Early Pregnancy In Pigs Research Articles

Norflurazon causes cell death and inhibits implantation-related genes in porcine trophectoderm and uterine luminal epithelial cells

Norflurazon, an inhibitor of carotenoid synthesis, is a pre-emergent herbicide that prevents growth of weeds. The norflurazon is known to hamper embryo development in non-mammals. However, specific toxic effects of norflurazon on mammalian maternal and fetal cells have not been elucidated. Thus, the hypothesis of this study is that norflurazon may influence the toxic effects between maternal and fetal cells during early pregnancy in pigs. We aimed to examine the toxic effects of norflurazon in porcine trophectoderm (Tr) and uterine luminal epithelium (LE) cells. Norflurazon, administered at 0, 20, 50 or 100 μM for 48 h was used to determine its effects on cell proliferation and cell-cycle arrest. For both uterine LE and Tr cell lines, norflurazone caused mitochondrial dysfunction by inhibiting mitochondrial respiration and ATP production, and down-regulated expression of mRNAs of mitochondrial complex genes. Norflurazon increased cell death by increasing intracellular calcium and regulating PI3K and MAPK cell signaling pathways, as well as endoplasmic reticulum (ER) stress, ER-mitochondrial contact, and autophagy-related target proteins. Norflurazone also inhibited expression of genes required for implantation of blastocysts, including SMAD2, SMAD4, and SPP1. These findings indicate that norflurazon may induce implantation failure in pigs and other mammals through adverse effects on both Tr and uterine LE cells.

43 Metabolomic Analysis of Uterine Luminal Fluid During the Peri-Implantation Period of Pregnancy in Pigs

Abstract During the peri-implantation period of pregnancy in pigs, the rapid elongation of conceptuses (embryonic/fetus and its extraembryonic membranes) is highly dependent on the composition of histotroph secreted from uterine luminal (LE) and glandular (GE) epithelial and stroma cells, as well as selective transport of nutrients. However, little is known about the metabolites in the uterine luminal fluid (ULF) associated with the uterine-conceptus communication during early pregnancy in pigs. Thus, this study was conducted to profile the metabolome in porcine ULF between days 10 and 16 of estrus cycle (C) and pregnancy (P). Gilts were observed for estrus and/or bred via artificial insemination at 12 and 24 h after onset of estrus (day 0). On days 10, 12, 14, or 16 of the estrous cycle and pregnancy (n = 6 gilts per day and status), uteri were flushed with 20 mL sterile PBS (pH 7.2) after gilts were subjected to a midventral laparotomy. Pregnancy was confirmed by the presence of one or more morphologically normal conceptuses. Recoverable uterine flushings (i.e., ULF) were then subjected to metabolomic analysis by LC-MS. Overall, 222 metabolites were detected in the ULF of which 102 were altered by status and 63 were altered by days (P < 0.05; fold change>1.5). Comparing to cyclic day 10 (10C), pregnancy stimulated increases in citric acid, lysine, arginine and other 19 metabolites in the ULF at pregnant day 10 (10P). At 12P when porcine conceptus initiates its morphological changes, ornithine, α-D-glucose, phenylalanine and other 13 metabolites were up-regulated as compared with 12C. At 14P when conceptus initiates implantation, 15 metabolites changed in ULF. At 16P, 123 metabolites were altered in ULF as compared with 16C. Interestingly, of 15 altered metabolites in ULF, 12 were downregulated including asparagine, nicotinamide riboside, and citrulline at 14P. Whereas 102 of 123 altered metabolites were upregulated including phosphorylcholine, 6-phosphate fructose and 6-phosphate glucose in ULF at 16P. To determine the absolute amount of amino acids in the ULF, we performed the targeted metabolomic analyses for 19 amino acids. As a result, arginine, asparagine, glutamic acid, glutamine, histidine, leucine/isoleucine, lysine, phenylalanine, proline, and valine were increased between 10P and 16P. They were less expressed in the ULF of estrus cycle as compared with pregnancy, and remained unchanged between 10C and 16C. Pathway analysis based on KEGG database indicating that arginine biosynthesis, alanine, aspartate and glutamate metabolism, and aminoacyl-tRNA biosynthesis have the highest degree of enrichment. These novel findings provide the foundation for future investigation of metabolomic and functional studies on both endometrium and conceptus required for elongation of porcine conceptus during peri-implantation period of pregnancy. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture

Serum exosomal miR-192 serves as a potential detective biomarker for early pregnancy screening in sows.

