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 present study aimed to investigate the conceptus-related changes during early pregnancy in the Black Bengal breed of goat. A total of 22 gravid genitalia of the Black Bengal goats were collected from local slaughterhouses. The crown-rump lengths (CRL) of the conceptuses were determined to estimate the gestational age (GA). The length and diameter of uterine horn and amniotic sac were measured, and volume of amniotic and allantoic fluid formed by individual conceptus were recorded. The results reveal that the CRL is positively correlated with GA of the conceptus (R2 = 0.89, p < 0.05); however, CRL was not influenced by number of conceptus. Both the left and the right uterine horn gradually increased in size with the advancement of pregnancy irrespective of conceptus number present in the horn. The size of the amniotic sac of conceptus gradually increased with the conceptus age but maintained spherical shape from 5 to 7 weeks of pregnancy. The amniotic fluid formed by individual conceptus rapidly increased from 5 weeks (3.4 ± 0.3 mL) to 7 weeks (21.0 ± 2.0 mL) and 9 weeks (111.5 ± 4.0 mL). The volume of allantoic fluid formed by individual conceptus was steadily increased until 7 weeks (60.0 ± 5.0 mL) and began to decline slowly thereafter (50.0 ± 5.0 mL at 9 weeks). Notably, there was no effect of conceptus number per pregnancy on individual amniotic and allantoic fluid volume. The cotyledons have first appeared on the allanto-chorionic surface from 4 to 5 weeks of pregnancy. The closed eye, nostril and hooves of the conceptus became visible at 7 weeks of pregnancy. The present study has shown the basic information on conceptus-related developmental changes during early pregnancy up to 9 weeks in Black Bengal goat.

The successful establishment and maintenance of pregnancy is achieved by well-coordinated interactions between the maternal uterus and the implanting conceptus. In pigs, the conceptus undergoes dramatic morphological and functional changes, and secretes various biological products such as estrogens and cytokines, interleukin-1beta (IL1B), interferon-gamma (IFNG), and IFN-delta (IFND) during the implantation period. The uterine endometrium in response to the conceptus-derived molecules and ovarian progesterone becomes receptive to the conceptus by changing cell adhesion molecule expression, epithelial cell depolarization and secretory activity. Conceptus-derived estrogen acts as the maternal pregnancy recognition signal which changes the direction of endometrial prostaglandin (PG) F2 secretion from the uterine vasculature into the uterine lumen. Estrogen also induces the expression of a variety of endometrial genes, including AKR1B1, FGF7, LPAR3, and SPP1. The function of cytokines, IL1B, IFNG, and IFND, in the endometrium is not fully understood, but some recent work shows that IL1B is involved in the synthesis and transport of endometrial PGs by regulating endometrial expression of PG-synthetic enzymes, PTGS1, PTGS2, and AKR1B1, and PG transporters, ABCC4 and SLCO2A1. Estrogen and IL1B also stimulate endometrial expression of IFN signaling molecules, suggesting that estrogen and IL1B act cooperatively on priming the endometrial function of conceptus IFNG and IFND. In turn, IFNG derived from the elongating conceptuses, induces many endometrial genes, including CXCL9, CXCL10, CXCL12, and SLA-DQ. The role of IFND at the maternal-conceptus interface is not well understood yet. Further analysis of the molecules derived from the endometrium and conceptus will provide insights into the cellular and molecular basis of maternal-conceptus interactions for the establishment of successful pregnancy in pigs.

Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants, but its expression in vivo is not well characterized. Objectives of this study were to determine IFNT gene isoforms expressed in the bovine uterus, and to identify differences in promoter sequences of IFNT genes that differ in their expression. Through the RNA-seq analysis of bovine conceptuses on days 17, 20 and 22 (day 0 = day of estrus), the expression of only two IFNT transcripts, IFNT1 and IFNTc1, were found, which were indeed classified into the IFNT gene clade. IFNT mRNAs were highest on day 17, and then decreased on days 20, and 22, which were also supported by the results of quantitative RT-PCR. Bovine ear-derived fibroblast (EF) cells were then cotransfected with luciferase reporter constructs carrying 5‘-upstream (positions -1000 to +51) regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids. CDX2, either alone or with other Ap-1, ETS2 and/or CREBBP transcription factors, was found to increase luciferase activity approximately 10 and 18 fold more than twice of those cotransfected with bIFNT1, c1-Luc construct. Furthermore, The degree of transcriptional activation by a combination of the AP1, ETS2, CREBBP and/or CDX2 expression vectors was similar to that of CDX2 along plasmid. However, expression patterns of these Luc activity differented. Whereas bIFNTc1-Luc showed lowest antivity had than bIFNT1-Luc reports. Although, lowest antivity had of the bIFNTc1 –Luc report, cotransfected with the bIFNTc1-Luc construct and AP1(JUN) or/and ETS2 expression plasmid, Luc activity was enhanced approximately 2 and 4-fold more than the bIFNT1-Luc. Furthermore, along CDX2 expression factor had high effect on activity of bIFNT1-Luc reporter than the c1 gene in EF cells. These results suggest that two forms of IFNT genes are expressed in utero and their transcriptional regulations differ.

Klotho (KL) is a single transmembrane protein composed of KL1 and KL2 repeats possessing β-glucuronidase activity and maintains calcium homeostasis in physiological state. It has been implicated in pigs that calcium is important for the establishment and maintenance of pregnancy, and our previous study has shown that transient receptor potential vanilloid type 6 (TRPV6), a calcium ion transporter, is predominantly expressed in the uterine endometrium during pregnancy in pigs. However, expression and function of KL in the uterine endometrium has not been determined in pigs. Thus, the present study determined expression and regulation of KL in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that levels of KL mRNA decreased between Days 12 to 15 of the estrous cycle, and its expression showed a biphasic manner during pregnancy. KL mRNA was expressed in conceptuses and in chorioallantoic tissues during pregnancy. Explant culture study showed that expression levels of KL were not affected by treatment of steroid hormones or interleukin-1beta during the implantation period. Furthermore, levels of KL mRNA in the uterine endometrium from gilts carrying somatic cell nuclear transfer (SCNT)- derived embryos were significantly lower than those from gilts carrying natural mating-derived embryos on Day 12 of pregnancy. These results exhibited that KL was expressed at the maternal-conceptus interface in a pregnancy statusand stage-specific manner, and its expression was affected by SCNT procedure, suggesting that KL may play an important role in the establishment and maintenance of pregnancy in pigs.

The majority of early embryonic mortality in pregnancy occurs during the peri-implantation stage, suggesting that this period is important for conceptus viability and the establishment of pregnancy. Successful establishment of preg-nancy in all mammalian species depends on the orchestrated molecular events that transpire at the conceptus- uterine interface during the peri-implantation period. This maternal-conceptus interaction is especially crucial in pigs because in them non-invasive epitheliochorial placentation occurs, in which the pre-implantation phase is prolonged. During the pre-implantation period, conceptus survival and the establishment of pregnancy are known to depend on the developing conceptus receiving an adequate supply of histotroph, which contains a wide range of nutrients and grow-th factors. Evidence links growth factors including epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), and colony-stimulating factor 2 (CSF2) to embryogenesis or implantation in various mammalian species; however, in the case of pig, little is known about such functions of these growth factors, especially their regulatory mechanisms at the maternal-conceptus interface. Our research group has presented evidence for promising growth factors affecting cellular activities of primary porcine trophectoderm (pTr) cells, and we have identified potential intracellular signaling pathways responsible for the activities induced by these factors. Therefore, this review focuses on promising growth factors at the maternal-conceptus interface regulating the develop-ment of the porcine conceptus and playing pivotal roles in implantation events during early pregnancy in pigs.

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.

The process of embryo implantation requires physical contact and physiological communication between the conceptus trophectoderm and the maternal uterine endometrium. During the peri-implantation period in pigs, the conceptus undergoes significant morphological changes and secretes estrogens, the signal for maternal recognition of pregnancy. Estrogens secreted from the conceptus act on uterine epithelia to redirect , luteolysin, secretion from the uterine vasculature to the uterine lumen to prevent luteolysis as well as to induce expression of endometrial genes that support implantation and conceptus development. In addition, conceptuses secrete cytokines, interferons, growth factors, and proteases, and in response to these signals, the uterine endometrium produces hormones, protease inhibitors, growth factors, transport proteins, adhesion molecules, lipid molecules, and calcium regulatory molecules. Coordinated interactions of these factors derived from the conceptus and the uterus play important roles in the process of implantation in pigs. To better understand mechanism of implantation process in pigs, this review provides information on signaling molecules at the conceptus-uterine interface during early pregnancy, including recently reported data reported.

