 28 of gestation, endometrial tissue explants treated with P4 (10-
lP

261 1000 nM) exhibited lower RARRES2 gene expression (Fig. 5G). During the mid-luteal phase of
na

262 the cycle, P4 at the dose of 10 nM decreased chemerin transcript content (Fig. 5I). The secretion

263 of chemerin protein by the endometrial tissue slices during all the studied periods of pregnancy
ur

264 and the oestrous cycle were at the same level in relation to the control regardless of the used P4
Jo

265 dose (Fig. 5B, D, F, H, J).

266

267 4.6. The effect of P4 on CMKLR1 gene and protein expression

268 On days 10 to 11 of pregnancy in the porcine endometrium, P4 (100 and 1000 nM) inhibited

269 the expression of CMKLR1 gene (Fig. 6A). During these days, CMKLR1 protein contents were

270 increased by P4 at the dose of 10 nM and reduced by the steroid at the dose of 100 nM (Fig. 6B).

271 On days 12 to 13 of pregnancy in the endometrial slices incubated with all tested doses of P4, the

272 expression of CMKLR1 gene was increased (Fig. 6C), whereas the receptor protein concentrations

12
273 were decreased (Fig. 6D). On days 15 to 16 of gestation, CMKLR1 mRNA content was elevated

274 by P4 at the dose of 1000 nM (Fig. 6E). However, the protein contents were reduced by P4 at all

275 tested doses (Fig. 6F). On days 27 to 28 of gestation, the expression of CMKLR1 gene was inhibited

276 by P4 (10 and 1000 nM; Fig. 6G). During this period of pregnancy, the receptor protein expression

277 was up-regulated by P4 at the dose10 nM (Fig. 6H). On days 10 to 11 of the oestrous cycle, P4 at

278 all the tested doses caused a stimulation of CMKLR1 gene expression (Fig. 6I), whereas did not

279 affect the receptor protein content (Fig. 6J).

of
280

ro
281 4.7. The effect of P4 on GPR1 gene and protein expression

282
-p
In the in vitro incubated endometrial tissue explants on days 10 to 11 of pregnancy, GPR1
re
283 gene (Fig. 7A) and protein (Fig. 7B) expression was enhanced by P4 at the doses of 10 nM and
lP

284 1000 nM, respectively. On days 12 to 13 of pregnancy, P4 at each of tested doses reduced the
na

285 contents of GPR1 mRNA (Fig. 7C), whereas GPR1 protein expression was not affected by the

286 steroid (Fig. 7D). On days 15 to 16 of gestation, P4 (1000 nM) enhanced both GPR1 gene and
ur

287 protein expression (Fig. 7E, F). On days 27 to 28 of pregnancy, the GPR1 gene expression was
Jo

288 reduced under the influence of P4 (10–1000 nM; Fig. 7G). During these days, GPR1 protein

289 contents were reduced by P4 at the doses of 100 and 1000 nM (Fig. 7H). On days 10 to 11 of the

290 cycle, P4 (10 nM) enhanced the gene expression of GPR1 (Fig. 7I), while had no effect on the

291 protein expression in the endometrium (Fig. 7J).

292

293 4.8. The effect of P4 on CCRL2 gene and protein expression

294 On days 10 to 11 of pregnancy, P4 (10 nM) induced the expression of CCRL2 gene (Fig. 8A) and

295 protein (Fig. 8B). On days 12 to 13 of pregnancy, P4 decreased CCRL2 gene

13
296 (10–1000 nM; Fig. 8C) and protein (10 nM; Fig. 8D) expression. In the porcine endometrium on

297 days 15 to 16 of pregnancy, CCRL2 mRNA contents were reduced by P4 at all tested doses (Fig.

298 8E), while the receptor protein concentrations were raised by the steroid (100 and 1000 nM; Fig.

299 8F). On days 27 to 28 of gestation, P4 at the dose of 1000 nM enhanced the receptor gene

300 expression (Fig. 8G), while the protein content was increased by P4 at the dose of 10 nM (Fig. 8H).

301 During the mid-luteal phase of the cycle, the gene expression of CCRL2 was not influenced by P4

302 (Fig. 8I), whereas the protein content was reduced by the steroid at the dose of 100 nM (Fig. 8J).

of
303

ro
304 All the results obtained are summarized in Table 2, while Table 3 presents the p and F values for

-p
305 the obtained results.

306
re
307 5. Discussion
lP

308 The function of chemerin in the female reproductive tract is not fully understood. This
na

309 adipokine affects pituitary gland and ovarian functions in the domestic pig [14,15,34,35].
ur

310 However, little is known about chemerin’s role in the porcine uterus. The chemerin system has
Jo

311 been identified in the human endometrium [36]. Carlino et al. [37] found that chemerin was highly

312 expressed in human stromal (ST) cells during decidualization, but not in decidual endothelial cells.

313 The presence of chemerin system mRNA and proteins in the porcine endometrium and

314 myometrium during the oestrous cycle and early pregnancy, as well as in trophoblasts and

315 conceptuses was demonstrated previously [27]. Chemerin concentrations fluctuated in the uterine

316 luminal flushings (ULF) [27] and blood plasma [13] of sows during early gestation and the

317 oestrous cycle. Our previous study also revealed that chemerin affects P4 and E2 synthesis in the

318 porcine endometrium [27], and that prostaglandins may influence chemerin system expression in

319 the porcine endometrium [38]. These results indicate that chemerin could act as a link between

14
320 metabolism and reproduction by affecting both processes to maintain homeostasis. We

321 hypothesized that changes in chemerin system expression in the porcine uterus could result mainly

322 from variations in hormone levels, especially steroid concentrations. The influence of steroid

323 hormones on chemerin system expression in the uterus remains insufficiently investigated. To the

324 best of our knowledge, this is the first study to indicate that P4 and E2 induce changes in the gene

325 and protein expression of chemerin receptors (CMKLR1, GPR1 and CCRL2), RARRES2 gene

326 expression, as well as chemerin secretion by incubated porcine endometrial slices in early stages

of
327 of gestation (days 10 to 11, 12 to 13, 15 to 16, and 27 to 28) and the mid-luteal phase of the oestrous

ro
328 cycle (days 10 to 11 of the cycle).