The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation. The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8). A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC] = 0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes. Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.

Chemerin affects the expression of angiogenesis-related factors in the porcine endometrium during early pregnancy and the oestrous cycle: an in vitro study.

The appropriate course of angiogenesis in the endometrium is crucial for pregnancy establishment and maintenance. Very little is known about the factors linking vessel formation and immune system functioning. We hypothesised that chemerin, an adipokine known for its involvement in the regulation of energy balance and immunological functions, may act as a potent regulator of endometrial angiogenesis during early pregnancy in pigs. Porcine endometrial tissue explants were obtained from pregnant pigs on days 10-11, 12-13, 15-16 and 27-28, and on days 10-12 of the oestrous cycle. The explants were in vitro cultured for 24h in the presence of chemerin (100, 200ng/mL) or in medium alone (control). We evaluated the in vitro effect of chemerin on the secretion of vascular endothelial growth factors A-D (VEGF-A-D), placental growth factor (PlGF), basic fibroblast growth factor (bFGF) and angiopoietin 1 and 2 (ANG-1, ANG-2) with the ELISA method. The protein abundance of angiogenesis-related factor receptors, VEGF receptors 1-3 (VEGFR1-3), FGF receptors 1 and 2 (FGFR1-2) and ANG receptor (TIE2) was evaluated with the Western blot (WB) method. We also analysed the influence of chemerin on the phosphorylation of AMPK using WB. We found that in the studied endometrial samples, chemerin up-regulated the secretion of VEGF-A, VEGF-B and PlGF, and protein expression of VEGFR3. The adipokine caused a decrease in VEGF-C, VEGF-D and ANG-1 release. Chemerin effect on bFGF and ANG-2 secretion, and protein content of VEGFR1, VEGFR2, FGFR1, FGFR2 and TIE2 were dependent on the stage of pregnancy. Chemerin was found to down-regulate AMPK phosphorylation. The obtained in vitro results suggest that chemerin could be an important factor in the early pregnant uterus by its influence on angiogenic factors' secretion and signalling. The obtained results on the role of chemerin in the process of endometrial angiogenesis may, in the long term perspective, contribute to the elaboration of more effective methods of modifying reproductive processes and maintaining energy homeostasis in farm animals.

Transcriptome profile of the anterior pituitary gland in pigs during maternal recognition of pregnancy

The activity of the pituitary gland determines the success of female reproduction. The knowledge regarding the reproductive-status-related changes in the transcriptome of the porcine pituitary is limited. This study aimed to determine and compare the transcriptome profile of the pituitary gland collected from pigs during maternal recognition of pregnancy, i.e. on days 12–13 of pregnancy and during the respective days of the estrous cycle. Analysis indicated 482 differentially expressed genes (DEGs) with an FC > 1.5 (P < 0.05) in the pituitary of pregnant vs. estrous-cyclic pigs. Among them, 68 were up-regulated and 414 were down-regulated. The evaluated DEGs were annotated into 39 gene ontology (GO) biological process terms, 13 GO cellular component terms, and 10 GO molecular function terms. Among the evaluated DEGs were selected genes coding for proteins potentially involved in the regulation of early pregnancy in pigs and used for gene interaction analysis and validation of microarray results. An analysis of the relationships among DEGs in pituitaries collected during maternal recognition of pregnancy showed that some of them are connected with, for example, TGFβ signaling pathway, PRL synthesis, adipocytokines pathway and immune response during maternal recognition of pregnancy. These findings expand the knowledge regarding the molecular mechanisms appearing in the porcine pituitary during the maternal recognition period of pregnancy.

