@article{liu_paudel_flowers_piedrahita_wang_2023, title={Adrenomedullin Stimulates Proliferation, Migration and Adhesion of Porcine Trophectoderm Cells Via CALCRL-AKT-TSC2-MTORC1 Cell Signaling Pathway}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad068.040}, abstractNote={Abstract
               Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr1) isolated from day -12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via AKT-TSC2-MTOR cell signaling pathway in pTr1 cells. The pTr1 cells were cultured in DMEM/F12 medium with 10% fetal bovine serum (FBS), 50 U/mL penicillin, 50 μg/mL streptomycin, 0.1 mM each for nutritionally nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, and 4 μg/mL insulin. Opti-MEM supplied with 2.5% (vol/vol) charcoal-stripped FBS was used for siRNA-mediated knockdown targeting non-treated control (siNTC) or specific genes including ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL). Cells were starved in FBS- and insulin-free medium for 24 hours before treatment. For proliferation assay, cell numbers were determined by staining with Janus-Green B after 48 h incubation. For migration assay, cells were treated with ADM after straight scratch in 6-well plates, and area of cell migration was calculated after 12 h treatment. For adhesion assay, cells were trypsinized in T-25 flasks and allowed for seeding in 96-well plates with density of 2×105 cells/0.2 mL/well, and the numbers of attached cells were determined after 12 h incubation. Western blot analyses were used to determine the expressions of target proteins at total and phosphorylated level. Porcine ADM at 10-7 M stimulated (P < 0.05) pTr1 cell proliferation, migration and adhesion by 1.4%-, 1.5%- and 1.2%-folds, respectively. These ADM-induced effects were abrogated (P < 0.05) by siADM and siCALCRL, as well as by rapamycin, the inhibitor of mechanistic target of rapamycin (MTOR). Using siRNA-mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph act on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation, migration and attachment of conceptuses. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Liu, Bangmin and Paudel, Sudikshya and Flowers, William L. and Piedrahita, Jorge A. and Wang, Xiaoqiu}, year={2023}, month={May} }
 @article{paudel_liu_cummings_wang_2023, title={Adrenomedullin: A Novel Peptide Hormone for Uterine Receptivity and Conceptus Elongation in Pigs?}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad068.049}, abstractNote={Abstract
               Adrenomedullin (ADM) is an evolutionarily conserved multi-functional peptide hormone in mammalian species. The implantation sites of conceptuses (embryo/fetus and its extra-embryonic membranes) in pregnant Adm+/− female mice are abnormally spaced leading to fetal crowding, intrauterine growth restriction (IUGR) and increases in pregnancy loss. As litter-bearing species, pigs exhibit a high incidence of early embryonic death (30-40%) and naturally occurring IUGR (15-25%), and significant numbers of stillborn piglets (3-9%). Therefore, it is imperative that the roles of ADM in regulation of uterine receptivity, as well as growth and development of conceptus in pigs be established. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components in uteri from cyclic and pregnant gilts, as well as conceptuses during the peri-implantation period of pregnancy. ADM receptor components include: calcitonin receptor-like receptor (CALCRL; G protein-coupled receptor bound by ADM), receptor activity modifying protein (RAMP2; dimerized with CALCRL to form ADM1 receptor) and RAMP3 (dimerized with CALCRL to form ADM2 receptor with lower specificity to ADM) and atypical chemokine receptor 3 (ACKR3; a decoy receptor that serves as a cell-autonomous molecular rheostat to dampen ADM signaling). Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, expression of CALCRL, RAMP2 and ACKR3 increased between days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line isolated from elongating porcine conceptuses recovered on day 2 post-breeding, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation and placentation in pigs. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture.}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Paudel, Sudikshya and Liu, Bangmin and Cummings, Magdalina J. and Wang, Xiaoqiu}, year={2023}, month={May} }
 @article{liu_duan_liu_wang_2023, title={Metabolomic Analysis of Uterine Luminal Fluid During the Peri-Implantation Period of Pregnancy in Pigs}, volume={101}, ISSN={["1525-3163"]}, DOI={10.1093/jas/skad068.046}, abstractNote={Abstract
