Background: The regulation of maternal immunity is critical for the establishment and maintenance of successful pregnancy. Among many cell types regulating the immune system, innate lymphoid cells (ILCs) are known to play an important role in innate immunity. Although some reports show that ILCs are present at the maternalconceptus interface in humans and mice, the expression and function of ILCs in the endometrium have not been studied in pigs. Methods: Thus, we determined the expression, localization, and regulation of ILC markers, CD127 (a common marker for ILCs), BCL11B (a ILC2 marker), and RORC (a ILC3 marker) at the maternal-conceptus interface in pigs. Results: The expression of BCL11B and RORC, but not CD127, in the endometrium changed during pregnancy in a stage-specific manner and the expression of CD127, BCL11B, and RORC was greatest on Day 15 during pregnancy. CD127, BCL11B, and RORC were also expressed in conceptus tissues during early pregnancy and in chorioallantoic tissues during the later stage of pregnancy. BCL11B and RORC proteins were localized to specific cells in endometrial stroma. The expression of CD127 and BCL11B, but not RORC, was increased by the increasing doses of interferon-γ (IFNG) in endometrial explants. Conclusions: These results suggest that ILCs present at the maternal-conceptus interface may play a role in the establishment and maintenance of pregnancy by regulating the innate immunity in pigs.

The cellular communication network factor (CCN) family proteins regulate many biological events such as angiogenesis, tumor growth, placentation, implantation, and embryogenesis. The expression and function of CCN1, CCN2, and CCN3 at the maternal-conceptus interface are established in humans and rodents, but little is known about the role of CCN4 to CCN6 in the reproductive organs in any other species. Several studies in transcriptome analysis in pigs have shown that the expression of CCN4 and CCN6 increases in the endometrium during early pregnancy. However, their expression, regulation, and function in the endometrium throughout the estrous cycle and pregnancy have not been fully understood in pigs. Thus, we determined the expression, localization, and regulation of CCN4 and CCN6 during the estrous cycle and at the maternal-conceptus interface in pigs. We found that the levels of CCN4, but not CCN6, changed during the estrous cycle. The levels of CCN4 were greater during mid- to late pregnancy than in the early stage, and the levels of CCN6 were greatest on Day 15 of pregnancy. CCN4 and CCN6 were detected in conceptus tissues during early pregnancy and in chorioallantoic tissues during the later stage of pregnancy. CCN4 mRNA was mainly localized to epithelial cells, CCN6 mRNAs to epithelial and stromal cells in the endometrium. In endometrial explant cultures, CCN4 expression was increased by progesterone, and CCN6 expression by interferon-γ. These results suggest that CCN4 and CCN6 may play roles in the establishment and maintenance of pregnancy by regulating the endometrial epithelial cell functions in pigs.

Secretory leukocyte protease inhibitor (SLPI), also known as neutrophil elastase and cathepsin-G protease inhibitor, functions in protection of epithelial cells from proteases. SLPI is expressed and secreted by many mucosal tissues, including lungs, seminal vesicles and cervix in women. SLPI plays an important role in protection of endometrial epithelial cells during pregnancy from degradation by degradation by proteases derived from trophoblast at the maternal-conceptus interface. In pigs, SLPI mRNA is known to be expressed in endometrial tissues, but the expression of SLPI in the endometrium throughout the estrous cycle and pregnancy has not been determined. Therefore, we analyzed the expression and regulation of SLPI mRNA in the endometrium throughout the whole stages of the estrous cycle and pregnancy in pigs. We obtained endometrial tissues from gilts on Days 0 (day of estrus), 3, 6, 9, 12, 15, and 18 of the estrous cycle and on Days 10, 12, 15, 30, 60, 90, and 114 of pregnancy. Real-time RT-PCR analysis showed that the expression of SLPI mRNA in the endometrium increases during midt-o late pregnancy. During the estrous cycle, levels of SLPPI mRNA in estrus and proestrus were higher than those in diestrus and metestrus. In situ hybridization analysis showed that SLPI mRNA was specifically localized to the glandular epithelial cells in the endometrium during pregnancy with strong signal intensity during mid-to late pregnancy. SLPI mRNA was not detectable in conceptus tissues on Days 12 and 15 of pregnancy, but SLPI mRNA was expressed in chorioallantoic tissues during mid-to term pregnancy with increasing levels toward term pregnancy. To determine the effects of steroid hormones, estrogen and progesterone, on the expression of SLPI mRNA, endometrial explant tissues from immature pigs were treated with increasing doses of estradiol-17β (E2) and progesterone (P4). Increasing doses of E2 and P4 increased the expression of SLPI mRNA in endometrial tissues. These results showed that SLPI was expressed in the endometrium in a pregnancy stage-and cell type-specific manner and the expression of SLPI was regulated by E2 and P4 in endometrial tissues, suggesting that SLPI may play an important role in regulating the endometrial epithelial cell function during mid-to late pregnancy in pigs. Further analysis to determine the roles of SLPI at the maternal-conceptus interface is still needed.

