Ferroptosis suppressor 1 regulates ferroptosis and mitochondrial function during mouse oocyte maturation

Ferroptosis suppressor 1 regulates ferroptosis and mitochondrial function during mouse oocyte maturation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ferroptosis suppressor 1 regulates ferroptosis and mitochondrial function during mouse oocyte maturation Hongzhen Ruan, Huifen Xiang, Yajing Liu, Peiwen Wang, Liuliu Dong, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4675534/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oocyte quality is critical for fertilization and embryo development. Recent studies have shown that ferroptosis may compromise oocyte quality. Ferroptosis suppressor protein 1 (FSP1) is a ferroptosis inhibitor with an undefined role in oocyte quality regulation during meiotic maturation. Here, we found that FSP1 is expressed throughout all stages of meiotic maturation and localizes to the cytoplasm of mouse oocytes. A decline in FSP1 expression was observed in the ovaries and oocytes of aged mice. Pharmacological inhibition of FSP1 caused a failure in germinal vesicle breakdown and polar body emission, accompanied by spindle abnormalities and chromosome misalignment. Moreover, FSP1 inhibition consistently activated the spindle assembly checkpoint, inducing meiotic arrest. Mechanistically, FSP1 inhibition increased Fe 2+ content, elevated dihydroethidium levels, promoted reactive oxygen species buildup, and heightened lipid peroxidation. Additionally, it dysregulated the expression of ferroptosis-related genes, suggesting that oocytes underwent ferroptosis. Furthermore, FSP1 inhibition provoked mitochondrial dysfunction, characterized by abnormal mitochondrial localization, reduced ATP levels, and elevated mitochondrial membrane potential. In summary, our findings demonstrate that FSP1 participates in oocyte meiotic maturation through its involvement in iron homeostasis and mitochondrial activity, and FSP1 inhibition results in ferroptosis-dependent meiotic failure. FSP1 oocyte meiosis ferroptosis mitochondria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. INTRODUCTION Oocyte quality is critical for female fertility, with compromised quality affecting sperm-egg binding and early embryonic development, influencing pregnancy outcome [ 1 , 2 ]. Numerous oocytes form during oogenesis, a complex and discontinuous meiotic process in female mammals. The undeveloped oocyte is halted in the initial meiotic prophase (G2) and exhibits a visible nucleus known as the germinal vesicle (GV). At sexual maturity, hormones stimulate the oocyte to undergo germinal vesicle breakdown (GVBD), marking the G2-M transition and resuming the first meiotic division [ 3 ]. The formation of the spindle, composed of microtubules, and the alignment of homologous chromosomes on the equatorial plate are critical events during the first meiotic metaphase (MI). The oocyte undergoes homologous chromosome segregation during the final phase of the first meiotic division, extrudes the first polar body, and stalls at the second meiotic metaphase (metaphase II, MII), developing into a mature oocyte capable of fertilization. [ 4 ]. Proper spindle assembly during meiosis is crucial for fertility as abnormal spindle morphology and chromosome alignment can cause oocyte meiotic defects, resulting in malformed embryos, miscarriages, and birth defects [ 5 , 6 ]. Therefore, exploring the regulatory mechanisms of oocyte development and identifying potential molecular regulatory targets have considerable theoretical and clinical applications. Ferroptosis represents a form of controlled cellular demise characterized by iron-mediated oxidation of lipids, which is mechanistically and morphologically distinct from apoptosis and alternative modes of regulated cell death [ 7 ]. Polyunsaturated fatty acids, iron, and reactive oxygen species (ROS) are the main positive factors triggering ferroptosis [ 8 ]. Polyunsaturated fatty acids highly expressed in the cell membrane form polyunsaturated phospholipids via long-chain acyl-CoA synthetase family 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) activity. These phospholipids then utilize free Fe 2+ as a catalyst for the Fenton reaction with O 2 , where electrons are transferred to H 2 O 2 to generate ROS, triggering lipid peroxidation and ferroptosis [ 9 ]. Ferroptosis assumes a crucial function in the development of female reproductive disorders [ 10 ]. Premature ovarian insufficiency, endometriosis, polycystic ovary syndrome, and trophoblastic dysfunction disorders are characterized by varying degrees of ferroptosis, suggesting it may be a therapeutic target for related diseases. For example, basonuclin zinc finger protein 1 ( BNC1 ) mutations in humans and mice cause ferroptosis-induced oocyte death and follicular atresia, leading to premature ovarian insufficiency. Inhibiting ferroptosis in mice attenuates Bnc1 mutation-induced premature ovarian insufficiency [ 11 ]. Evidence indicates that ferroptosis is strongly associated with impaired oocyte meiotic maturation [ 12 – 15 ]; however, our knowledge of ferroptosis in oocyte meiotic maturation remains limited. Ferroptosis suppressor protein 1 (FSP1), or apoptosis inducing factor mitochondria associated (AIFM2), is a highly conserved flavoprotein initially identified as a p53-responsive gene due to its structural similarity with the apoptosis-inducing factor (AIF). Recent evidence indicates that FSP1 functions as a glutathione-independent inhibitor of ferroptosis that participates in ubiquinone (CoQ10) reduction, which prevents lipid oxidation [ 16 , 17 ]. N-myristoylated, plasma membrane-localized FSP1 promotes the recruitment of CoQ10 to the cell membrane and its NADPH-catalyzed reduction to ubiquinol (CoQ10H2). Consequently, CoQ10H2 traps free radicals to hinder lipid peroxidation and inhibit cellular ferroptosis [ 18 , 19 ]. The FSP1-CoQ10-NADPH axis, in conjunction with glutathione and glutathione peroxidase 4 (GPX4), acts as