               During the peri-implantation period of pregnancy in pigs, the rapid elongation of conceptuses (embryonic/fetus and its extraembryonic membranes) is highly dependent on the composition of histotroph secreted from uterine luminal (LE) and glandular (GE) epithelial and stroma cells, as well as selective transport of nutrients. However, little is known about the metabolites in the uterine luminal fluid (ULF) associated with the uterine-conceptus communication during early pregnancy in pigs. Thus, this study was conducted to profile the metabolome in porcine ULF between days 10 and 16 of estrus cycle (C) and pregnancy (P). Gilts were observed for estrus and/or bred via artificial insemination at 12 and 24 h after onset of estrus (day 0). On days 10, 12, 14, or 16 of the estrous cycle and pregnancy (n = 6 gilts per day and status), uteri were flushed with 20 mL sterile PBS (pH 7.2) after gilts were subjected to a midventral laparotomy. Pregnancy was confirmed by the presence of one or more morphologically normal conceptuses. Recoverable uterine flushings (i.e., ULF) were then subjected to metabolomic analysis by LC-MS. Overall, 222 metabolites were detected in the ULF of which 102 were altered by status and 63 were altered by days (P < 0.05; fold change>1.5). Comparing to cyclic day 10 (10C), pregnancy stimulated increases in citric acid, lysine, arginine and other 19 metabolites in the ULF at pregnant day 10 (10P). At 12P when porcine conceptus initiates its morphological changes, ornithine, α-D-glucose, phenylalanine and other 13 metabolites were up-regulated as compared with 12C. At 14P when conceptus initiates implantation, 15 metabolites changed in ULF. At 16P, 123 metabolites were altered in ULF as compared with 16C. Interestingly, of 15 altered metabolites in ULF, 12 were downregulated including asparagine, nicotinamide riboside, and citrulline at 14P. Whereas 102 of 123 altered metabolites were upregulated including phosphorylcholine, 6-phosphate fructose and 6-phosphate glucose in ULF at 16P. To determine the absolute amount of amino acids in the ULF, we performed the targeted metabolomic analyses for 19 amino acids. As a result, arginine, asparagine, glutamic acid, glutamine, histidine, leucine/isoleucine, lysine, phenylalanine, proline, and valine were increased between 10P and 16P. They were less expressed in the ULF of estrus cycle as compared with pregnancy, and remained unchanged between 10C and 16C. Pathway analysis based on KEGG database indicating that arginine biosynthesis, alanine, aspartate and glutamate metabolism, and aminoacyl-tRNA biosynthesis have the highest degree of enrichment. These novel findings provide the foundation for future investigation of metabolomic and functional studies on both endometrium and conceptus required for elongation of porcine conceptus during peri-implantation period of pregnancy. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture}, journal={JOURNAL OF ANIMAL SCIENCE}, author={Liu, Bangmin and Duan, Likun and Liu, Xiaojing and Wang, Xiaoqiu}, year={2023}, month={May} }
 @article{liu_paudel_flowers_piedrahita_wang_2023, title={Uterine histotroph and conceptus development: III. Adrenomedullin stimulates proliferation, migration and adhesion of porcine trophectoderm cells via AKT-TSC2-MTOR cell signaling pathway}, volume={55}, ISSN={0939-4451 1438-2199}, url={http://dx.doi.org/10.1007/s00726-023-03265-6}, DOI={10.1007/s00726-023-03265-6}, abstractNote={Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr2) isolated from Day-12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via activation of mechanistic target of rapamycin (MTOR) cell signaling pathway in pTr2 cells. Porcine ADM at 10–7 M stimulated (P < 0.05) pTr2 cell proliferation, migration and adhesion by 1.4-, 1.5- and 1.2-folds, respectively. These ADM-induced effects were abrogated (P < 0.05) by siRNA-mediated knockdown of ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL), as well as by rapamycin, the inhibitor of MTOR. Using siRNA-mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph acts on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation, migration and attachment of conceptuses.}, number={6}, journal={Amino Acids}, publisher={Springer Science and Business Media LLC}, author={Liu, Bangmin and Paudel, Sudikshya and Flowers, William L. and Piedrahita, Jorge A. and Wang, Xiaoqiu}, year={2023}, month={Apr}, pages={743–756} }
 @article{paudel_liu_cummings_quinn_bazer_caron_wang_2021, title={Temporal and spatial expression of adrenomedullin and its receptors in the porcine uterus and peri-implantation conceptuses}, volume={105}, ISSN={["1529-7268"]}, DOI={10.1093/biolre/ioab110}, abstractNote={Abstract
               Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10−7 M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs.}, number={4}, journal={BIOLOGY OF REPRODUCTION}, author={Paudel, Sudikshya and Liu, Bangmin and Cummings, Magdalina J. and Quinn, Kelsey E. and Bazer, Fuller W. and Caron, Kathleen M. and Wang, Xiaoqiu}, year={2021}, month={Oct}, pages={876–891} }