Interleukin-10 and its receptors at the maternal-conceptus interface: expression, regulation, and implication for T helper 2 cytokine predominance and maternal immune tolerance in the pig, a true epitheliochorial placentation species†.

The appropriate balance between pro-inflammatory and anti-inflammatory cytokines is important for the maternal immune tolerance during pregnancy in mammals. Among the various cytokines, interleukin (IL)-10 (IL10) plays an essential role in anti-inflammatory responses, while IL12 is involved in pro-inflammatory responses during pregnancy. However, the roles of IL10 and IL12 in the endometrium during pregnancy have not been studied in pigs. Thus, we investigated the expression of IL10, IL12 (IL12A and IL12B), and their receptors (IL10RA, IL10RB, IL12RB1, and IL12RB2) at the maternal-conceptus interface. IL10, IL12, and their receptors were expressed in the endometrium during the estrous cycle and pregnancy in a pregnancy stage-specific manner. During pregnancy, IL10 expression increased on Day 15, whereas the expression of IL12A and IL12B decreased after the implantation period. IL10 protein was localized to luminal epithelial (LE), stromal cells, and macrophages; IL10RA protein to LE, endothelial, stromal, and T cells; and IL10RB mRNA to LE cells in the endometrium. IL10 and IL10RA proteins and IL10RB mRNA were also localized to chorionic epithelial (CE) cells. In endometrial explants, the expression of IL10RA and IL10RB was induced by estradiol-17ß, IL-1ß, and/or interferon-γ. Heme oxygenase 1, an IL10-inducible factor, was expressed in the endometrium with the highest levels on Day 30 of pregnancy and was localized to LE and CE cells. These results in pigs suggest that conceptus-derived signals change the endometrial immune environment by regulating the expression of IL10 and IL10 receptors at the maternal-conceptus interface and that IL10 may provide anti-inflammatory conditions for the maternal immune tolerance.

Calcium-binding proteins S100A8, S100A9, and S100A12: expression and regulation at the maternal-conceptus interface in pigs†.

Among the many calcium-binding proteins, S100A8, S100A9, and S100A12 play important roles in inflammation, innate immunity, and antimicrobial function, but their expression, regulation, and function at the maternal-conceptus interface in pigs are not fully understood. Therefore, we determined the expression and regulation of S100A8, S100A9, S100A12, and their receptor AGER at the maternal-conceptus interface in pigs. We found that S100A8, S100A9, and S100A12 mRNAs were expressed in the endometrium during the estrous cycle and pregnancy, with the greatest levels on Day (D) 12 of pregnancy, and AGER appeared at greater levels on D15 and D30 of pregnancy than on other days. The expression of S100A8, S100A9, and S100A12 was predominantly localized to epithelial cells in the endometrium, and they were detected in early-stage conceptus and later chorioallantoic tissues during pregnancy. AGER expression was localized to endometrial epithelial and stromal cells and chorionic epithelial cells. In endometrial explant tissues, the expression of S100A8, S100A9, and S100A12 was induced by estrogen, S100A8 by interleukin-1ß, and AGER by interferon-γ. We further found that on D12 of pregnancy, the expression of S100A8, S100A9, and S100A12 decreased significantly in the endometria of gilts carrying conceptuses derived from somatic cell nuclear transfer. These results indicate that the expression of S100A8, S100A9, and S100A12 is dynamically regulated in response to conceptus-derived signals at the maternal-conceptus interface, suggesting that S100A8, S100A9, and S100A12 could play a critical role in regulating endometrial epithelial cell function and conceptus implantation to support the establishment and maintenance of pregnancy in pigs.

Serial gene co-expression network approach to mine biological meanings from integrated transcriptomes of the porcine endometrium during estrous cycle.