an independent parallel system essential for maintaining phospholipid redox homeostasis by inhibiting ferroptosis and phospholipid peroxidation. iFSP1 is a selective FSP1 inhibitor [ 20 ] that binds to FSP1 at the plasma membrane, impeding its NADH oxidase activity [ 21 ]. Hence, iFSP1 is often used in FSP1-related studies, and its inhibitory effects are well described. Interestingly, a recent study in pigs showed that FSP1 inhibition impairs early embryonic development [ 22 ], implying that FSP1 has a vital role in gametogenesis and embryonic development. Nevertheless, the function of FSP1 during oocyte meiosis and the underlying mechanism is still unknown and require a detailed exploration to elucidate our understanding of the events decisive to oocyte quality. In our investigation, we examined the function of FSP1 during mouse oocyte meiosis utilizing the iFSP1 inhibitor. We demonstrated that FSP1 was situated in the cytoplasm during mouse oocyte meiosis. Moreover, we found that FSP1 inhibition impaired oocyte maturation in conjunction with ferroptosis, accompanied by mitochondrial dysfunction. Our findings offer initial proof that FSP1 is crucial in mouse oocyte meiosis. 2. MATERIALS AND METHODOLOGIES 2.1. Experimental animals The 3-week-old female, 8-week-old male, and 8-month-old female Kunming (KM) mice were obtained from the Laboratory Animal Center of Anhui Medical University (Hefei, China) and Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China). The mice were housed at the Laboratory Animal Center of Anhui Medical University in a controlled environment with a steady 24°C temperature and a 12-h light-dark cycle. They were granted unlimited access to nourishment and hydration for the duration of the investigation. All procedures involving animals were sanctioned by the Ethical Committee of Anhui Medical University. 2.2. Experimental reagents and antibodies The primary reagents utilized in this research are detailed in Supplementary Table 1. Antibody information is provided in Supplementary Table 2. 2.3. In vitro oocyte maturation and iFSP1 treatment Following adaptation, female mice were euthanized, and their ovaries were extracted and immediately transferred to an M16 medium supplemented with 50 µM 3-isobutyl-1-methylxanthine. The GV-expressing oocytes were collected under a stereomicroscope and relocated to a balanced M16 culture medium. The specimens were maintained in an incubator at 37°C with 5% CO 2 and complete humidity for 0 h (GV stage), 2 h (GVBD stage), 8 h (MI stage), and 12 h (MII stage). A stock solution of iFSP1 was prepared by dissolving it in dimethylsulfoxide. Treatment cohorts were subjected to the M16 medium with incremental concentrations of iFSP1: 5 µM, 10 µM, and 15 µM, and the control cohort to an equivalent volume of dimethylsulfoxide in the M16 medium. 2.4. Immunohistochemistry Mouse ovarian sections were available from 3-week-old and 8-month-old female KM mice. Sections underwent deparaffinization in xylene and subsequent rehydration via a gradient of diminishing alcohol concentrations (100%-70%). Endogenous peroxidase was inhibited in 3% H 2 O 2 for 30 min. Antigen retrieval was performed in citrate buffer (pH 6.0) at 100°C for 2 min. The sections were rinsed in phosphate-buffered saline (PBS) and then in 5% BSA for 1 h to reduce nonspecific binding. The sections were exposed to anti-FSP1 as the primary antibody (1:500) at 4°C overnight. The samples underwent washing and were then exposed to the secondary antibody for 1 h. The sections were treated with diaminobenzidine and hematoxylin, dehydrated, and mounted in neutral resin. 2.5. Immunofluorescence The fertilized oocytes were placed in 4% paraformaldehyde with 0.5% Triton X-100, then incubated for 50 min at ambient temperature. Following this, the oocytes were rinsed utilizing PBS with 2% bovine serum albumin (BSA) for 1 h at ambient temperature. An overnight incubation followed at 4°C with the primary antibodies at a 1:100 dilution ratio. The oocytes were rinsed 3× with PBS coupled with 0.1% Tween 20 and 0.01% Triton X-100. Next, they underwent incubation with anti-α-tubulin-FITC (1:100) or Cy3-conjugated goat anti-rabbit IgG (1:100) as the secondary antibody at 37°C for 1 h. After DAPI staining for 10 min, the oocytes were positioned in the middle of a slide with a droplet of antifade mounting medium for fluorescence examination. Fluorescence intensity was analyzed with ImageJ (NIH, Bethesda, MA, USA) or ZEN3.4 (Zeiss, Jena, Germany) software. Mouse ovarian sections underwent dewaxed with xylene and rehydration in an alcohol gradient for immunofluorescence. Fluorescence intensity was investigated utilizing ZEN3.4 (Zeiss). 2.6. Western blotting Total protein was extracted from tissues using RIPA lysis buffer (P0013, Beyotime) supplemented with SDS-PAGE sample loading buffer (P0295, Beyotime). The protein samples were heat-denatured at 95°C for 10 min, electrophoretically separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (IPVH00010, Millipore) with 0.45-µm pores. The membranes underwent a blocking phase in 5% bovine serum albumin for 2 h and a washing process, followed by an incubation with the primary antibodies. Following another wash, the membrane underwent a 2-h incubation at an ambient temperature with secondary antibodies the next day. The detection and densitometric evaluation of the immunoblots was performed using a chemiluminescence detection kit (P0018, Beyotime), and the bands were visually documented using the Tanon 5200 image-capturing system (Tanon). 