Date of Award
Fall 2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Abbott, Allison
Second Advisor
Blumenthal, Edward
Third Advisor
Yang, Pinfen
Abstract
Gene regulation mediated by microRNAs (miRNAs) at the post-transcriptional level is important for producing functional sperm in diverse species including C. elegans. However, the specific regulatory roles of miRNAs in this process are largely unknown. The central goal of my dissertation is to address this question. I proposed to study the function of miRNAs enriched in male gonads, which are approximately 95% germ cells and 5% somatic cells. I isolated adult gonads of males and hermaphrodites for small RNA sequencing and the analysis revealed a differential miRNA expression profile between hermaphrodite and male gonads. 29 male gonad-enriched miRNAs have the potential to function in regulating spermatogenesis. Many of them had no available loss of function mutations. Therefore, I used CRISPR/Cas9 genome editing to generate miRNA mutants with single miRNA or a miRNA cluster deleted. To identify miRNAs that regulate sperm formation or function, male and hermaphrodite fertility in miRNA mutants were assessed by mating assays, brood size assays, and sperm quantification. None of the miRNA mutants had male sterility, suggesting that single miRNA or miRNA clusters are not required for sperm function. However, four miRNA mutants produced fewer spermatids, suggesting possible defects in spermatogenesis. Three of these miRNA mutants also had lower brood size in hermaphrodites, likely due to defects in spermatogenesis. Genetic analysis of multiple miRNA mutants suggested interaction among these miRNAs. Further analysis suggested that meiotic progression defects in these mutants may account for the observed decreased sperm production. Computational analysis was used to generate miRNA-target regulatory network, which revealed potential targets for further study, suggesting complex genetic miRNA interaction to regulate germ cell development and differentiation during spermatogenesis. Taken together, this study furthered understanding of specific roles of miRNA in the male germline and somatic gonad that promote male fertility and fecundity.