Supplementary Material for: Differentially expressed miRNAs in testes of dominant and subordinate Nile tilapia males and identification of oni-miR-499 as regulator of amh gene expression.

Introduction: Gonadal development and reproduction are under the control of the endocrine system, which acts along the brain-pituitary-gonad (BPG) axis. Besides well-known regulators of the BPG axis, such as the gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), the Anti-Müllerian hormone (Amh) came into the focus of research on the BPG axis. Amh is expressed differently in the gonads of dominant and subordinate Nile tilapia (Oreochromis niloticus) males and could be involved in the regulation of the differently developed gonads. In addition, the regulatory networks and the control of gene expression depend on microRNAs (miRNAs), an often not considered epigenetic mechanism in hormonal research. Methods: We used a long-term, stable social hierarchy of Nile tilapia males as an experimental system to identify differentially expressed miRNAs in the testes of dominant and subordinate animals. A Dual Luciferase Reporter Assay and in vitro analysis of amh expression in primary testis cells were used to demonstrate predicted interactions. Results: We identified 23 differentially expressed miRNAs in the testes of dominant and subordinate males and predicted the targets in the pools of differentially expressed genes. Using these data, we placed the identified GO terms and KEGG pathways in the context of differently developed gonads under social control. The most differentially expressed miRNA, oni-miR-499, is up-regulated in the testes of dominants and regulates amh expression. Conclusion: We conclude that oni-miR-499 affects testis development via amh expression in Nile tilapia. Many miRNAs and biological processes identified in our study could be conserved mechanisms of testis development.