2.7. RNA isolation and reverse transcription-quantitative PCR (RT-qPCR) Seventy-five oocytes each from control and iFSP1-treated groups treated for 8 h were collected. Subsequently, total RNA extraction was conducted using the RNAprep Pure Micro Kit as per the instructions, which consisted mainly of cell lysis and elution from the adsorption column. Reverse transcription was performed to obtain cDNA as per the instructions of the PrimeScript™ RT reagent Kit (Perfect Real Time). cDNA levels of target genes were measured utilizing the LightCycler 480 SYBR Green I Master. The reaction system consisted of 1 µl cDNA, 10 µl SYBR Green I Master, 2 µl primers, and 7 µl DEPC water. Reactions were performed on a LightCycler® 96 (Roche, Germany) instrument. The relative expression levels of genes to Gapdh were quantified utilizing the 2-ΔΔCT method, with Gapdh serving as a control. The specific information is listed in Supplementary Table 3. 2.8. Fe 2+ and lipid peroxidation level detection Fe 2+ levels were identified utilizing the FerroOrange fluorescent probe, and lipid peroxidation levels were determined with the BODIPY 581/591 C11 fluorescent probe. For detection, oocytes were harvested, rinsed, placed in the staining medium, and maintained at 37°C for 25 min. The excess staining solution was removed by rinsing with PBS, and the oocytes were placed into confocal petri dishes. 2.9. Mitochondrial localization and membrane potential detection Mitochondrial localization in oocytes was determined with Mito-Tracker Red. The mitochondrial membrane potential of oocytes was assessed with a TMRE-mitochondrial membrane potential assay kit. In brief, oocytes were harvested, rinsed, placed in the dye medium, and maintained at 37°C for 25 min. The excess staining solution was removed by rinsing with PBS, and the oocytes were placed into confocal petri dishes. 2.10. Superoxide anion and ROS detection ROS levels within mouse oocytes were assessed utilizing DCFH-DA (dichloro-dihydro-fluorescein diacetate), and superoxide anions were assessed utilizing dihydroethidium (DHE). Control and iFSP1-exposed oocytes cultivated for 8 h were gathered and placed in an M16 medium containing 20 µM DCFH-DA or DHE for 25 min at 37°C. The oocytes were rinsed utilizing an M16 medium without DCFH-DA or DHE to remove the indicators that failed to penetrate the cytoplasm. 2.11. Annexin-V assay Initial stages of programmed cell death in oocytes were identified utilizing an annexin V-FITC apoptosis detection kit. The outer membrane was eliminated employing Tyrode’s medium, and the oocytes were placed in an annexin V-FITC binding solution and maintained at 37°C for 25 min. The oocytes were rinsed 3× with PBS and placed in confocal petri dishes. 2.12. Adenosine triphosphate detection Adenosine triphosphate (ATP) levels in oocytes were quantified employing an ATP assay kit. A total of 30 oocytes from the control or iFSP1-treated group were gathered, placed into 100 µL of lysis solution, and disrupted on ice for 3 min. Afterward, 100 µL of the cell extract was transferred into a 96-well plate containing the test mixture and blended completely. The chemiluminescence intensity was quantified employing a SpectraMax iD3 multifunctional microplate reader and SoftMax Pro 7 software (Molecular Devices, LLC.). 2.13. Statistical analysis All results were denoted as mean ± standard deviation (SD) and derived from at least 3 separate replicate experiments. Data were assessed and visualized with GraphPad Prism. The variations across the 2 cohorts were determined utilizing the t test, and those across multiple cohorts were assessed utilizing the analysis of variance (ANOVA). The Bonferroni post hoc analysis was carried out utilizing the findings of the chi-square test. Statistical significance was identified when p < 0.05. 3. RESULTS 3.1. Expression and localization of FSP1 in mouse ovaries and oocytes Uterine, testicular, and ovarian tissues were collected for Western blotting to determine FSP1 expression in mouse reproductive organs. The FSP1 protein was expressed in all 3 tissues, with significantly higher levels in ovaries than in uteruses and testicles (Fig. 1 A, B). Moreover, immunofluorescence analysis of mouse ovarian tissues revealed that FSP1 was expressed across all stages of follicular development: primordial, primary, secondary, and mature follicles (Fig. 1 C). We also compared FSP1 expression between the ovaries of 3-week-old and 8-month-old female mice to investigate the association between FSP1 and age-related changes. We observed reduced FSP1 expression in the ovaries of 8-month-old female mice (Fig. 1 D, E), indicating that FSP1 expression decreases with age. Strikingly, oocytes of 8-month-old mice showed lower FSP1 expression than those of 3-week-old mice (Fig. 1 F, G). Thus, we collected oocytes at the GV, GVBD, MI, and MII phases to delineate FSP1 expression patterns during mouse oocyte meiosis. We demonstrated that FSP1 had consistent expression throughout oocyte maturation, from the GV to MII stage (Fig. 1 H, I). In addition, oocytes immunofluorescently stained with anti-FSP1 antibody exhibited a punctate distribution of FSP1 within the cytoplasm (Fig. 1 J). These data suggest that FSP1 has high but age-dependent expression in mouse ovaries. Its persistent expression throughout the stages of oocyte meiotic maturation implies that FSP1 has an indispensable role in oocyte maturation. 3.2. FSP1 inhibition impairs oocyte cell cycle and meiotic maturation We used the iFSP1 inhibitor to investigate whether FSP1 expression affects oocyte quality regulation during meiotic maturation. We incubated oocytes for 2 h in the M16 medium with escalating iFSP1 levels (0 µM, 5 µM, 10 µM, or 15 µM). We showed that FSP1 inhibition at 5 µM and 10 µM iFSP1 concentrations did not significantly affect oocytes at GVBD and PBE stages compared with those from the control group (Fig. 2 A-D). However, 15 µM iFSP1 significantly impaired the occurrence of the GVBD stage and diminished the frequency of PBE in comparison with oocytes from the control cohort (Fig. 2 A-D). Hence, we selected 15 µM iFSP1 for subsequent experiments to explore how the iFSP1-induced loss of FSP1 activity compromises mouse oocyte GVBD and PBE stages. Following a 12 h treatment with iFSP1, oocytes were collected for cell cycle analysis to gain detailed insights into meiosis under FSP1 inhibition. Most oocytes in the FSP1-inhibited cohort were halted at the GVBD and MI phases, whereas the bulk of oocytes in the control cohort progressed to the MII stage (Fig. 2 E and 2 F). The results suggest that pharmacological FSP1 inhibition impairs oocyte cell cycle progression, provoking meiotic failure. 