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

## TL;DR

This study shows that salubrinal can reduce endoplasmic reticulum stress in mouse spermatogenic cells exposed to heat stress, potentially offering a treatment for heat-related infertility.

## Contribution

The study demonstrates that salubrinal mitigates heat-induced ER stress in spermatogenic cells, suggesting its therapeutic potential for infertility.

## Key findings

- Heat stress increases ER stress markers in spermatogenic cells.
- Salubrinal treatment reduces ER stress and cytotoxic effects in heat-stressed cells.
- Salubrinal may support spermatogenic cell survival under heat stress.

## Abstract

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.

This study investigates the protective role of salubrinal against heat‐induced endoplasmic reticulum (ER) stress in mouse spermatogenic cells (GC1 and GC2). By modulating the ER stress pathway, salubrinal alleviates cellular stress and supports spermatogenic cell survival, suggesting its potential as a therapeutic candidate for heat‐related infertility.