Performing Department
(N/A)
Non Technical Summary
It is the goal of animal scientists to improve the efficiency at which agriculturally-important animals produce products useful for man. Most commonly, enhanced production has been achieved through a combination of two strategies: selection of genes that are optimal for production and provision of a postnatal environment that maximizes the opportunity for those genes to be expressed. There is a third strategy that is only now being explored - regulation of the processes of prenatal development to produce a neonate poised for optimal production. Developmental programming is the phenomenon whereby changes in the environment of the developing organism or of the gametes from which it is derived reprograms aspects of development to modify postnatal phenotype. Exploitation of the phenomenon of developmental programming offers new opportunities for enhancing animal growthwhile also avoiding some negative perceptions by consumers of some other methods to increase muscle growth (e.g., growth promotants that could be considered as hormones by consumers).While the concept of developmental programming is well established, strategies to use this phenomenon to enhance animal production are poorly developed. We still do not understand how specific components of the maternalenvironment act on the developing embryo or fetus to program characteristics of the animal after birth. Here we will do experiemnts to distinguish between two potential mechanisms. One possibility is that choline induces epigenetic changes in DNA or histones that persist into fetal and postnatal life and cause changes in somatic growth. Another possibility is that choline acts to modify processes involved in lineage formation affect organ formation and postnatal growth. To evaluate the first possibility, we will test whether there are aa set of epigenetic marks modified by choline that persist into fetal and postnatal life To test the second possibility, we will test whetther choline causes changes in the distribution of the day 21 embryo into specific cell lineages.Gaining an increased knowledge of how a molecule like choline programs postnatal development could provide insights that inform other strategies for programming.One pertinent question is whether it is the changes in the epigenome of the preimplantation period that drive actions of choline on postnatal phenotype or whether other modifications induced by choline are important. Understanding how the maternal environment of the preimplantation embryo affects its development in the short- and long-term makes the proposed research directly relevant to the goal of the Animal Reproduction Program to study embryonic and fetal development including interaction between the conceptus and its uterine environment.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
The long-term goal of the proposed research is to develop methods to program postnatal growth through manipulation of the environment of the preimplantation embryo. The goal of the current proposal is to understand how one programming agent, choline, acts on the bovine preimplantation embryo to modify postnatal growth. One possibility is that choline induces epigenetic changes in DNA or histones that persist into fetal and postnatal life and cause changes in somatic growth. Another possibility is that choline acts to modify processes involved in lineage formation affect organ formation and postnatal growth. Both of these hypotheses will be tested in the current proposal. There are three objectives:Characterize actions of choline to change the epigenetic landscape of the inner cell mass and trophectoderm of the bovine blastocyst with respect to DNA methylation, histone methylation and gene expression.Using skeletal muscle and liver as model tissues, test the hypothesis that epigenetic changes in DNA and histone methylation caused by preimplantation exposure of the embryo to choline persist through fetal and postnatal development.Employ single-cell RNA-Seq to test whether treatment of the preimplantation embryo with choline alters the relative numbers of various cell lineages in the embryo during gastrulation at Day 21 of development. ?
Project Methods
Objective 1:Characterize actions of choline to change the epigenetic landscape of the inner cell mass and trophectoderm of the bovine blastocyst with respect to DNA methylation, histone methylation and gene expression.Female embryos will be produced in vitro using oocytes harvested from Brahman cows by transvaginal ultrasound guided aspiration (oocyte pickup; OPU) that are fertilized with X-sorted semen using a single Brahman bull. Embryos will be cultured from the zygote to blastocyst stages in culture medium containing either vehicle (1.8 mM NaCl) or choline (1.8 mM extra choline chloride). Blastocysts will be graded for morphological quality using standards of the International Embryo Technology Society (Robertson and Nelson, 1998). Grade 1 blastocysts will be bisected into halves consisting of TE or ICM. Pools of ICM and TE will be separately prepared from 10 female blastocysts and used for transcriptome analysis, whole genome bisulfite sequencing for DNA methylation and CUT&RUN (TheCleavageUnderTargets andReleaseUsingNuclease method that builds upon ChIP-seq) approach to identify DNA associated with histone H3K4me3 (histone methylation associated with gene activation) and H3K27me3 (repressive histone mark). The resultant data will be analyzed to identify modifications caused by choline. Additional analyses will be conducted to determine the relationship between gene expression and each epigenetic feature and to evaluate how differentiation of the blastocyst into ICM and TE is associated with alterations in the transcriptome and epigenome.Objective 2:Using skeletal muscle and liver as model tissues, test the hypothesis that epigenetic changes in DNA and histone methylation caused by preimplantation exposure of the embryo to choline persist through fetal and postnatal development.Analysis of gene expression, DNA methylation and genomic regions associated with H3K4me3 and H3K27me3 will be assessed at four points of development - day 90 and 180 of gestation and day 90 and 180 of postnatal life. Brahman embryos will be produced in vitro in culture medium + 1.8 mM choline and grade 1 blastocysts transferred at the blastocyst stage into synchronized recipients. Fetal tissues will be collected after slaughter of the dam. Postnatal samples of skeletal muscle and liver will be collected by biopsy. All tissues will then be analyzed by RNA-Seq, whole genome bisulfite sequencing, and Chip-Seq using antibodies against H3K4me3 and H3K27me3. Changes caused by choline treatment will be identified and data will be assessed to determine whether there are a set of epigenetic marks that are conserved between stages of development including as early as the blastocyst period.Objective 3: Employ single-cell RNA-Seq to test whether treatment of the preimplantation embryo with choline alters the relative numbers of various cell lineages in the embryonic disc and trophoblast of the elongating embryo during gastrulation at Day 16 of development.Embryos produced in vitro in culture medium + 1.8 mM choline will be transferred to recipient females. After slaughter of recipients at day 21 of gestation, the embryo will be recovered manually from the uterus. The embryo proper and trophoblast will be separately subjected to single cell RNA-Seq to identify specific cell types and quantify their relative abundance in vehicle-treated and choline-treated embryos.