Performing Department
Biomedical Sciences
Non Technical Summary
In livestock production, altered fetal growth can have significant negative impacts on the survival of the newborn in addition to many long-term consequences, such as smaller offspring, poor feedlot performance and fertility, and reduced meat quality. A common category of altered fetal growth that can create these problems is IUGR: intrauterine growth restriction. IUGR has directly resulted in pregnancies with higher incidences of newborn deaths and/or problems with the consequences already mentioned in addition to functional placental insufficiency. The placenta is responsible for exchange of nutrients (such as glucose and amino acids) between the fetus and the dam, in addition to transporting oxygen, other gases and waste products. As such, the placenta is an important regulator of fetal growth. Additionally, glucose is the primary energy source for both the placenta and the fetus to grow during pregnancy. Our lab is therefore interested in investigating whether a lack of placental exchange of glucose between the fetus and the dam would result in an IUGR pregnancy. Understanding the mechanisms of how glucose transport across the placenta could be used in the future for potential therapies in compromised pregnancies.Two major glucose transporters in the placenta are SLC2A1 and SLC2A3. To address the relative importance of both, our lab has developed SLC2A1 and SLC2A3 deficient pregnancies using a comprehensive gene-targeting approach. This proposal aims to test the central hypothesis that that SLC2A1 deficiency will have a greater impact on fetal growth than does SLC2A3 deficiency. To test this hypothesis, we propose two specific aims: Specific Aim 1 will generate, validate and use SLC2A1 deficient pregnancies for harvesting at 75 days of gestation for analysis and testing whether SLC2A1 deficiency results in IUGR at mid-gestation. Specific Aim 2 will compare the impacts of SLC2A1 and SLC2A3 deficiencies at mid-gestation using various analytical methods, including investigating the genetics of placental and fetal metabolism pathways affected (RNAseq). This proposal is significant because it addresses major knowledge gaps in understanding mechanisms and genes involved with placental insufficiency. Given the critical need for adequate nutrition during pregnancy and how large a role glucose transport plays in that, is work will expand our knowledge of IUGR pathology and its impacts on placental and fetal metabolism. This proposal will advance the field by providing insights into how we may improve IUGR pregnancy outcomes and fetal success in the future. This project is highly responsive to the goals of AFRI Education and Workshop Development as it supports the goal: 1): satisfy human food and fiber needs; as many livestock succumb to morbidities and mortalities brought about by IUGR pregnancies which reduce red meat production. By addressing current gaps in knowledge regarding the pathology of IUGR with this proposed research, we will support development of future therapeutic approaches to ameliorating and/or potentially rescuing at-risk offspring so more healthy animals can enter production. Improvement of offspring survival and lifelong health (and therefore productivity) also directly addresses the goal 4): sustain the economic viability of farm operations; which will allow the producer more return on their investment by improving herd survival. We are also addressing the goal 5): enhance the quality of life for farmers and society as a whole; by contributing to food securitythrough improved productivity and survival of livestock. Finally, this proposal is responsive to both the Animal Nutrition, Growth and Lactation (A1231) and AnimalReproduction (A1211) Program Areas, since successful fetal growth is so tightly interconnected with postnatal survival, growth, and production traits. Lastly, my specific aims are also highly responsive to the Predoctoral Fellowship Program Area (A7101), in that they outline a paradigm for comprehensive mentorship, implementation of goal-driven evaluation plans, and foster my development as a part of the next generation of research scientists. The proposed study will also support my education goals during my PhD program which include gaining both project leadership and technical skills in my field of interest. These experiences will contribute to our field through scientific presentations, peer-reviewed manuscripts, but also translational knowledge for the producer, as well as enable me to eventually develop my own high-impact research program in the future.