Abstract

   Chemerin (CHEM) is a hormone mainly expressed in adipocytes involved in
   the regulation of energy homeostasis and inflammatory response. CHEM
   expression has been demonstrated in the structures of the porcine
   hypothalamic-pituitary-gonadal axis, as well as in the uterus,
   trophoblasts and conceptuses of pigs. In this study, we performed
   high-throughput proteomic analyses (liquid chromatography with tandem
   mass spectrometry, LC-MS/MS) to examine the influence of CHEM (400
   ng/mL) on differentially regulated proteins (DRPs) in the porcine
   endometrial tissue explants during implantation (15 to 16 days of
   gestation). Among all 352 DRPs, 164 were up-regulated and 188 were
   down-regulated in CHEM-treated group. DRPs were assigned to 47 gene
   ontology (GO) terms (p-adjusted < 0.05). Validation of four DRPs
   (IFIT5, TGFβ1, ACO1 and PGRMC1) by Western blot analysis confirmed the
   veracity and accuracy of the LC-MS/MS method used in the present study.
   We suggest that CHEM, by modulating various protein expressions, takes
   part in the endometrial cell proliferation, migration and invasion at
   the time of implantation. It also regulates the endometrial immune
   response, sensitivity to P4 and the formation of new blood vessels.
   Additionally, CHEM appears to be an important factor involved in
   endothelial cell dysfunction during the pathogenesis of preeclampsia.
   The identification of a large number of DRPs under the influence of
   CHEM provides a valuable resource for understanding the molecular
   mechanisms of this hormone action during implantation, which is a
   prerequisite for better control of pig reproduction.

   Keywords: chemerin, pig, implantation, uterus, proteome, early
   pregnancy, DRPs

1. Introduction

   Secretory products from maternal uterine epithelia and precise
   cell-specific molecular signaling between embryos and the endometrium
   are critical to conceptus growth and development, pregnancy recognition
   signaling and implantation [[36]1]. Regulation of the uterine
   receptivity and conceptus adhesion to the endometrium is critical for
   placentation. Alternations of these signaling cascades can lead to an
   abnormal exchange of nutrients and pregnancy failure. A comprehensive
   understanding of the molecular mechanisms governing conceptus
   implantation is critical for the improvement of the fertility and
   reproductive health of pigs [[37]2,[38]3]. The roles of various
   feto-maternal signals, such as progesterone, estrogens, prostaglandins,
   interleukins, interferons and growth factors have been demonstrated to
   be involved in the implantation process [[39]4]. In addition to the
   above molecules, there are many more unique proteins within the uterine
   environment of pigs that take part in the establishment of pregnancy.
   For example, studies indicated that thyroid hormones,
   thyroid-stimulating hormone receptor and thyroid hormone receptors (α1,
   α2, and β1) are present in the endometrium and influence implantation
   taking part in crosstalk between the competent blastocyst and the
   receptive endometrium [[40]5,[41]6]. Plenty of studies have shown that
   the hormones produced by adipose tissue, like leptin [[42]7] and
   chemerin [[43]8], also take part in the control of reproduction.

   Chemerin (CHEM) is a hormone mainly expressed in adipocytes [[44]9].
   CHEM exerts its physiological functions through binding to three G
   protein-coupled receptors: chemokine like receptor 1 (CMKLR1), G
   protein-coupled receptor 1 (GPR1) and CC motif chemokine receptor-like
   2 (CCRL2) [[45]10]. It plays a pleiotropic role as a cytokine and
   adipokine (a hormone produced by the adipose tissue). CHEM regulates
   inflammatory response and energy homeostasis [[46]11]. Recent studies
   indicated that CHEM also takes part in the control of reproduction by
   the modulation of, among others, uterine and ovarian steroidogenesis
   during both the early pregnancy and the estrous cycle
   [[47]8,[48]12,[49]13,[50]14]. CHEM expression has been demonstrated in
   the structures of the porcine hypothalamic-pituitary-gonadal axis, as
   well as in the uterus, trophoblasts and conceptuses of pigs
   [[51]15,[52]16,[53]17,[54]18,[55]19].

   CHEM, as a cytokine and adipokine, is a very interesting research
   object and can play an essential role in the implantation process.
   Gudelska et al. [[56]19] confirmed that the implantation/embryo
   attachment was the stage of the highest CHEM protein expression in the
   porcine endometrium, focusing our attention on the role of CHEM in this
   process. Moreover, Yang et al. [[57]20] reported that the expression of
   CHEM in the human decidua correlates with early spontaneous abortion.
   The above reports suggest that CHEM may be an important factor in the
   maintenance of early pregnancy. However, there is a lack of information
   on the exact roles that CHEM may play during implantation.

   Recent studies have shown molecular changes in the porcine endometrium
   during the peri-implantation period and revealed sixteen differentially
   expressed proteins involved in the recognition and establishment of
   early gestation in pigs [[58]21]. It has been hypothesized that CHEM
   may affect the proteome of the porcine uterus during implantation, and
   consequently influence the production of many factors essential for the
   proper course of gestation. Accordingly, in this work, using the tandem
   mass tag (TMT)-based quantitative proteomics method based on
   TMT-isobaric mass tag labeling and liquid chromatography with tandem
   mass spectrometry (LC–MS/MS) analysis, we present for the first time
   the proteome profile of the porcine endometrial explants affected by
   CHEM during the peri-implantation period (days 15 to 16 of gestation).

