The effect of salubrinal on the endoplasmic reticulum stress pathway in heat-stressed spermatogonial cells in vitro

In this study, we aimed to investigate the effect of salubrinal (SAL) on endoplasmic reticulum stress via an experimental in vitro heat stress model (HSM) of spermatogenic cells. In order to achieve this, mouse spermatogonium (GC1) and spermatocyte (GC2) cell lines were used. The IC50 dose of SAL was calculated using an MTT assay. Each cell line was separated into four different groups: control (GC1C, GC2C), SAL-treated (GC1SAL, GC2SAL), experimental HSM (GC1HSM, GC2HSM), and SAL-treated HSM (GC1HSMSAL, GC2HSMSAL). Control cells were incubated under standard culture conditions. HSM group cells were incubated at 43 °C for 60 min. In the SAL group, cells were incubated with 20 μm SAL-containing culture medium for 24 h. Following treatment, all groups were stained with immunofluorescence probes for p-PERK, ATF6, GRP78, p-IRE1α, p-eIF2α, and HSP70 antibodies. Moreover, the mRNA levels of GRP78, PERK, and eIF2α were evaluated via qRT-PCR. We observed that HSM cells showed cytotoxic effects as all markers showed elevated immunoreactivity levels, which were attributed to ER stress. SAL treatment decreased levels of ER stress. Furthermore, GRP78, PERK, and eIF2α mRNA levels were upregulated in the HSM group and although there was a downregulation following SAL treatment, the difference was not statistically significant. In light of these findings, we concluded that heat stress triggers ER stress in spermatogenic cells, and SAL might affect ER stress markers. Further studies on ER-related stress mechanisms in spermatogenic cells will be critical in developing therapeutic strategies with advanced molecular analyses in the future.