FUNCTIONAL ANALYSIS OF ADRENOMEDULLIN IN ELONGATION OF PORCINE CONCEPTUSES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028117
• Grant No.: 2022-67015-36491
• Cumulative Award Amt.: $300,000.00
• Proposal No.: 2021-06781
• Project Start Date: Jan 1, 2022
• Project End Date: Dec 31, 2025
• Grant Year: 2022
• Program Code: [A1211]- Animal Health and Production and Animal Products: Animal Reproduction

Project Director
Wang, X.

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Animal Science

Non Technical Summary
Large litters in commercial breeding herds often increase the birth of "runt" piglets. This represents major constraint to improving reproductive efficiency and profitability in swine production enterprises. Asynchronous development between uterus and conceptus (embryo/fetus and its extra-embryonic membranes) limits surface area of placentation at maternal-conceptus interface, compromises conceptus development and results in low-birth-weight piglets with poor survival in the neonatal period of life. Thus, understanding fundamental roles of conceptus- and uterine-secreted factors that govern conceptus-uterine synchrony required for implantation, establishment of pregnancy, and placental expansion is prerequisite for ultimately developing counteracting measures. Here, we hypothesize that adrenomedullin (ADM), a highly conserved peptides among mammals, plays a critical role in the control of uterine capacity and conceptus development in pigs. Our objective is to utilize RNA interference technology to knockdown the ADM receptor CALCRL to determine the impact of ADM on in vitro cell behaviors of porcine trophectoderm (pTr; future placenta), and early embryonic development and survival in vivo. Completion of this research will fill a major gap in our knowledge of ADM function in conceptus development and survival in pigs. We anticipate that the outcomes will provide strong support for a future Standard Grant proposal, addressing the molecular mechanisms by which ADM regulates uterine capacity and conceptus development, thereby improving reproductive efficiency and profitability in swine production enterprises.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3510	1020	100%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1020 - Physiology;

Keywords

adrenomedullin

conceptus

elongation

implantation

mtor

pig

pregnancy

uterus

Goals / Objectives
The long-term goals of research are to understand the fundamental roles of conceptus- and uterine-secreted factors that govern synchronous uterine and conceptus development required for implantation, establishment of pregnancy, and placental development required for fetal growth and survival. We aim to develop new strategies to optimize placental and fetal growth that will improve overall health and survivability of piglets before and after birth to reduce neonatal death losses. The central hypothesis is that adrenomedullin (ADM) functions as an essential mediator of synchronous uterine and conceptus development by activating MTORC1 and MTORC2 cell signaling to stimulate proliferation, migration, and attachment of trophectoderm (Tr) cells required for porcine conceptuses to undergo implantation, provide signals for establishment of pregnancy, and undergo placentation required for support of fetal growth during pregnancy. In this Seed Grant proposal, we will test the modified sub-hypothesis that ADM activates CALCRL-mediated MTORC1 cell signaling to stimulate proliferation, migration and adhesion of Tr cells required for conceptus elongation and gene expression required for implantation in pigs. This will be accomplished by meeting two specific aims. In Aim 1, we will determine the functional roles of ADM on proliferation, migration and adhesion of pTr cells in vitro. In Aim 2, we will determine whether perturbation of CALCRL expression in vivo compromises elongation of porcine conceptus Tr.