2. Materials and Methods

2.1. Collection of Samples

   According to the Polish (Journal of Laws, 2015; item 266) and European
   (Directive 2010/63/Eu) regulations on the protection of animals used
   for scientific or educational purposes, the experiments did not require
   the consent of the competent ethics committee for animal experiments.
   The present study was conducted on five female crossbreed pigs (Large
   White × Polish Landrace, seven to eight months of age and 120 to 130 kg
   in weight), acquired from a private breeding farm. The animals were on
   days 15 to 16 of pregnancy (the beginning of implantation). Females
   were verified for estrus behavior in the presence of a boar daily.
   Insemination, by natural mating, was conducted on days one to two of
   the estrous cycle. The day after coition was indicated as the first day
   of pregnancy. Uteri were gathered immediately after slaughter, placed
   in ice-cold sterile phosphate-buffered saline (PBS), supplemented with
   100 IU/mL penicillin, 100 μg/mL streptomycin and carried to the
   laboratory on ice.

2.2. Tissue Cultures

   Uterine tissue cultures were performed as described by Smolinska et al.
   [[59]22]. The explants were preincubated for 2 h at a rocking platform
   in a water bath at 37 °C (95% O[2] and 5% CO[2]) and then treated for
   24 h with human recombinant CHEM (cat. no. 268e10032; RayBiotech Life,
   Peachtree Corners, GA, USA) at the concentration of 400 ng/mL. Tissue
   explants incubated with medium alone were used as controls. The dose of
   CHEM was selected based on ULFs CHEM concentrations indicated in our
   previous study [[60]19]. All cultures were performed in duplicates, in
   five independent experiments (n = 5). After incubation, the endometrial
   slices were frozen in liquid nitrogen and stored at −80 °C until the
   total protein extraction. The viability of tissue explants was
   monitored by quantifying lactate dehydrogenase (LDH) activity as we
   described in Dobrzyn et al. [[61]23]. The average activity of LDH in
   the cultured tissue explants after the treatment period was 128.7 ±
   35.6 U/L (0.67% of maximal release of LDH after the total endometrial
   cells destruction).

2.3. Protein Extraction

   Protein extraction was performed by homogenizing 15–20 mg of
   endometrial tissue in 150–200 μL of lysis buffer (30 mM TrisHCl pH 8, 8
   M urea and 2% CHAPS) on ice three times for 15 s using TissueRuptor
   (Qiagen, Hilden, Germany). Protein extracts were incubated on ice for
   30 min and then centrifuged (10,000× g for 5 min at 4 °C). The
   supernatants were quantified using a Bradford dye-binding procedure
   with 0.5 mg/mL BSA as a standard. All analyses were performed in
   duplicate. Probes were stored at −80 °C for further analysis.

2.4. Protein’s Digestion

   A proteomics analysis was performed by the Mass Spectrometry Laboratory
   at the Institute of Biochemistry and Biophysics of the Polish Academy
   of Sciences. 100 µg of each endometrial sample and pulled internal
   standard was diluted with 200 µL 8 M urea solution in a 200 mM
   triethylammonium bicarbonate buffer (TEAB, pH 8.5). Cysteine bridges
   were reduced by incubation with 20 mM tris(2-carboxyethyl)phosphine
   (TCEP) (1 h at 37 °C). Proteins were transferred into Vivacon 30 kDa
   molecular weight cut-off filter (Sartorius Stedim, Göttingen, Germany)
   and digested according to a filter-aided sample preparation (FASP)
   protocol with minor modifications. Samples were spun at 14,500× g for
   30 min and washed with 100 µL urea solution before cysteine blocking by
   15 min incubation with 50 mM s-methylmethanethiosulfonate (MMTS) at RT.
   Proteins were washed three times with 8 M urea buffer and 200 mM TEAB.
   After each addition, the samples were centrifuged until the cut-off
   filter was dry. Digestion was carried out using a trypsin/LysC mix
   (Promega, Madison, WI, USA) in 1:25 enzyme-to-protein ratio overnight
   at 37 °C. Peptides were eluted from spin filters by two washes with 200
   mM TEAB and one wash with 500 mM NaCl. Dried peptides were resuspended
   in 85 µL of 200 mM TEAB buffer and labelled with TMT10plex (Thermo
   Fisher Scientific, Waltham, MA, USA) tags in 41 µL acetonitrile for 1 h
   on vortex. Internal standards were labelled with TMT10-126 label
   reagent. The reaction was quenched by the addition of 8 µL 5%
   hydroxylamine. The labelling efficiency was checked, and combined
   samples for each TMT set were desalted using two 30 mg Oasis HLB
   columns (Waters, Milford, MA, USA). Briefly, cartridges were
   preconditioned with 1 mL methanol and 1 mL MS-grade water. After the
   sample was loaded and rinsed with 1 mL water, peptides were eluted from
   columns with 400 µL of 80% acetonitrile (ACN) and 0.1% formic acid
   (FA). Aliquots were dried and resuspended in 500 µL 10 mM ammonium
   hydroxide.