The estrous cycle is a complex process regulated by several hormones. To understand the dynamic changes in gene expression that takes place in the swine endometrium during the estrous cycle relative to the day of estrus onset, we performed RNA-sequencing analysis on days 0, 3, 6, 9, 12, 15, and 18, resulting in the identification of 4495 differentially expressed genes (DEGs; Q ≤ 0.05 and |log2FC| ≥ 1) at various phases in the estrous cycle. These DEGs were integrated into multiple gene co-expression networks based on different fold changes and correlation coefficient (R2) thresholds and a suitable network, which included 899 genes (|log2FC| ≥ 2 and R2 ≥ 0.99), was identified for downstream analyses based on the biological relevance of the Gene Ontology (GO) terms enriched. The genes in this network were partitioned into 6 clusters based on the expression pattern. Several GO terms including cell cycle, apoptosis, hormone signaling, and lipid biosynthetic process were found to be enriched. Furthermore, we found 15 significant KEGG pathways, including cell adhesion molecules, cytokine-cytokine receptor signaling, steroid biosynthesis, and estrogen signaling pathways. We identified several genes and GO terms to be stage-specific. Moreover, the identified genes and pathways extend our understanding of porcine endometrial regulation during estrous cycle and will serve as a good resource for future studies.

Structure-based functional analysis of a PadR transcription factor from Streptococcus pneumoniae and characteristic features in the PadR subfamily-2.

Since the first discovery of phenolic acid decarboxylase transcriptional regulator (PadR), its homologs have been identified mostly in bacterial species and constitute the PadR family. PadR family members commonly contain a winged helix-turn-helix (wHTH) motif and function as a transcription factor. However, the PadR family members are varied in terms of molecular size and structure. As a result, they are divided into PadR subfamily-1 and PadR subfamily-2. PadR subfamily-2 proteins have been reported in some pathogenic bacteria, including Listeria monocytogenes and Streptococcus pneumoniae, and implicated in drug resistance processes. Despite the growing numbers of known PadR family proteins and their critical functions in bacteria survival, biochemical and biophysical studies of the PadR subfamily-2 are limited. Here, we report the crystal structure of a PadR subfamily-2 member from Streptococcus pneumoniae (SpPadR) at a 2.40 Å resolution. SpPadR forms a dimer using its N-terminal and C-terminal helices. The two wHTH motifs of a SpPadR dimer expose their positively charged residues presumably to interact with DNA. Our structure-based mutational and biochemical study indicates that SpPadR specifically recognizes a palindromic nucleotide sequence upstream of its encoding region as a transcriptional regulator. Furthermore, comparative structural analysis of diverse PadR family members combined with a modeling study highlights the structural and regulatory features of SpPadR that are canonical to the PadR family or specific to the PadR subfamily-2.

Crystal structure of the Pseudomonas aeruginosa PA0423 protein and its functional implication in antibiotic sequestration.

Pseudomonas aeruginosa is a widely found opportunistic pathogen. The emergence of multidrug-resistant strains and persistent chronic infections have increased. The protein encoded by the pa0423 gene in P. aeruginosa is proposed to be critical for pathogenesis and could be a virulence-promoting protease or a bacterial lipocalin that binds a lipid-like antibiotic for drug resistance. Although two functions of proteolysis and antibiotic resistance are mutually related to bacterial survival in the host, it is very unusual for a single-domain protein to target unrelated ligand molecules such as protein substrates and lipid-like antibiotics. To clearly address the biological role of the PA0423 protein, we performed structural and biochemical studies. We found that PA0423 adopts a single-domain ß-barrel structure and belongs to the lipocalin family. The PA0423 structure houses an internal tubular cavity, which accommodates a ubiquinone-8 molecule. Furthermore, we reveal that PA0423 can directly interact with the polymyxin B antibiotic using the internal cavity, suggesting that PA0423 has a physiological function in the antibiotic resistance of P. aeruginosa.

Uterine epithelial expression of the tumor necrosis factor superfamily: a strategy for immune privilege during pregnancy in a true epitheliochorial placentation species.

