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.

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.

Pregnancy-associated plasma protein-A (PAPP-A) is a 200 kDa metalloprotease identified as an IGFBP-4 protease and likely an important regulator of IGF bioavailability. PAPPA mRNA is detected in bovine granulosa and theca cells and the PAPP-A protein is also found in follicular fluid. Proteolytic activity supposed to be due to the PAPP-A in bovine follicular fluid is induced by follicle stimulating hormone (FSH) treatment and PAPP-A-like activity appears concomitantly with increased E2 during follicular development. However, the effects of FSH, E2 and progesterone on the expression of PAPP-A in bovine corpus luteum of pregnancy have been evaluated in a limited number of studies. This study was performed to expression of PAPP-A and progesterone during early pregnancy in bovine corpus luteum. To determine the function of PAPP-A gene during early pregnancy, we collected the bovine pregnancy corpus luteum samples on 30, 60 and 90 day of pregnancy. The mRNA expression of PAPP-A, progesterone-receptor, VEGF and IGFBP4 gene was conducted by real-time PCR. And proteins expression of PAPP-A and progesterone antibody was detected by Immunohistochemistry and ELISA. The VEGF and PAPP-A mRNA expression was progressively increased on day 90 in the pregnancy corpus luteum. The mRNA expression of progesterone-receptor, IGFBP4 in the corpus luteum progressively was increased from 30 to 60 day, but decreased on 90 day of pregnancy. The proteins expressions of progesterone and PAPP-A were similar pattern in mRNA expression. Our results indicate that the IGFBP4 protease role of PAPP-A increases in response to pregnancy 90 day corpus luteum and suggest that progesterone is an connected for the expression of PAPP-A genes in luteal cells. Therefore, we suggest that PAPP-A stimulated with progesterone play a crucial role for IGF-I system in bovine early pregnancy. And could be used to predict the condition of normal pregnancy.