Caspases are a family of cysteine protease enzymes composed of more than 10 members that play essential roles in apoptosis and inflammation. It has been reported that caspases play a critical role in regulating apoptosis at the maternal-conceptus interface in many species. However, the expression and regulation of caspases have not been determined in the endometrium in pigs. Therefore, we analyzed the expression, localization, and regulation of caspases in the endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that caspases were expressed in the endometrium during the estrous cycle and pregnancy. The expression of CASP6, CASP7, and CASP8 during the estrous cycle and CASP3, CASP6, CASP7, and CASP10 changed during pregnancy. Levels of CASP3 mRNA in the endometrium were higher on Day 12 of pregnancy than the estrous cycle and levels of CASP7 mRNAs were highest on Day 15 of estrous cycle and pregnancy. Immunohistochemistry analysis showed that CASP3 protein was localized to endometrial epithelial cells on Days 12 and 15 the estrous cycle and pregnancy, but cleaved CASP3 was localized only to luminal epithelial (LE) cells on Days 12 and 15 of pregnancy in the endometrium. CASP7 protein was localized to endometrial LE cells only on Day 15 of pregnancy. CASP3, CASP6, CASP7, CASP8, and CASP10 mRNAs were detectable in conceptus on D12 and D15 of pregnancy, and chorioallantoic tissues expressed CASP3, CASP6, CASP7, CASP8, CASP8, and CASP10 with increasing levels toward term pregnancy, except CASP3 mRNA. The effect of steroid hormones and interleukin-1βß (IL1B) on CASP3 expression and the effect of interferon-γ(IFNG) on CASP7 expression was determined by endometrial explant cultures and we found that CASP3 expression was increased by IL1B and CASP7 expression was increased by IFNG in a dose-dependent manner. These results showed that caspases were expressed in the endometrium during the estrous cycle and pregnancy in a stage- and/or pregnancy-specific dependent manner and some caspases were regulated by IL1B or IFNG in the endometrial tissues, suggesting that caspase may play an important role in regulating apoptosis for the establishment and maintenance of pregnancy at the maternal-conceptus interface in pigs.

Pro-inflammatory cytokines, interleukin-1β (IL1B), IL6, and tumor necrosis factor-alpha (TNF), are known to play important roles in regulating the endometrial function in the uterus during the estrous cycle and pregnancy in several species. However, the expression and function of these cytokines and their receptors in the uterine endometrium during the estrous cycle have not been studied in pigs. Thus, this study determined the expression and regulation of IL1B, IL6, TNF and their respective receptors, IL1R1, IL1RAP, IL6R, GP130, TNFRSF1A, and TNFRSF1B during the estrous cycle in pigs. To analyze levels of each gene expression in the uterine endometrium we obtained from endometrial tissues on Days 0, 3, 6, 9, 12, 15, and 18 of the estrous cycle. Real-time RT-PCR analysis showed that levels of IL1B, IL1RAP, IL6R, GP130, TNF, TNFRSF1A, and TNFRSF1B mRNAs were highest on Day 15 or 18 of the estrous cycle, which corresponds to the proestrus period. Levels of IL1R1 were highest on Day 0, while levels of IL6 were biphasic with high levels on Day 6 and Day 15. The abundance of IL1B, IL6, IL6R, and TNF mRNAs was decreased by progesterone, while levels of GP130 were increased by progesterone in endometrial tissue explants. These results showed that expression of pro-inflammatory cytokines and their receptors changed stage-specifically during the estrous cycle and regulated by progesterone in the uterine endometrium in pigs, suggesting that these pro-inflammatory cytokines may be involved in the regulation endometrial function during the estrous cycle in pigs.