3.3. FSP1 inhibition impairs oocyte spindle assembly and chromosomal arrangement Because the precise assembly of the spindle and the correct chromosomal arrangement are critical for oocyte maturation, we sought to explore the spindle formation at MI in iFSP1-treated oocytes. Oocytes in the control cohort displayed characteristic barrel-shaped MI spindles with well-aligned chromosomes (Fig. 3 A). Conversely, iFSP1-treated oocytes exhibited disorganized spindle morphology and misaligned chromosomes, marked by diminished spindle dimensions and haphazard chromosomal distribution (Fig. 3 A). A statistical analysis confirmed a notably elevated frequency of irregular spindles (Fig. 3 B) and misaligned chromosomes in the iFSP1-treated cohort (Fig. 3 C). Additionally, our analysis suggested that the iFSP1-treated oocytes showed a conspicuously decreased spindle length (Fig. 3 D) but observed no significant differences in spindle width relative to the control cohort (Fig. 3 E). Moreover, iFSP1-treated oocytes displayed a markedly elevated chromosome width (Fig. 3 F) and the ratio of chromosome width to spindle length versus the control group (Fig. 3 G). 3.4. FSP1 inhibition disturbs subcellular localization of Pericentrin, p-MAPK, and p-Aurora A Since mouse oocytes lack centrosomes, they rely on acentriolar microtubule-organizing centers (MTOCs), which contain essential pericentriolar material, for spindle assembly. Pericentrin, p-MAPK, and p-Aurora A are MTOC-related proteins that play a critical role in the spindle formation during meiosis, so we assessed their expression by immunofluorescence under FSP1 inhibition in oocytes. Whereas control oocytes had Pericentrin and p-MAPK localized to the MI spindle poles (Fig. 4 A, B), their localization in iFSP1-treated oocytes showed an irregular distribution. Likewise, control oocytes displayed a distinct positive signal for p-Aurora A protein at spindle poles, while iFSP1-treated oocytes exhibited a scattered and irregularly distributed p-Aurora A signal (Fig. 4 C). In summary, FSP1 inhibition disorganizes the localization of various MTOC-associated proteins in mouse oocytes, indicating protein dysfunction. 3.5. FSP1 inhibition persistently activates the spindle assembly checkpoint Aberrant spindle phenotype and the altered chromosome arrangement in oocytes after FSP1 inhibition suggest that the spindle assembly checkpoint (SAC) is activated to arrest meiotic progression. To validate our assumption, we investigated iFSP1-treated mouse oocytes for the localization of mitotic checkpoint Bub1-related kinase or MAD3/Bub1b (BubR1), an essential element of the SAC. We observed bright BubR1 signals in control and iFSP1-treated oocytes after 4.5 h culture (Fig. 5 A). Moreover, after 10.5 h culture, no positive BubR1 signal was detected in control oocytes, only segregated homologous chromosomes. By contrast, BubR1 was apparent as a bright fluorescence signal on the chromosomes in iFSP1-treated oocytes, and the chromosomes were unsegregated. Indeed, statistical data confirmed that the fraction of activated BubR1 in the iFSP1-administered cohort was notably elevated relative to the control cohort (Fig. 5 B). These results indicate that repressed FSP1 activity in mouse oocytes sustains SAC activation, blocking oocyte meiotic progression. 3.6. FSP1 inhibition induces ferroptosis in mouse oocytes The FSP1 protein is a suppressor of ferroptosis; therefore, we investigated whether ferroptosis contributes to oocyte maturation failure in the absence of FSP1 activity. Considering the importance of Fe 2+ in lipid peroxidation and ferroptosis, we first utilized the Fe 2+ -selective fluorescent probe FerroOrange to determine Fe 2+ levels in oocytes. The iFSP1-treated cohort showed markedly elevated Fe 2+ levels relative to the control cohort (Fig. 6 A, B). In addition, iFSP1-treated oocytes had significantly elevated ROS and DHE levels (Fig. 6 C-F), implying that oocytes experience excessive oxidative stress under FSP1 inhibition. We also detected increased levels of oxidized lipids in iFSP1-treated oocytes, indicated by higher fluorescence of oxidized (green) BODIPY 581/591 C11 reporter in treated oocytes compared to controls (Fig. 6 G and 6 I). Concurrently, a marked reduction in non-oxidized (violet) BODIPY 581/591 C11 fluorescence was revealed in the iFSP1-treated group, suggesting enhanced lipid peroxidation (Fig. 6 G and 6 H). The ratio of oxidized lipids to reduced lipids markedly elevated in the iFSP1-treated oocytes, further substantiating ferroptosis induction under FSP1 inhibition (Fig. 6 J). Moreover, iFSP1-treated oocytes showed aberrant expression of ferroptosis marker genes: prostaglandin-endoperoxide synthase 2 ( Ptgs2 ), lysophosphatidylcholine acyltransferase 3 ( Lpcat3 ), acyl-CoA synthetase long-chain family member 4 ( Acs4 ), solute carrier family 7 ( Slc7a11 ), and cytochrome p450 oxidoreductase ( Por ) (Fig. 6 K). Finally, to distinguish ferroptosis from apoptosis, we also assessed apoptosis levels by staining oocytes with annexin V (Fig. 6 L). The absence of an annexin V signal confirmed that FSP1 inhibition did not cause apoptosis in iFSP1-treated oocytes (Fig. 6 M). In conclusion, these findings suggest that FSP1 inhibition affects mouse oocyte development by inducing ferroptosis. 