The maternal immune system tolerates semi-allogeneic placental tissues during pregnancy. Fas ligand (FASLG) and tumor necrosis factor superfamily 10 (TNFSF10) are known to be components of maternal immune tolerance in humans and mice. However, the role of FASLG and TNFSF10 in the tolerance process has not been studied in pigs, which form a true epitheliochorial type placenta. Thus, the present study examined the expression and function of FASLG and TNFSF10 and their receptors at the maternal-conceptus interface in pigs. The endometrium and conceptus tissues expressed FASLG and TNFSF10 and their receptor mRNAs during pregnancy in a stage-specific manner. During pregnancy, FASLG and TNFSF10 proteins were localized predominantly to endometrial luminal epithelial cells with strong signals on Day 30 to term and on Day 15, respectively, and receptors for TNFSF10 were localized to some stromal cells. Interferon-γ (IFNG) increased the expression of TNFSF10 and FAS in endometrial tissues. Co-culture of porcine endometrial epithelial cells over-expressing TNFSF10 with peripheral blood mononuclear cells yielded increased apoptotic cell death of lymphocytes and myeloid cells. In addition, many apoptotic T cells were found in the endometrium on Day 15 of pregnancy. The present study demonstrated that FASLG and TNFSF10 were expressed at the maternal-conceptus interface and conceptus-derived IFNG increased endometrial epithelial TNFSF10, which, in turn, induced apoptotic cell death of immune cells. These results suggest that endometrial epithelial FASLG and TNFSF10 may be critical for the formation of micro-environmental immune privilege at the maternal-conceptus interface for the establishment and maintenance of pregnancy in pigs.

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 been known 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-1ß 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.

Leukemia inhibitory factor and its receptor: expression and regulation in the porcine endometrium throughout the estrous cycle and pregnancy.

OBJECTIVE: Leukemia inhibitory factor (LIF) binds to a heterodimeric receptor composed of LIF receptor (LIFR) and glycoprotein 130 (GP130) to transmit signals into the cell. LIF plays an important role in reproduction by regulating immune response, decidualization, and implantation in several species. However, the expression of LIF and LIFR in the endometrium throughout the estrous cycle and pregnancy in pigs is not fully understood. METHODS: We analyzed the expression of LIF and LIFR in the endometrium on days 0 (estrus), 3, 6, 9, 12, 15, and 18 of the estrous cycle, and days 12, 15, 30, 60, 90, and 114 of pregnancy, in conceptuses on days 12 and 15, and in chorioallantoic tissues on days 30, 60, 90, and 114 of pregnancy in pigs. We also determined the effects of estrogen and progesterone on the expression of LIF and LIFR in endometrial tissues. RESULTS: The expression of LIF increased in the endometrium during the late diestrus phase of the estrous cycle and during mid- to late- pregnancy, while the expression of LIFR increased during early pregnancy. The expression of LIF was induced by increasing doses of estrogen, whereas the expression of LIFR was induced by increasing doses of progesterone. CONCLUSION: These results indicate that the expression of LIF and its receptor LIFR in the endometrium is regulated in a stage-specific manner during the estrous cycle and pregnancy, suggesting that LIF and its receptor signaling system may play critical roles in regulating endometrial function in pigs.

Unique epithelial expression of S100A calcium binding protein A7A in the endometrium at conceptus implantation in pigs

Objective: S100A7A, a member of the S100 protein family, is involved in various biological processes, including innate immunity, antimicrobial function, and epithelial tumorigenesis. However, the expression and function of S100A7A in the endometrium during the estrous cycle and pregnancy are not well understood in pigs. Therefore, this study determined the expression and regulation of S100A7A at the maternal-conceptus interface in pigs. Methods: We obtained endometrial tissues from pigs throughout the estrous cycle and pregnancy, conceptus tissues during early pregnancy, and chorioallantoic tissues during mid- to late pregnancy and analyzed the expression of S100A7A in these tissues. We also determined the effects of steroid hormones, estradiol-17ß (E2) and progesterone, and interleukin-1ß (IL1B) on S100A7A expression in endometrial tissues. Results: We found that S100A7A was expressed in the endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-dependent manner and was localized to endometrial LE and superficial GE cells with strong intensity in LE cells on Day 12 of pregnancy. Early stage conceptuses and chorioallantoic tissues from Day 30 to term pregnancy also expressed S100A7A. The expression of S100A7A was increased by E2 and IL1B in endometrial tissues. Conclusion: S100A7A was expressed at the maternal-conceptus interface at the initiation of implantation in response to conceptus-derived estrogen and IL1B and could be a unique endometrial epithelial marker for conceptus implantation in pigs. These findings provide an important insight into the understanding of conceptus-endometrial interactions for the successful establishment of 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.

