Candidate non-coding RNAs (miRNAs) and their Functional Role in Inflicting Male Infertility Kothandaraman Narasimhan, Ralf Henkel Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa Introduction: The objective of this study was to elucidate the relationship between regulatory roles of miRNAs and genes coding for key functions associated with male infertility (MI). Through computational and network analysis we identified new sets of miRNAs and associated genes that were unique for MI-related abnormalities. Molecular determinants leading to defective spermatogenesis and role of endocrine disruptors (EDCs) were also investigated. Fig. 5 Fig. 6 Material and Methods: Manual and computational tools based on Systems Biology approaches were used to create a comprehensive database for all the miRNAs and miRNA-regulated genes reported in studies associated with human MI. From a total of 133 studies we tabulated 49 miRNAs and 122 genes that have been reported as of March 2017. miRNA annotation tools such as miRBase, TAM were used for the current study. Standard miRNA enrichment analysis tools were used. Functional analysis was carried out using miRDB. Figure 5. miRNAs and genes associated with oxidative stress (OS)-induced male infertility were shortlisted. miRNAs do play a role in accelerating damage induced by OS in inflicting MI. Key processes involved in spermatogenesis such as gamete generation and abnormal spermatogenesis are targeted by overrepresented miRNAs in male infertility such as miR-34b, miR-499-3p, miR-499-5p, miR-146a and miR-34c-5p. Figure 6. Male infertility is affected due to a wide range of environmental factors. Seven miRNAs were identified to cause severe abnormalities due to defective spermatid development and differentiation and azoospermia. A major affected biological process includes spermatogenesis initiating from germ cell development and resulting in azoospermia possibly targeting cell division in the germ line cells by affecting genes MEI1 and ING2. Figure 7. Oxidative stress triggered miRNAs and their role in Male infertility. OS and reactive oxygen species (ROS) are causative factors leading to MI. Functional analysis identified OS and H2O2 metabolism triggered miRNAs and affected genes in MI. Key processes regulated by miRNAs include epithelial cell differentiation involved in prostate gland development involving NOTCH1 and PSAP genes. Prosaposin (PSAP) gene, an integral membrane protein with specific lysosome hydroxylase activities, has a direct role in cellular OS, prostate development and is regulated by more than one miRNAs (miR-301b, miR-454-3p, miR-130a-3p and miR-301a-3p). Fig. 7 Results: Table 1. Genes along with their regulatory miRNAs and affected biological functions is scored in the table. Figure 8. Environment-gene, miRNA and male infertility. Genes associated with MI due to EDCs were analysed for the role of miRNAs in mediating the expression pattern of candidate genes that were known to cause MI. Different classes of genes were found to be regulated by miRNAs previously reported to be associated with male MI. Some of the key genes affected by miRNAs include CYP family of genes involved in detoxification and steroid metabolism as well as MTHFR genes involved in methylation that is capable of altering the epigenome of male gametes. Fig. 8 Figure 1. Key interaction partners in miRNA–gene interaction in male infertility. Interaction network developed for genes and miRNA regulated genes by using 49 miRNAs that have been reported till now for MI. miRNAs that were found to play key roles in the pathogenic landscape of MI include miR-133b, miR-429, miR-449a, miR-375 and miR-564, respectively. Key interacting partners as well as interlinking genes are shown (big orange dots). Fig. 1 Figure 9. Endocrine Disrupting Compounds induced miRNAs and their role in sperm function. Three miRNAs, miR-7, miR-23b and miR-21 were reported for EDC-associated MI. Our analysis resulted in the elucidation of functional roles for these three miRNAs as they were found to be involved in cytoskeleton maintenance and motility. Two genes SEPT1 and HOOK2 were directly found to be involved in this process controlled by these three miRNAs. Fig. 9 Figure 2. miRNAs and their role in defective spermatogenesis. A whole spectrum of abnormalities were associated with the miRNAs and the genes they control that are responsible for normal spermatogenesis. The majority of the malformations were associated with sperm motility and sperm count. Candidate miRNAs that were associated with this were miR-383, miR-429, miR-141, mir-122, miR-155, miR-145, and miR-7-1*, respectively. Fig. 2 Conclusions: The diverse spectrum of roles for miRNA in causing male infertility and their trigger initiated through oxidative stress and environment factors (EDCs) is shown in the current study. We also show the role of miRNAs in steroid hormone biosynthesis and their effects on spermatogenesis. This study also identified miRNA-regulated genes responsible for (1) abnormal sperm development, (2) spermatogenic failure, (3) defects in motility and sperm count and (4) miRNA-gene mediated altered synthesis of testosterone and androgen. Exogenous factors could trigger miRNA’s that could lead to aberrant gene expression leading to increased OS and altered H2O2 metabolism leading to defective spermatogenesis. miRNA’s and associated genes were also found to target prostate development and affect the male reproductive phenotype. Discovery of new miRNA’s and unravelling their regulatory role could be pivotal in widening the network of miRNA’s and the genes they regulate in male reproduction. Fig. 3 Fig. 4 Figure 3-4. Role of miRNA on testosterone and androgen biosynthesis. Through miRNA-mRNA integrative analysis, 2 genes, TBPL1 (Fig. 3) and HSD17B3 (Fig. 4), were identified that were specifically associated with sperm maturation processes such as spermatid nucleus differentiation and acrosome assembly. The expression of TBPL1 is regulated by miRNAs miR155-5p, miR-145-5p and miR-196a-5p, while miR-133b was found to regulate HSD17B3 gene, which is involved testosterone and androgen synthesis. The role of miRNAs and genes they regulate which are involved in biosynthetic pathways required for normal spermatogenesis and synthesis of testosterone/androgen were shown for the first time.