329
-p
The results of the present study confirm this observation and corroborate previous reports
re
330 postulating the presence of a relationship between steroid hormones and chemerin system
lP

331 expression in different tissues. Our previous findings suggest that steroid hormones could be
na

332 responsible for the varied expression of chemerin system components in the porcine uterus [27].
ur

333 Wang et al. [39] used 5α-dihydrotestosterone (DHT) to induce the polycystic ovary syndrome
Jo

334 (PCOS) in a rat model, and found that this androgen increased chemerin and CMKLR1 protein

335 levels in the ovaries. Luque-Ramirez et al. [40] observed that chemerin levels were negatively

336 correlated with free-E2 concentrations in human serum, and they concluded that E2 could affect

337 chemerin secretion. E2 and P4 were also found to increase chemerin secretion by ST cells in

338 pregnant, fertile non-pregnant, and menopausal women [37]. In the present study, E2 increased

339 chemerin secretion by endometrial tissue explants on days 12 to 13 of pregnancy, but decreased

340 chemerin secretion on days 15 to 16 of gestation. However, P4 had no significant effect on

341 chemerin release from endometrial tissue. This study also demonstrated that the extent to which

342 E2 and P4 influence the expression of chemerin receptors is mediated by the steroid dose and the

15
343 period of pregnancy/oestrous cycle. Steroid hormones could also regulate endometrial sensitivity

344 to chemerin by influencing the expression of adipokine receptors. By controlling the abundance of

345 chemerin receptors, steroid hormones could indirectly regulate adipokine’s influence on the

346 endometrium, thus ensuring the proper course of early pregnancy and creating a supportive

347 environment for developing embryos.

348 In the current study, P4 and E2 exerted varied effects on gene and protein expression. In

349 mammals, the correlation between gene expression and the abundance of the corresponding

of
350 proteins can be as low as 40%, depending on numerous factors [41]. The above could explain the

ro
351 presence of noticeable differences in RNA and protein expression patterns in this experiment.

352
-p
Moreover, the differences in the stability of mRNAs and proteins could explain the variations in
re
353 the expression of genes and the corresponding proteins [42]. P4 and E2 also exerted varied effects
lP

354 on chemerin system expression in the endometrial tissues sampled from pregnant and non-
na

355 pregnant pigs (during the mid-luteal phase of the oestrous cycle). The differences in expression
ur

356 patterns could also be attributed to the effects exerted by other factors which are responsible for
Jo

357 embryo development, including other steroid hormones, prostaglandins, growth factors, or even

358 other adipokines.

359 In the present study, P4 and E2 modulated the expression of chemerin system components, but

360 a clear stimulatory or inhibitory pattern was not noted. P4 and E2 act through both genomic and

361 non-genomic mechanisms. Nuclear progesterone receptors A (PR-A) and B (PR-B) are responsible

362 for the most widely recognised physiological and biological actions of P4 [43–45]. The efficacy or

363 effects of ligand binding by these receptors may vary. According to Mulac-Jericevic et al. [46],

364 PR-A and PR-B play different roles in the uterus during gestation. In the current study, P4 affected

365 the expression of chemerin receptors during early pregnancy, when PRs are present in the surface

16
366 and glandular epithelium, and in the stroma. P4 influences the PR-dependent process of

367 decidualization, which relies on the expression of genes that participate in differentiation, cell

368 cycle transit, survival, and apoptosis. Chemerin is associated with many of these processes

369 [34,35,49,50], which could suggest that P4 regulates chemerin’s effects on the porcine uterus by

370 modulating chemerin receptor levels in the endometrium during early pregnancy and the mid-

371 luteal phase of the oestrous cycle. Oestrogens could induce such effects by binding to oestrogen

372 receptors α (ERα) and β (ERβ) which belong to the group of nuclear receptors. Geisert et al. [53]

of
373 found changes in ER levels in the uterine epithelium during the oestrous cycle and early pregnancy,

ro
374 and reported the highest expression levels on days 5 to 12 of the cycle and gestation [53]. P4 and

375
-p
E2 can also be bound by membrane receptors to elicit rapid, non-genomic action and induce signal
re
376 transduction, mainly through kinase pathways [for a review, see 54]. The above could imply that
lP

377 non-genomic action is somewhat different from oestrogens’ impact via nuclear receptors. The
na

378 varied action of P4 and E2 could also be attributed to their auto- and para-regulatory effects on the

379 expression of their receptors [55,56]. The combined effects of P4 and E2 on the expression of the
ur

380 chemerin system in the uterine endometrium could result from various mechanisms exploited by
Jo

381 different P4 and E2 receptors.

382 The secretion of E2 increased in P4-stimulated endometrial and myometrial tissues harvested

383 during the oestrous cycle and pregnancy [57]. P4 directly affects endometrial and myometrial

384 tissues, but it could also act as a substrate for the production of other steroid hormones, including

385 E2 [58]. During steroidogenesis, cytochrome P450C17 converts P4 to androstenedione, which is then

386 converted to testosterone (T) by 17β-hydroxysteroid dehydrogenase or used for E1 synthesis by

387 cytochrome P450 aromatase (P450AROM). Testosterone can be further converted to E2 by P450AROM

388 [58]. Thus, the effects of P4 could be directly or indirectly modulated by the products of

17
389 steroidogenic enzyme activity in the porcine endometrium. The above assumption could be

390 validated by the fact that the steroidogenic acute regulatory protein (StAR) and the key

391 steroidogenic enzymes (P450 side-chain cleavage enzyme, cytochrome P450C17, P450AROM, 17β-

392 hydroxysteroid, and 3β-hydroxysteroid dehydrogenases) have been previously identified in the

393 porcine endometrium [59,60, Kiezun et al., unpublished]. In the present study, only certain

394 similarities were observed in the effects of P4 and E2. These effects could be attributed to the direct

395 action of P4, but also to the influence of other biologically active metabolites.

of
396 The maternal recognition of pregnancy and embryo implantation are crucial periods for

ro
397 pregnancy establishment. Up to 30% of conceptuses may not survive during this period [61]. Two-

398
-p
way communication between the mother and the conceptus is highly dependent on E2 which is
re
399 secreted by the embryos on days 11 to 12 of their development [62]. P4 has been shown to exert a
lP

400 significant and dose-dependent inhibitory effect on lymphocyte cytotoxicity in vitro [63]. In our
na

401 previous study, chemerin concentrations in the porcine ULF peaked on days 12 to 13 of pregnancy
ur

402 [27], which could be due to higher concentrations of steroid hormones and other pregnancy-related
Jo

403 factors in ULF, such as calcium, prostaglandins, and total protein levels [64]. During the maternal

404 recognition of pregnancy, chemerin secretion increased under the influence of E2, which

405 corroborates previous findings [27]. In the present study, E2 increased the abundance of GPR1 and

406 CCRL2 receptor proteins, but decreased CMKLR1 protein levels. In the analysed period of

407 gestation, P4 had only a down-regulating effect on the abundance of CMKLR1 and CCRL2

408 proteins. In turn, during embryo implantation (days 15 to 16 of pregnancy), the expression of

409 CMKLR1 protein was inhibited by P4. E2 exerted an identical effect during this period of gestation.

410 The above could suggest that the concentrations of CMKLR1 protein, the major pro-inflammatory

18
411 chemerin receptor [65], decrease under the influence of E2 and P4 to minimise pro-inflammatory

412 effects in the porcine uterus in these crucial stages of early pregnancy.