Analysis of cysteine-X-cysteine motif chemokine ligands 9, 10, and 11, their receptor CXCR3, and their possible role on the recruitment of immune cells at the maternal-conceptus interface in pigs.

Chemokines play critical roles in the establishment and maintenance of pregnancy in animals. Cysteine-X-cysteine motif chemokine ligand 9 (CXCL9), CXCL10, and CXCL11 are involved in recruiting immune cells by binding to their shared receptor, CXC receptor 3 (CXCR3), in a variety of tissues. This study examined the expression and regulation of chemokines CXCL9, CXCL10, and CXCL11, their receptor CXCR3, and their role at the maternal-conceptus interface in pigs. The endometrium expressed CXCL9, CXCL10, CXCL11, and CXCR3 stage specifically during pregnancy, with the greatest abundance on Day 15 of pregnancy. It was noted that their expression was primarily localized to stromal cells, endothelial cells, or vascular smooth muscle cells in the endometrium. Interferon-γ increased the abundance of CXCL9, CXCL10, CXCL11 mRNAs, but not CXCR3, in endometrial explants. Furthermore, recombinant CXCL9 (rCXCL9), rCXCL10, and rCXCL11 proteins increased migration of cultured peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Recombinant CXCL9 and rCXCL10 caused migration of CD4+, CD8+, CD4+CD8+ T cells, and natural killer (NK) cells, and rCXCL11 increased migration of CD4+ T and NK cells in PBMCs. The present study demonstrated that interferon-γ-induced CXCL9, CXCL10, and CXCL11, and their receptor CXCR3 were expressed in the uterus in stage- and cell-type specific manners and increased the migration of T and NK cells, which showed the greatest endometrial infiltration on Day 15 of pregnancy. These results suggest that CXCL9, CXCL10, and CXCL11 may play an important role in the recruitment of immune cells into the endometrium during the implantation period in pigs.

Expression and regulation of prostaglandin transporters, ATP-binding cassette, subfamily C, member 1 and 9, and solute carrier organic anion transporter family, member 2A1 and 5A1 in the uterine endometrium during the estrous cycle and pregnancy in pigs.

OBJECTIVE: Prostaglandins (PGs) function in various reproductive processes, including luteolysis, maternal pregnancy recognition, conceptus development, and parturition. Our earlier study has shown that PG transporters ATP-binding cassette, subfamily C, member 4 (ABCC4) and solute carrier organic anion transporter family, member 2A1 (SLCO2A1) are expressed in the uterine endometrium in pigs. Since several other PG transporters such as ABCC1, ABCC9, SLCO4C1, and SLCO5A1 are known to be present in the uterine endometrium, this study investigated the expression of these PG transporters in the porcine uterine endometrium and placenta. METHODS: Uterine endometrial tissues were obtained from gilts on day (D) 12 and D15 of the estrous cycle and days 12, 15, 30, 60, 90, and 114 of pregnancy. RESULTS: ABCC1, ABCC9, SLCO4C1, and SLCO5A1 mRNAs were expressed in the uterine endometrium, and levels of expression changed during the estrous cycle and pregnancy. Expression of ABCC1 and ABCC9 mRNAs was localized mainly to luminal and glandular epithelial cells in the uterine endometrium, and chorionic epithelial cells during pregnancy. Conceptuses during early pregnancy and chorioallantoic tissues from mid to late pregnancy also expressed these PG transporters. Estradiol-17ß increased the expression of ABCC1 and SLCO5A1, but not ABCC9 and SLCO4C1 mRNAs and increasing doses of interleukin-1ß induced the expression of ABCC9, SLCO4C1, and SLCO5A1 mRNAs in endometrial explant tissues. CONCLUSION: These data showed that several PG transporters such as ABCC1, ABCC9, SLCO4C1, and SLCO5A1 were expressed at the maternal-conceptus interface, suggesting that these PG transporters may play an important role in the establishment and maintenance of pregnancy by regulating PG transport in the uterine endometrium and placenta in pigs.