The migration, adhesion, and proliferation of conceptuses during pregnancy are tightly controlled processes that are mediated by various factors including cytokines, growth factors, and hormones. Among many factors, chemokines play key roles in lymphocyte trafficking, cellular proliferation, vascularization, and embryogenesis in many mammalian species. Especially, it has been shown that C-X-C chemokine ligand 12 (CXCL12) plays an important role in early pregnancy by promoting trophoblast invasion, proliferation, and differentiation through its receptor, C-X-C chemokine receptor 4 (CXCR4) in humans. However, expression and function of CXCL12 in the uterine endometrium during pregnancy have not been well studied in pigs. Thus, we determined expression of CXCL12 and its receptor, CXCR4, in the uterine endometrium during the estrous cycle and pregnancy in pigs. We obtained endometrial tissues from gilts on day (D) 12 and D15 of the estrous cycle and D12, D15, D30, D60, D90, and D114 of pregnancy, conceptus tissues from D12 and D15 of pregnancy, and chorioallantoic tissues from D30, D60, D90, and D114 of pregnancy. Real-time RT-PCR analysis showed that levels of CXCL12 and CXCR4 mRNAs changed in the uterine endometrium during pregnancy. Levels of CXCL12 and CXCR4 mRNAs on D15 of pregnancy were higher than those on D15 of the estrous cycle. After D15 of pregnancy levels of CXCL12 and CXCR4 mRNAs gradually decreased toward term of pregnancy, and CXCL12 and CXCR4 were expressed in the chorioallantoic tissues during the mid- to late pregnancy. CXCL12 and CXCR4 mRNAs were expressed in chorioallantoic tissues during mid- to late pregnancy, and RT-PCR analysis showed that CXCL12 and CXCR4 mRNAs were detectable in conceptus on D12 and D15 of pregnancy. Immunohistochemistry showed that CXCL12 proteins were localized to endometrial luminal and glandular epithelial cells during the estrous cycle and pregnancy, and to chorionic epithelial cells during mid- to late pregnancy. Abundance of CXCL12 mRNAs, but not CXCR4, in the uterine endometrium was increased by the treatment of IFNG. These results showed that CXCL12 and CXCR4 were expressed in the uterine endometrium, conceptus, and chorioallantoic tissues and IFNG increased endometrial CXCL12 expression in pigs, suggesting that CXCL12 and its receptor may play a key role in regulation of the establishment and maintenance of pregnancy by affecting the conceptus development in pigs. [supported by the Next Generation BioGreen 21 Program (#PJ01110301), Rural Development Administration]

For the establishment and maintenance of successful pregnancy the maternal immune system must tolerate semi-allogenic fetus during pregnancy. Several mechanisms explaining immune tolerance have been proposed. Among those, it has been suggested that the CD40/CD40L system is involved in immune tolerance in several tissues. However, expression and function of CD40/CD40L in the maternal-fetal interface during pregnancy have not been studied in pigs. Thus, this study determined expression and localization of CD40 and CD40L in the uterine endometrium during pregnancy in pigs. We obtained uterine endometrial tissue samples from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy. Quantitative real-time PCR analysis showed that levels of CD40L mRNA expression during pregnancy increased on D15 of pregnancy and decreased thereafter whereas levels of CD40 mRNA was highest on D30 of pregnancy. Localization of CD40 and CD40L proteins by immunohistochemistry showed that CD40 was localized to vascular endothelial cells with strongest signal intensity on D15 of pregnancy, and CD40L was localized to luminal epithelial cells on D15 of pregnancy and amniotic membrane during mid- to late pregnancy. To determine the effect of IFNG on CD40 and CD40L expression, we took advantage of endometrial explant culture using tissues from D12 of the estrous cycle, and found that CD40 was up-regulated by IFNG in a dose-dependent manner. These results showed that CD40 and CD40L were expressed in the uterine endometrium in a cell-type and stage-specific fashion during pregnancy, and IFNG induced CD40, indicating that the CD40/CD40L system may be important for establishment and maintenance of pregnancy in pigs. [Supported by the Next Generation BioGreen21 Program (#PJ01110301), Rural Development Administration]