413 Conclusions

414 This is the first study to examine the effect of steroid hormones on the expression of the

415 chemerin system in the uterus of the domestic pig during early pregnancy and the mid-luteal phase

416 of the oestrous cycle. The presented data suggest that P4 and E2 modulate chemerin gene

of
417 expression, chemerin secretion, and the expression of chemerin receptor (CMKLR1, GPR1 and

ro
418 CCRL2) genes and proteins, depending on the steroid dose and the hormonal status of animals in

-p
419 different periods of early pregnancy and/or the oestrous cycle. These results validate the hypothesis
re
420 that the hormonal environment could modulate chemerin system expression in endometrial tissues.
lP

421
na

422 Declaration of interest

423 None.
ur
Jo

424

425 Acknowledgements

426 This research was supported by Polish National Science Centre [grant number

427 2017/25/B/NZ9/00040].

428

429

19
430 Bibliography

431 [1] Wittamer V, Franssen JD, Vulcano M, Mirjolet JF, Le Poul E, Migeotte I, et al. Specific

432 recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human

433 inflammatory fluids. J Exp Med. 2003;198(7):977-85.

434 https://doi.org/10.1084/jem.20030382.

435 [2] Meder W, Wendland M, Busmann A, Kutzleb C, Spodsberg N, John H, et al.

436 Characterization of human circulating TIG2 as a ligand for the orphan receptor ChemR23.

of
437 FEBS Lett. 2003;555(3):495-9. https://doi.org/10.1016/s0014-5793(03)01312-7.

ro
438 [3] Rourke JL, Dranse HJ, Sinal CJ. Towards an integrative approach to understanding the role

439 of chemerin in human health

-p
and disease. Obes Rev. 2013;14(3):245-62.
re
440 https://doi.org/10.1111/obr.12009.
lP

441 [4] Zhao L, Yamaguchi Y, Shen WJ, Morser J, Leung LLK. Dynamic and tissue-specific
na

442 proteolytic processing of chemerin in obese mice. PLoS One. 2018;13(8):e0202780.

ur

443 https://doi.org/10.1371/journal.pone.0202780.
Jo

444 [5] Nagpal S, Patel S, Jacobe H, DiSepio D, Ghosn C, Malhotra M, et al. Tazarotene-induced

445 gene 2 (TIG2), a novel retinoid-responsive gene in skin. J Invest Dermatol.

446 1997;109(1):91-5. https://doi.org/10.1111/1523-1747.ep12276660.

447 [6] Huang J, Zhang J, Lei T, Chen X, Zhang Y, Zhou L, et al. Cloning of porcine chemerin,

448 ChemR23 and GPR1 and their involvement in regulation of lipogenesis. BMB Rep.

449 2010;43(7):491-8. https://doi.org/10.5483/bmbrep.2010.43.7.491.

450 [7] Kennedy AJ, Davenport AP. International Union of Basic and Clinical Pharmacology CIII:

451 Chemerin Receptors CMKLR1 (Chemerin1) and GPR1 (Chemerin2) Nomenclature,

20
452 Pharmacology, and Function. Pharmacol Rev. 2018;70(1):174-96.

453 https://doi.org/10.1124/pr.116.013177.

454 [8] Goralski KB, McCarthy TC, Hanniman EA, Zabel BA, Butcher EC, Parlee SD, et al.

455 Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism. J Biol

456 Chem. 2007;282(38):28175-88. https://doi.org/10.1074/jbc.M700793200.

457 [9] Chamberland JP, Berman RL, Aronis KN, Mantzoros CS. Chemerin is expressed mainly

458 in pancreas and liver, is regulated by energy deprivation, and lacks day/night variation in

of
459 humans. Eur J Endocrinol. 2013;169(4):453-62. https://doi.org/10.1530/EJE-13-0098.

ro
460 [10] Reverchon M, Cornuau M, Ramé C, Guerif F, Royère D, Dupont J. Chemerin inhibits IGF-

461
-p
1-induced progesterone and estradiol secretion in human granulosa cells. Hum Reprod.
re
462 2012;27(6):1790-800. https://doi.org/10.1093/humrep/des089.
lP

463 [11] Shimizu N, Soda Y, Kanbe K, Liu HY, Jinno A, Kitamura T, et al. An orphan G protein-
na

464 coupled receptor, GPR1, acts as a coreceptor to allow replication of human

465 immunodeficiency virus types 1 and 2 in brain-derived cells. J Virol. 1999;73(6):5231-9.

ur

466 https://doi.org/10.1128/JVI.73.6.5231-5239.1999.
Jo

467 [12] Banas M, Zegar A, Kwitniewski M, Zabieglo K, Marczynska J, Kapinska-Mrowiecka M,

468 et al. The expression and regulation of chemerin in the epidermis. PLoS One.

469 2015;10(2):e0117830. https://doi.org/10.1371/journal.pone.0117830.

470 [13] Smolinska N, Kiezun M, Dobrzyn K, Rytelewska E, Kisielewska K, Gudelska M, et al.

471 Expression of Chemerin and Its Receptors in the Porcine Hypothalamus and Plasma

472 Chemerin Levels during the Oestrous Cycle and Early Pregnancy. Int J Mol Sci.

473 2019;20(16):3887. https://doi.org/10.3390/ijms20163887.

21
474 [14] Kisielewska K, Rytelewska E, Gudelska M, Kiezun M, Dobrzyn K, Bogus-Nowakowska

475 K, et al. Expression of chemerin receptors CMKLR1, GPR1 and CCRL2 in the porcine

476 pituitary during the oestrous cycle and early pregnancy and the effect of chemerin on

477 MAPK/Erk1/2, Akt and AMPK signalling pathways. Theriogenology. 2020a;157:181-98.

478 https://doi.org/10.1016/j.theriogenology.2020.07.032.

479 [15] Kisielewska K, Rytelewska E, Gudelska M, Kiezun M, Dobrzyn K, Bogus-Nowakowska

480 K, et al. Relative abundance of chemerin mRNA transcript and protein in pituitaries of pigs

of
481 during the estrous cycle and early pregnancy and associations with LH and FSH secretion

ro
482 during the estrous cycle. Anim Reprod Sci. 2020b;219:106532.