Successful pregnancy requires well-coordinated interactions between the maternal uterus and the developing embryo in pigs. In pigs, implantation begins around Day 12 of pregnancy. During this period, conceptus undergoes a dramatic morphological change and secretes various factors such as estrogens, interleukin-1 beta (IL1B), and interferons. Estrogens produced by conceptuses act as the signal for maternal recognition of pregnancy, and the mechanism of estrogen action is explained by the endocrine and exocrine theory. The uterine endometrium becomes receptive to the conceptus by changing cell adhesion molecules, polarizing epithelial cells and increasing secretory activity. Some changes of uterine activity are affected by the ovarian hormone, progesterone, but the presence of conceptus in the uterus also induces changes of endometrial functions, including most importantly maternal recognition of pregnancy. Many factors, such as hormones, cytokines, enzymes, extracellular matrix proteins, and transport proteins are reported to be present at the maternal-fetal interface and function in the establishment of pregnancy in pigs. However, understanding of the cellular and molecular events occurring in the endometrium is not complete. In recent studies we made some progress on understanding of expression and function of genes involved in maternal-fetal interaction for the establishment and maintenance of pregnancy in the uterine endometrium in pigs. Firstly, we found that lysophosphatidic acid (LPA) was present at the maternal-and fetal interface at the time of implantation and LPA receptor 3 was uniquely expressed in the endometrium during early pregnancy. Secondly, we observed that salivary lipocalin (SAL1), a lipid-binding protein, was uniquely expressed in the uterine endometrium at the time of embryo implantation, and its expression was regulated by IL1B. Furthermore, expression of IL1B receptors are regulated by estrogen and IL1B, and IL1B functions in expression of genes related to prostaglandin synthesis and transport. Thirdly, we found that calcium regulatory molecules TRPV6 and S100G were dynamically regulated in the uterine endometrium during pregnancy, suggesting that regulation of calcium ion concentration may important for the embryo implantation and the maintenance of pregnancy. Finally, we observed that an MHC class II molecule, SLA-DQ, is expressed in the uterine endometrium at the time of conceptus implantation and its expression is essential for successful pregnancy, indicating that appropriate maternal-fetal immune interaction is required for the maintenance of pregnancy. Further analysis of these molecules will provide insights into the cellular and molecular basis of maternal-and fetal interaction during pregnancy 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.