Progesterone regulates endometrial functions to support implantation, placentation, and fetal/placental development in the uterus. It is known that actions of progesterone are mediated by nuclear progesterone receptor (PGR), using the signaling pathway referred to the genomic pathway. However, all physiological progesterone actions cannot be explained by the genomic pathway via PGR, and it is understood that there are non-genomic actions of progesterone though membrane progesterone receptors, progesterone receptor membrane components (PGRMCs) and progestin and adipoQ receptors (PAQRs). The expression and localization of PGRMCs and PAQRs has been reported in female reproductive tissues of several species such as human, mouse and cattle. Previously, we have shown that PGRMCs and PAQRs are expressed in the porcine uterine endometrium during the estrous cycle and pregnancy. However, the regulatory mechanism for expression of PGRMCs and PAQRs in the uterine endometrium has not been studied in pigs. Thus, to understand the regulatory mechanism of PGRMC1, PGRMC2, PAQR5, PAQR6, PAQR7, PAQR8, and PAQR9 expression in the uterine endometrium during the estrous cycle and pregnancy in pigs, we determined the effect of steroid hormones estrogen and progesterone on expression of PGRMCs and PAQRs using the endometrial tissue explants for immature pigs. Levels of PGRMC1, PGRMC2, PAQR5, PAQR6, PAQR7, PAQR8, and PAQR9 mRNAs were increased by increasing doses of progesterone, but not by estradiol in the uterine endometrium. Blocking PGR by treatment of RU486, a progesterone receptor antagonist, increased levels of endometrial PGRMCs and PAQRs mRNA. These data showed that membrane progesterone receptors were induced by progesterone in the uterine endometrium, suggesting that these membrane progesterone receptors may play an important role in mediating progesterone actions in the uterine endometrium for regulation of the estrous cyclicity and pregnancy. [Supported by the Next Generation Biogreen 21 Program (# PJ01119103), Rural Development Administration, Republic of Korea]

For successful embryo implantation, the communication of the maternal endometrium with the conceptus trophec-toderm is required essentially. In pigs, conceptuses undergo morphological change in length to enlarge the physical contact area with the maternal endometrium and secrete estrogen to induce the maternal recognition of pregnancy during the peri-implantation period. Conceptus-derived estrogen prevents luteolysis by conversion in direction of PGF2α secretion from the uterine vasculature to the uterine lumen as well as it affects on expression of the uterine endo-metrial genes. In addition to estrogen, conceptuses release various signaling molecules, including cytokines, growth factors, and proteases, and, in response to these signaling molecules, the maternal uterine endometrium also syn-thesizes many signaling molecules, including hormones, cytokines, growth factors, lipid molecules, and utilizes ions such as calcium ion by calcium regulatory molecules. These reciprocal interactions of the conceptus trophectoderm with the maternal uterine endometrium make development and successful implantation of embryos possible. Thus, signaling molecules at the maternal-conceptus interface may play an important role in the implantation process. This review summarized syntheses and functions of signaling molecules at the maternal-conceptus interface to further understand mechanisms of the embryo implantation process in pigs.

Interleukin-12 (IL12) and IL23 are members of the IL12 family and secreted from antigen presenting cells (APCs) such as dendritic cells and macrophages. IL12 and IL23 are composed of two subunits of a sharing subunit, IL12B, and a unique subunit, IL12A for IL12 and IL23A for IL23. IL12 is involved in induction of T helper (Th) type 1 response, whereas IL23 is associated with the differentiation of naive T cells into Th17 cells. It has shown that IL12, a proinflammatory cytokine, is down-regulated during pregnancy for successful establishment and maintenance pregnancy and increases in plasma levels in women with preeclampsia. IL23 decreases IL12 expression to change the immune microenvironment at the maternal fetal interface in humans and mice. In the present study we determined the expression of IL12 and IL23 and their receptors in the endometrium and placenta during pregnancy. Real-time RT-PCR analysis showed levels of IL12A and IL12B mRNAs in the endometrium were high during early pregnancy, but maintained low during mid- to term pregnancy. During pregnancy, levels of IL12RB1 mRNA in the endometrium showed a biphasic pattern with the highest levels on Days 15 and 60 of pregnancy, while levels of IL12RB2 mRNA did not change. Levels of IL23A and IL23R mRNAs in the endometrium decreased toward term pregnancy. Immunohistochemical analysis showed that IL12A protein was localized specifically to scattered cells in endometrial stroma, but barely detected during mid- to term pregnancy. Conceptuses from early pregnancy expressed IL12, IL23, and their receptors, except IL12RB2, and corioallantoic tissues during mid- to late pregnancy expressed IL12, IL23, and their receptors, but not IL23R. These results showed that IL12 and IL23 and their receptors were expressed at the maternal-conceptus interface, suggesting that IL12 and IL23 may play a key role in regulating maternal immune environment for the establishment and maintenance of pregnancy in pigs.