Project Methods
Aim 1 will determine the functional roles of ADM on proliferation, migration and adhesion of pTr cells in vitro. In Aim 1.1, we will determine effects of porcine ADM (pADM) on proliferation, migration and adhesion of pTr cells. The established pTr cells will be seeded at 30% confluency in 24-microwell plates and treated with 10-9, 10-8, 10-7 or 10-6 M pADM (Bachem, Torrance, CA; n=4 wells per treatment) for 48 and 96 h (37°C, 5% CO2) in DMEM/F-12 containing 5% FBS, 50 U/ml penicillin, 50 ?g/ml streptomycin, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, and 4 ?g/ml insulin. The medium will be replaced every 2 days. After 48 and 96 h treatments, cell numbers will be determined. Cell migration will be assayed using pTr cell seeded in a confluence layer on 8-µm pore transwell inserts, and pADM added to lower wells (n=4) in FBS-free DMEM/F-12. After 24 h, the cells that migrate to the bottom surface of the membrane will be counted using a Zeiss Axio Imager M1 micrscope with an Axiocan HR camera and Axiovision 4 software. For cell adhesion assay, the pTr cells will be added into a 96-well plate and allowed to attach for 1, 2, and 6 h in the presence of pADM at indicated doses. After each time-point, the number of attached pTr cells will be measured. In Aim 1.2, we will determine the impact of siRNA knockdown of CALCRL, the shared component of ADM receptors on proliferation, migration and adhesion of pTr cells. The pTr cells will be pre-incubated with siCALCRL or non-targeting siRNA control (siNTC; Dharmacon) for 48 h before being subjected to assays to assess effects on proliferation, migration and adhesion in the presence of 10-7 M pADM for additional 48 and 96 h. The treatments (n=4) include: 1) siNTC as negative control; 2) siNTC+pADM as positive control; and 3) siCALCRL+pADM. This will determine whether proliferation, migration and adhesion of pTr cells requires ADM binding to its receptors ADM1 (CALCRL/RAMP2) and ADM2 (CALCRL/RAMP3). In Aim 1.3, we will determine if ADM stimulates proliferation, migration and adhesion of pTr cells via activation of CALCRL-induced MTOCR1 cell signaling. We will first determine if ADM stimulation of proliferation, migration and adhesion of pTr cells requires MTORC1 using rapamycin, the inhibitor of MTOR. Thus, the treatments (n=4) include: 1) control; 2) ADM; 3) rapamycin; and 4) ADM+rapamycin. We will then determine if ADM activates MTORC1 cell signaling cascades in pTr cells via activation of CALCRL using siRNA knockdown. After 0, 0.25, 0.5, 1, 2, and 24 h in culture (n=4 replicates per treatment per time point), steady-state levels of proteins in pTr cells will be assessed by Western blot. In Aim 1.4, we will determine the impact of ADM on gene expression of pTr cells required for elongation and implantation. After 48 h siRNA and additional 48 h pADM culture, steady-state levels of mRNAs in pTr cells will be assessed by qPCR analyses for genes associated with Tr cell proliferation, migration and adhesion required for elongation and implantation, i.e., Ki67, TGFB, IL1B2, FGF4, FGFR2IIIB, BMP4, ITGB3, SPP1, CYP19A1 and IFNδ. In Aim 2, we will determine whether perturbation of CALCRL expression compromises elongation of porcine conceptus Tr in vivo. We will use morpholino antisense oligonucleotides (MAO) to knockdown translation of CALCRL mRNA in vivo, and determine alterations in phenotype of conceptuses, as well as MTORC1 cell signaling and gene expression by Tr cells using RNA-seq.

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:The target audience consists of the scientific community engaged in research to improve reproductive efficiency in livestock enterprises. The information generated from this research will be shared with the scientific community through publications in peer-reviewed journal articles, as well as with various stakeholders, including pig producers, extension personnel, undergraduate and graduate students through classroom instruction. Graduate students conducting research for their MS and PhD degrees are also a significant part of this audience. Changes/Problems:1.Unforeseen Technical Challenges: we encountered unforeseen technical challenges related to cell culture room contamination. The poor facility conditions in the building led to a persistent contamination issue, significantly impacting our ability to conduct crucial experiments involving cell cultures. Despite rigorous sterilization efforts, the contamination persisted, necessitating extensive decontamination procedures and, in some cases, the repetition of experiments. Although this challenge has already been solved, it delays our project timeline. 2.Pandemic-related disruptions: The post-effect of COVID-19 pandemic further cause a worldwide shortage of manufacturing. For instance, the 96 well PCR plates were backordered for almost 4 months, and the delivery of equipment(s) we proposed to buy and budgeted in the grant was delayed by 9 months, thereby further delaying our project timeline. What opportunities for training and professional development has the project provided?Three graduate students and three undergraduate students received training through their research experience and collaboration with fellow scientists. Each participant developed professionally, which included presentations at scientific meetings by the graduate students at local, state, national, and international levels. How have the results been disseminated to communities of interest?The results of the research have been disseminated to the scientific community through publications in peer-reviewed papers and presentations at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals?We will conduct experiments to determine the impact of ADM on gene expression of pTr2 cells required for elongation and implantation. We will conduct the experiments to determine whether perturbation of CALCRL expression compromises elongation of porcine conceptus Tr in vivo. In addition, we will perform metabolomic profiling for understanding the uterine environment during the peri-implantation period of pregnancy in pigs.

