Date of Graduation
Davis College of Agriculture, Natural Resources and Design
Agricultural and Resource Economics
P Brett Kenney
Reproductive development in female teleosts like rainbow trout starts with release of the sex steroid estrogen, necessary for synthesis of the egg yolk protein vitellogenin. Increase in estrogen secretion during these reproductive phases results in increased energy demands. Skeletal muscle serves as an immediate endogenous source to address energy demands during vitellogenesis and spawning due to which it undergoes rapid degradation resulting in water accumulation, poor fillet and low egg quality. An imbalance in protein turnover is observed not only because of rapid increase in degradative pathways but also due to decreased protein synthesis. These changes in protein metabolism in skeletal muscle were identified within 24 hours of E2 exposure and confirmed in both in vivo and in vitro models. Normally during adult myogenesis myogenic precursor cells proliferate and differentiate further to form a mature muscle fiber resulting in either hyperplasty or hypertrophy or both in fishes. Signaling pathways and MRFs need to function in coordination with epigenetic factors and non-coding RNAs to positively or negatively regulate muscle synthesis. Interestingly, estrogen also influences the expression of miRNA and in turn their target genes. However, the effects of estrogen regulating protein imbalance in rainbow trout skeletal muscle is not completely understood. Hence studies to understand the influence of estrogen were carried out using juvenile, sexually immature fish. Juvenile rainbow trout were treated with either estrogen or vehicle and their skeletal muscle collected after 24 hours and 72 hours of treatment to understand the role of miRNA and DNA methylation in regulation of myogenesis. Findings from the miRNA study revealed diluted effects of estrogen after 72 hours of treatment, hence further studies were carried out with samples from 24-hour post treatment.;miRNAs play a crucial role in regulation of gene expression along with epigenetics. These are small non-coding RNAs with length ranging from 21--23bp mostly functioning as post-transcriptional regulators of gene expression. Generally, they bind to the 3' region of transcribed mRNA with a complementary region called seed sequence. These genes are called target genes of miRNA. A single miRNA can target more than one mRNA. Binding of miRNA to its target gene results in either translational repression or deadenylation or both. Deadenylation results in loss of RNA stability, which leads to its degradation. miRNAs express in tissue specific manner and those that express specifically in muscle are called myomiRNA. Various myomiRNA have been identified and studied for their functions. Estrogen influence the expression of miRNA and in turn their target genes. Therefore, rainbow trout skeletal muscle samples were sequenced for small RNAs to understand the effects of estrogen. Differentially expressed miRNA were identified in estrogen treated samples when compared to control. A total of 36 miRNAs were either upregulated or down regulated in E2 exposed skeletal muscle of which two were novel. Target genes of these differentially expressed miRNAs were identified followed by gene ontology enrichment. These target genes are involved in various biological and molecular functions including their role in signaling pathways, cell cycle, DNA methylation, signal transduction and transcription factor binding. Genes regulated by miRNAs were also involved specifically in proliferation and differentiation of myogenic precursor cells and degradative pathways. E2 induced expression of miR-17 and miR-20, which are important in regulation of cell cycle. Further gene expression analysis of myogeneic regulatory factor, Pax7 specific to MPCs confirms increase in their number with decreased expression of MyoD, specific to differentiation. Similarly, miR-23a revealed its role in maintenance of mitochondrial outer membrane permeability and post-transcriptional regulation of atrophic genes. Decreased expression of miR-23a with increase in permeability was supported by increased gene expressions including fbxo32, caspases 3a and 9 that are involved in atrophy and apoptosis respectively. Expression analysis of one of the novel genes, miR-nov-285, was performed to know its expression pattern in different tissues, highest expression was observed in testis. C-5 methylation of cytosine was one of the GO term that was enriched during the target gene analysis of this novel miRNA. Further regulation of Dnmt1 gene expression by this novel miRNA was confirmed by luciferase assay. All together these observations indicate that E2 influences differential expression of various miRNAs which in turn regulate gene expression contributing to muscle metabolism.;Reduced expression of MyoD was observed in our previous study and direct regulation of this differentiation factor was not observed by any of the differentially expressed miRNA. Since gene expression is also regulated transcriptionally by epigenetic mechanisms including DNA methylation, efforts were made to understand its role in reduced MyoD expression. Generally, DNA methylation reduces gene expression by directly blocking transcription factor binding or by recruiting other epigenetic contributors like histone deacetylases or histone methyltransferases. (Abstract shortened by ProQuest.).
Koganti, Prasanthi P., "Comprehensive Analyses of miRNA and DNA Methylation in 17beta-Estradiol Exposed Juvenile Rainbow Trout Skeletal Muscle" (2017). Graduate Theses, Dissertations, and Problem Reports. 5998.