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Placental mediation of fetal growth and development is a critical component of offspring performance, both in terms of fertility and end products (such as meat or fiber). Placental insufficiency in livestock can have deleterious effects on animal productivity. While impairments cannot yet be reprogrammed, therapeutic strategies that can be implemented in utero have powerful potential. As such, a better understanding of placental insufficiency, and itseffects on placental metabolism, provides one of the first stepping stones to therapeutic applications. A key player in placental metabolism (and influencer on fetal growth) is glucose, a significant amount of which is transported across the placenta by key transportersSLC2A1 andSLC2A3.Dr. Anthony's laboratory has already employed lentiviral-mediated RNAi to produce SLC2A3-deficient pregnancies in sheep with success.Thus, I propose to utilize our lab's model of in-vivo lentiviral mediated RNA interferenceto generate and study placenta-specific SLC2A1-deficient sheep pregnancies. This will be followed by in vitro analysis to examine SLC2A1 deficiency on placental metabolic pathways, and finally RNAseq analysis to compare transcriptionalimpacts of bothSLC2A1 andSLC2A3 deficient pregnancies on placental metabolism.My research goalis therefore to demonstrate the necessity of glucose uptake/utilization/transfer in the context of location of transporters on the placenta. This includes comparison of SLC2A1 (fetal side) and SLC2A3 (maternal side) glucose transporters, with the aim of divulging whichtransporter is more critical to placental and fetal metabolism. Understanding these relationshipswill have direct implications for the development of better maternal nutrition management strategies for livestock producers.To address this goal,we will generate placenta-specific SLC2A1-deficient pregnancies, using lentiviral-mediated RNAi in sheep, to assess the relative importance of this glucose transporter prior to mid-gestation (d 75 gestational age; GA). This will allow us to compare phenotypic outcomes with the SLC2A3-deficient pregnancies, and to assess the relative importance of the two primary facilitative glucose transporters found in the ruminant placenta. In this application, two Specific Aims are proposed:Specific Aim 1will generate, validate and useSLC2A1 RNAi lentivirus to create SLC2A1-deficient pregnancies, which will be harvested at 75 dGA for analysis; andSpecific Aim 2will use RNAseq, qPCR, Western blotting and immunohistochemistry to assess the relative impact of SLC2A1 deficiency versus SLC2A3 deficiency, compared to SC (scramble control) RNAi placenta, on placental metabolic pathways.While we predict that both transporters are required for normal fetal growth,thecentral hypothesisof this application is that SLC2A1 deficiency will have a greater impact on fetal growth than does SLC2A3 deficiency. This hypothesis is supported byin vivoassessment of placental glucose uptake/utilization/transfer, demonstrating that the glucose concentration in the umbilical circulation was the primary factor in determining the rate of glucose transfer to the fetus.It ismylong-term goalto improve pregnancy by interrogating the relationship between placental function and fetal development, and apply the knowledge gained to positively impact pregnancy and postnatal performance of livestock. These research goals will also result in presentations at scientific meetings and peer-reviewed literature.
Project Methods
All tissues used for analysis in this proposal will be collected fromin vivoexperiments (NIH R21 HD094952), focusing on the ramifications of placental glucose transporter deficiencies (SLC2A1 and SLC2A3) in sheep. Briefly, thein vivostudy will utilize lentiviral-mediated RNAi to directly diminish placental glucose transporter expression through infection of the trophectoderm cells of expanded sheep blastocysts. Development, production and surgical transfer of single blastocysts infected with lentiviral constructs will be accomplished as extensively described by our laboratory, and has already been used to generate the SLC2A3-deficient pregnancies. All tissues and virus will be handled following BSL2 guidelines, as per our existing Institutional Biosafety protocol (17-039B). The use of research animals for these purposes has been approved by the Colorado State University IACUC (KP 147). After confirmation of pregnancy at 50 days of gestation (dGA), fetal growth and umbilical velocimetry will be assessed at 70 dGA by Doppler ultrasonography. At 75 dGA, terminal surgeries will be performed to harvest uterine artery and vein, as well as umbilical artery and vein blood samples simultaneously, followed by euthanasia of the ewe and fetus, and tissue collection.Specific Aim 1:To generate, validate and utilize SLC2A1 RNAi lentivirus to create SLC2A1-deficient pregnancies, and determine the impact of placental SLC2A1 deficiency at mid-gestation.Approach:Currently, our laboratory is testing four different sheep SLC2A1 RNAi constructs, utilizing sheep cells engineered to overexpress SLC2A1, to determine the most efficacious construct. The most effective SLC2A1 RNAi lentivirus will then be used to generate SLC2A1-deficient placenta, and resulting control (SC RNAi) and SLC2A1 RNAi pregnancies will be harvested at mid-gestation (75 dGA). The pregnancies will be generated using established techniquesin our laboratory, and described briefly above.Tissue collection:At d75 of gestational age, ewes (SLC2A1 n = 8, SC n = 8) will undergo terminal surgery to collect blood and tissue samples. Blood samples will be collected from both umbilical and uterine arteries and veins. The entire pregnant uterus will be collected and weighed, followed by dissecting out placentomes for counting and weighing, and finally separation of maternal caruncle and fetal cotyledon portions. Samples of fetal (cotyledonary) and maternal (caruncular) tissue will be snap frozen in liquid nitrogen and stored at -80C. Three representative placentomes per