Impacts
What was accomplished under these goals? 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). Given that the MTOR inhibitor rapamycin at 50 nM abrogated ADM-derived proliferative effects on pTr2 cells (Paudel et al. 2021), we then used rapamycin at the same dosage to evaluate whether the effects of ADM on migration and adhesion of pTr2 cells are also due to activation of MTOR. After 12 h incubation, exogenous ADM at 10-7 M increased (P<0.05) pTr2 cell migration by 1.3-fold in ADM group as compared to the nontreated control. This ADM-derived migration of pTr2 cells were abrogated (P<0.05) by 37% via inhibition of MTOR using rapamycin (rapamycin+ADM versus ADM). Interestingly, as compared to the nontreated control, rapamycin at 50 nM decreased (P<0.05) migration of pTr2 cells by 31% and 22% with or without exogenous ADM at 10-7 M, respectively. In addition, exogenous ADM at 10-7 M increased (P<0.05) the number of adherent pTr2 cells by 1.2-fold as compared to the nontreated control after 2 h incubation; whereas rapamycin reduced (P<0.05) such ADM-driven attachment of pTr2 cells by 25% (rapamycin+ADM versus ADM). Likewise, as compared to the nontreated control, rapamycin at 50 nM with or without exogenous ADM at 10-7 M decreased (P<0.05) the number of adherent pTr2 cells by 14%. To further investigate whether and how ADM activates MTOR, particularly MTORC1 cell signaling pathway, we determined the protein expressions of total (t-) and phosphorylated (p-) MTOR, 4EBP1, P70S6K, S6, TSC2, and AKT in pTr2 cells at 30 and 60 min of ADM incubation using siRNA-mediated knockdown and Western blot analyses. After siRNA transfection and additional ADM treatment, the relative phosphorylation of MTOR (p-MTOR/t-MTOR) decreased (P<0.05) by 39% and 80% in siCALCRL-treated pTr2 cells as compared to siNTC at 30 and 60 min of ADM incubation, respectively. As the direct downstream effectors of MTORC1, the relative phosphorylation of 4EBP1, P70S6K and S6 (target of P70S6K) were further determined. The p-4EBP1/t-4EBP1 ratio decreased (P<0.05) by 27% and 56% in siCALCRL-treated pTr2 cells at 30 and 60 min of ADM incubation, respectively; but increased (P<0.05) by 1.5-fold in siNTC controls between 30 and 60 min of ADM incubation. Likewise, the p-P70S6K/t-P70S6K ratio decreased (P<0.05) by 79% and 86% in siCALCRL-treated pTr2 cells at 30 and 60 min of ADM incubation, respectively; but increased (P<0.05) by 1.6-fold in siNTC controls between 30 and 60 min of incubation with ADM. As the immediate target of P70S6K, the relative phosphorylation of S6 (p-S6/t-S6) decreased (P<0.05) by 39% and 43% in siCALCRL-treated pTr2 cells at 30 and 60 min of ADM incubation, respectively. Moreover, we determined the relative phosphorylation of two upstream regulators of MTORC1, TSC2 and AKT. The p-TSC2/t-TSC2 ratio decreased (P<0.05) by 49% and 46% in siCALCRL-treated pTr2 cells at 30 and 60 min of ADM incubation, respectively; whilst the p-AKT/t-AKT ratio decreased (P<0.05) by 79% and 89%. In addition, ADM increased (P<0.05) both p-TSC2/t-TSC2 and p-AKT/t-AKT by 1.6- and 2.3-folds, respectively, in siNTC controls between 30 and 60 min of incubation. 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.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Paudel S, Liu B, Cummings MJ, Wang X. Adrenomedullin: a novel peptide hormone for uterine receptivity and conceptus elongation in pigs? Program 2023 American Society of Animal Science Southern Section Annual Meeting (SSASAS 2023). January 21-24, 2023. Raleigh, NC. Abstract ID: 1366884 (Platform Presentation)
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Liu B, Paudel S, Flowers WL, Piedrahita JA, Wang X. Adrenomedullin stimulates proliferation, migration and adhesion of porcine trophectoderm cells via CALCRL-AKT-TSC2-MTORC1 cell signaling pathway. Program 2023 American Society of Animal Science Southern Section Annual Meeting (SSASAS 2023). January 21-24, 2023. Raleigh, NC. Abstract ID: 1367530 (Platform Presentation)
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Liu B, Duan L, Liu X, Wang X. Metabolomic analyses uncover stage-specific regulatory pathways in the porcine uterine fluids during peri-implantation period of pregnancy. Triangle Consortium for Reproductive Biology 31st Annual Meeting (TCRB 2023). February 25, 2023. East Carolina State University, Greenville, NC. Abstract No. P25 (Poster Presentation)
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Liu B, Duan L, Liu X, Wang X. Stage-specific metabolomic profiling of conceptus during the peri-implantation period of pregnancy in pigs. Program 2023 Society for the Study of Reproduction 56th Annual Meeting (SSR 2023). July 11-14, 2023. Ottawa, Canada. Abstract No. P46 (Poster Presentation)
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Liu B, Paudel S, Flowers WL, Piedrahita JA, Wang X. Uterine histotroph and conceptus development: III. Adrenomedullin stimulates proliferation, migration and adhesion of porcine trophectoderm cells via AKT-TSC2-MTOR cell signaling pathway. Amino Acids 2023; 55(6):743-756. PMID: 37036518
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Liu B, Duan L, Liu X, Wang X. Metabolomic analysis of uterine luminal fluid during the peri-implantation period of pregnancy in pigs. Program 2023 American Society of Animal Science Southern Section Annual Meeting (SSASAS 2023). January 21-24, 2023. Raleigh, NC. Abstract ID: 1367507 (Platform Presentation)