2.5. Reversed-Phase Peptide Fractionation at High pH

   TMT labelled peptides were fractionated using high-pH reverse-phase
   chromatography on an XBridge Peptide BEH C18 column (4.6 × 250 mm, 130
   Å, 5 µm; Waters, Milford, MA, USA). Separation was performed at 1
   mL/min flow rate for 27 min on a Waters Acquity UPLC H-class system.
   Mobile phases consisted of water (A), ACN (B) and 100 mM ammonium
   hydroxide solution (C). The percentage of phase C was kept at a
   constant 10% through the entire gradient. Fractions were collected
   every 1 min starting from the third minute of the run. The following
   gradient was applied: 3 to 5% solvent B for 0.5 min, 5 to 22% for 17
   min, 22 to 28% for 2 min, 28 to 45% for 1.5 min, 45 to 90% for 0.5 min,
   2.5 min isocratic hold at 90% and final column equilibration at 3%
   phase B for 3 min. The peptide elution profile was monitored at 214 nm
   by UV detector. Twenty-five fractions from each TMT set were dried in
   Speedvac and reconstituted in 100 uL 0.1% trifluoroacetic acid (TFA)
   and 2% acetonitrile before LC-MS/MS analysis.

2.6. Mass Spectrometry

   Fractions were analyzed using an LC-MS/MS system comprised of an
   ACQUITY UPLC M-Class System (Waters, Milford, MA, USA) directly coupled
   to a QExactive mass spectrometer (Thermo Fisher Scientific, Waltham,
   MA, USA). Peptides were trapped on a C18 pre-column (180 µm × 20 mm;
   Waters, Milford, MA, USA) using 0.1% FA in water as a mobile phase and
   transferred to a nanoAcquity BEH C18 column (75 µm × 250 mm, 1.7 µm;
   Waters, Milford, MA, USA) using ACN gradient (0–35% ACN in 160 min) in
   the presence of 0.1% FA at a flow rate of 250 nL/min. Data acquisition
   was carried out using a data-dependent method with the top 12
   precursors selected for MS2 analysis after collisional induced
   fragmentation (CID) with normalized collision energy (NCE) of 27. Full
   MS scans covering the mass range of 300–1600 m/z (mass-to-charge ratio)
   were acquired at a resolution of 70,000 with a maximum injection time
   of 60 ms and an AGC target value of 1e6. MS2 scans were acquired with a
   maximum injection time of 120 ms and an AGC target value of 5e5 with a
   resolution of 35,000. The isolation window was set to 1.2 m/z and a
   dynamic exclusion was set to 30 s.

2.7. Data Analysis

   Offline recalibration, as well as peptides and proteins identification,
   was performed in the MaxQuant/Andromeda software suite (version
   1.6.17.0) [[62]24] using a Sus scrofa full Uniprot database (versions
   2020_10). The search included tryptic and LysC-generated peptides, and
   Metylthio (C) was set as a fixed modification and oxidation (M) as a
   variable one. Reporter MS2 quantification was specified in order to
   obtain values for quantitative analysis. TMT 10plex correction factors
   were specified for each labelling set according to the manufacturer’s
   instructions. A reverse database was used for target/decoy statistical
   results validation. Protein groups along with quantitative data were
   further analyzed in Perseus (version 1.6.13) [[63]25]. Hits from the
   reversed database and proteins only identified by site and contaminants
   were removed. TMT reporter values were normalized with internal
   reference scaling (IRS) [[64]26]. Reporter intensities were log2
   transformed and proteins with less than 3 values in at least one
   biological group were filtered out. Missing values were replaced with
   data from normal distribution (width 0.3, down shift 1.8) separately
   for each column. The significance threshold for p-value resulting from
   a Student’s t-test was 0.05 and the fold-change cut-off was ±1.2.

2.8. Network and Functional Analysis

   To analyze the involvement of DRPs in the common biological processes,
   an enrichment ontology and pathway analysis was performed with the use
   of g:Profiler software (version e104_eg51_p15_3922dba) [[65]27] based
   on GO [[66]28], and KEGG [[67]29] databases (p-adjusted < 0.05).