ewe will be selected for morphological and immunochemical analysis following fixation. The fetus will be weighed and fetal measurements of crown-rump-length, head circumference, abdominal circumference, and femur and tibia length will be recorded to compare to previous ultrasonographic measures on 70 dGA. Fetal liver and pancreas will also be dissected out for weighing and preservation.In vitroanalysis:Uterine and umbilical arterial and venous blood samples will be collected and analyzed for glucose, ghrelin, glucagon, lactate, IGF1, insulin, progesterone, and amino acids. Placental expression of SLC2A1 and SLC2A3 will be examined by real-time (RT) qPCR and Western blot analysis in order to confirm successfulin vivodepletion of SLC2A1. Real-time qPCR will be performed on select tissues (fetal liver, pancreas, placenta) to explore expression of metabolic factors and their receptors (such as insulin, IGFs and IGFBPs, and amino acid transporter systems A, L and y+; 13). Finally, histomorphometric and immunohistochemical analysis of fixed placentomes will be performed to examine placental expression of SLC2A1. These analyses will jointly aid in our understanding of the degree of our SLC2A1 knockdown and its effects on fetal metabolism.Anticipated Pitfalls, results and alternative approaches:We do not anticipate any technical pitfalls in the generation and use of lentiviral-mediated RNA interferencein vivo, due to our past experience with all of the techniques involved. It is possible that a 50% or greater reduction in SLC2A1 could result in pregnancy loss before the 75 dGA endpoint, but we believe that to be unlikely based on our SLC2A3 results. Monitoring pregnancy establishment and maintenance via estrus detection and ultrasound assessment of pregnancy viabilityshould allow us to shift the endpoint if needed. However, attainment of a notable phenotype in our SLC2A3 ovine pregnancies that were carried out without complication to the same gestational age gives us confidence that our SLC2A1 model will be attained with minimal losses. The possibility exists of a successful knockdownin vivowithout overt phenotypic differences, such as in fetal measures. However, this still highlights the importance of our other analyses (qPCR, Western-blot, RNAseq) of blood and tissues to determine the true effects of SLC2A1-deficient pregnancies.Efforts & Evaluationfrom Specific Aim 1 will result in the presentation of findings at scientific meeting platforms and the drafting of manuscripts for submission to peer review journals which are milestones for project success.Specific Aim 2:To examine changes in the placental transcriptome resulting from SLC2A1 and SLC2A3 RNAi, as compared to controls (SC RNAi).Samples:Placental (fetal cotyledon) tissues harvested from Specific Aim 1, and those harvested from the SLC2A3-deficient pregnancies will be used for RNAseq and bioinformatic analysis. RNAseq will be conducted on SLC2A1 RNAi (n=5), SLC2A3 (n=5) and SC RNAi (n=6; 3 from the SLC2A1 cohort and 3 from the SLC2A3 cohort) samples as briefly described below.Library preparation, RNA sequencing and bioinformatics analysis:RNA will be extracted from fetal portions of placental tissues (cotyledons) using a RNeasy Mini Kit (Ref# 74104; QIAGEN, Hilden, Germany). Samples will then be submitted to the University of Colorado Genomics and Microarray Core (Aurora, CO) for sequencing. RNA will be assessed for quality by the Agilent TapeStation. Individual libraries will be constructed using NuGEN mRNA library prep kit (cat# 0508; NuGEN Technologies, Inc., Redwood City, CA). Samples will be analyzed on the Illumina NovaSEQ6000 platform (University of Colorado Anschutz Medical Campus Research Core Facilities; Aurora, CO). Raw sequence reads will be aligned to the reference ovine and bovine genomes (12, 14) using HISAT2 version 2.2.0 (23) and DESeq2 to perform the differential expression analysis. Networks and functional analysis will be performed using Ingenuity Pathway Analysis software (IPA; Qiagen, Redwood City, CA) and IPathwayGuide (Advaita Bio; Plymouth, MI; 24).In vitroanalysis:After transcriptome and bioinformatic analysis, we expect to potentially identify up to 30 genes that could be critically impacted by SLC2A1 and SLC2A3 deficiency, which we would like to validate with further real-time qPCR and Western blot analysis.Anticipated Pitfalls, results and alternative approaches:One challenge that may occuris that the sheep genome is not well annotated, which may make RNA-seq data analysis and alignment difficult. However, our lab's experience alongside the collaboration of Dr. Cushman (see letter of collaboration), as well as the potential for approaching this possible hurdle by aligning to both the ovine and bovine genome, will aid us in overcoming the issue. Bovine genome annotations will therefore help to strengthen gene loci identification and improve our analysis.Efforts & Evaluationfrom Specific Aim 2 will result in the presentation of findings at scientific meeting platforms and the drafting of manuscripts for submission to peer review journals which are milestones for project success. Additionally,open access will be provided to the new pathways and/or models derived from the bioinformatic data. Finally, this specific Aim should teach the PD how to manage large datasets and develop new collaborations.