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:The target audience consists of the scientific community engaged in research to improve reproductive efficiency in livestock enterprises. The information generated from this research will be shared with the scientific community through publications in peer-reviewed journal articles, as well as with various stakeholders, including pig producers, extension personnel, undergraduate and graduate students through classroom instruction. Graduate students conducting research for their MS and PhD degrees are also a significant part of this audience. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students and three undergraduate students received training through their research experience and collaboration with fellow scientists. Each participant developed professionally, which included presentations at scientific meetings by the graduate students at local, state, national, and international levels. How have the results been disseminated to communities of interest?The results of the research have been disseminated to the scientific community through publications in peer-reviewed papers and presentations at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals?We will conduct experiments to determine if ADM stimulates proliferation, migration and adhesion of pTr2 cells via activation of CALCRL-induced MTORC1 cell signaling. In addition, we will determine the impact of ADM on gene expression of pTr2 cells required for elongation and implantation. We will also conduct the experiments to determine whether perturbation of CALCRL expression compromises elongation of porcine conceptus Tr in vivo. We will prepare one manuscript for publication of results from the experiments in 2022 on the effects of ADM on pTr2 cell behaviors.

Impacts
What was accomplished under these goals? The functional roles of ADM on proliferation, migration and adhesion of porcine trophectoderm (pTr2) cells in vitro were determined. We first characterized the phenotype of pTr2 cells via double immunofluorescence microscopy, which demonstrated positive staining for cytokeratin and negative staining for vimentin. The expression of KGFR, which is commonly found in porcine Tr and endometrial epithelial cells, and IFND, a marker of porcine Tr cells, was detected in pTr2 cells via RT-PCR analysis. Next, we evaluate the siRNA-mediated knockdown efficiencies of endogenous ADM and its receptor component CALCRL, the mRNA and protein levels of both targets were determined using qRT-PCR and Western blot analyses. The results showed that treatment of pTr2 cells with siADM resulted in a 47% decrease (P<0.01) in the mRNA and a 50% decrease (P<0.01) in the protein levels of ADM compared to negative control siNTC. Similarly, the mRNA and protein levels of CALCRL in pTr2 cells were reduced (P<0.01) by 88% and 58%, respectively, after treatment with siCALCRL. We determined that ADM stimulates proliferation of pTr2 cells via its receptor component CALCRL. Effects of ADM on pTr2 cell proliferation were confirmed using siRNA-mediated knockdown of endogenous ADM (siADM) and its receptor component CALCRL (siCALCRL). Compared with siNTC control, exogenous ADM at 10-7 M increased (P<0.05) pTr2 cell proliferation by 1.4-fold in siNTC+ADM group at 48 h. On the other hand, inhibition of endogenous ADM by siADM decreased (P<0.05) pTr2 cell proliferation by 33% and 29% in the absence (siADM versus siNTC) and presence (siADM+ADM versus siNTC+ADM) of