3.7. Inhibited FSP1 protein causes mitochondria dysfunction in oocytes Since ferroptosis is typically accompanied by abnormalities in mitochondrial function, we investigated the impact of FSP1 inhibition on mitochondrial performance in oocytes. We discovered that mitochondria were uniformly distributed in the cytoplasm of the control group, whereas a notable aggregation of mitochondria was evident in the iFSP1-treated cohort (Fig. 7 A). Statistically, the treated cohort displayed a marked elevate in the proportion of abnormal mitochondrial distribution (Fig. 7 B). Mitochondrial function under FSP1 inhibition was further assessed by measuring mitochondrial membrane potential with the TMRE probe. Oocytes treated with iFSP1 showed a significant increase in the membrane potential versus control oocytes, indicating impaired mitochondrial function (Fig. 7 C and 7 D). Indeed, significantly reduced ATP content in iFSP1-treated oocytes confirmed enhanced mitochondrial stress under suppressed FSP1 activity (Fig. 7 E). We also investigated the expression of dynamin-related protein 1 (DRP1), an essential mediator of mitochondrial fission. The DRP1 protein had even cytosolic distribution in control oocytes but became concentrated around the outer mitochondrial membrane in iFSP1-treated oocytes (Fig. 7 F), corroborating elevated mitochondrial stress. These findings demonstrate that FSP1 inhibition promotes ferroptosis and mitochondrial dysfunction in mouse oocytes. 4. DISCUSSION 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 Fe 2+ 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 Fe 2+ 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 Fe 2+ 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. Declarations Author Contribution Hongzhen Ruan: Writing – original draft, Visualization, Formal analysis, Data curation, Conceptualization. Huifen Xiang: Validation, Data curation. Huilei Chen: Visualization, Funding acquisition.Yajing Liu: Visualization, Formal analysis, Data curation. Yunxia Cao: Supervision, Project administration, Funding acquisition. Zhiming Ding: Writing – review & editing, Writing – original draft, Supervision, Project administration, Conceptualization. Dan Liang: Supervision,Resources, Funding acquisition.Peiwen Wang : Visualization, Software, Formal analysis.Liuliu Dong: Validation, Methodology. Yaxin Chen and Yingying Zhang and Cong Ma : Validation, Formal analysis. Mengyao Wang and Caiyun Wu: Visualization, Validation.Hongzhen Ruan,Huifen Xiang and Yajing Liu contribute equally and should be considered as co-first authors. References Miao Y, Cui Z, Gao Q, Rui R, Xiong B. Nicotinamide Mononucleotide Supplementation Reverses the Declining Quality of Maternally Aged Oocytes. CELL REP. 2020;32(5):107987. Reader KL, Stanton JL, Juengel JL. The Role of Oocyte Organelles in Determining Developmental Competence. BIOLOGY-BASEL 2017, 6(3). Bhattacharya S, Basu D, Ak N, Priyadarshini A. Molecular mechanism of oocyte maturation. Soc Reprod Fertil Suppl. 2007;63:45–55. Li J, Qian WP, Sun QY. Cyclins regulating oocyte meiotic cell cycle progression†. BIOL REPROD. 2019;101(5):878–81. Mikwar M, MacFarlane AJ, Marchetti F. Mechanisms of oocyte aneuploidy associated with advanced maternal age. MUTAT RES-REV MUTAT. 2020;785:108320. Zhang FL, Li WD, Zhu KX, Zhou X, Li L, Lee TL, Shen W. Aging-related aneuploidy is associated with mitochondrial imbalance and failure of spindle assembly. CELL DEATH DISCOV. 2023;9(1):235. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060–72. Stockwell BR, Friedmann AJ, Bayir H, Bush AI, Conrad M, Dixon SJ, Fulda S, Gascón S, Hatzios SK, Kagan VE et al. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. CELL 2017, 171(2):273–285. Poindessous V, Lazareth H, Crambert G, Cheval L, Sampaio JL, Pallet N. STAT3 drives the expression of ACSL4 in acute kidney injury. ISCIENCE. 2024;27(6):109737. Zhang J, Su T, Fan Y, Cheng C, Xu L. LiTian: Spotlight on iron overload and ferroptosis: Research progress in female infertility. LIFE SCI. 2024;340:122370. Wang F, Liu Y, Ni F, Jin J, Wu Y, Huang Y, Ye X, Shen X, Ying Y, Chen J, et al. BNC1 deficiency-triggered ferroptosis through the NF2-YAP pathway induces primary ovarian insufficiency. NAT COMMUN. 2022;13(1):5871. Hu W, Zhang Y, Wang D, Yang T, Qi J, Zhang Y, Jiang H, Zhang J, Sun B, Liang S. Iron Overload-Induced Ferroptosis Impairs Porcine Oocyte Maturation and Subsequent Embryonic Developmental Competence in vitro. FRONT CELL DEV BIOL. 2021;9:673291. Huang J, Fan H, Li C, Yang K, Xiong C, Xiong S, Feng S, Chen S, Wang B, Su Y, et al. Dysregulation of ferroptosis-related genes in granulosa cells associates with impaired oocyte quality in polycystic ovary syndrome. FRONT ENDOCRINOL. 2024;15:1346842. Liu M, Wu K, Wu Y. The emerging role of ferroptosis in female reproductive disorders. BIOMED PHARMACOTHER. 2023;166:115415. Ni Z, Li Y, Song D, Ding J, Mei S, Sun S, Cheng W, Yu J, Zhou L, Kuang Y, et al. Iron-overloaded follicular fluid increases the risk of endometriosis-related infertility by triggering granulosa cell ferroptosis and oocyte dysmaturity. CELL DEATH DIS. 2022;13(7):579. Bersuker K, Hendricks JM, Li Z, Magtanong L, Ford B, Tang PH, Roberts MA, Tong B, Maimone TJ, Zoncu R, et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature. 2019;575(7784):688–92. Doll S, Freitas FP, Shah R, Aldrovandi M, Da SM, Ingold I, Goya GA, Xavier DST, Panzilius E, Scheel CH, et al. FSP1 is a glutathione-independent ferroptosis suppressor. Nature. 2019;575(7784):693–8. Li W, Liang L, Liu S, Yi H, Zhou Y. FSP1: a key regulator of ferroptosis. TRENDS MOL MED. 2023;29(9):753–64. Zhang S, Gou S, Zhang Q, Yong X, Gan B, Jia D. FSP1 oxidizes NADPH to suppress ferroptosis. CELL RES. 2023;33(12):967–70. Xavier DST, Schulte C, Alves AN, Maric HM, Friedmann AJ. Molecular characterization of AIFM2/FSP1 inhibition by iFSP1-like molecules. CELL DEATH DIS. 2023;14(4):281. Cheu JW, Lee D, Li Q, Goh CC, Bao MH, Yuen VW, Zhang MS, Yang C, Chan CY, Tse AP, et al. Ferroptosis Suppressor Protein 1 Inhibition Promotes Tumor Ferroptosis and Anti-tumor Immune Responses in Liver Cancer. CELL MOL GASTROENTER. 2023;16(1):133–59. Wang YQ, Qu HX, Dong YW, Qi JJ, Wei HK, Sun H, Jiang H, Zhang JB, Sun BX, Liang S. Inhibition of FSP1 impairs early embryo developmental competence in pigs. Theriogenology. 2024;214:257–65. Wasielak-Politowska M, Kordowitzki P. Chromosome Segregation in the Oocyte: What Goes Wrong during Aging. INT J MOL SCI 2022, 23(5). Artini PG, Obino ME, Vergine F, Sergiampietri C, Papini F, Cela V. Assisted reproductive technique in women of advanced fertility age. MINERVA GINECOL. 