ATF4 contributes to proper maintenance of BTB-associated junctional organization under stress conditions in sertoli cells of mice

Spermatogenesis is highly sensitive to cellular stress conditions and requires precise regulation of Sertoli cell function to support germ cell development, yet the underlying mechanisms remain poorly understood. Activated transcription factor 4 (ATF4) is a key regulator of cellular stress adaptation; however, its role in spermatogenesis remains unclear. Here, we demonstrate that ATF4 is highly expressed in mouse testes and is predominantly localised to the nuclei of Sertoli cells. Sertoli cell-specific knockout of Atf4 did not impair spermatogenesis or sperm motility under physiological conditions. However, under high-fat diet (HFD)-induced metabolic stress conditions, Atf4 knockout mice showed a modest reduction in litter size, accompanied by decreases in sperm count and motility. Moreover, loss of ATF4 altered blood-testis barrier (BTB)-associated junctional organization in HFD-fed conditional knockout males. Mechanistically, loss of Atf4 exacerbated endoplasmic reticulum (ER) stress and was associated with altered transforming growth factor-β (TGF-β) signalling-related responses, accompanied by reduced expression and disorganization of BTB-associated junction proteins. In vitro, tunicamycin (Tm)-induced ER stress similarly disrupted cellular architecture and junction-associated molecular organization in isolated primary Sertoli cells. Restoration of TGF-β signalling partially alleviated these alterations in a heterologous cellular system. Taken together, our results reveal that ATF4 contributes to maintaining Sertoli cell function and junction-associated molecular responses under stress conditions. These findings provide new insight into the mechanisms linking metabolic stress to impaired male reproductive function.