Urinary metabolomics reveals the biological characteristics of early pregnancy in pigs

BackgroundEmbryo implantation in sows is an important event during pregnancy. During this process, blastocysts undergo dramatic morphologic changes, and the endometrium becomes receptive. Studies have shown that developmental changes associated with the crosstalk between peri-implantation embryos and embryo-uterine are driven by various biomolecules secreted by the endometrium and embryos. In sows, changes in the uterus are also reflected in circulating body fluids and urine. Metabolomics reveals the metabolic state of cells, tissues, and organisms. In this study, we collected urine samples from large white sows during the peri-implantation period. The levels of urinary metabolites at different periods were analyzed using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) analysis techniques.ResultsA total of 32 samples were collected from 8 sows during the estrus period and at each phase of early pregnancy (9, 12, and 15 days of gestation). A total of 530 metabolites were identified with high confidence in all samples. Compared with samples collected during the estrus phase, 269 differential metabolites were found in samples obtained during early pregnancy.ConclusionsThe identified metabolites included lipids and lipid-like molecules, organic acids and their derivatives, organic oxygen compounds, organoheterocyclic compounds, benzenoids, among others. Metabolites, such as choline and pregnanediol-3-glucuronide, play important roles in pregnancy in sows and other animals. These results reveal the metabolic changes in urine of sows during early pregnancy phase. The differential urinary metabolites can be used for assessing peri-implantation status in sows. Understanding these metabolic changes may promote the management of pregnant sows through various interventions such as provision of proper nutrition.

Global proteomic analysis of serum during early pregnancy in the pig using data-independent acquisition mass spectrometry with verification by parallel reaction monitoring.

The current pregnancy diagnosis is generally not ideal in accuracy and efficiency, and the physiological process of early pregnancy in pig remains unclarified. This study aimed to evaluate protein expression profiles and identify typical proteins of early pregnancy for more understanding of physiological processes. Data-independent acquisition-based (DIA) quantitative proteomic analysiswas performed to compare the serum proteome profiles on days 0, 5, 12, 16, and 19 of gestation in Tibetan pig.Parallel reaction monitoring (PRM) was subsequently performed to verify relative expression level. 396 proteins were detected, of which 113 differentially expressed proteins (DEPs) were identified. Functional annotation and pathway analysis indicated that the DEPs were mainly involved in catalytic activity, metabolic processes and the proteasome. Four candidate DEPs (talin 1, profilin, carbonic anhydrase, and HGF activator) showed consistent expression trends in both DIA and PRM approaches. The DIA based proteomic methods indicate the involvement of numerous serum proteins in early pregnancy physiological function in pigs. The combination of DIA-PRM based global proteomic analysis may provide insights for function study and pregnancy diagnosis biomarkers. The global proteomic analyses performed here have increased the knowledge of early pregnancy in Tibetan swine and provide potential methods for pregnancy detection.

Integrated Insight into the Molecular Mechanisms of Spontaneous Abortion during Early Pregnancy in Pigs.

