Regulatory Role of Tubulin Beta 1 in Oocyte Endoplasmic Reticulum Stress and Mitochondrial Integrity via Transforming Growth Factor‐β1 Signaling Pathway in Mice

Enhancing the developmental competence and quality of oocytes cultured in vitro remains challenging, in part due to limited understanding of the structural and molecular regulators of oocyte maturation. Microtubules, as key components of the cytoskeleton, are essential for chromosome segregation and meiotic progression. Tubulin beta 1 class VI (TUBB1) plays crucial roles in cellular stability, yet its involvement in regulating endoplasmic reticulum (ER) stress and mitochondrial function in oocytes via immune checkpoint pathways remains unclear. This study aimed to investigate the role of TUBB1 in regulating ER stress and mitochondrial integrity in oocytes via the transforming growth factor-β1 (TGF-β1)/SMAD family member 3 (Smad3) pathway, and its potential implications for improving assisted reproductive technologies. First, oocytes isolated from ICR female mice were subjected to oxidative stress using hydrogen peroxide (H₂O₂) to investigate TUBB1 expression. Next, lentiviral vectors and a TGF-β1/Smad3 activator (SRI-011381 hydrochloride) were utilized to modulate pathway activity. ER stress, mitochondrial integrity, oxidative stress, and apoptosis were subsequently assessed. H₂O₂ exposure significantly reduced TUBB1 expression, confirming its susceptibility to oxidative stress. TUBB1 silencing exacerbated ER stress (elevated Bax, GRP78, Caspase-12; decreased Bcl-2), increased apoptosis rates, and caused mitochondrial dysfunction evidenced by disrupted mitochondrial morphology; increased Ca²⁺ levels, higher ROS and MDA, and lower SOD activity. Conversely, TUBB1 overexpression or direct activation of the TGF-β1/Smad3 pathway reversed these detrimental effects, significantly reducing apoptosis, normalizing mitochondrial distribution, and alleviating ER and oxidative stress. Furthermore, activation of the TGF-β1/Smad3 pathway in TUBB1-silenced oocytes (rescue group) effectively restored mitochondrial and ER stress parameters to normal levels. Our findings demonstrate that TUBB1 plays a critical role in modulating ER and mitochondrial stress responses in oocytes via the TGF-β1/Smad3 signaling pathway. Targeted regulation of TUBB1 may serve as a promising therapeutic approach to improve oocyte quality in assisted reproductive technologies.