483
-p
https://doi.org/10.1016/j.anireprosci.2020.106532.
re
484 [16] Rytelewska E, Kisielewska K, Kiezun M, Dobrzyn K, Gudelska M, Rak A, et al.
lP

485 Expression of chemerin and its receptors in the ovaries of prepubertal and mature gilts.
na

486 Mol Reprod Dev. 2020. https://doi.org/10.1002/mrd.23391.

487 [17] Mattern A, Zellmann T, Beck-Sickinger AG. Processing, signaling, and physiological
ur

488 function of chemerin. IUBMB Life. 2014;66(1):19-26. https://doi.org/10.1002/iub.1242.

Jo

489 [18] Takahashi M, Takahashi Y, Takahashi K, Zolotaryov FN, Hong KS, Kitazawa R, et al.

490 Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in

491 3T3-L1 adipocytes. FEBS Lett. 2008;582(5):573-8.

492 https://doi.org/10.1016/j.febslet.2008.01.023.

493 [19] Bozaoglu K, Segal D, Shields KA, Cummings N, Curran JE, Comuzzie AG, et al.

494 Chemerin is associated with metabolic syndrome phenotypes in a Mexican-American

495 population. J Clin Endocrinol Metab. 2009;94(8):3085-8. https://doi.org/10.1210/jc.2008-

496 1833.

22
497 [20] Hart R, Greaves DR. Chemerin contributes to inflammation by promoting macrophage

498 adhesion to VCAM-1 and fibronectin through clustering of VLA-4 and VLA-5. J Immunol.

499 2010;185(6):3728-39. https://doi.org/10.4049/jimmunol.0902154.

500 [21] Wittamer V, Bondue B, Guillabert A, Vassart G, Parmentier M, Communi D. Neutrophil-

501 mediated maturation of chemerin: a link between innate and adaptive immunity. J

502 Immunol. 2005;175(1):487-93. https://doi.org/10.4049/jimmunol.175.1.487.

503 [22] Zabel BA, Allen SJ, Kulig P, Allen JA, Cichy J, Handel TM, et al. Chemerin activation by

of
504 serine proteases of the coagulation, fibrinolytic, and inflammatory cascades. J Biol Chem.

ro
505 2005;280(41):34661-6. https://doi.org/10.1074/jbc.M504868200.

506 [23]
-p
Peng L, Yu Y, Liu J, Li S, He H, Cheng N, et al. The chemerin receptor CMKLR1 is a
re
507 functional receptor for amyloid-β peptide. J Alzheimers Dis. 2015;43(1):227-42.
lP

508 https://doi.org/10.3233/JAD-141227.
na

509 [24] Rourke JL, Muruganandan S, Dranse HJ, McMullen NM, Sinal CJ. Gpr1 is an active

510 chemerin receptor influencing glucose homeostasis in obese mice. J Endocrinol.

ur

511 2014;222(2):201-15. https://doi.org/10.1530/JOE-14-0069.

Jo

512 [25] Yang YL, Ren LR, Sun LF, Huang C, Xiao TX, Wang BB, et al. The role of GPR1

513 signaling in mice corpus luteum. J Endocrinol. 2016;230(1):55-65.

514 https://doi.org/10.1530/JOE-15-0521.

515 [26] Rourke JL, Dranse HJ, Sinal CJ. CMKLR1 and GPR1 mediate chemerin signaling through

516 the RhoA/ROCK pathway. Mol Cell Endocrinol. 2015;417:36-51.

517 https://doi.org/10.1016/j.mce.2015.09.002.

518 [27] Gudelska M, Dobrzyn K, Kiezun M, Rytelewska E, Kisielewska K, Kaminska B, et al. The

519 expression of chemerin and its receptors (CMKLR1, GPR1, CCRL2) in the porcine uterus

23
520 during the oestrous cycle and early pregnancy and in trophoblasts and conceptuses.

521 Animal. 2020;14(10):2116-28. https://doi.org/10.1017/S175173112000097X.

522 [28] Akins EL, Morrissette MC. Gross ovarian changes during estrous cycle of swine. Am J Vet

523 Res. 1968;29(10):1953-7.

524 [29] Anderson LL. Growth, protein content and distribution of early pig embryos. Anat Rec.

525 1978;190(1):143-53. https://doi.org/10.1002/ar.1091900112.

526 [30] Smolinska N, Dobrzyn K, Kiezun M, Szeszko K, Maleszka A, Kaminski T. Effect of

of
527 adiponectin on the steroidogenic acute regulatory protein, P450 side chain cleavage

ro
528 enzyme and 3β-hydroxysteroid dehydrogenase gene expression, progesterone and

529
-p
androstenedione production by the porcine uterus during early pregnancy. J Physiol
re
530 Pharmacol. 2016;67(3):443-56.
lP

531 [31] Blitek A, Kiewisz J, Waclawik A, Kaczmarek MM, Ziecik AJ. Effect of steroids on
na

532 HOXA10 mRNA and protein expression and prostaglandin production in the porcine

533 endometrium. J Reprod Dev. 2010;56(6):643-8. http://doi.org/10.1262/jrd.10-046k.

ur

534 [32] Dobrzyn K, Smolinska N, Szeszko K, Kiezun M, Maleszka A, Rytelewska E, et al. Effect
Jo

535 of progesterone on adiponectin system in the porcine uterus during early pregnancy. J

536 Anim Sci. 2017;95(1):338-52. https://doi.org/10.2527/jas.2016.0732.

537 [33] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time

538 quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8.

539 https://doi.org/10.1006/meth.2001.1262.

540 [34] Rytelewska E, Kiezun M, Kisielewska K, Gudelska M, Dobrzyn K, Kaminska B, et al.

541 Chemerin as a modulator of ovarian steroidogenesis in pigs: an in vitro study.

542 Theriogenology. 2021;160:95-101. https://doi.org/10.1016/j.theriogenology.2020.10.040.

24
543 [35] Rytelewska E, Kiezun M, Zaobidna E, Gudelska M, Kisielewska K, Dobrzyn K, et al.

544 Chemerin as a modulator of angiogenesis and apoptosis processes in the corpus luteum of

545 pigs: an in vitro study†. Biol Reprod. 2021;105(4):1002-1015.

546 https://doi.org/10.1093/biolre/ioab126.

