Melatonin Protects Mouse Type A Spermatogonial Stem Cells against Oxidative Stress via The Mitochondrial Thioredoxin System

Abstract

Objective: Mitochondrial oxidative stress is an important factor in infertility. The mitochondrial thioredoxin systemplays an important role in this condition. N-acetyl-5-methoxy tryptamine (melatonin) plays a role in reducing oxidativestress and apoptosis in spermatogonial stem cells (SSCs). In this study, we explore the probable protective effects ofmelatonin on the mitochondrial thioredoxin system [thioredoxin 2 (Trx2)/Txnip] in SSCs under oxidative stress.Materials and Methods: In this experimental study, SSCs were co-cultured two-dimensionally (2D) with Sertoli cellsin DMEM culture medium that contained 10% fetal bovine serum (FBS), 1% antibiotics, and 10 ng/ml glial cell-derivedneurotrophic factor (GDNF) for 30 days. The cultured cells were subsequently divided into four groups: control; melatonin(250 μM, 24 hours); melatonin (250 μM, 24 hours)+hydrogen peroxide (H2O2, 50 μM, 24 hours); and H2O2 (50 μM, 24hours). Intracellular reactive oxygen species (ROS) production was determined by flow cytometry. Malondialdehyde(MDA) levels were measured by Fluorometry. The expressions of apoptotic and antioxidant genes and nuclear factorerythroid 2-related factor 2 (Nrf2), Trx2, and nicotinamide nucleotide transhydrogenase (NNT) proteins were determinedby quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Adenosine triphosphate (ATP) levelswere measured by fluorometry.Results: Melatonin reduced H2O2-induced ROS levels and apoptosis in the SSCs. Melatonin also increased mRNAexpression of Nrf2, Trx2, NNT, Sirtuin 3 (Sirt3), and decreased mRNA expression of Txnip, and increased proteinexpressions of Nrf2, Trx2, NNT thereby increasing activity of the mitochondrial thioredoxin system. In addition, melatoninincreased ATP levels.Conclusion: Melatonin increased Trx2 expression through the Nrf2 pathway. This study suggests that melatonin mayprotect SSCs from oxidative stress in diseases related to infertility.

Keywords

• melatonin
• oxidative stress
• spermatogonia