Oocyte in vitro maturation is crucial for in vitro embryo production technology, which provides oocytes resources for in vitro fertilization and somatic cell nuclear transfer. Previous studies proved that SIRT2, a member of the sirtuin family, plays a role in oocyte meiosis, but its role in sheep oocyte maturation and its regulating mechanism remains unknown. Firstly, we confirmed the role of Sirt2 in sheep oocytes maturation by supplementation of SIRT2 inhibitor and activator. To further explore the specific mechanism, we performed knockdown of Sirt2 in granulosa cells and then cocultured them with oocytes. Moreover, we determined the effects of Sirt2 on granulosa cell oxidative apoptosis, cell migration, and diffusion, and examined its effects on granulosa cell mitochondrial function, mitophagy, and steroid hormone levels. The results showed that supplementation of SIRT2 inhibitor decreased the oocytes maturation rate (69.28% ± 1.28 vs. 45.74% ± 4.74, p < 0.05), while resveratrol, a SIRT2 activator, increased its maturation rate (67.44% ± 1.68 vs. 78.52 ± 1.28, p < 0.05). Knockdown of Sirt2 in sheep granulosa cells also reduced the oocytes maturation rate (47.98% ± 1.43 vs. 33.60% ± 1.77, p < 0.05), and led to decreased cell migration and expansion ability, oxidative apoptosis, abnormal mitochondrial gene expression, decreased mitochondrial membrane potential and ATP level, and increased mitophagy level. Overexpression of Sirt2 improved mitochondrial membrane potential and ATP level and improved mitochondrial function. Furthermore, we found that Sirt2 knockdown in granulosa cells promotes the secretion of P₄ through regulating p-ERK1/2. In conclusion the present study showed that SIRT2 is critical for sheep oocyte maturation through regulating the function of ovarian granulosa cells, especially affecting its mitochondrial function.

Background/Objectives: Cellular oxidative stress is crucial for GV stage oocyte vitrification quality. PI3K and the aquaporin family have been shown to facilitate various cellular processes related to redox homeostasis and energy balance; yet, the mechanisms underlying the involvement of aquaporin 7 (AQP7) in vitrified oocyte oxidative stress remain unclear. The purpose of the present investigation was to evaluate the role of AQP7 in vitrified oocytes and the mechanisms involved. Methods: AQP7 inhibitors were employed to investigate the effect of AQP7 on oxidative stress in mouse-vitrified oocytes, whereas PI3K activators were harnessed to ascertain whether AQP7 serves as a functional molecule involved in this process. Results: Our results indicate that AQP7 inhibition in vitrified oocytes results in a significant decrease in glutathione (GSH) levels associated with cellular oxidation and an elevation in H₂O₂ levels. This was accompanied by exacerbated mitochondrial dysfunction, weakened cytoskeletal proteins, accelerated early apoptosis. Consequently, both survival and maturation rates were markedly reduced. Interestingly, PI3K/AKT activation increased AQP7 expression, restored abnormal mitochondrial distribution, as well as calcium homeostasis, and rescued the oocyte survival/maturation rate. Conclusions: Our results provide new insights indicating that PI3K/AKT/AQP7 decreases oxidative stress by regulating mitochondrial morphology, function, and distribution, thereby rescuing oocyte maturation in vitrification.

Oocyte vitrification imposes oxidative stress that compromises maturation competence. Aquaporin-7 (AQP7) has been implicated in cellular redox regulation, but its specific role in cryopreserved oocytes remains unclear. Here, germinal vesicle (GV) stage oocytes were vitrified and warmed with AQP7 inhibitor Z433927330 (0.5, 5, 50 μM). AQP7 inhibition disrupted redox balance, compromised mitochondrial function. Consequently, it severely compromised developmental competence, leading to significantly reduced cleavage (39.90% ± 6.17 vs. 52.93% ± 3.37) and blastocyst formation rates (1.67% ± 2.89 vs. 5.17% ± 2.49) in vitro. To confirm, we performed microinjection-mediated AQP7 knockdown and overexpression and assessed their effects on maturation. AQP7 knockdown further reduced the maturation rate of vitrified oocytes (20.22% ± 3.14 vs. 36.31% ± 2.10), whereas overexpression partially restored it (43.98% ± 4.71 vs. 33.74% ± 2.21). The mitochondrial-targeted antioxidant MitoQ partially rescued the maturation rate (53.13% ± 2.75 vs. 43.52% ± 2.71). Thus, AQP7 is essential for the maturation of vitrified sheep oocytes by safeguarding intracellular redox homeostasis, thereby preventing mitochondrial dysfunction and cytoskeletal damage, and loss of embryonic developmental potential.