Due to the high rate of spontaneous abortion (SAB) in porcine pregnancy, there is a major interest and concern on commercial pig farming worldwide. Whereas the perturbed immune response at the maternal–fetal interface is an important mechanism associated with the spontaneous embryo loss in the early stages of implantation in porcine, data on the specific regulatory mechanism of the SAB at the end stage of the implantation remains scant. Therefore, we used high-throughput sequencing and bioinformatics tools to analyze the healthy and arresting endometrium on day 28 of pregnancy. We identified 639 differentially expressed lncRNAs (DELs) and 2357 differentially expressed genes (DEGs) at the end stage of implantation, and qRT-PCR was used to verify the sequencing data. Gene set variation analysis (GSVA), gene set enrichment analysis (GSEA), and immunohistochemistry analysis demonstrated weaker immune response activities in the arresting endometrium compared to the healthy one. Using the lasso regression analysis, we screened the DELs and constructed an immunological competitive endogenous RNA (ceRNA) network related to SAB, including 4 lncRNAs, 11 miRNAs, and 13 genes. In addition, Blast analysis showed the applicability of the constructed ceRNA network in different species, and subsequently determined HOXA-AS2 in pigs. Our study, for the first time, demonstrated that the SAB events at the end stages of implantation is associated with the regulation of immunobiological processes, and a specific molecular regulatory network was obtained. These novel findings may provide new insight into the possibility of increasing the litter size of sows, making pig breeding better and thus improving the efficiency of animal husbandry production.

Pyridaben leads to inhibition of cell growth and induction of cell death through intracellular mechanisms in early pregnancy

Recently, infertility has become a major global issue. It is crucial to identify environmental factors that lead to infertility. The prevalent use of pesticides in agriculture results in the exposure of livestock and humans to these pesticides. Studies have reported the harmful effects of pesticides on pregnancy. Pyridaben, a pesticide that inhibits mitochondrial complex 1, has been reported to have detrimental effects on neurons, spermatogenesis, hormonal balance, and embryonic development. However, the effect of pyridaben on the female reproductive system has not yet been studied. Therefore, in this study, we evaluated the effects of pyridaben on early pregnancy in porcine reproductive cell lines, which are known to mimic the female reproductive system. Results demonstrated that pyridaben decreased cell growth in porcine endometrial luminal epithelial and porcine trophectoderm cell lines through inhibition of cell signal transduction. Further, pyridaben increased subG1 phase and late apoptosis through the induction of reactive oxygen species production, mitochondrial dysfunction, calcium unbalances, pro-apoptotic signals, and endoplasmic reticulum (ER) stress. Moreover, we found that pyridaben induced autophagy and inhibition of placentation through the regulation of ER-mitochondria axis proteins. Overall, pyridaben was found to be harmful in early pregnancy in pigs and may have similar effects in human pregnancy.

Embryonic and uterine development during early pregnancy in pigs

Embryonic and uterine development during early pregnancy in pigs

Effect of progesterone antagonist RU486 on uterine progesterone receptor mRNA expression,embryonic development and ovarian function during early pregnancy in pigs

Effect of progesterone antagonist RU486 on uterine progesterone receptor mRNA expression,embryonic development and ovarian function during early pregnancy in pigs

Role of uterine immune cells in early pregnancy in pigs

The immune system discriminates 'self' from 'non-self', and eliminates that which it determines to be non-self. Mammalian pregnancy appears to represent a failure of self-non-self discrimination, yet it is a highly successful reproductive strategy. We present evidence that the immune system of the female pig responds to the challenges of both mating and the presence of conceptuses. Mating induces an influx of inflammatory leukocytes into the endometrial stroma and uterine lumen. This response, while partially under endocrine control, is amplified by as yet unidentified factors in seminal plasma. In addition to preventing microbial infection, this mating-induced immune response may enhance reproductive performance. During the first month of pregnancy when intimate contact between maternal and fetal tissues is being established, the number of uterine lymphocytes decrease in the luminal epithelium and increase in the endometrial stroma at sites of conceptus attachment. The majority of these lymphocytes express the CD2 and CD8 surface markers, consistent with either T or natural killer (NK) cell lineage. Dispersed endometrial cells obtained during early gestation exhibit a pregnancy-specific increase in NK-like lytic activity. Our ongoing efforts to determine whether these NK-like lymphocytes are the ones that localize to sites of conceptus attachment are discussed. We speculate on how the reactions of pig uterine leukocytes to seminal plasma and conceptuses might contribute to successful pregnancy.