547 [36] Jin CH, Yi KW, Ha YR, Shin JH, Park HT, Kim T, et al. Chemerin Expression in the

548 Peritoneal Fluid, Serum, and Ovarian Endometrioma of Women with Endometriosis. Am

549 J Reprod Immunol. 2015;74(4):379-86. https://doi.org/10.1111/aji.12405.

of
550 [37] Carlino C, Trotta E, Stabile H, Morrone S, Bulla R, Soriani A, et al. Chemerin regulates

ro
551 NK cell accumulation and endothelial cell morphogenesis in the decidua during early

552 pregnancy. J Clin

-p
Endocrinol Metab. 2012;97(10):3603-12.
re
553 https://doi.org/10.1210/jc.2012-1102.
lP

554 [38] Dobrzyn K, Kiezun M, Zaobidna E, Kisielewska K, Rytelewska E, Gudelska M, et al. The
na

555 In Vitro Effect of Prostaglandin E2 and F2α on the Chemerin System in the Porcine

556 Endometrium during Gestation. Int J Mol Sci. 2020;21(15):5213.

ur

557 https://doi.org/10.3390/ijms21155213.
Jo

558 [39] Wang Q, Kim JY, Xue K, Liu JY, Leader A, Tsang BK. Chemerin, a novel regulator of

559 follicular steroidogenesis and its potential involvement in polycystic ovarian syndrome.

560 Endocrinology. 2012;153(11):5600-11. https://doi.org/10.1210/en.2012-1424.

561 [40] Luque-Ramírez M, Martínez-García MÁ, Montes-Nieto R, Fernández-Durán E, Insenser

562 M, Alpañés M, et al. Sexual dimorphism in adipose tissue function as evidenced by

563 circulating adipokine concentrations in the fasting state and after an oral glucose challenge.

564 Hum Reprod. 2013;28(7):1908-18. https://doi.org/10.1093/humrep/det097.

25
565 [41] de Sousa Abreu R, Penalva LO, Marcotte EM, Vogel C. Global signatures of protein and

566 mRNA expression levels. Mol Biosyst. 2009;5(12):1512-26.

567 https://doi.org/10.1039/B908315D.

568 [42] Gry M, Rimini R, Strömberg S, Asplund A, Pontén F, Uhlén M, et al. Correlations between

569 RNA and protein expression profiles in 23 human cell lines. BMC Genomics. 2009;10:365.

570 https://doi.org/10.1186/1471-2164-10-365.

571 [43] Wei LL, Norris BM, Baker CJ. An N-terminally truncated third progesterone receptor

of
572 protein, PR(C), forms heterodimers with PR(B) but interferes in PR(B)-DNA binding. J

ro
573 Steroid Biochem Mol Biol. 1997;62(4):287-97. https://doi.org/10.1016/s0960-

574 0760(97)00044-7.
-p
re
575 [44] Mulac-Jericevic B, Conneely OM. Reproductive tissue selective actions of progesterone
lP

576 receptors. Reproduction. 2004;128(2):139-46. https://doi.org/10.1530/rep.1.00189.

na

577 [45] Wetendorf M, DeMayo FJ. Progesterone receptor signaling in the initiation of pregnancy

578 and preservation of a healthy uterus. Int J Dev Biol. 2014;58(2-4):95-106.

ur

579 https://doi.org/10.1387/ijdb.140069mw.
Jo

580 [46] Mulac-Jericevic B, Lydon JP, DeMayo FJ, Conneely OM. Defective mammary gland

581 morphogenesis in mice lacking the progesterone receptor B isoform. Proc Natl Acad Sci U

582 S A. 2003;100(17):9744-9. https://doi.org/10.1073/pnas.1732707100.

583 [47] Geisert RD, Pratt TN, Bazer FW, Mayes JS, Watson GH. Immunocytochemical

584 localization and changes in endometrial progestin receptor protein during the porcine

585 oestrous cycle and early pregnancy. Reprod Fertil Dev. 1994;6(6):749-60.

586 https://doi.org/10.1071/RD9940749.

26
587 [48] Steinhauser CB, Bazer FW, Burghardt RC, Johnson GA. Expression of progesterone

588 receptor in the porcine uterus and placenta throughout gestation: correlation with

589 expression of uteroferrin and osteopontin. Domest Anim Endocrinol. 2017;58:19-29.

590 https://doi.org/10.1016/j.domaniend.2016.07.002.

591 [49] Yang H, Li F, Kong X, Yuan X, Wang W, Huang R, et al. Chemerin regulates proliferation

592 and differentiation of myoblast cells via ERK1/2 and mTOR signaling pathways. Cytokine.

593 2012;60(3):646-52. https://doi.org/10.1016/j.cyto.2012.07.033.

of
594 [50] Rodríguez-Penas D, Feijóo-Bandín S, García-Rúa V, Mosquera-Leal A, Durán D, Varela

ro
595 A, et al. The Adipokine Chemerin Induces Apoptosis in Cardiomyocytes. Cell Physiol

596
-p
Biochem. 2015;37(1):176-92. https://doi.org/10.1159/000430343.
re
597 [51] Sukjumlong S, Persson E, Dalin AM, Janson V, Sahlin L. Messenger RNA levels of
lP

598 estrogen receptors alpha and beta and progesterone receptors in the cyclic and
na

599 inseminated/early pregnant sow uterus. Anim Reprod Sci. 2009;112(3-4):215-28.

600 https://doi.org/10.1016/j.anireprosci.2008.04.019.
ur

601 [52] Srisuwatanasagul S. Steroid receptor and their roles in pig uterus. Thai J Vet Med.
Jo

602 2011;41(1):43–9.

603 [53] Geisert RD, Brenner RM, Moffatt RJ, Harney JP, Yellin T, Bazer FW. Changes in

604 oestrogen receptor protein, mRNA expression and localization in the endometrium of

605 cyclic and pregnant gilts. Reprod Fertil Dev. 1993;5(3):247-60.

606 https://doi.org/10.1071/RD9930247.

607 [54] Wilkenfeld SR, Lin C, Frigo DE. Communication between genomic and non-genomic

608 signaling events coordinate steroid hormone actions. Steroids. 2018;133:2-7.

609 https://doi.org/10.1016/j.steroids.2017.11.005.

27
610 [55] Sukjumlong S, Dalin AM, Sahlin L, Persson E. Immunohistochemical studies on the

611 progesterone receptor (PR) in the sow uterus during the oestrous cycle and in inseminated

612 sows at oestrus and early pregnancy. Reproduction. 2005;129(3):349-59.

613 https://doi.org/10.1530/rep.1.00514.