2018;70(6):738–49. Seshadri S, Morris G, Serhal P, Saab W. Assisted conception in women of advanced maternal age. BEST PRACT RES CL OB. 2021;70:10–20. Armstrong DT. Effects of maternal age on oocyte developmental competence. Theriogenology. 2001;55(6):1303–22. Duncan FE, Hornick JE, Lampson MA, Schultz RM, Shea LD, Woodruff TK. Chromosome cohesion decreases in human eggs with advanced maternal age. Aging Cell. 2012;11(6):1121–4. Ferreira AF, Soares M, Almeida-Santos T, Ramalho-Santos J, Sousa AP. Aging and oocyte competence: A molecular cell perspective. WIRES MECH DIS. 2023;15(5):e1613. Ahmed TA, Ahmed SM, El-Gammal Z, Shouman S, Ahmed A, Mansour R, El-Badri N. Oocyte Aging: The Role of Cellular and Environmental Factors and Impact on Female Fertility. ADV EXP MED BIOL. 2020;1247:109–23. Vollenhoven B, Hunt S. Ovarian ageing and the impact on female fertility. F1000Res 2018, 7. Wang X, Wang L, Xiang W. Mechanisms of ovarian aging in women: a review. J OVARIAN RES. 2023;16(1):67. Labit H, Fujimitsu K, Bayin NS, Takaki T, Gannon J, Yamano H. Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C. EMBO J. 2012;31(15):3351–62. Luo S, Tong L. Structural biology of the separase-securin complex with crucial roles in chromosome segregation. CURR OPIN STRUC BIOL. 2018;49:114–22. Vogt E, Kirsch-Volders M, Parry J, Eichenlaub-Ritter U. Spindle formation, chromosome segregation and the spindle checkpoint in mammalian oocytes and susceptibility to meiotic error. MUTAT RES-FUND MOL M. 2008;651(1–2):14–29. So C, Menelaou K, Uraji J, Harasimov K, Steyer AM, Seres KB, Bucevičius J, Lukinavičius G, Möbius W, Sibold C, et al. Mechanism of spindle pole organization and instability in human oocytes. Science. 2022;375(6581):j3944. Wu T, Dong J, Fu J, Kuang Y, Chen B, Gu H, Luo Y, Gu R, Zhang M, Li W, et al. The mechanism of acentrosomal spindle assembly in human oocytes. Science. 2022;378(6621):q7361. Baumann C, Wang X, Yang L, Viveiros MM. Error-prone meiotic division and subfertility in mice with oocyte-conditional knockdown of pericentrin. J CELL SCI. 2017;130(7):1251–62. Kim S, Leem J, Oh JS, Kim JS. Cytotoxicity of 9,10-Phenanthrenequinone Impairs Mitotic Progression and Spindle Assembly Independent of ROS Production in HeLa Cells. TOXICS 2022, 10(6). Ou XH, Li S, Xu BZ, Wang ZB, Quan S, Li M, Zhang QH, Ouyang YC, Schatten H, Xing FQ, et al. p38α MAPK is a MTOC-associated protein regulating spindle assembly, spindle length and accurate chromosome segregation during mouse oocyte meiotic maturation. Cell Cycle. 2010;9(20):4130–43. Peng L, He Y, Wang W, Chu Y, Lin Q, Rui R, Li Q, Ju S. PAK1 Is Involved in the Spindle Assembly during the First Meiotic Division in Porcine Oocytes. INT J MOL SCI 2023, 24(2). Chen Y, Zhang J, Tian Y, Xu X, Wang B, Huang Z, Lou S, Kang J, Zhang N, Weng J, et al. Iron accumulation in ovarian microenvironment damages the local redox balance and oocyte quality in aging mice. REDOX BIOL. 2024;73:103195. Fu C, Cao N, Zeng S, Zhu W, Fu X, Liu W, Fan S. Role of mitochondria in the regulation of ferroptosis and disease. FRONT MED-LAUSANNE. 2023;10:1301822. Han L, Pei J, Tao H, Guo X, Wei Y, Yang Z, Zhang H. The potential role of ferroptosis in the physiopathology of deep tissue injuries. INT WOUND J 2023, 21(2). Adegboro AG, Afolabi IS. Molecular mechanisms of mitochondria-mediated ferroptosis: a potential target for antimalarial interventions. FRONT CELL DEV BIOL. 2024;12:1374735. Afsar A, Zhang L. Putative Molecular Mechanisms Underpinning the Inverse Roles of Mitochondrial Respiration and Heme Function in Lung Cancer and Alzheimer's Disease. BIOLOGY-BASEL 2024, 13(3). Pfeifer GP. DNA Damage and Parkinson's Disease. INT J MOL SCI 2024, 25(8). Huang J, Yan Z, Song Y, Chen T. Nanodrug Delivery Systems for Myasthenia Gravis: Advances and Perspectives. PHARMACEUTICS 2024, 16(5). Bahety D, Böke E, Rodríguez-Nuevo A. Mitochondrial morphology, distribution and activity during oocyte development. TRENDS ENDOCRIN MET; 2024. Kang X, Yan L, Wang J. Spatiotemporal Distribution and Function of Mitochondria in Oocytes. REPROD SCI. 2024;31(2):332–40. Cao B, Qin J, Pan B, Qazi IH, Ye J, Fang Y, Zhou G. Oxidative Stress and Oocyte Cryopreservation: Recent Advances in Mitigation Strategies Involving Antioxidants. CELLS-BASEL 2022, 11(22). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.docx SupplementaryTable2.docx SupplementaryTable3.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4675534","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":325635824,"identity":"17913639-541f-4d3a-b2a5-d995185471ae","order_by":0,"name":"Hongzhen Ruan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYLCChAoJOzb25gMHP/6zkWNjbz9AWMuDMxbJ/DzHEg9LsKUZ8/GcSSCog/FhWwXjzBk+ygd42A4nzpNwMMCr3HxG8rMHCWckmA1u8DAckOBJS2+TYEhg+FGxDacWmRtp5gZAv/AZ3O49cKBAwia3TbrxAGPPmds4tUhIJJhJgG25cy7hgIRBWm6bzIEEZsY2fFrSv0kktkkwbriRY3CAJ+FwOptEggEBLTlmYC0zZ4C0HDicQFgLz5sykMNAgZxwWLIhzbANGMgH8fqFPX2b5I+KOlBUHv74scFGXr69/eCDHxW4tWAHB0hUPwpGwSgYBaMADQAAqC1cqn6NWYAAAAAASUVORK5CYII=","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":true,"prefix":"","firstName":"Hongzhen","middleName":"","lastName":"Ruan","suffix":""},{"id":325635826,"identity":"6591b5e3-283f-43f3-9873-a7ee020aa534","order_by":1,"name":"Huifen Xiang","email":"","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":false,"prefix":"","firstName":"Huifen","middleName":"","lastName":"Xiang","suffix":""},{"id":325635828,"identity":"730c8ea0-1b8e-403b-82b8-5ef0b11a1ced","order_by":2,"name":"Yajing Liu","email":"","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":false,"prefix":"","firstName":"Yajing","middleName":"","lastName":"Liu","suffix":""},{"id":325635829,"identity":"c6e04b25-0cc6-46ad-86ca-d80732786e6a","order_by":3,"name":"Peiwen Wang","email":"","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":false,"prefix":"","firstName":"Peiwen","middleName":"","lastName":"Wang","suffix":""},{"id":325635832,"identity":"7f0858b7-00de-4dd2-8788-a50a5bdc007b","order_by":4,"name":"Liuliu