Role of Small RNAs in Male Infertility
PI: D. A. Paduch
Male infertility is common but our poor understanding of the biology of human fertility has limited options for diagnosis and therapy. Micro RNAs (miRNA) and small interfering RNAs (siRNAs) are key posttranscriptional regulators of mRNAs, with each miRNA capable of affecting the expression of multiple genes. Small RNAs have been identified in human testis, and hold promise not only in the development of new diagnostic tests for male infertility, but also in improved treatment options. Little is known, however, about the specific regulatory functions of miRNAs in human spermatogenesis. The leading hypothesis of this project is based on extensive preliminary results obtained by this group showing that 70% of miRNA expressed from human testis are highly conserved in humans and rodents. We hypothesize that differentially expressed miRNAs in men with infertility lead to changes in levels of target mRNAs involved in key regulatory pathways in cell biology. The current proposal aims to use RNA from men with normal spermatogensis and men with infertility for multiplexed deep sequencing to identify differentially expressed miRNAs, and to detect target mRNAs through correlation analysis of actual mRNA expression profiles obtained from the same patients. Using the same testis samples for both miRNA and mRNA profiling is a unique approach in the study of human reproduction and will overcome the poor reliability of in silico predictive algorithms to identify miRNA:mRNA pairs. Subsequent experiments will focus on a subset of conserved and X-linked miRNA that target genes known to be important in reproduction. Messenenger RNA profiles obtained from fertile and infertile patients will be used to experimentally identify critical miRNA:mRNA targets, and selected targets will be confirmed further in transgenic models as part of this U54. At the end of the proposed funding period, this work will have identified and confirmed a set of approximately 20-30 miRNA:mRNA interactions that are critical in male infertility, verified their target specificity in vitro, delineated their mechanisms of action, and assigned them to known regulatory pathways. This will lay a solid groundwork for the development of in vivo models, that can be used to test whether delivery of exogenous miRNAs, or their inhibitors, can rescue the infertile phenotype. This work will also provide insights for the development of RNA-based therapies for infertility and other testicular disorders and will lead to significant advancements in understanding of male reproduction.