2.9. Validation of LC-MS/MS Results by Western Blot

   In order to validate the LC-MS/MS results, we have performed a Western
   blot analysis for four chosen proteins (IFIT5, ACO1, TGFβ1, PGRMC1)
   selected from the list of proteins with the expression significantly
   changed under the influence of CHEM. The samples for Western blot
   analysis were homogenized on ice, with T-PER Tissue Protein Extraction
   Reagent (Thermo Fisher Scientific, Waltham, MA, USA) enriched with
   protease inhibitors (Sigma-Aldrich, Saint Louis, MO, USA). The lysates
   were cleared by centrifugation (10,000× g, 5 min, 4 °C). Western blot
   analysis was conducted according to Smolinska et al. [[68]15], with
   modifications. Actin protein was used as a control for equal loading
   and to quantify the relative abundance of the examined proteins. 40 μg
   of total protein isolates were solubilized in a sample buffer (100 mM
   Tris-HCl, 4% SDS, 20% glycerol, 0.2% bromophenol blue and 200 mM
   dithiothreitol, pH = 6.8) and incubated for 3 min at 99.9 °C. Next,
   samples were separated by SDS-PAGE electrophoresis in a 12.5%
   polyacrylamide gel (100 V) and transferred onto a polyvinylidene
   fluoride membrane (PVDF) (Roche Diagnostics, Mannheim, Germany). A 5%
   BSA solution in TBST was used to block nonspecific binding. Next, the
   membranes were incubated at 4 °C overnight with specific primary
   antibodies: IFIT5 (1:500; ab220954; Abcam, Cambridge, UK), PGRMC1
   (1:500; sc-393015; Santa Cruz Biotechnology, Dallas, TX, USA), ACO1
   (1:1000; orb330127; Biorbyt, Cambridge, UK), TGFβ1 (1:500; PA1-29032;
   Thermo Fisher Scientific, Waltham, MA, USA), and actin (1:2000; A2066;
   Sigma-Aldrich, Saint Louis, MO, USA). After washing, membranes were
   incubated with HRP-conjugated secondary antibodies (1.5 h, RT): goat
   anti-rabbit IgG (AP156P; Merck Millipore, Burlington, MA, USA) for
   IFIT5, ACO1, TGFβ1 (1:5000) and actin (1:20,000), and goat anti-mouse
   IgG for PGRMC1 (1:5000; 115-035-003; Jackson ImmunoResearch, West
   Grove, PA, USA). The visualization of immunocomplexes was carried out
   using chemiluminescence HRP substrate (Merck Millipore, Burlington, MA,
   USA) according to the manufacturer’s protocol and visualized with an
   Azure 280 Imaging System (Azure Biosystems, Dublin, CA, USA). The
   results of the analysis were quantified by optical density (OD)
   analysis of immunocomplexes with Image Studio Lite v.5.2 (LI-COR,
   Lincoln, NE, USA). Data were presented as a ratio of examined protein
   relative to actin protein in arbitrary OD units. The normality of
   Western blot data distributions was confirmed using a Shapiro–Wilk test
   (p > 0.05), and the results were statistically checked by a Student’s
   t-test (p < 0.05) using Statistica software (Statsoft Inc., Tulsa, OK,
   USA). Data were presented as mean ± SEM (n = 5).

3. Results

3.1. Differentially Regulated Proteins and Functional Annotations (GO and
KEGG)

   A total of 1499 proteins were identified in the current study
   ([69]Table S1). After relative quantification, filters (fold change
   cutoff 1.2 and p < 0.05) were applied to the results to obtain the
   final list of 352 differentially regulated proteins (DRPs). DRPs were
   visualized in the Volcano plot ([70]Figure 1).

Figure 1.

   [71]Figure 1
   [72]Open in a new tab

   Volcano plot of the proteins identified by LC–MS/MS in CHEM vs. CTR
   comparison. The volcano plot shows the fold-change (x-axis) versus the
   significance (y-axis) of the identified differentially regulated
   proteins (DRPs) under the influence of CHEM. The significance
   (non-adjusted p-value) and the fold-change are converted to
   −Log10(p-value) and Log2(fold-change), respectively. The vertical and
   horizontal dotted lines show the cut-off of fold-change = ±1.2, and of
   p-value = 0.05, respectively. The chosen DRPs are labeled by the gene
   symbols.

   Among all DRPs, 164 were up-regulated and 188 were down-regulated in
   the CHEM-treated group ([73]Table S2). The fold change values for DRPs
   ranged from −2.16 (SCD5) to 3.01 (HLA-DQB1). To discover possible
   functions of 352 annotated DRPs identified in the CHEM-treated porcine
   endometrial explants, the proteins were classified into three main
   categories: ‘biological processes’ (BP), ‘cellular components’ (CC) and
   ‘molecular function’ (MF) ([74]Table S3). DRPs were assigned to 47 gene
   ontology (GO) terms (p-adjusted < 0.05); within these GO terms, 24 were
   ascribed to BP, 14 to CC and 9 to MF category ([75]Figure 2).

Figure 2.

   [76]Figure 2
   [77]Open in a new tab

   The g:Profiler analysis of differentially regulated proteins (DRPs).
   Manhattan plot that illustrates the enrichment analysis results. The
   functional terms are grouped and color-coded by data sources, i.e.,
   molecular function (MF) are red, biological processes (BP) are orange,
   cellular components (CC) are green, and Kyoto Encyclopedia of Genes and
   Genomes (KEGG) are pink.

   Most of the DRPs from the BP category were allocated to ‘cell motility’
   (GO:0048870; 43 DRPs), ‘localization of cell’ (GO:0051674; 43 DRGs),
   ‘cell migration’ (GO:0016477; 42 DRPs) and ‘carboxylic acid metabolic
   process’ (GO:0019752; 29 DRPs). Within the BP category, the most
   interesting DRPs were enriched to several subcategories: leukocyte
   cell-cell adhesion’ (GO:0007159; 11 DRPs), ‘regulation of cell
   migration’ (GO:0030334; 23 DRPs) and ‘regulation of T cell
   proliferation’ (GO:0042129; 6 DRPs) ([78]Figure 3).