Transcriptomic analysis of interferon-γ-regulated genes in endometrial explants and their possible role in regulating maternal endometrial immunity during the implantation period in pigs, a true epitheliochorial placentation species

The implantation process requires precisely controlled interactions between the maternal uterine endometrium and the implanting conceptus. Conceptus-derived secretions affect endometrial cells to facilitate the adhesion and attachment of trophoblasts, and endometrial secretions support the growth and development of the conceptus. In pigs, the conceptus secretes a large amount of type II interferon, interferon-γ (IFNG), during the implantation period. However, the role of IFNG in the implantation process has not been fully understood in pigs. Thus, to determine the role of IFNG in the endometrium during early pregnancy in pigs, we treated endometrial explant tissues with increasing doses of IFNG and analyzed the transcriptome regulated by IFNG using an RNA-sequencing analysis. Data analyses identified 276 differentially regulated genes, their Gene Ontology terms, and 94 signature genes in a Gene Set Enrichment Analysis. Furthermore, we analyzed the expression of IFNG-regulated genes, including CIITA, KYNU, IDO1, WARS, and MHC class II molecules, in the endometrium throughout pregnancy and found that levels of those genes in the endometrium were highest on Day 15 of pregnancy, corresponding to the time of peak IFNG secretion by porcine conceptuses. In addition, immunohistochemical analyses revealed that CIITA, KYNU, and IDO proteins were expressed in a cell type- and pregnancy status-specific manner in the endometrium. These results show that genes overrepresented in endometrial tissues in response to IFNG were mainly related to immune responses, suggesting that conceptus-derived IFNG could play critical roles in regulating the maternal immune response for the establishment of pregnancy in pigs.

Flufenoxuron disturbs early pregnancy in pigs via induction of cell death with ER-mitochondrial dysfunction

The use of pesticides can result in unintended side effects, such as environmental pollution and animal diseases; in serious cases, it may cause abortion. Flufenoxuron is an inhibitor of chitin synthesis that is used widely as a pesticide on farmland. It is difficult to break down and therefore accumulates in the body, and has also been detected in breast milk. Moreover, the effects of flufenoxuron in pregnancy remain elusive. Therefore, we investigated the effects of flufenoxuron on early pregnancy. Our results suggested that flufenoxuron inhibits cell development and cell cycle progression in porcine trophectoderm (pTr) cell and porcine endometrial luminal epithelial (pLE) cell lines through the repression of signal transduction pathways. Flufenoxuron induced programmed cell death through DNA fragmentation and apoptotic signals. In addition, flufenoxuron induced ROS production, ER stress, and mitochondrial malfunction; consequently, the cytosolic and mitochondrial calcium levels were increased. Expression of proteins on the ER-mitochondrial axis was increased by flufenoxuron. Cell migration was decreased by flufenoxuron treatment between pLE and pTr cells. In addition, the expression of pregnancy-related genes was decreased flufenoxuron. Collectively, our results indicated that flufenoxuron may be harmful to livestock and women in the early stages of pregnancy.

Analysis of interferon-γ receptor IFNGR1 and IFNGR2 expression and regulation at the maternal-conceptus interface and the role of interferon-γ on endometrial expression of interferon signaling molecules during early pregnancy in pigs.

It has long beenknown that pig conceptuses produce interferon-γ (IFNG) at the time of implantation, but the role of IFNG and its mechanism of action at the maternal-conceptus interface are not fully understood. Accordingly, we analyzed the expression and regulation of IFNG receptors IFNGR1 and IFNGR2 in the endometrium during the estrous cycle and pregnancy in pigs. Levels of IFNGR1 and IFNGR2 messenger RNA (mRNA) expression changed in the endometrium, with the highest levels during mid pregnancy for IFNGR1 and on Day 12 of pregnancy for IFNGR2. The expression of IFNGR1 and IFNGR2 mRNAs was also detected in conceptuses during early pregnancy and chorioallantoic tissues during mid to late pregnancy. IFNGR1 and IFNGR2 mRNAs were localized to endometrial epithelial and stromal cells and to the chorionic membrane during pregnancy. IFNGR2 protein was also localized to endometrial epithelial and stromal cells, and increased epithelial expression of IFNGR2 mRNA and protein was detectable during early pregnancy than the estrous cycle. Explant culture studies showed that estrogen increased levels of IFNGR2, but not IFNGR1, mRNAs, while interleukin-1β did not affect levels of IFNGR1 and IFNGR2 mRNAs. Furthermore, IFNG increased levels of IRF1, IRF2, STAT1, and STAT2 mRNAs in the endometrial explants. These results in pigs indicate that IFNGR1 and IFNGR2 are expressed in a stage of pregnancy- and cell-type specific manner in the endometrium and that sequential cooperative action of conceptus signals estrogen and IFNG may be critical for endometrial responsiveness to IFNs for the establishment of pregnancy in pigs.