The majority of early conceptus mortality in pregnancy occurs during the peri-implantation stage, suggesting that this period is important for conceptus viability and the establishment of pregnancy. Successful establishment of pregnancy in all mammalian species depends on the orchestrated molecular events that transpire at the conceptus-uterine interface during the peri-implantation phase. This maternal-conceptus interaction is especially crucial in pigs because in them non-invasive epitheliochorial placentation occurs, in which the pre-implantation phase is prolonged. During the pre-implantation period, conceptus survival and the establishment of pregnancy are known to depend on the developing conceptus receiving an adequate supply of histotroph, which contains a wide range of nutrients and growth factors. Evidence links epidermal growth factor (EGF), insulin- like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), and colony-stimulating factor 2 (CSF2) to embryogenesis or implantation in various mammalian species; however, in the case of pig, little is known about such functions of these growth factors, especially their regulatory mechanisms at the maternal-conceptus interface. Therefore, the objectives of this study were to determine: 1) the temporal and cell-specific expression of EGF, IGF-I, VEGF, and CSF2 signaling systems in the porcine endometrium during the estrous cycle and early pregnancy; 2) the potential intracellular signaling pathways responsible for the activities of these four factors in primary porcine trophectoderm (pTr) cells; and 3) the changes in cellular activities induced by these promising factors. First, the functional effect and cellular signaling cascades in pTr cells induced by EGF, which exhibits potential growth-promoting activities on the conceptus and endometrium, were investigated. EGFR mRNA and protein were abundant in endometrial luminal epithelia (LE) and glandular epithelia (GE), stratum compactum stroma, and conceptus trophectoderm on Days 13-14 of pregnancy, but not in any other cells of the uterus. EGF treatment of pTr cells increased the abundance of phosphorylated (p)-AKT1, p-ERK1/2 MAPK and p-P90RSK in the nucleus and/or cytoplasm when compared with the levels in control cells. Furthermore, EGF-stimulated phosphorylation of AKT1 and ERK1/2 MAPK were inhibited in pTr cells transfected with an EGFR siRNA, and compared with control siRNA- transfected pTr cells, the EGFR siRNA-transfected pTr cells exhibited an increase in the expression of gene encoding interferon (IFN)-δ and transforming growth factor (TGF) β-1; by contrast, no effect was detected on the expression of the gene encoding IFN-γ. Moreover, EGF stimulated the proliferation and migration of pTr cells, but these stimulatory effects were blocked by pharmacological inhibitors such as SB203580 (a p38 inhibitor), U0126 (a MAPK inhibitor), rapamycin (an MTOR inhibitor), and LY294002 (a PI3K inhibitor). Second, IGF-I was examined. IGF-1 is another promising growth factor that is known to play key roles in reproductive processes; however, little is known about IGF-I-induced functional effects and regulatory mechanisms during peri-implantation in pigs. In this study, endometrial type I IGF receptor (IGF-IR) mRNA was determined to increase substantially during early pregnancy relative to the level during the estrous cycle, and the mRNAs of both IGF-I and IGF-IR were abundant in endometrial LE and GE, stroma and conceptus trophectoderm on Day 12 of pregnancy. Moreover, IGF-I treatment potently increased the amounts of p-AKT1 and, ERK1/2 MAPK in the nucleus and cytoplasm and of RPS6 in the cytosol when compared with the amounts in untreated pTr cells, and IGF-I-induced activation of AKT1 and ERK1/2 was blocked by LY294002. Furthermore, IGF-I strongly stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited by SB203580, U0126, rapamycin and LY294002. Third, this study focused on VEGF, which was identified as a potential mediator of the fetal-maternal dialog that regulates the development of the peri-implantation porcine conceptus. In addition to its known angiogenic effects, VEGF has been suggested to play roles in the development of the early embryo, but VEGF-induced effects on the peri-implantation conceptus remain unknown. Results of this study revealed that endometrial VEGF, VEGF receptor (VEGFR)-1, and VEGFR-2 mRNA levels in endometrial LE and GE, endothelial blood vessels, and scattered cells in the stroma were more abundant during the peri-implantation period of pregnancy than during the estrous cycle. Moreover, VEGF treatment of pTr cells increased the abundance of p-AKT1, p-ERK1/2, p-p70RSK, p-RPS6 and p-4EBP1, and the addition of LY294002 suppressed VEGF-induced phosphorylation of ERK1/2 and AKT1. Furthermore, VEGF potently stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited in the presence of SB203580, U0126, rapamycin and LY294002. The fourth promising cytokine studied was CSF2, which is also known as granulocyte-macrophage colony-stimulating factor (GM-CSF). CSF2 plays a role in facilitating mammalian early embryonic development. In this study, endometrial CSF2 mRNA expression was determined to be increased during the peri-implantation period relative to the mRNA level during the estrous cycle. In pTr cells, CSF2 significantly induced the activation of AKT1, ERK1/2, MTOR, p70RSK, and RPS6, but not of STAT3, and the addition of LY294002 abolished CSF2-induced increases in p-ERK1/2, p-MTOR, and p-AKT1 levels. Furthermore, CSF2 strongly stimulated pTr cell proliferation, an effect that was blocked by U0126, rapamycin and LY294002. Collectively, these results provide new insights into the potential mediators that regulate the development of the peri-implantation conceptus at the fetal-maternal interface. These results indicate that endometrial- and/or conceptus derived EGF, IGF-I, VEGF, and CSF2 critically affect the growth and development of porcine trophectoderm cells, and that these stimulatory effects are coordinately regulated by multiple cellular signaling cascades including the PI3K-AKT and ERK1/2 MAPK pathways during early pregnancy in pigs.