Figure 3.

   [79]Figure 3
   [80]Open in a new tab

   Visualization of the differentially regulated proteins (DRPs)
   determined to be statistically significant and their enrichment in the
   ontology terms detected by g:Profiler tool. (A) The circular plot
   illustrates the five most interesting GO biological processes enriched
   by DRPs. The outer track shows upregulated (red dots) and downregulated
   DRPs (blue dots) annotated in the particular GO terms. The inner track
   depicts the ratio of up- and downregulated proteins engaged in GO
   terms. A more intense red color shows the greater involvement of
   overexpressed proteins. (B) The circular plot shows the selected GO
   terms enriched by DRPs evaluated in the endometrium treated with CHEM.

   Most of the proteins assigned to the CC category were annotated to
   ‘cell junction’ (GO:0030054; 44 DRPs) and ‘anchoring junction’
   (GO:0070161; 31 DRPs). The proteins were CC of e.g., cell-cell
   junction, cytoskeleton, cell leading edge, and polymeric cytoskeletal
   fiber.

   The most enriched GO term in the MF category was ‘identical protein
   binding’ (GO:0042802; 59 DRPs). The MF aspect encompassed proteins
   annotated to ‘phospholipid binding’ (GO:0005543; 19 DRPs),
   ‘protein-containing complex binding’ (GO:0044877; 15 DRPs), ‘structural
   constituent of cytoskeleton’ (GO:0005200; 5 DRPs).

   The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis
   revealed the modulation of two signaling pathways, activated by DRPs.
   The enrichment analysis qualified well-documented pathways that are
   linked with CHEM activation, e.g., ‘hypertrophic cardiomyopathy’
   (KEGG:05410; 9 DRPs) and ‘dilated cardiomyopathy’ (KEGG:05414; 6 DRPs).

3.2. Western Blot

   To validate the obtained LC-MS/MS results, four DRPs were selected for
   western blot analysis. In the endometrial tissue treated in vitro with
   CHEM, when compared to the control, the abundances of Interferon
   Induced Protein With Tetratricopeptide Repeats 5 (IFIT5) and
   Transforming Growth Factor Beta 1 (TGFβ1) were up-regulated (p < 0.05).
   CHEM administration was related to a decreased protein content of
   Aconitase 1 (ACO1) and membrane-associated progesterone receptor
   component 1 (PGRMC1) (p < 0.05). The expression patterns of the
   selected DRPs agreed with the LC-MS/MS results ([81]Figure 4).
   Validation of the results confirmed the veracity and accuracy of the
   LC-MS/MS method used in the present study.

Figure 4.

   [82]Figure 4
   [83]Open in a new tab

   Western blot validation of the LC-MS results for differentially
   regulated proteins under the CHEM influence in the endometrium.
   Validation was performed for IFIT5, ACO1, TGFβ1 and PGRMC1 proteins
   with reference protein (actin) (p-value < 0.05). Data are presented as
   the mean ± standard error of the mean (n = 5). Bars with different
   letters are significantly different at p < 0.05. K—samples from the
   control groups; CHEM—samples from the CHEM-treated groups.
   IFIT5—Interferon Induced Protein With Tetratricopeptide Repeats 5;
   ACO1—Aconitase 1; TGFβ1—Transforming Growth Factor Beta 1;
   PGRMC1—membrane-associated progesterone receptor component 1.

4. Discussion

   In the present study, for the first time, an LC-MS/MS analysis was
   performed to investigate DRPs in the cultured porcine endometrial
   explants exposed to CHEM (400 ng/mL) collected during the implantation
   period. In the current study, among all 352 DRPs, 164 were up-regulated
   and 188 were down-regulated in the CHEM-treated group. DRPs were
   assigned to 47 functional annotations. The above proteins were
   classified by GO functional annotation to processes connected to, among
   others, the regulation of cell migration and motility, the
   integrin-mediated signaling pathway, and the leukocyte cell-cell
   adhesion. Furthermore, we have confirmed the results obtained using
   high-throughput LC-MS/MS by the evolution of the CHEM effect on four
   chosen DRPs abundance in the porcine endometrium by the western blot
   method.