614 [56] Sukjumlong S, Kaeoket K, Dalin AM, Persson E. Immunohistochemical studies on

615 oestrogen receptor alpha (ER alpha) and the proliferative marker Ki-67 in the sow uterus

616 at different stages of the oestrous cycle. Reprod Domest Anim. 2003;38(1):5-12.

of
617 https://doi.org/10.1046/j.1439-0531.2003.00383.x.

ro
618 [57] Franczak A, Kotwica G. Secretion of estradiol-17beta by porcine endometrium and

619
-p
myometrium during early pregnancy and luteolysis. Theriogenology. 2008;69(3):283-9.
re
620 https://doi.org/10.1016/j.theriogenology.2007.09.023.
lP

621 [58] Miller WL, Auchus RJ. The molecular biology, biochemistry, and physiology of human
na

622 steroidogenesis and its disorders. Endocr Rev. 2011;32(1):81-151.

623 https://doi.org/10.1210/er.2010-0013.
ur

624 [59] Wojciechowicz B, Kotwica G, Zglejc K, Waszkiewicz E, Franczak A. Expression of 17β-

Jo

625 hydroxysteroid dehydrogenase and the effects of LH, FSH and prolactin on oestrone and

626 17β-oestradiol secretion in the endometrium of pigs during early pregnancy and the

627 oestrous cycle. Reprod. Fertil. Dev. 2017;29(5),975–84. https://doi.org/10.1071/RD15430

628 [60] Gudelska M, Dobrzyn K, Kiezun M, Kisielewska K, Rytelewska E, Kaminski T, et al.

629 Chemerin Affects P4 and E2 Synthesis in the Porcine Endometrium during Early

630 Pregnancy. Int J Mol Sci. 2022;23(2):945. https://doi.org/10.3390/ijms23020945.

631 [61] Geisert R, Schmitt R. Early embryonic survival in the pig: Can it be improved? J. Anim.

632 Sci. 2002;80:E54–E65.

28
633 [62] Geisert RD, Zavy MT, Moffatt RJ, Blair RM, Yellin T. Embryonic steroids and the

634 establishment of pregnancy in pigs. J Reprod Fertil Suppl. 1990;40:293-305.

635 [63] Szekeres-Bartho J, Kilaŕ F, Falkay G, Csernus V, Török A, Pacsa AS. The mechanism of

636 the inhibitory effect of progesterone on lymphocyte cytotoxicity: I. Progesterone-treated

637 lymphocytes release a substance inhibiting cytotoxicity and prostaglandin synthesis. Am J

638 Reprod Immunol Microbiol. 1985;9(1):15-8. https://doi.org/10.1111/j.1600-

639 0897.1985.tb00334.x.

of
640 [64] Geisert RD, Thatcher WW, Renegar RH. Relationship between porcine blastocyst

ro
641 development and uterine secretions. Biol. Reprod. 1981;24(1):p.122A.

642 [65]
-p
Ernst MC, Sinal CJ. Chemerin: at the crossroads of inflammation and obesity. Trends
re
643 Endocrinol Metab. 2010;21(11):660-7. https://doi.org/10.1016/j.tem.2010.08.001.
lP

644 [66] Lord E, Ledoux S, Murphy BD, Beaudry D, Palin MF. Expression of adiponectin and its
na

645 receptors in swine. J. Anim. Sci. 2005;83(3):565-78.

646 https://doi.org/10.2527/2005.833565x.
ur

647 [67] Spagnuolo-Weaver M, Fuerst R, Campbell ST, Meehan BM, McNeilly F, Adair B, et al.
Jo

648 A fluorimeter-based RT-PCR method for the detection and quantitation of porcine

649 cytokines. Journal of immunological methods, 1999;230(1-2),19–27.

650 https://doi.org/10.1016/s0022-1759(99)00114-3.

651

652

653

29
654 Figure legends

655 Figure 1. The impact of oestradiol (E2) on retinoic acid responder 2 gene (RARRES2)

656 expression and chemerin secretion in the porcine endometrium during the early pregnancy

657 and mid-luteal phase of the oestrous cycle. The effect of E2 (1, 10 and 100 nM) on the relative

658 RARRES2 gene expression (A, C, E, G, I) and chemerin secretion (B, D, F, H, J) in the in vitro

659 incubated endometrial explants on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and

660 10 to 11 of the oestrous cycle. Quantitative real-time PCR was used to determine mRNA contents

of
661 (n=5, each sample in duplicate), whereas ELISA test was used to designate protein concentrations

ro
662 (n=5, each sample in duplicate). Results were delineated as means ± SEM (n=5). Bars with

663
-p
different subscripts indicate statistically significant differences (p<0.05).
re
lP

664 Figure 2. The impact of oestradiol (E2) on chemokine like receptor 1 (CMKLR1) gene and

665 protein expression in the porcine endometrium during the early pregnancy and mid-luteal
na

666 phase of the oestrous cycle. The effect of E2 (1, 10 and 100 nM) on the relative CMKLR1 gene
ur

667 (A, C, E, G, I) and protein (B, D, F, H, J) expression in the in vitro incubated endometrial explants
Jo

668 on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and 10 to 11 of the oestrous cycle.

669 Quantitative real-time PCR was used to determine mRNA contents (n=5, each sample in

670 duplicate), whereas Western blot was used to designate protein abundance (n=5). Upper panels:

671 representative immunoblots, lower panels: densitometric analysis of CMKLR1 protein relative to

672 actin protein. Results were delineated as means ± SEM (n=5). Bars with different subscripts

673 indicate statistically significant differences (p<0.05).

674 Figure 3. The impact of oestradiol (E2) on G protein-coupled receptor 1 (GPR1) gene and

675 protein expression in the porcine endometrium during the early pregnancy and mid-luteal

30
676 phase of the oestrous cycle. The effect of E2 (1, 10 and 100 nM) on the relative GPR1 gene (A,

677 C, E, G, I) and protein (B, D, F, H, J) expression in the in vitro incubated endometrial explants on

678 days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and 10 to 11 of the oestrous cycle.

679 Quantitative real-time PCR was used to determine mRNA contents (n=5, each sample in

680 duplicate), whereas Western blot was used to designate protein abundance (n=5). Upper panels:

681 representative immunoblots, lower panels: densitometric analysis of GPR1 protein relative to actin

682 protein. Results were delineated as means ± SEM (n=5). Bars with different subscripts indicate

of
683 statistically significant differences (p<0.05).

ro
684 Figure 4. The impact of oestradiol (E2) on C-C chemokine receptor-like 2 (CCRL2) gene and

685
-p
protein expression in the porcine endometrium during the early pregnancy and mid-luteal
re
686 phase of the oestrous cycle. The effect of E2 (1, 10 and 100 nM) on the relative CCRL2 gene (A,
lP

687 C, E, G, I) and protein (B, D, F, H, J) expression in the in vitro incubated endometrial explants on
na

688 days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and 10 to 11 of the oestrous cycle.
ur

689 Quantitative real-time PCR was used to determine mRNA contents (n=5, each sample in
Jo

690 duplicate), whereas Western blot was used to designate protein abundance (n=5). Upper panels:

691 representative immunoblots, lower panels: densitometric analysis of CCRL2 protein relative to

692 actin protein. Results were delineated as means ± SEM (n=5). Bars with different subscripts

693 indicate statistically significant differences (p<0.05).

