Document Type : Original Article
Authors
1
Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
2
Medical Biology Research Centre, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
3
Department of Biostatistics, School of Health, Kermanshah University of Medical Sciences, Kermanshah, Iran
4
Medical Technology Research Centre, Institute of Health Technology, Kermanshah University of Medical Sciences, Kermanshah, Iran
Abstract
Objective: Mitochondrial oxidative stress is an important factor in infertility. The mitochondrial thioredoxin system
plays an important role in this condition. N-acetyl-5-methoxy tryptamine (melatonin) plays a role in reducing oxidative
stress and apoptosis in spermatogonial stem cells (SSCs). In this study, we explore the probable protective effects of
melatonin 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 cells
in DMEM culture medium that contained 10% fetal bovine serum (FBS), 1% antibiotics, and 10 ng/ml glial cell-derived
neurotrophic 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, 24
hours). 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 factor
erythroid 2-related factor 2 (Nrf2), Trx2, and nicotinamide nucleotide transhydrogenase (NNT) proteins were determined
by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Adenosine triphosphate (ATP) levels
were measured by fluorometry.
Results: Melatonin reduced H2O2-induced ROS levels and apoptosis in the SSCs. Melatonin also increased mRNA
expression of Nrf2, Trx2, NNT, Sirtuin 3 (Sirt3), and decreased mRNA expression of Txnip, and increased protein
expressions of Nrf2, Trx2, NNT thereby increasing activity of the mitochondrial thioredoxin system. In addition, melatonin
increased ATP levels.
Conclusion: Melatonin increased Trx2 expression through the Nrf2 pathway. This study suggests that melatonin may
protect SSCs from oxidative stress in diseases related to infertility.
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