Lysophosphatidic Acid (LPA) Receptor 3-Mediated LPA Signal Transduction Pathways: A Possible Relationship with Early Development of Peri-Implantation Porcine Conceptus.

Lysophosphatidic acid (LPA) is a phospholipid with a variety of fatty acyl groups that mediates diverse biological effects on various types of cells through specific G protein-coupled receptors. LPA appears to play a significant role in many reproductive processes, including luteolysis, implantation, and placentation. Our previous study in pigs demonstrated that LPA and the LPA receptor system are present at the maternal-conceptus interface and that LPA increases uterine endometrial expression of prostaglandin-endoperoxide synthase 2 (PTGS2) through LPA receptor 3 (LPAR3). However, the role of LPA in conceptuses during early pregnancy has not been determined. Therefore, this study examined the effects of LPA in cell proliferation, migration, and activation of the intracellular signaling pathway in porcine conceptuses by using an established porcine trophectoderm (pTr) cell line isolated from Day 12 conceptuses. All examined LPA species with various fatty acid lengths increased proliferation and migration of pTr cells as the dosage increased. Immunoblot analyses found that LPA activated intracellular signaling molecules, extracellular signal-regulated kinase 1/2 (ERK1/2), ribosomal protein S6 kinase 90 kDa (P90RSK), ribosomal protein S6 (RPS6), and P38 in pTr cells. Furthermore, LPA increased expression of PTGS2 and urokinase-type plasminogen activator (PLAU), and the LPA-induced increases in PTGS2 and PLAU expression were inhibited by LPAR3 siRNA. Collectively, these results showed that LPA promotes proliferation, migration, and differentiation of pTr cells by activating the ERK1/2-P90RSK-RPS6 and P38 pathways, indicating that the LPA-LPAR3 system may be involved in the development of trophoblast during early pregnancy in pigs.

Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

Many chemokines are present at the maternal-fetal interface and play important roles in the establishment and maintenance of pregnancy. Our study demonstrates that a chemokine, chemokine (C-C motif) ligand 28 (CCL28), is expressed in the uterine endometrium during early pregnancy in pigs. Thus, we investigated expression of CCL28 and its receptors, chemokine (C-C motif) receptor type 3 (CCR3) and 10 (CCR10), in the uterine endometrium during the estrous cycle and pregnancy and the function of CCL28 at the maternal-fetal interface during early pregnancy. Levels of CCL28 mRNAs were highest on Day 10 of pregnancy and decreased thereafter during pregnancy and CCL28 was localized mainly to endometrial glandular epithelial cells. The presence of the CCL28 protein in uterine flushings was confirmed on Day 12 of the estrous cycle and pregnancy. Endometrial tissues expressed CCR3 and CCR10 during pregnancy. The CCR10 protein was localized to endometrial luminal and glandular epithelial cells, chorionic epithelial cells, and the allantoic membrane during pregnancy. Conceptuses during early pregnancy expressed CCL28 and CCR10, but not CCR3, and chorioallantoic tissues expressed CCR10 at increasing levels towards term. Treatment with recombinant CCL28 increased the proliferation and migration of a porcine trophectoderm cell line. These results indicated that the CCL28 chemokine and its receptors, CCR3 and CCR10, are expressed at the maternal-conceptus interface, and CCL28 induces the proliferation and migration of trophectoderm cells through CCR10, suggesting that CCL28 may play a critical role in the establishment and maintenance of pregnancy in pigs.