694 Figure 5. The impact of progesterone (P4) on retinoic acid responder 2 gene (RARRES2)

695 expression and chemerin secretion in the porcine endometrium during the early pregnancy

696 and mid-luteal phase of the oestrous cycle. The effect of P4 (10, 100 and 1000 nM) on the relative

697 RARRES2 gene expression (A, C, E, G, I) and chemerin secretion (B, D, F, H, J) in the in vitro

698 incubated endometrial explants on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and

31
699 10 to 11 of the oestrous cycle. Quantitative real-time PCR was used to determine mRNA contents

700 (n=5, each sample in duplicate), whereas ELISA test was used to designate protein concentrations

701 (n=5, each sample in duplicate). Results were delineated as means ± SEM (n=5). Bars with

702 different subscripts indicate statistically significant differences (p<0.05).

703 Figure 6. The impact of progesterone (P4) on chemokine like receptor 1 (CMKLR1) gene and

704 protein expression in the porcine endometrium during the early pregnancy and mid-luteal

705 phase of the oestrous cycle. The effect of P4 (10, 100 and 1000 nM) on the relative CMKLR1

of
706 gene (A, C, E, G, I) and protein (B, D, F, H, J) expression in the in vitro incubated endometrial

ro
707 explants on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and 10 to 11 of the oestrous

708
-p
cycle. Quantitative real-time PCR was used to determine mRNA contents (n=5, each sample in
re
709 duplicate), whereas Western blot was used to designate protein abundance (n=5). Upper panels:
lP

710 representative immunoblots, lower panels: densitometric analysis of CMKLR1 protein relative to
na

711 actin protein. Results were delineated as means ± SEM (n=5). Bars with different subscripts
ur

712 indicate statistically significant differences (p<0.05).

Jo

713 Figure 7. The impact of progesterone (P4) on G protein-coupled receptor 1 (GPR1) gene and

714 protein expression in the porcine endometrium during the early pregnancy and mid-luteal

715 phase of the oestrous cycle. The effect of P4 (10, 100 and 1000 nM) on the relative GPR1 gene

716 (A, C, E, G, I) and protein (B, D, F, H, J) expression in the in vitro incubated endometrial explants

717 on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and 10 to 11 of the oestrous cycle.

718 Quantitative real-time PCR was used to determine mRNA contents (n=5, each sample in

719 duplicate), whereas Western blot was used to designate protein abundance (n=5). Upper panels:

720 representative immunoblots, lower panels: densitometric analysis of GPR1 protein relative to actin

32
721 protein. Results were delineated as means ± SEM (n=5). Bars with different subscripts indicate

722 statistically significant differences (p<0.05).

723 Figure 8. The impact of progesterone (P4) on C-C chemokine receptor-like 2 (CCRL2) gene

724 and protein expression in the porcine endometrium during the early pregnancy and mid-

725 luteal phase of the oestrous cycle. The effect of P4 (10, 100 and 1000 nM) on the relative CCRL2

726 gene (A, C, E, G, I) and protein (B, D, F, H, J) expression in the in vitro incubated endometrial

727 explants on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy and 10 to 11 of the oestrous

of
728 cycle. Quantitative real-time PCR was used to determine mRNA contents (n=5, each sample in

ro
729 duplicate), whereas Western blot was used to designate protein abundance (n=5). Upper panels:

730
-p
representative immunoblots, lower panels: densitometric analysis of CCRL2 protein relative to
re
731 actin protein. Results were delineated as means ± SEM (n=5). Bars with different subscripts
lP

732 indicate statistically significant differences (p<0.05).

na

733
ur
Jo

33
737 Table 1 Characteristics of primers and real-time PCR reaction conditions used in the study
Gene Primers sequences Accession Primer Reference
Reaction Conditions
number (nM)
Activation: 95˚C, 10 min
F: 5’-TGGAGGAGTTCCACAAGCAC-3’ 500
40 cycles of:
RARRES2 EU660865 1. Denaturation: 95˚C, 15 s [13]
R: 5’-GCTTTCTTCCAGTCCCTCTTC-3’ 500 2. Annealing: 60˚C, 1 min
3. Extension: 72˚C, 1 min
Activation: 95˚C, 10 min
F: 5’-GAGCAGCAGCTACTTACTTCC-3’ 200
40 cycles of:

f
NM_001001
1. Denaturation: 95˚C, 15 s

oo
CCRL2 [13]
617.1
R: 5’-CTGCCCACTGACCGAGTTC-3’ 200 2. Annealing: 60˚C, 1 min
3. Extension: 72˚C, 1 min

r
-p
Activation: 95˚C, 10 min
F: 5’-GGACTACCACTGGGTGTTCG-3’ 200
40 cycles of:

re
CMKLR1 EU660866 1. Denaturation: 95˚C, 15 s [13]
R: 5’-GCCATGTAAGCCAGTCGGA-3’ 2. Annealing: 60˚C, 1 min

lP
200
3. Extension: 72˚C, 1 min
Activation: 95˚C, 10 min

na
F: 5'- ACCGACTTGGAGGAGAAAGC -3' 200
40 cycles of:
GPR1 FJ234899.1 1. Denaturation: 95˚C, 15 s [13]
ur
R: 5'- ATTGAGGAACCAGAGCGTGG -3' 200 2. Annealing: 60˚C, 1 min
3. Extension: 72˚C, 1 min
Jo

Activation: 50˚C, 2 min; 95˚C,

F: 5'-GCACTGGTGGCAAGTCCAT-3' 300
10 min
PPIA AY266299 40 cycles of: [66]
R: 5'-AGGACCCGTATGCTTCAGGA-3' 300 1. Denaturation: 95˚C, 15 s
2. Annealing: 60˚C, 1 min
Activation: 95˚C, 10 min
F: 5'-ACATCAAGGAGAAGCTCTGCTACG-3' 500
40 cycles of:
ACTB U07786 1. Denaturation: 95˚C, 15 s [67]
R: 5'-GAGGGGCGATGATCTTGATCTTCA-3' 500 2. Annealing: 61˚C, 1 min
3. Extension: 72˚C, 1 min
738 RARRES2: chemerin; CCRL2: C-C motif chemokine receptor like 2; CMKLR1: chemokine-like receptor 1; GPR1: G protein-coupled receptor 1;
739 PPIA: cyclophilin A; ACTB: β-actin; F: forward; R: reverse

34
740 Table 2 Summary of the steroid hormones effect on the chemerin system expression
741 in the porcine endometrium during early pregnancy and mid-luteal phase of the oestrous cycle.
742 The summary of oestradiol (E2) and progesterone (P4) effect on chemerin gene expression and
743 protein release, and chemerin receptors (chemokine-like receptor 1 – CMKLR1,
744 G protein-coupled receptor 1 – GPR1, C-C motif chemokine receptor like 2 -CCRL2) gene and
745 protein expression.

