Chemerin Effect on the Endometrial Proteome of the Domestic Pig during Implantation Obtained by LC-MS/MS Analysis

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: DRPs; chemerin; early pregnancy; implantation; pig; proteome; uterus.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.