The therapeutic effect of melatonin combined with miR-19b on PERK/eIf2a/ATF4/CHOP signaling in endoplasmic reticulum stress induced leydig and sertoli cells

Sertoli and Leydig cells are the main cell types in the testes and play important roles in the regulation of spermatogenesis, the sperm production process. Dysfunction or any damage to Sertoli and Leydig cells may adversely affect the production of healthy spermatozoa, impair sperm motility and quality, and cause infertility. Leydig cells are responsible for the endocrine function of the testes. High protein synthesis occurs in these cells and therefore Leydig cells are sensitive to endoplasmic reticulum (ER) stress. In addition, melatonin controls growth, proliferation, energy metabolism, oxidative stress and inflammatory processes in Sertoli cells. The processes from primordial germ cells to the proliferation of spermatogonia and from the formation of motile sperm to fertilisation are regulated by various miRNA families. miR-17-92 cluster, of which miR-19b is a member, is required for the maturation of spermatogonial stem cells and spermatogenesis. In addition, known targets of miR-19b include apoptosis and cell cycle genes. In this thesis, we aimed to determine the effects of miR-19b and melatonin on the PERK/eIF2α/ATF4/CHOP UPR pathway and molecular mechanisms of apoptosis in mouse Leydig and Sertoli cells under ER stress. For this purpose, six experimental groups were formed in our study: Control, Tunikamycin, Tunikamycin+Melatonin, Tunikamycin+miR-19b mimic inhibitor, Tunikamycin+miR-19b mimic, Tunikamycin+Melatonin+miR-19b mimic. MTT test, which is an in vitro cytotoxicity test, was used to determine the application concentrations and times. NucBlue (Hoechst) and Acridine orange/ethidium bromide (AO/EB) dyes were used to observe nuclear changes in apoptotic cells and fluorescent staining was performed using CM-H2DCFDA dye to observe oxidative stress. Proteins were analysed by Western blotting and gene expression levels were determined by qRT-PCR. Our results show that melatonin and miR-19b are effective on ER stress and apoptosis signaling in leydig cells, which have an important function in reproduction. Melatonin treatment alone decreased the expression of PERK/eIF2α/ATF4/CHOP and IRE1α/XBP/sXBP1 UPR pathway genes to control levels in Leydig cells. In addition, it significantly decreased the expressions of Bax, Bim, Bak, APAF1, caspase 3, and cytochrome-c from mitochondrial apoptosis pathway genes. miR-19b alone or in combination with melatonin caused a decrease in the expression of apoptosis (Bax, Bim, APAF1, caspase 3) genes. In addition, a significant increase in Bcl-2 expression after miR-19b administration alone caused Bcl-2/Bax balance and showed its anti-apoptotic role. According to our study results, the combination of melatonin and mir-19b may be a therapeutic approach in reversing testicular apoptosis and may offer new approaches in the treatment of infertility by modulating the pathways targeted by miR-19b. However, it needs to be supported by further in-vitro and in-vivo studies.