exogenous ADM, respectively. When endogenous ADM was inhibited by siADM, exogenous ADM at 10-7 M simulated (P<0.05) pTr2 cell proliferation by 1.5-fold at 48 h (siADM+ADM versus siADM). Moreover, inhibition of CALCRL, the shared component of ADM receptors, abrogated the ADM-derived proliferation of pTr2 cells by 41% in siCALCRL+ADM group as compared to siNTC+ADM control. Next, we determined the effects of ADM on migration of pTr2 cells using a 12-h wound-healing assay coupled with siRNA-mediated knockdown. After 48 h transfection and additional 12 h ADM treatment, exogenous ADM at 10-7 M increased (P<0.05) pTr2 cell migration by 1.5-fold in siNTC+ADM group as compared to siNTC control. Inhibition of endogenous ADM by siADM decreased (P<0.05) pTr2 cell migration by 46% and 23% in the absence (siADM versus siNTC) and presence (siADM+ADM versus siNTC+ADM) of exogenous ADM, respectively. When endogenous ADM was inhibited by siADM, exogenous ADM at 10-7 M simulated (P<0.05) pTr2 cell migration by 2.1-fold at 12 h (siADM+ADM versus siADM). In addition, inhibition of CALCRL abrogated (P<0.05) the ADM-derived migration of pTr2 cells by 42% in siCALCRL+ADM group as compared to siNTC+ADM control. We then investigated the effects of ADM on attachment of pTr2 cells using siRNA-mediated knockdown. After 48 h transfection with siRNAs and 2 h attachment with or without exogenous ADM at 10-7 M, the number of adherent cells was increased (P<0.05) by 1.2-fold in both siNTC+ADM and siADM+ADM groups as compared to siNTC and siADM controls, respectively. Cell adhesion was not affected between siNTC and siADM groups regardless of the absence (siADM versus siNTC) and presence (siADM+ADM versus siNTC+ADM) of exogenous ADM. However, inhibition of CALCRL decreased (P<0.05) the ADM-derived attachment of pTr2 cells by 20% in siCALCRL+ADM as compared to siNTC+ADM. Together, these results suggest that ADM acts on its receptor component CALCRL to stimulate proliferation, migration and adhesion of pTr2 cells.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Liu B, Duan L, Liu X, Wang X. Metabolomic analysis of uterine luminal fluid during the peri-implantation period of pregnancy in pigs. Program 2022 Society for the Study of Reproduction 55th Annual Meeting (SSR 2022). July 26-29, 2022. Spokane, WA. Abstract No. P286 (Poster Presentation)
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Liu B, Paudel S, Cummings MJ, Wang X. Adrenomedullin stimulates proliferation, migration and adhesion of porcine trophectoderm cells via Akt-TSC2-MOTR cell signaling pathway. Program 2022 Triangle Consortium for Reproductive Biology 30th Annual Meeting (TCRB 2022). March 5, 2022. East Carolina State University, Greenville, NC. Abstract No. P16 (Poster Presentation)
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Liu B, Paudel S, Cummings MJ, Piedrahita JA, Wang X. Functional roles of adrenomedullin in conceptus development during peri-implantation period of pregnancy in pigs. Program 2022 NCSU Animal Science Research Poster Session. April 8, 2022. Raleigh, NC. (Poster Presentation)
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Liu B, Paudel S, Cummings MJ, Wang X. Adrenomedullin stimulates proliferation, migration and adhesion of porcine trophectoderm cells via CALCRL-AKT-TSC2-MTORC1 cell signaling pathway. Program 2022 Society for the Study of Reproduction 55th Annual Meeting (SSR 2022). July 26-29, 2022. Spokane, WA. Abstract No. P270 (Poster Presentation)