Dong","email":"","orcid":"","institution":"Bengbu Medical University, No. 2600 Donghai Avenue, Bengbu 233030, Anhui, China","correspondingAuthor":false,"prefix":"","firstName":"Liuliu","middleName":"","lastName":"Dong","suffix":""},{"id":325635833,"identity":"61fe4261-437a-4d6b-bdb1-aacbfa0a678b","order_by":5,"name":"Yaxin Chen","email":"","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":false,"prefix":"","firstName":"Yaxin","middleName":"","lastName":"Chen","suffix":""},{"id":325635835,"identity":"a94b7d05-01af-454c-a92e-5b78bb523460","order_by":6,"name":"Yingying Zhang","email":"","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Zhang","suffix":""},{"id":325635838,"identity":"0d67a076-cdf7-4c3e-b08f-1df86eadf05f","order_by":7,"name":"Cong Ma","email":"","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Ma","suffix":""},{"id":325635839,"identity":"f964f4a6-33b2-46a2-ab98-59d1a2c11992","order_by":8,"name":"Mengyao Wang","email":"","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":false,"prefix":"","firstName":"Mengyao","middleName":"","lastName":"Wang","suffix":""},{"id":325635840,"identity":"fb9cac69-fe67-4dcd-978b-df1a77e190ce","order_by":9,"name":"Caiyun Wu","email":"","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":false,"prefix":"","firstName":"Caiyun","middleName":"","lastName":"Wu","suffix":""},{"id":325635842,"identity":"2e221079-5bee-4adb-8c65-0eed5395218a","order_by":10,"name":"Yunxia Cao","email":"","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":false,"prefix":"","firstName":"Yunxia","middleName":"","lastName":"Cao","suffix":""},{"id":325635844,"identity":"71f65a0c-720f-412a-b287-e2d768beccd3","order_by":11,"name":"Dan Liang","email":"","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Liang","suffix":""},{"id":325635845,"identity":"e7850270-a410-4762-963e-27d32c3612c5","order_by":12,"name":"Zhiming Ding","email":"","orcid":"","institution":"Reproductive Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, No.218 Jixi Road, Hefei 230022, China","correspondingAuthor":false,"prefix":"","firstName":"Zhiming","middleName":"","lastName":"Ding","suffix":""}],"badges":[],"createdAt":"2024-07-02 16:14:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4675534/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4675534/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61344802,"identity":"6116751e-b7d4-4606-92e4-1bf5cb9755e6","added_by":"auto","created_at":"2024-07-29 17:46:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9427435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFerroptosis suppressor 1 (FSP1) is expressed in mouse oocytes\u003c/strong\u003e(A) Mouse uterine, testicular, and ovarian tissues were collected for Western blotting. (B) Data were denoted as mean ± SD of at least 3 independent experiments. (C) The FSP1 protein detected with immunofluorescence in mouse ovarian sections. Scale bar, 50 μm. (D) Reduced FSP1 protein levels were determined with Western blotting in the ovaries of 8-month-old mice. (E) Data were denoted as mean ± SD. 3W, n = 9; 8M, n = 9. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. (F) Decreased FSP1 protein expression detected with immunohistochemistry in 8-month-old mouse oocytes. Scale bar, 50 μm. (G) Data were denoted as mean ± SD of at least 3 independent experiments. 3W, n = 3; 8M, n = 3. ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. (H) Mouse oocytes were incubated for 0, 2, 8, and 12 h and progressed until germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) stages for Western blotting. Protein samples were examined with FSP1 or GAPDH antibodies. (I) Data were denoted as mean ± SD of at least 3 independent experiments. GV, n = 150; GVBD, n = 150; MI, n = 150; MII, n = 150.\u003cem\u003e \u003c/em\u003ens,\u003cem\u003e P\u003c/em\u003e \u0026gt; 0.05. (J) Mouse oocytes were incubated for 0, 2, 8, and 12 h and developed to GV, GVBD, MI, and MII stages for immunofluorescence staining with FSP1 (violet) or anti-α-tubulin (α-tubulin) antibody (green). Scale bar, 25μm.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-4675534/v1/de9d8e6ab303fe222fc15b0c.png"},{"id":61344362,"identity":"ec2505d5-bbae-40a9-81d0-6468e30537c1","added_by":"auto","created_at":"2024-07-29 17:38:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17534782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibited FSP1 protein impairs oocyte meiotic maturation and cell cycle progression\u003c/strong\u003e(A) Oocytes at the GV stage after 2 h incubation in an M16 medium with 0, 5, 10, and 15 μM iFSP1 (FSP1 inhibitor). Scale bar, 100 μm. (B) GVBD rates of control and iFSP1-treated oocytes. Control group (92.00% ± 4.16%, n = 75); 5 μM group (90.00% ± 5.77%, n = 79); 10 μM group (69.67% ± 11.14%, n = 76); 15 μM group (38.67% ± 4.70%, n = 74). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. (C) Oocytes at the GV stage after 12 h incubation in an M16 medium with 0, 5, 10, and 15 μM iFSP1. Scale bar, 100 μm. (D) PBE rates of the 2 groups. Control group (84.00% ± 4.73%, n = 75); 5 μM group (74.00% ± 4.93%, n = 79); 10 μM group (75.67% ± 4.67%, n = 76); 15 μM group (17.67% ± 5.78%, n = 74). ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (E) Oocytes at various meiotic stages. α-tubulin, green; DNA, blue. Scale bar, 25 μm. (F) The fraction of oocytes at diverse meiotic stages after exposing oocytes at the GV stage to 0 μM or 15 μM iFSP1 for 12 h. Control group, n = 75; 15 μM group, n = 74. