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The Piwi Pathway in Bovine Gametes and Preimplantation Embryos

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Title: The Piwi Pathway in Bovine Gametes and Preimplantation Embryos
Author: Russell, Stewart
Department: Department of Biomedical Sciences
Program: Biomedical Sciences
Advisor: LaMarre, Jonathan
Abstract: Epigenetic reprogramming is required during both embryo and gamete development to restore the potential for cellular pluripotency. Changes in DNA methylation during reprogramming can allow the expression of integrated endogenous transposable elements (TEs) that may lead to genomic instability should widespread reintegration occur. Small RNA mediated mechanisms have therefore evolved to constrain TE expression during reprogramming. PIWI proteins are a subfamily of the Argonaute clade of proteins which have functions in small RNA (sRNA) mediated TE and gene silencing. PIWI proteins direct silencing through RNA-mediated interactions with complementary nucleic acid targets. TE expression has been documented in early bovine embryos, although mechanisms regulating their expression are not widely studied. This period is further complicated by extensive targeted decay of maternally-derived mRNAs which may utilize related mechanisms of gene silencing. Based on these observations, it was hypothesized that the PIWI pathway is present in bovine gametes and embryos, and regulates endogenous TEs and mRNAs during reprogramming. PIWIL1 expression was characterized in bovine gonads, oocytes, and embryos. The testis-specific expression of full-length PIWIL1 showed localization to spermatocytes and spermatids, and association with piRNAs of 29-32 nucleotides in length. Additionally, two shorter, 3ʹ truncated PIWIL1 transcript isoforms were found to be expressed in testis, oocytes and embryos, and showed dynamic regulation throughout embryogenesis. Full-length PIWIL2 and PIWIL3 transcripts were also quantified throughout embryogenesis. To address potential roles for the PIWI pathway, sRNAs from oocytes and zygotes were profiled through next-generation sequencing and compared to those from testis, ovary and sperm. An analysis “pipeline” was developed to identify putative piRNAs from the sequencing libraries. The piRNA-“like” RNAs (pilRNAs) were characterized based on their size and canonical biogenesis features and were abundant in oocytes and embryos. When mapped to bovine repeats, up to one third of embryonic pilRNAs targeted TE classes including LINEs, SINEs, and ERVs. Through comparisons with published embryonic mRNA transcriptomes this analysis also revealed the potential for maternally-deposited, zygotic pilRNAs to direct mRNA degradation. These data provide one of the first characterizations of the PIWI pathway outside of classical models, and provide evidence for novel functions in embryo TE and gene regulation.
Date: 2016-08
Rights: Attribution-NonCommercial-NoDerivs 2.5 Canada

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Attribution-NonCommercial-NoDerivs 2.5 Canada Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivs 2.5 Canada