Abstract

Melatonin is a key neuroendocrine hormone that promotes spermatogenesis and sperm motility, but the underlying mechanisms remains poorly understood. In this study, we aimed to investigate the possible roles of m 6 A (N 6 --methyl-adenosine) in mediating melatonin-regulated spermatogonia activity alterations. In this study, mouse-derived GC-1 spermatogonia (spg) cell line was used as the in vitro cellular model. The viability, proliferation rates and apoptosis of spermatogonia were detected via CCK-8, Edu staining and flow cytometry respectively. Total m 6 A level was quantitated by dot blot, while mRNA and proteins contents in spermatogonia were measured by qRT-PCR and western blot respectively. Differentially expressed mRNAs were characterized by deep RNA sequencing method. Results showed that melatonin significantly promoted viability and proliferation rate while inhibited apoptosis in the GC-1 spg cells. The total m 6 A levels in GC-1 spg cells were also greatly increased by melatonin treatment, accompanied by remarkable expressional elevation of the m 6 A writer KIAA1429. Moreover, the regulation of GC-1 spg cell viability, proliferation and apoptosis by melatonin were greatly abrogated by KIAA1429 silencing but effectively strengthened by KIAA1429 overexpression. In addition, KIAA1429 overexpression regulates multiple biological process and signaling pathways in spermatogonia such as the PI3K/AKT signaling. The PI3K inhibitor LY294002 effectively mitigated the regulation of spermatogonia activity by KIAA1429 overexpression under melatonin treatment. Taken together, melatonin promotes spermatogonia activity via enhancing KIAA1429 expression and m 6 A RNA methylation to activate the downstream PI3K/AKT signaling pathway.