Determining the role of sperm microRNAs in early development and non-genetic inheritance

ABSTRACT Historically, sperm were largely disregarded as contributors of non-genetic information due to their significantly reduced cytoplasmic contribution to the zygote relative to eggs. However, recent studies have demonstrated that sperm can transmit non-genetic information to offspring, thereby modulating inherited phenotypes. Specifically, the microRNA (miRNA) content of sperm is altered by changes to paternal diet and exposure to stress and various toxins. While several studies have causally demonstrated that the microinjection of specific sperm miRNAs can induce changes in offspring phenotypes, the molecular mechanism of action of sperm miRNAs in early development remains unknown. The primary functions of miRNAs are to downregulate messenger RNA (mRNA) transcripts via destabilization, decay, and translational repression. My preliminary data shows that a single miRNA has the ability to downregulate dozens of mRNA transcripts during early embryonic development. I hypothesize that sperm miRNAs play an important role in modulating early development by altering embryonic gene expression via the contribution of double-stranded miRNAs to the zygote during fertilization. To test this hypothesis, I will determine the effects of sperm miRNAs on early development by injecting miRNAs into mouse eggs chemically induced to develop in the absence of sperm (parthenotes). This system will allow me to study how a single component of sperm, miRNAs, mechanistically function during early embryonic development in the absence of all other contents of sperm. I will microinject parthenotes with individual sperm miRNAs upregulated upon stress exposure (Aim 1a) and important for embryonic development (Aim 1a) and quantitate the transcriptome throughout early development using single- embryo RNA-sequencing (RNA-seq) to determine how miRNAs regulate embryonic gene expression. In Aim 2a, I will selectively digest either single- or double-stranded sperm miRNAs to systematically analyze which sperm miRNAs are delivered to zygotes as duplexes using small RNA-seq. In Aim 2b, I will use small RNA- seq to probe whether sperm acquire precursor miRNAs during maturation. Duplex molecules will have increased stability and enhanced functionality to regulate embryonic gene expression due to their increased efficiency of loading into AGO. Together, my project will provide novel insight into the functions of sperm miRNAs by providing a comprehensive and mechanistic understanding of how they regulate early embryonic development.