Female fertility experiences a notable decline with increasing age, particularly beyond 35 [23]. Assisted reproductive technologies can improve reproductive outcomes to some extent, but their success rates also decline [24, 25] largely due to age-related decrease in ovarian reserve and declining oocyte quality [26–28]. Oocyte maturation is a complex and continuous process, complicating our understanding of the molecular mechanisms that regulate oocyte quality throughout meiotic maturation. Since FSP1 is crucial for mammalian gametogenesis and embryonic development, we examined the impact of FSP1 in the meiotic maturation of mouse oocytes utilizing the selective inhibitor iFSP1.
The FSP1 protein reduces CoQ10 to prevent lipid oxidation independently of the GPX4 pathway, inhibiting ferroptosis via an unknown regulatory mechanism [16, 17]. Because the function of ferroptosis in oocyte meiotic maturation is elusive, we explored in this study the function of FSP1 in oocyte quality regulation during meiotic maturation. We found that FSP1 was highly and stably expressed in oocytes at all meiotic stages, suggesting that FSP1 may be involved in oocyte meiosis. Ovarian aging and declining oocyte quality are pivotal factors in female fertility [29–31]. Our study revealed reduced FSP1 expression in the ovaries and oocytes of 8-month-old mice compared to 3-week-old mice, implying a potential role for FSP1 in modulating oocyte quality. Indeed, FSP1 is essential for oocyte meiotic maturation since iFSP1-inhibited FSP1 activity affected the meiotic progression, causing the majority of treated oocytes to arrest in the GVBD and MI phases.
The correct spindle assembly and the proper chromosomal arrangement are crucial events during meiosis. At the MI stage, chromosomes interact with spindle microtubules to form kinetochore-microtubule attachments, where homologous chromosomes are attached to microtubules emanating from opposite spindle poles, respectively. Correct and stable attachments trigger the activation of the anaphase-promoting complex/cyclosome, orchestrating securin and cyclin B degradation. Following these events, separase initiates the cleavage of the cohesion that binds chromosome arms, facilitating the segregation of homologous chromosomes [32–34]. Consequently, any element influencing the assembly of spindles could potentially impede the proper distribution of chromosomes, resulting in meiotic stoppage. We noticed numerous misaligned spindles and chromosomes following FSP1 inhibition in the mouse oocytes. Since they do not possess centrosomes, spindles are assembled with the help of acentriolar MTOCs, which contain essential centromeric and pericentromeric material [35, 36]. We hypothesized that FSP1 inhibition affects these MTOCs, causing spindle and chromosomal misalignment during oocyte divisions. Indeed, the localization patterns of 3 MTOC-associated proteins (p-MAPK, p-Aurora A, and Pericentrin) were altered [37–40] in the iFSP1-treated oocytes. Irregular spindle formation and misaligned chromosomes are often accompanied by faulty kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) monitors the defective attachments and blocks the MI to AI transition until the microtubules and chromosomes reestablish correct connections. We found that FSP1 inhibition triggers SAC and meiotic arrest, indicating that abnormal spindle assembly due to FSP1 inhibition is a major contributor to impaired oocyte maturation.
Since FSP1 inhibits ferroptosis through CoQ10 action, we asked whether the impaired oocyte maturation caused by FSP1 inhibition is associated with ferroptosis. Elevated Fe2+ levels we quantified in iFSP1-treated oocytes suggest oocytes undergo ferroptosis upon FSP1 inhibition and could relate to decreased oocyte quality. Iron accumulation-induced ferroptosis hinders porcine oocyte meiosis and reduces oocyte quality. In addition, iron-overloaded follicular fluid triggers ferroptosis in granulosa cells and oocyte dysmaturity. Moreover, high Fe2+ levels are found in oocytes of aging mice, and these oocytes have substantially reduced quality[41]. Thus, this evidence points to a clear association between iron accumulation and oocyte quality, agreeing with our results. However, whether a relationship exists between age-dependent reduction in FSP1 expression and elevated Fe2+ in oocytes of aged mice remains elusive. Furthermore, significantly increased ROS, DHE, and oxidized lipid levels in the iFSP1-treated oocytes substantiate that FSP1 inhibition triggers ferroptosis, and this finding is also supported by dysregulated expression of ferroptosis-related genes in the treated oocytes. Evidence shows that FSP1 inhibition induces glutathione-independent ferroptosis and promotes oxidative stress via mitochondrial dysfunction, ultimately affecting the developmental competence of early porcine embryos. These results support FSP1 as part of a crucial GPX4-independent ferroptosis-inhibiting pathway in mammalian oocytes and demonstrate its absence triggers ferroptosis, impairing oocyte quality.
Mitochondria occupy a crucial position in iron metabolism [42] and undergo morphological changes during ferroptosis, encompassing enhanced membrane density and diminished or absent mitochondrial cristae [43]. Iron overload causes mitochondrial dysfunction, evidenced by reduced mitochondrial respiration, elevated mitochondrial ROS levels, depolarization of the mitochondrial membrane potential, and mitochondrial swelling [44]. Our study uncovered that FSP1 inhibition provoked mitochondrial dysfunction in oocytes, indicated by reduced ATP levels and increased mitochondrial membrane potential. Mitochondria are exceptionally adaptable cellular components that engage in merging and dividing processes to preserve their structural soundness and equilibrium. Iron overload disrupts mitochondrial dynamics and interferes with the equilibrium among fission and fusion. After FSP1 inhibition in oocytes, the distribution of the fission-regulating protein DRP1 was altered, suggesting disturbed mitochondrial dynamics. Mitochondrial dysfunction produces excess ROS and reduces ATP content [45]. Excess ROS damages lipids, nucleic acids, and proteins, promoting DNA damage and protein dysfunction [46]. An inequity among ROS levels and antioxidant defenses induces oxidative stress, and accumulated ROS triggers ferroptosis [47]. Correct mitochondrial function is essential for proper maturation and oocyte competence [48]. During oocyte maturation, the mtDNA copy number increases dramatically, and the distribution of mitochondria changes considerably to produce enough energy for meiosis [49]. Abnormal mitochondrial function leads to abnormal spindle assembly and failed polar body extrusion [50]. This evidence confirms that oocyte maturation defects observed under FSP1 inhibition are due to mitochondrial dysfunction.
In conclusion, our study provides substantial evidence that FSP1 regulates oocyte meiotic maturation by affecting iron homeostasis and mitochondrial function. In addition, it demonstrates that pharmacological inhibition of FSP1 results in ferroptosis-dependent meiotic failure.