Changes in calcium levels in the endometrium throughout pregnancy and the role of calcium on endometrial gene expression at the time of conceptus implantation in pigs.

Calcium plays an essential role in regulating many cellular functions, including proliferation, differentiation, and apoptosis. In spite of its importance in the establishment and maintenance of pregnancy, changes in calcium levels at the maternal-conceptus interface during pregnancy and its action on endometrial gene expression are not well understood. Thus, we examined changes in calcium levels in the endometrium during pregnancy, calcium deposition at the maternal-conceptus interface during pregnancy, and the role of calcium on the expression of endometrial genes related to conceptus implantation during early pregnancy in pigs. The amounts of endometrial calcium increased during mid- to late pregnancy, and calcium deposition was mainly localized to endometrial and chorionic epithelial cells at the maternal-conceptus interface during pregnancy and conceptus tissues during early pregnancy. The amounts of total recoverable calcium in uterine flushings were greater on Day 12 of pregnancy than Day 12 of the estrous cycle, and estrogen increased absorption of calcium ions by endometrial tissues. Increasing endometrial calcium levels by treatment with A23187, a calcium ionophore, decreased the expression of the estrogen-responsive endometrial genes AKR1B1, ESR1, FGF7, IL1RAP, LPAR3, S100G, SPP1, and STC1 and increased the expression of genes related to prostaglandin synthesis and transport, namely PTGES, PTGS2, and SLCO5A1. These data suggest that calcium ions at the maternal-conceptus interface play a critical role in the establishment and maintenance of pregnancy in pigs by regulating the expression of endometrial genes involved in conceptus implantation, as well as the attachment of endometrial epithelial and conceptus trophectoderm/chorionic epithelial cells during pregnancy.

In vitro effect of orexin A on the transcriptomic profile of the endometrium during early pregnancy in pigs

Orexin A belongs to the group of hypothalamic-derived peptides that are involved in a number of processes, such as regulation of energy metabolism, control of food intake and regulation of the reproductive system, by influencing the hypothalamic-pituitary-ovarian axis. Orexin A is also present in the endometrium, myometrium and placenta, which indicates that it may function as an important local regulator of the reproductive functions. The aim of this study was to explore the effects of orexin A on global gene expression in the endometrium of pigs during early gestation, on days 15 to 16 of pregnancy (implantation period). Orexin A altered the expression of 1,242 genes. In this group, 1,104 genes had a fold change greater than 1.2 (P < 0.05). In the group of genes with a fold change that was greater than 1.2, the expression of 457 genes were increased and 647 decreased because of the effects of orexin A. An analysis of the interactions between differentially expressed genes demonstrated that orexin A interacts with genes that potentially encode intracellular signalling pathways and factors regulating reproductive functions in the endometrium. The data from the present study indicate that orexin A affects a number of genes and processes in the endometrium of pregnant pigs and may be regarded as an important regulator of implantation, depending on maternal nutritional status.