   Progesterone (P[4]) is an important factor regulating the functioning
   of the endometrium. It participates in the preparation of the
   endometrium for implantation, and then maintains the pregnancy by
   inhibiting uterine muscle contractions, stimulating uterine growth and
   modulating the mother’s immune system [[84]30]. P[4] acts through the
   classical signaling pathway via nuclear P[4] receptors (PRs) and also
   through non-classical signaling pathways [[85]31]. In our in vitro
   study, one of the DRPs that down-regulated under the influence of CHEM
   was PGRMC1, which is a membrane-associated non-classical P[4] receptor
   [[86]32]. The PGRMC1 protein has been suggested to be important in
   early pregnancy because its expression is present in the human decidua
   at the maternal-fetal interface and associated membranes, with the
   highest expression being in the smooth muscle of placental blood
   vessels, villous capillaries, and in the syncytiotrophoblasts. In
   addition, it was shown that the expression of PGRMC1 protein depends on
   the stage of the estrous cycle and increases in endometrial cells
   undergoing steroid-dependent terminal differentiation [[87]33]. Another
   study indicated that the mRNA level of PGRMC1 significantly increased
   during gestation in the bovine endometrium [[88]34]. It can be assumed
   that PGRMC1 is involved in cell differentiation. Lyzikova et al.
   [[89]35] pointed to PGRMC1 as a potential factor involved in recurrent
   miscarriage. In the cases of recurrent miscarriage, endometrial protein
   expression of PGRMC1 is inversely correlated with infiltrated immune
   cells. It is suggested that PGRMC1 may regulate the immune response of
   the uterus [[90]35]. The latest reports confirm that CHEM is involved
   in the regulation of steroidogenesis, including P[4] secretion in the
   porcine endometrium [[91]8]. In the light of these facts, we may assume
   that CHEM, except a direct impact on P[4] secretion, in modulating
   endometrial PGRMC1 content may also be responsible for the proper
   sensitivity of tissues to the action of P[4], which is necessary for
   the maintenance of pregnancy.

   Regulation of the cell migration process is crucial in the control of
   trophoblast attachment [[92]36]. Stanniocalcin 1 (STC1) is a
   glycoprotein assigned to the ‘regulation of cell migration’ category
   (GO:0030334), that reduces calcium levels and increases phosphate
   levels in the cells and tissues. STC1 is involved in many processes,
   including angiogenesis, mineral homeostasis, cell proliferation,
   inflammation, and apoptosis [[93]37,[94]38]. In our experiment, CHEM
   increased the level of this protein. Song et al. [[95]37] showed that
   the STC1 gene and protein are present in the porcine endometrium and
   ULFs (uterine luminal flushing) during the estrus cycle and pregnancy.
   In addition, STC1 mRNA expression has been shown to increase in the
   luminal epithelium (LE) between days 12 and 20 of pregnancy, possibly
   due to the increasing concentration of P[4], which positively
   correlates with STC1 expression [[96]37]. Studies on the human
   endometrium demonstrated that STC1 showed a similar expression with
   receptivity markers during the window of implantation [[97]39]. The
   authors of the above studies suggest that STC1 may be a unique marker
   of implantation. CHEM, by stimulating STC1 content, may affect
   implantation through the effects on uterine receptivity.

   The extracellular matrix (ECM) builds the intercellular network of
   collagen and proteoglycans through which it is possible to spread
   nutrients, metabolites, cytokines and growth factors. Degradation of
   ECM is essential for tissue development, morphogenesis and repair.
   Tissue inhibitors of metalloproteinases (TIMPs), by regulating the
   activity of matrix metalloproteinases (MMPs), are the most important
   mediators in ECM remodeling [[98]40]. Our in vitro study has shown that
   CHEM diminished TIMP2 protein levels in the porcine endometrium during
   implantation. Ledgard et al. [[99]41] indicated that TIMP2 protein was
   present in ULFs in lower amounts in pregnant compared with non-pregnant
   animals. The authors suggested that TIMP2 is involved in the maternal
   recognition of pregnancy [[100]41]. On the other hand, another study
   has shown that the presence of conceptus induced endometrial TIMP2 mRNA
   expression. Additionally, increased TIMP2 protein abundance in ULFs
   during embryo elongation was observed [[101]42]. The presence of TIMP2
   was also reported in the human decidua and placenta
   [[102]43,[103]44,[104]45]. Tropomyosin alpha-4 chain (TPM4),
   up-regulated in this study by CHEM, is likewise associated with the
   cytoskeletal remodeling essential for pregnancy establishment. It has
   been shown that TPM4 protein is increased in the decidualized stromal
   cells during the late secretory phase of the menstrual cycle in women.
   The presence of this protein has also been confirmed in the decidual
   cells on the fetal-maternal interface in the first trimester of
   pregnancy [[105]46]. Taking into consideration that CHEM modulated
   TIMP2 and TPM4 protein abundance, we can imply that CHEM may be
   involved in the control of endometrial remodeling during trophoblast
   invasion.

   Subsequent DRPs identified in this study under CHEM influence,
   classified in the ‘regulation of cell migration’ category (GO:0030334),
   are TGFβ1 and its receptor Endoglin (ENG). TGFβ1 is one of the many
   growth factors identified in the porcine endometrium during the
   peri-implantation period [[106]47,[107]48]. Much research supports the
   finding that the porcine conceptus and endometrium are sources of
   secreted TGFβ1 during implantation, which suggests its possible roles
   in early embryo growth and maternal-conceptuses interactions
   [[108]47,[109]48,[110]49,[111]50,[112]51]. The obtained results
   revealed the stimulatory effect of CHEM on TGFβ1 and ENG protein
   expression. Transgenic mice with inappropriate expression of TGFβ1
   receptors in the uterus during early pregnancy were characterized by a
   delay in the attachment phase of implantation [[113]52]. A study on the
   murine uterus has shown that the epithelial-derived TGFβ1 takes part in
   stromal differentiation at the time of implantation [[114]53]. Other
   studies conducted on pregnant pigs on day 15 of gestation indicated
   that TGFβ1 plays also an immunosuppressive function [[115]54].
   Interestingly, Chadchan et al. [[116]55] have observed up-regulated ENG
   protein and mRNA expression throughout the window of endometrial
   receptivity for mice embryo implantation. TGFβ1 and its receptor ENG
   are important for the normal implantation process, and we suggest that
   CHEM, by the stimulation of TGFβ1 and ENG levels, is an important
   regulating factor at the conceptus-maternal interface during the
   peri-implantation period in pigs.

