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%
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.