Date of Award:

8-2020

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Animal, Dairy, and Veterinary Sciences

Committee Chair(s)

Abby D. Benninghoff

Committee

Abby D. Benninghoff

Committee

Kenneth L. White

Committee

S. Clay Isom

Committee

Irina A. Polejaeva

Committee

Gregory J. Podgorski

Abstract

Cloning animals using somatic cell nuclear transfer (scNT) was first successfully demonstrated with the birth of Dolly the sheep, but the process of cloning remains highly inefficient. By improving our understanding of the errors that may occur during cloned cattle embryo development, we could obtain a greater understanding of how specific molecular events contribute to successful development. The central dogma of biology refers to the process of DNA being transcribed into messenger RNA (mRNA) and the translation of mRNA into proteins, which ultimately carry out the functions encoded by genes. The epigenetic code is defined as the array of chemical modifications, or "marks", to DNA molecules that do not change the genome sequence but do allow for control of gene expression. During early development, genome reprogramming involves the removal of epigenetic marks from the sperm and egg and re-establishment of marks for the embryonic genome that code for proper gene expression to support embryo development. The point during this process at which the embryo's genes are turned on is known as embryonic genome activation (EGA). Small non-coding RNAs (sncRNAs), including microRNAs (miRNAs), may also contribute to the this process. For example, miRNA molecules do not code for proteins themselves, but rather bind to mRNAs and effectively block their translation into protein. We hypothesized that aberrant expression of sncRNAs in cloned embryos may lead to anomalous abundance of mRNA molecules, thus explaining poor development of cloned embryos. First, we used RNA sequencing to examine the total population of sncRNAs in cattle embryos produced by in vitro fertilization (IVF) and found a dramatic shift in populations at the EGA. Next, we collected both sncRNA and mRNA from scNT cattle embryos, and again performed sequencing of both RNA fractions. We found that few sncRNAs were abnormally expressed in scNT embryos, with all differences appearing after EGA at the morula developmental stage. However, notable differences in the populations of sncRNAs were evident when comparing embryos by developmental stage. For populations of mRNA, we observed dramatic differences when comparing scNT and IVF cattle embryos, with the highest number of changes occurring at the EGA (8-cell stage) and after (morula stage). While changes in specific miRNA molecules (miR-34a and miR-345) were negatively correlated with some of their predicted target mRNAs, this pattern was not widespread as would be expected if these sncRNAs are functionally binding to all of the predicted mRNA targets. Collectively, our observations suggest that other mechanisms leading to altered expression of mRNA in cloned embryos may be responsible for their relatively poor development.

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