Are Full-Length mRNA In Bos taurus Spermatozoa Transferred to the Oocyte During Fertilization?

Abstract

This thesis focuses on the discovery of full-length mRNA transcripts in Bos taurus spermatozoa. The primary aim of this study is to identify and validate full-length mRNA primarily from RNA-Sequencing of bovine spermatozoa. The secondary aim is to determine if full-length spermatozoal transcripts are delivered to the oocyte at fertlilization, allowing for future studies to track their inheritance from paternal sources to the embryo. The main hypothesis of this thesis is that full-length mRNA transcripts exist within the spermatozoal transcript profile in Bos taurus. The secondary hypothesis is that if spermatozoal mRNA is functional after fertilization, then full-length transcripts should be present in the early stage embryo. To examine these hypotheses, this thesis is divided into three main chapters. The first is a literature review, discussing the process of spermatogenesis, the unique properties of spermatozoal mRNAs, including some hypothesized functions of spermatozoal mRNAs. A summary of a new technique, RNA-Sequencing, will be discussed in this review as well as comparisons to previous literature techniques for identifying mRNA transcripts of interest. The second chapter is the manuscript published in the journal Biology of Reproduction in January 2013, co-first-authored by Christopher Card. This manuscript uses the technique RNA-Seq to examine the transcript profile of nine Bos taurus bulls, and highlights several transcripts of interest for further study. This study found 6,166 total transcripts, and performed Gene Ontology analysis of the transcripts to categorize them into functional categories for further examination, the top most category of interest being translation. The third chapter of this thesis is a manuscript in preparation, formatted for submission to the journal of Molecular Reproduction and Development. This manuscript evaluates twenty four target mRNA transcripts to see if they are full-length. These transcripts were identified through four main methods: their location on the Y chromosome, their high expression in the RNA-Seq data set from chapter 2, their presence in Gene Ontology categories of interest from chapter 2, and their discovery from previous literature studies. Sixteen transcripts are found to be full-length, eight are degraded, and four have alternative polyadenylation ends. In conclusion, several full-length transcripts were found in this study, which have the potential to create functional proteins downstream in the fertilized oocyte. Several transcripts were also proved to be degraded in the mature spermatozoa. This has confirmed the need for this type of study, and elucidates new transcript targets for further research to pursue.