Characterization of C-C motif chemokine ligand 4 in the porcine endometrium during the presence of the maternal–fetal interface

Chemokines and their receptors play a crucial role in embryo implantation at the maternal–fetal interface during pregnancy. In this study, we investigated the role of CCL4 in development of the porcine endometrium in the early gestational period. Porcine CCL4 showed high similarity with the human counterpart, and mRNA expression of CCL4 and its receptor (CCR5) was predominantly present in the endometrium during early pregnancy. Treatment with CCL4 increased proliferation of porcine uterine luminal epithelial (pLE) cells by activation of PI3K and MAPK signal transduction. In addition, CCL4 recovered the endoplasmic-reticulum stress–reduced proliferation and decreased the unfolded protein response in pLE cells. Besides, the lipopolysaccharide-activated NF-κB pathway was suppressed in response to CCL4 in pLE cells. Inhibition of CCR5 decreased the proliferation of pLE cells and activation of the PI3K and MAPK pathways by CCL4. Furthermore, CCL4 enhanced conceptus-maternal interactions between porcine trophectoderm (pTr) cells and pLE cells during early pregnancy by activating expression of migration and implantation-related genes. Collectively, the results suggest that CCL4 may improve successful implantation in early pregnancy in pigs.

Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

Cysteine-X-cysteine (CXC) motif chemokine ligand 12 (CXCL12) and its receptor, CXC chemokine receptor type 4 (CXCR4), are involved in regulating the proliferation, migration, and survival of trophoblast cells and the maternal immune response in humans and mice. The present study examined the expression, regulation, and function of CXCL12 and CXCR4 at the maternal-conceptus interface during pregnancy in pigs. The endometrium expressed CXCL12 and CXCR4 mRNAs with the greatest CXCL12 abundance on Day 15 of pregnancy. CXCL12 protein was localized mainly in endometrial epithelial cells, while CXCR4 protein was localized in subepithelial stromal cells, vascular endothelial cells, and immune cells in blood vessels in the endometrium during the estrous cycle and pregnancy. CXCL12 protein was detected in uterine flushing on Day 15 of pregnancy. The conceptus during early pregnancy and chorioallantoic tissues during mid-to-late pregnancy expressed CXCL12 and CXCR4. Interferon-γ increased the abundance of CXCL12, but not CXCR4 mRNA in endometrial explants. Recombinant CXCL12 (rCXCL12) protein dose-dependently increased migration of cultured porcine trophectoderm cells and peripheral blood mononuclear cells (PBMCs). Furthermore, rCXCL12 caused migration of T cells, but not natural killer cells, in PBMCs. This study revealed that interferon-γ-induced CXCL12 and its receptor, CXCR4, were expressed at the maternal-conceptus interface and increased the migration of trophectoderm cells and T cells at the time of implantation in pigs. These results suggest that CXCL12 may be critical for the establishment of pregnancy by regulating trophoblast migration and T cell recruitment into the endometrium during the implantation period in pigs.

Differential expression and functional roles of chemokine (C-C motif) ligand 23 and its receptor chemokine (C-C motif) receptor type 1 in the uterine endometrium during early pregnancy in pigs

Many chemokines expressed by cells of the uterine endometrium of mammals are involved in cell-cell interactions. However, little is known about expression and functional roles of chemokine (C-C motif) ligand 23 (CCL23) in the uterine endometrium. Results of this study demonstrated that CCL23 and its receptor, chemokine (C-C motif) receptor type 1 (CCR1), are up-regulated in porcine endometria during pregnancy. CCL23 and CCR1 mRNAs were strongly expressed in endometrial glandular (GE) and luminal (LE) epithelial cells. Treatment of porcine uterine luminal epithelial (pLE) cells with recombinant CCL23 increased the abundances of PCNA and cyclin D1, and enhanced proliferation and cell cycle progression in pLE cells. CCL23 also stimulated phosphorylation of cell signaling molecules including AKT and MAPKs in pLE cells. Furthermore, ER stress-related molecules were reduced by CCL23. These results suggest that CCL23-CCR1 signaling is important for endometrial development and establishment of pregnancy in pigs.