E2 P4
DAYS (PHASE) 1 10 100 10 100 1000
nM nM nM nM nM nM
GENE ↑ ↑ − ↑ − ↑
CHEMERIN
− − − − − −
(the transuterine migration

PROTEIN

of
GENE ↑ − − − ↓ ↓
of embryos)

CMKLR1
10 to 11

↓ − − ↑ ↓ −

ro
PROTEIN
GENE ↑ − − ↑ − −
GPR1
PROTEIN
-p↓ − − − − ↑
re
GENE ↓ ↓ ↓ ↑ − −
CCRL2
lP

PROTEIN ↓ − − ↑ − −
GENE ↑ ↑ − ↑ − ↑
na
EARLY PREGNANCY

CHEMERIN
(the maternal recognition of

PROTEIN ↑ − − − − −
ur

GENE ↑ ↑ ↑ ↑ ↑ ↑
CMKLR1
pregnancy)

Jo
12 to 13

PROTEIN ↓ ↓ ↓ ↓ ↓ ↓
GENE ↓ − ↓ ↓ ↓ ↓
GPR1
PROTEIN − − ↑ − − −
GENE ↓ ↓ ↓ ↓ ↓ ↓
CCRL2
PROTEIN − ↑ − ↓ − −
GENE − − − − − ↑
CHEMERIN
(the beginning of

PROTEIN − ↓ ↓ − − −
implantation)
15 to 16

GENE ↑ − ↓ − − ↑
CMKLR1
PROTEIN − ↓ − ↓ ↓ ↓
GENE − ↑ − − − ↑
GPR1
PROTEIN ↑ − ↑ − − ↑

35
 GENE ↓ ↓ ↓ ↓ ↓ ↓
CCRL2
PROTEIN − ↑ ↑ − ↑ ↑
GENE ↓ ↓ ↓ ↓ ↓ ↓
CHEMERIN
(the end of implantation) PROTEIN − − − − − −
GENE ↓ − ↓ ↓ − ↓
CMKLR1
27 to 28

PROTEIN − − ↑ ↑ − −
GENE ↓ ↓ ↓ ↓ ↓ ↓
GPR1
PROTEIN − ↓ − − ↓ ↓

of
GENE − ↓ ↓ − − ↑
CCRL2

ro
PROTEIN − ↑ ↑ ↑ − −
GENE − − − ↓ − −
CHEMERIN
-p
THE OESTROUS CYCLE

PROTEIN − − − − − −
re
(the mid-luteal phase)

GENE ↑ ↑ ↑ ↑ ↑ ↑
lP

CMKLR1
10 to 11

PROTEIN ↓ ↓ ↓ − − −
GENE − − − ↑ − −
na

GPR1
PROTEIN − − ↑ − − −
ur

GENE − − ↑ − − −
CCRL2
Jo

PROTEIN ↓ ↓ − − ↓ −
746 ↑: stimulatory effect; ↓: inhibitory effect; −: no effect; E2: oestradiol; P4: progesterone;
747 CMKLR1: chemokine-like receptor 1; GPR1: G protein-coupled receptor 1; CCRL2: C-C motif chemokine
748 receptor like 2.

749

36
750 Table 3 The main factors and the interactions affecting CMKLR1, GPR1 and CCRL2 protein abundance in the endometrial tissue
751 explants under the influence of E2 and P4

CMKLR1 GPR1 CCRL2

F P F P F P

10 TO 11 F3,16 = 5.09 0.011583 F3,16 = 5.12 0.011329 F3,16 = 5.19 0.010785

PREGNANCY
DAYS OF

f
12 TO 12 F3,16 = 9.92 0.000619 F3,16 = 2.88 0.068395 F3,16 = 5.09 0.011528

r oo
E2 15 TO 16 F3,16 = 2.97 0.063306 F3,16 = 7.56 0.002277 F3,16 = 63.64 0.000000

-p
re
27 TO 28 F3,16 = 12.99 0.000148 F3,16 = 2.66 0.083409 F3,16 = 15.46 0.000055

lP
DAYS 10 TO 11 OF THE
F3,16 = 36.90 0.000000 F3,16 = 6.15 0.005549 F3,16 = 4.97 0.012632

na
OESTROUS CYCLE

10 TO 11
ur
F3,16 = 19.73 0.000013 F3,16 = 2.68 0.081540 F3,16 = 3.96 0.027430
PREGNANCY

Jo
DAYS OF

12 TO 12 F3,16 = 12.71 0.000167 F3,16 = 0.33 0.806279 F3,16 = 2.43 0.102912

P4 15 TO 16 F3,16 = 97.87 0.000000 F3,16 = 10.44 0.000476 F3,16 = 8.69 0.001184

27 TO 28 F3,16 = 14.46 0.000081 F3,16 = 8.90 0.001057 F3,16 = 31.79 0.000001

DAYS 10 TO 11 OF THE
F3,16 = 4.27 0.021502 F3,16 = 9.16 0.000921 F3,16 = 2.86 0.069871
OESTROUS CYCLE
752 E2: oestradiol; P4: progesterone; CMKLR1: chemokine-like receptor 1; GPR1: G protein-coupled receptor 1; CCRL2: C-C motif
753 chemokine receptor like 2

37
 754
38
Jo
ur
na
lP
re
-p
ro
of
 755
39
Jo
ur
na
lP
re
-p
ro
of
 756
40
Jo
ur
na
lP
re
-p
ro
of
 757
41
Jo
ur
na
lP
re
-p
ro
of
 758
42
Jo
ur
na
lP
re
-p
ro
of
 759
43
Jo
ur
na
lP
re
-p
ro
of
 760
44
Jo
ur
na
lP
re
-p
ro
of
 761
45
Jo
ur
na
lP
re
-p
ro
of
1 • E2 modulate chemerin secretion by the porcine endometrium.

2 • CMKLR1, GPR1 and CCRL2 expression change under the influence of E2 and P4.

3 • The effect of E2 and P4 depends on the hormonal status of animals.

of
ro
-p
re
lP
na
ur
Jo