Characterization of Spermatozoal Small Non-coding RNAs using Functional Genomics and Immunocytochemistry

Date of Award

2013

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Clore, Amy

Keywords

Immunocytochemistry, Small Non-Coding RNAs, Functional Genomics

Area of Concentration

Biology

Abstract

Fertilization is the cornerstone of early mammalian development which leads to formation of zygote and then embryo. In addition to providing half of the zygotic genome, the sperm also contributes macromolecules such as small non-coding RNAs (sncRNAs) into the egg. However, identities of these paternal sncRNAs, as well as molecular and cellular mechanisms by which these molecules regulate embryonic development, are not well understood. This gap in the knowledge base is an important problem because it is preventing advancement in developmental biology and reproductive biotechnology. The central hypothesis is that sperm contain diverse sets of sncRNAs, including microRNAs (miRNAs) that play vital roles in embryonic genome activation at the onset of mammalian development. The overall approach was a comprehensive literature review followed by experiments using miRNA microarray to validate the expression of a series of miRNAs in sperm from bulls of high and low fertility. For my portion of this larger project, immunocytochemistry was employed for the examination of PIWI proteins and AGO2 protein in mature sperm and RNA isolation and bioinformatics was used to survey differences in miRNA expression between high and low fertility bull samples. Overall the results showed that: 1) sperm contain ample amounts of sncRNAs, including miRNAs, that may be essential for embryo development 2) proteins that bind to the sncRNAs, such as PIWIL1, PIWIL2 and AGO2, are well-conserved across mammals and appear to be present in the mature bull sperm. The findings may ultimately be important for better understanding of dynamic molecular and events in sperm and embryos that are critical for mammalian development, and for developing reproductive technologies such as biomarkers for predicting male fertility.

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