Detection of factors related to ROS and ERS in the reproductive organs of hibernating Daurian ground squirrels ( <i>Spermophilus dauricus</i> )

This study investigated the modulation of cellular stress in the male reproductive system of Daurian ground squirrels (Spermophilus dauricus) across distinct phases of hibernation under extreme environmental conditions. Morphological and volumetric changes in the testes and epididymides were assessed through paraffin-embedded sections and hematoxylin-eosin (H&E) staining, while oxidative stress (OS) and endoplasmic reticulum stress (ERS) markers were quantified using western blotting, colorimetric assays, and immunofluorescence histochemistry. Relative protein expression within key signaling pathways was also evaluated. Compared to the summer active stage, (1) reproductive hormone concentrations and testicular and epididymal visceral mass increased during hibernation and post-hibernation, indicating sustained reproductive activity. (2) Malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels remained stable in the testes and epididymides during hibernation, suggesting effective suppression of oxidative stress during this stage. (3) Superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) increased in the testes during pre-hibernation, potentially supporting germ cell differentiation. (4) Protein expression of unfolded protein response (UPR) markers, including CHOP, p-PKR, GRP78, p-IRE1, and p-PERK, was elevated during hibernation in both the testes and epididymides, implicating UPR activation in the preservation of reproductive tissue integrity and subsequent recovery. Despite stage-specific variations, the testes and epididymides of Daurian ground squirrels maintained a well-regulated cellular stress response throughout hibernation, preventing structural degeneration of the reproductive system. Furthermore, hibernation markedly reduced the abundance of common internal reference proteins such as β-actin and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), underscoring the need to use total protein normalization for accurate quantification in hibernation-related western blot analyses.