Prostaglandin transporters ABCC4 and SLCO2A1 in the uterine endometrium and conceptus during pregnancy in pigs.

Prostaglandins (PGs) are involved in many reproductive activities including luteolysis, maternal recognition of pregnancy, endometrial gene expression, conceptus development, and parturition in domestic animals. However, mechanisms by which PGE2 and PGF2alpha are modulated in the uterine endometrium and expression of ABCC4 and SLCO2A1, responsible for efficient transport of PGs across the cell membrane, in the endometrium during the estrous cycle and pregnancy are not fully understood in pigs. Therefore, we determined expression of ABCC4 and SLCO2A1, genes involved in transport of PGE2 and PGF2alpha in the uterine endometrium during the estrous cycle and pregnancy in pigs. ABCC4 and SLCO2A1 mRNAs were expressed in the uterine endometrium, most abundantly on Day 12 of pregnancy and during late pregnancy. Expression of ABCC4 mRNA and protein was localized mainly to uterine luminal epithelial (LE) and glandular epithelial (GE) cells, and expression of SLCO2A1 mRNA and protein was expressed primarily in uterine LE and blood vessels. Expression of ABCC4 and SLCO2A1 mRNAs was also detected in conceptuses during early pregnancy. In addition, explant culture experiments showed that increasing doses of interleukin 1B (IL1B) with estrogen and progesterone increased levels of ABCC4 and SLCO2A1 mRNAs in the uterine endometrium. These results indicate that expression of genes responsible for transport of PGE2 and PGF2alpha are dynamically regulated in the uterine endometrium during pregnancy and that ABCC4 and SLCO2A1 play critical roles in supporting the establishment and maintenance of pregnancy by regulating PG transport at the maternal-fetal interface in pigs.

Comprehensive analysis of prostaglandin metabolic enzyme expression during pregnancy and the characterization of AKR1B1 as a prostaglandin F synthase at the maternal-conceptus interface in pigs.

Prostaglandins (PGs) are important lipid mediators regulating various reproductive processes in many species. In pigs, the expression pattern of PGE2 and PGF2α metabolic enzymes and the regulatory mechanism controlling PGE2 and PGF2α levels in the uterus during pregnancy are not completely understood. This study determined endometrial expression of the genes (PLA2G4A, PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, CBR1, and HPGD) involved in PGE2 and PGF2α metabolism during the estrous cycle and pregnancy and measured levels of PGE2 and PGF2α in uterine endometrial tissues and uterine flushings at the time of conceptus implantation in pigs. Except PTGES3, expression of the genes studied changed in a pregnancy-stage-specific manner, and localization of PTGES, AKR1B1, CBR1, and HPGD mRNAs were cell-type specific in the uterine endometrium. Levels of both PGE2 and PGF2α in uterine endometrial tissues and uterine lumen were higher on Day 12 of pregnancy than those of the estrous cycle and affected by different morphology of spherical and filamentous conceptuses. Furthermore, we determined that endometrial expression of AKR1B1, known to encode a PGF2α synthase in other species, was increased by estrogen and interleukin-1beta and that AKR1B1 exhibited PGF2α synthase activity in the porcine uterine endometrium. These results in pigs indicate that the PGE2 and PGF2α metabolic enzymes are expressed stage specifically in the endometrium during pregnancy and regulate the abundance of PGE2 and PGF2α in the uterus at the time of implantation and that AKR1B1 may act as a major PGF synthase in the endometrium during early pregnancy.

Complement system molecules: Expression and regulation at the maternal-conceptus interface during pregnancy in pigs.