   In pigs, the maternal and fetal sides are connected by the placental
   microvasculature, thus there is a maternal-fetal exchange between the
   trophoblast and uterine luminal epithelium [[117]56]. The dominant
   lymphocytes that accumulate at the interface between mother and fetus
   are the natural killers (NK). They play a key role in the maternal and
   fetal immune recognition and angiogenesis of the placenta. Any
   abnormalities in the migration of NK cells may lead to disturbances in
   the transformation of the uterine spiral arteries and adequate
   fetoplacental perfusion [[118]57]. Carlino et al. [[119]58]
   demonstrated that CHEM mRNA and protein are up-regulated during
   decidualization in the human endometrium and might contribute to NK
   cell accumulation and vascular remodeling during early gestation. Zhang
   et al. [[120]59] proved that increased levels of TGFβ1 (described
   above) inhibit the activation of NK and their angiogenic properties.
   Moreover, epithelial cell adhesion molecule (EpCAM), down-regulated in
   this study by CHEM, is a transmembrane glycoprotein that promotes cell
   proliferation, migration and invasion [[121]60,[122]61]. Münz et al.
   [[123]60] indicated that the inhibition of EpCAM expression strongly
   decreased proliferation and metabolism in human carcinoma cells.
   Studies on gliomas have shown that EpCAM overexpression significantly
   correlates with microvessel density, which indicates the participation
   of this protein in angiogenesis [[124]62]. Other cancer studies also
   link EpCAM to the formation of new blood vessels
   [[125]63,[126]64,[127]65]. Interestingly, EpCAM is expressed in the
   human [[128]66] and mouse [[129]67] endometrial epithelia and rat
   embryos [[130]68]. Endothelial cell-selective adhesion molecule (ESAM)
   is the next cell adhesion molecule identified in this study to be
   up-regulated under the CHEM influence. ESAM is primarily expressed in
   the embryo-developing vasculature and takes part in cell-cell
   interactions [[131]69]. It has been proven that tumor growth in ESAM
   knockout mice is retarded. The above phenomenon was related to less
   migratory and angiogenic activity [[132]70]. Another enhanced cell
   adhesion molecule identified in this study is platelet endothelial cell
   adhesion molecule (PECAM1). PECAM1 is suggested to play a role in
   angiogenesis and leukocyte-endothelial interactions in the process of
   leukocyte margination during inflammation [[133]71,[134]72]. It is
   worth noting that EpCAM, ESAM, and PECAM1 are three of the 24 DRPs
   identified in this study, classified in the ‘cell-cell junction’ GO
   category (GO:0005911). During penetration of the uterine basal lamina
   by the trophoblasts, junctional complexes are formed between the
   syncytial trophoblast and maternal endothelial cells of blood vessels
   [[135]1]. CHEM, by modulating the TGFβ1, EpCAM, ESAM, and PECAM1
   proteins content in the endometrium, may control endothelial cell
   migration and tube formation intensity, which are crucial for
   endometrial cell differentiation during implantation. The above
   reports, together with the DRPs discussed in the previous paragraph,
   confirm the important role of CHEM in the endometrial remodeling and
   preparation for embryo implantation.