S100 protein family is small calcium-binding proteins with two EF-hand motifs and comprises more than 20 proteins in human. Although S100A proteins are known to play important roles in proinflammatory responses including damage-associated molecular pattern (DAMP) signaling and in the establishment of pregnancy, the expression of S100As have not been determined in the uterine endometrium during the estrous cycle in pigs. Thus, this study was performed to investigate expression and localization of S100A8, S100A9, and S100A12 in the uterine endometrial tissues during the estrous cycle in pigs. Real-time RT-PCR analysis showed that S100A8, S100A9, and S100A12 mRNAs were expressed in the uterine endometrium during the estrous cycle with higher levels on days 15 and 18 of the estrous cycle than the other days of cycle. Immunohistochemistry analysis showed that S100A9 and S100A12 proteins were mainly localized to the immune cells in the uterine endometrium. Especially, S100A9- and S100A12-positive immune cells were detected in the uterine blood vessels on day 15 of the estrous cycle, and also localized to stroma near to luminal epithelium on days 0 and 18 of the estrous cycle. These results showed that S100As were expressed in the uterine endometrium during the estrous cycle in a cyclic stage-specific manner, and these proteins were localized to the immune cells in the endometrium. These suggest that immune cells expressing S100A proteins may be recruited into the endometrium during the estrous cycle and play an important role in regulating endometrial function in pigs.

The implantation process in pigs is initiated when the conceptus begins secretion of estrogen, the signal for maternal recognition of pregnancy, and cytokines including interleukin-1β(IL1B), interferon delta (IFND) and interferon gamma (IFNG). Our previous study showed that IFNG receptors, IFNGR1 and IFNGR2, were expressed in the uterine endometrium during the estrous cycle and early pregnancy. However, the molecular and cellular mechanism of IFNG in the uterine endometrium in pigs is poorly understood. To determine the role of IFNG on the uterine endometrium during the implantation period, we took advantage of RNA-Seq analysis using explant tissues treated with IFNG in the presence of estrogen and progesterone, and found that many genes including CXCL9, CXCL10, CXCL11, IDO1, IL15, IL15RA, TNFSF10 (TRAIL), and WARS were up-regulated by IFNG. Additional analysis in the uterine endometrial tissues from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy determined the expression of these IFNG-regulated genes in pigs by quantitative real-time PCR Results showed that expression of CXCL9, CXCL10, and IDO1 dramatically increased on D15 of pregnancy, and expression of CXCL11 and TNFSF10 was high during mid- to term pregnancy. These results indicate that IFNG regulates immune-associated genes in the uterine endometrium in a stage-specific fashion during pregnancy, and may play a critical role to support the establishment and maintenance of pregnancy at the fetomaternal interface in pigs.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane and participates in transport of various nutrients including glucose, amino acids, and ions. In addition, Na+/K+-ATPase is also involved in regulation of intracellular calcium ion concentration by coupling with Na+/Ca+ exchanger expressed at the maternal-fetal interface in pigs. Na+/K+-ATPase consists of α, β, and FXYD subunits, but only α and β subunits are required for primary functions. FXYD subunit is an auxiliary protein for αβ complex of Na+/K+-ATPase. However, it has not been determined that subunits of Na+/K+-ATPase are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs. In this study, we determined expression of alpha (ATP1A1-4), beta (ATP1B1-3), and FXYD (FXYD1-7) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that all alpha, beta, and FXYD subunits, except ATP1A3, were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific fashion. In situ hybridization analysis exhibited that ATP1A1, ATP1A4, and ATP1B1 were localized to luminal (LE) and glandular epithelium (GE) during the estrous cycle and early pregnancy, and during mid to term pregnancy. ATP1A1 mRNA was localized to LE, GE, and areolae of the chorioallantois, especially at high levels to LE in areolae regions. ATP1B3 mRNA was detected only in LE during the estrous cycle and pregnancy with highest levels on day (D) 12 of pregnancy. Transcripts of all subtypes of FXYD subunit were primarily localized to LE and GE during the estrous cycle and early pregnancy and to chorionic membrane (CM) during mid to term pregnancy. RT-PCR analysis showed that all subtypes of Na+/K+-ATPase subunits, except ATP1A2, ATP1A3, and ATP1B2 mRNAs, were expressed in conceptuses on D12 and D15 of pregnancy. These results indicate that Na+/K+-ATPase subunits are expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner. These suggest that Na+/K+-ATPase subunits may be involved in the establishment and maintenance of pregnancy by coordinate regulation of absorption and secretion of nutrients such as glucose, amino acids, and ions at the maternal-fetal interface in pigs.