The complement system, composed of complement components and complement control proteins, plays an essential role in innate immunity. Complement system molecules are expressed at the maternal-conceptus interface, and inappropriate activation of the complement system is associated with various adverse pregnancy outcomes in humans and rodents. However, the expression, regulation, and function of the complement system at the maternal-conceptus interface in pigs have not been studied. In this study, we investigated the expression, localization, and regulation of complement system molecules at the maternal-conceptus interface in pigs. Complement components and complement control proteins were expressed in the endometrium, early-stage conceptus, and chorioallantoic tissues during pregnancy. The expression of complement components acting on the early stage of complement activation increased in the endometrium on Day 15 of pregnancy, with greater levels on that day compared with the estrous cycle. Localization of several complement components and complement control proteins was cell-type specific in the endometrium. The expression of C1QC, C2, C3, C4A, CFI, ITGB2, MASP1, and SERPING1 was increased by IFNG in endometrial explant tissues. Furthermore, cleaved C3 fragments were detected in endometrial tissues and uterine flushings on Day 15 of the estrous cycle and Day 15 of pregnancy, with greater levels on Day 15 of pregnancy. These results suggest that complement system molecules in pigs expressed at the maternal-conceptus interface play important roles in the establishment and maintenance of pregnancy by regulating innate immunity and modulating the maternal immune environment during pregnancy.

Analysis of legumain and cystatin 6 expression at the maternal-fetal interface in pigs.

Cathepsins (CTSs), a family of lysosomal cysteine proteases, and their inhibitors, cystatins (CSTs), play a critical role in endometrial and placental tissue remodeling during the establishment and maintenance of pregnancy in many species including rodents, sheep, cow, and pigs. In this study, we determined expression of legumain (LGMN), a cathepsinmember, and its inhibitor, CST6, at the maternal-fetal interface in pigs. Expression of both LGMN and CST6 mRNAs increased during mid- to late pregnancy in the uterine endometrium. LGMN and CST6 mRNAs localized to luminal epithelial cells (LE) and glandular epithelial cells (GE) and to the chorionic membrane (CM), with a strong intensity in GE and the CM for LGMN and in the CM for CST6 during pregnancy. LGMN protein was detected at molecular weights (MW) of approximately 50,000 and 37,000, and the abundance of the37,000-MW LGMN protein increased during mid- to latepregnancy. CST6 protein was also highly expressed in the uterine endometrium in mid- to latepregnancy. LGMN protein localized to LE, GE, and the CM during pregnancy. LGMN and CST6 were aberrantly expressed in the uterine endometrium from gilts with somatic cell nuclear transfer-derived conceptuses at term compared to those of gilts carrying conceptuses derived from natural mating. These results demonstrated that LGMN and CST6 were expressed in the uterine endometrium in a cell-type and stage-specific manner, suggesting that the LGMN and CST6 system at the maternal-fetal interface may play an important role in the establishment and maintenance of pregnancy in pigs.

Analysis of ENPP2 in the Uterine Endometrium of Pigs Carrying Somatic Cell Nuclear Transfer Cloned Embryos.

Somatic cell nuclear transfer (SCNT) is a useful tool for animal cloning, but the efficiency of producing viable offspring by SCNT is very low. To improve this efficiency in the production of cloned pigs, it is critical to understand the interactions between uterine function and cloned embryos during implantation. Lysophosphatidic acid (LPA) is a lipid mediator that plays an important role in the establishment of pregnancy in pigs; however, LPA production in the uterine endometrium of pigs carrying SCNT-cloned conceptuses has not been determined. Therefore, we investigated expression of ENPP2, an LPA-generating enzyme, in the uterine endometrium of gilts with conceptuses derived from SCNT during the implantation period. Uterine endometrial tissue and uterine flushing were obtained from gilts carrying SCNT-derived conceptuses and from gilts carrying conceptuses resulting from natural mating on d 12 of pregnancy. Our results demonstrated no difference in the level of ENPP2 mRNA expression in the uterine endometrium between gilts carrying SCNT-derived conceptuses and gilts carrying naturally-conceived conceptuses, but secretion of ENPP2 protein into the uterine lumen did decrease significantly in pigs with SCNT-derived conceptuses. These results indicate that expression and secretion of ENPP2, which are critical for appropriate LPA production and successful pregnancy, are dysregulated in the uterine endometrium of pigs carrying SCNT-derived conceptuses.