   TGFβ1 and Vascular Cell Adhesion Molecule 1 (VCAM1) up-regulated in
   this study by CHEM, are both classified in the ‘leukocyte cell-cell
   adhesion’ category (GO:0007159). Both proteins play a key role in the
   activation of NK cells, which may have a direct impact on the
   pathogenesis of preeclampsia [[136]59,[137]73]. Preeclampsia is a
   common disorder that develops during pregnancy (4–5% of human
   pregnancies). It is characterized by elevated blood pressure and an
   intensified systemic inflammatory reaction. It has been suggested that
   the abnormal invasion of trophoblasts into the uterine blood vessels
   and the immunological intolerance between the fetal-placental and
   maternal tissues are responsible for the occurrence of this disease
   [[138]74]. Recent reports confirmed the role of CHEM in the
   pathogenesis of preeclampsia. Tan et al. [[139]75] indicated that
   placental CHEM expression and release increased in women with this
   disorder. It has been shown that CHEM reduced vascular endothelial
   factor-A and inhibited trophoblast migration and invasion. Moreover,
   according to the same authors, trophoblast CHEM overexpression in mice
   contributed to fetal growth restriction [[140]75]. In the present
   study, CHEM, by increasing the expression of TGFβ1 protein in the
   endometrium, may contribute to the inadequate activation of NK cells
   and the development of preeclampsia. Cell adhesion molecules, due to
   their role in the adhesion of leukocytes to endothelial cells and the
   migration of leukocytes to the perivascular tissue, are believed to be
   subsequent indicators of endothelial dysfunction in preeclampsia
   [[141]76]. VCAM1 has become the subject of many studies in terms of its
   involvement in the pathophysiology of preeclampsia. Various research
   suggests an increase in VCAM1 plasma concentration in preeclamptic
   patients compared with normal pregnancy
   [[142]76,[143]77,[144]78,[145]79]. It can be assumed that CHEM may
   regulate cytotrophoblast invasion in the spiral arteries and
   endothelial cell dysfunction during the pathogenesis of preeclampsia.
   Two other proteins associated with the development of preeclampsia and
   affected by CHEM in this study are Tubulointerstitial nephritis
   antigen-like 1 (TINAGL1) and Endoplasmic Reticulum Aminopeptidase 2
   (ERAP2). TINAGL1 is a matricellular protein up-regulated in this study
   by CHEM interacting with structural matrix proteins and promoting cell
   adhesion [[146]80]. The presence of the TINAGL mRNA and protein has
   been demonstrated in the mouse uterus [[147]81] and embryos [[148]82].
   The expression of TINAGL1 was increased just before implantation in
   embryos and the postimplantation period in the decidual endometrium
   [[149]81,[150]82]. Mary et al. [[151]83] reported that TINAGL1 was
   present in the human placenta and down-regulated under preeclamptic
   conditions. ERAP2 is known for its involvement in processes regulating
   immune recognition, angiogenesis, and blood pressure. Moreover, studies
   reported that ERAP2 abundance is lower in the placenta of women with
   preeclampsia [[152]84,[153]85]. Additionally, the reports of other
   teams also confirmed the correlation between the CHEM serum
   concentration with preeclampsia [[154]86,[155]87]. The above suggest
   CHEM involvement in the broadly understood pathogenesis of this
   disorder.

   Considering the above-described roles of proteins differentially
   regulated under the influence of CHEM, we suggest that the adipokine
   takes part in the endometrial cell proliferation, migration and
   invasion at the time of implantation. It also regulates the endometrial
   immune response, sensitivity to P[4], and the formation of new blood
   vessels[.] Additionally, CHEM appears to be an important factor
   involved in endothelial cell dysfunction during the pathogenesis of
   preeclampsia. Our results support the hypothesis that a balanced
   expression of CHEM is important for matrix remodeling and controlled
   trophoblast attachment during the implantation period. The obtained
   results provide a platform for a better understanding of the influence
   of CHEM on the porcine endometrium during implantation. However, future
   studies must be carried out to identify the exact mechanism of CHEM
   action.

Supplementary Materials

   The following supporting information can be downloaded at:
   [156]https://www.mdpi.com/article/10.3390/cells11071161/s1, Table S1.
   Total proteome changes in the porcine endometrial tissues induced by
   chemerin treatment; Table S2. The differentially regulated proteins in
   endometrial tissues after chemerin treatment; Table S3. Gene Ontology
   and Kyoto Encyclopedia of Genes and Genomes signaling pathways
   enrichment analysis results.
   [157]Click here for additional data file.^ (1.1MB, zip)

Author Contributions

   Conceptualization, K.O. and N.S.; methodology, K.O. and N.S.;
   validation, K.O.; formal analysis, K.O. and B.Ś.; investigation, K.O.,
   K.D., M.K. and N.S.; resources, T.K. and N.S.; data curation, K.O. and
   A.M.; writing—original draft preparation, K.O.; writing—review and
   editing, K.O., N.S. and T.K.; visualization, K.O; supervision, T.K. and
   N.S.; project administration, N.S.; funding acquisition, N.S. All
   authors have read and agreed to the published version of the
   manuscript.

Funding

   This research was funded by the National Science Center (Poland) grant
   number [2017/25/B/NZ9/00040]. The equipment used was sponsored in part
   by the Centre for Preclinical Research and Technology (CePT), a project
   co-sponsored by European Regional Development Fund and Innovative
   Economy, The National Cohesion Strategy of Poland. Kinga Orzechowska is
   a recipient of a scholarship from the Program Interdisciplinary
   Doctoral Studies in Biology and Biotechnology
   (POWR.03.05.00-00-Z310/17), which is funded by the European Social
   Fund. “Development Program of the University of Warmia and Mazury in
   Olsztyn”, POWR.03.05.00-00-Z310/17, co-financed by the European Union
   under the European Social Fund from the Operational Program Knowledge
   Education Development.

Institutional Review Board Statement

   The study was conducted according to the Polish Act of the protection
   of animals used for educational or scientific purposes of 15 January
   2015 and directive 2010/63/EU (Journal of Laws Dz.U. 2015 No. item 266)
   of the European Parliament of 22 September 2010 (2010/63/EU) on the
   protection of animals used for scientific objectives, so this study did
   not require the consent of the competent ethics committee for animal
   experiments.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The mass spectrometry proteomics data have been deposited to the
   ProteomeXchange Consortium via the PRIDE [[158]88] partner repository
   with the dataset identifier PXD031795. Other data are available on
   request from the corresponding author.

Conflicts of Interest

   The authors declare that they have no conflict of interest.

Footnotes

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References