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-4675534/v1/2494ff86044bde497c271f96.png"},{"id":61344355,"identity":"3c5ae756-3a9e-4320-9bb3-14a4b584f871","added_by":"auto","created_at":"2024-07-29 17:38:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":714548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eiFSP1 treatment interferes with oocyte spindle assembly and chromosomal arrangement\u003c/strong\u003e(A) Representative images of spindle morphology and chromosomal arrangement in the control and iFSP1-treated oocytes. α-tubulin, green; DNA, blue. Scale bar, 10 μm. (B) The percentage of oocytes with abnormal spindle in Control (16.00% ± 2.80%, n = 75) and iFSP1-treated (70.43% ± 1.77%, n = 74) groups. ****\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001. (C) The percentage of oocytes with abnormal chromosomes in both cohorts. Control (11.50% ± 1.85%, n = 75); iFSP1 treatment (68.63% ± 3.39%, n = 47). ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001. (D) Spindle length in the 2 groups. Control (46.51 ± 1.15 μm, n = 47); iFSP1 treatment (32.36 ± 0.88 μm, n = 47). ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. (E) Spindle width in the 2 groups. Control (26.66 ± 0.87 μm, n = 47); iFSP1 treatment (24.40 ± 0.70 μm, n = 47). ns, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05. (F) Oocyte chromosome width in both groups. Control (18.86 ± 0.80 μm, n = 47); iFSP1 treatment (24.82 ± 0.88 μm, n = 47). ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. (G) The percentage of chromosome width to spindle length in control and iFSP1-treated oocytes. Control (0.41 ± 0.02, n=47); iFSP1 treatment (0.78 ± 0.04, n = 47). ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-4675534/v1/c258615a874769e9c89541c1.png"},{"id":61344358,"identity":"23dcd826-254e-41f3-a08d-24a407883d6b","added_by":"auto","created_at":"2024-07-29 17:38:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2103163,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFSP1 inhibition affects the normal function of MTOCs in oocytes\u003c/strong\u003e(A) The localization of p-MAPK after iFSP1 treatment. p-MAPK, violet; α-tubulin, green; DNA, blue. Scale bar, 10 μm. (B) The localization of Pericentrin after iFSP1 treatment. Pericentrin, violet; α-tubulin, green; DNA, blue. Bar, 10 μm. (C) The localization of p-Aurora A after iFSP1 treatment. p-Aurora A, violet; α-tubulin, green; DNA, blue. Scale bar, 10 μm.\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-4675534/v1/574c7f3a908368807367ac80.png"},{"id":61344364,"identity":"b42b4558-6433-4e98-83c7-caf87a005263","added_by":"auto","created_at":"2024-07-29 17:38:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":998438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFSP1 repression continuously activates SAC\u003c/strong\u003e(A) Immunofluorescence images of BubR1 in control and iFSP1-treated oocytes incubated for 4.5 h and 10.5 h. BubR1, violet; DAPI, blue. Scale bar, 10 μm. (B) The percentage of oocytes with positive BubR1 signal after 10.5 h culture. Control (72.00% ± 1.16%, n = 75); iFSP1 treatment (91.33% ± 2.91%, n = 92). **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"F5.png","url":"https://assets-eu.researchsquare.com/files/rs-4675534/v1/d77135a22de83384a1c5cd6e.png"},{"id":61344361,"identity":"59c88f71-86f2-4e2f-85f7-9d47e99936f2","added_by":"auto","created_at":"2024-07-29 17:38:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3053550,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMouse oocytes undergo ferroptosis under FSP1 inhibition\u003c/strong\u003e(A) Representative images of the FerroOrange probe in the control and iFSP1-treated oocytes. FerroOrange, violet. Scale bar, 25 μm. (B) Relative fluorescence intensity statistics of FerroOrange in both groups. Control, n = 57; iFSP1 treatment, n = 63, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. (C) Representative images of DHE in the control and iFSP1-treated oocytes. DHE, violet. Scale bar, 25 μm. (D) Relative fluorescence intensity statistics of DHE in both groups. Control, n = 72; iFSP1 treatment, n = 60. ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. (E) Representative images of DCFH-DA in the control and iFSP1-treated oocytes. DCFH-DA, violet. Scale bar, 25 μm. (F) Relative fluorescence intensity statistics of DCFH-DA in both groups. Control, n = 78; iFSP1 treatment, n = 63. ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. (G) Representative images of reduced and oxidized lipids in the control and iFSP1-treated oocytes. Reduced lipids, violet; oxidized lipids, green. Scale bar, 25 μm. (H) Relative fluorescence intensity statistics of reduced lipids in both groups. Control, n = 57; iFSP1 treatment, n = 77. ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. (I) Relative fluorescence intensity statistics of oxidized lipids in both groups. Control, n = 57; iFSP1 treatment, n = 77. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. (J) The ratio of oxidized to reduced lipids. Control (0.66 ± 0.02, n = 57); iFSP1 treatment (0.86 ± 0.02, n = 77). ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. (K) The relative mRNA levels of ferroptosis marker genes \u003cem\u003ePtgs2, lpcat3, Acsl4, Slc7a11\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand\u003cem\u003e Por\u003c/em\u003e were quantified with RT-qPCR in both groups. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. (L) Representative images of annexin V in the control and iFSP1-treated oocytes. Annexin V, violet. Scale bar, 25 μm. (M) Relative fluorescence intensity statistics of annexin V in positive oocytes from both groups. Control, n = 65; iFSP1 treatment, n = 72. ns, \u003cem\u003eP \u003c/em\u003e\u0026gt;\u003cem\u003e \u003c/em\u003e0.05.\u003c/p\u003e","description":"","filename":"F6.png","url":"https://assets-eu.researchsquare.com/files/rs-4675534/v1/7423b2e0df7d6fe684808e5c.png"},{"id":61344803,"identity":"64cad124-23aa-4c85-a4a3-d6cb5bb30f10","added_by":"auto","created_at":"2024-07-29 17:46:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2008289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibited FSP1 protein causes mitochondrial dysfunction in oocytes\u003c/strong\u003e(A) Representative images of Mito-Tracker in the control and iFSP1-treated oocytes. Mito-Tracker, violet. Scale bar, 25 μm. (B) The proportion of oocytes with abnormal mitochondrial distribution. Control, n = 75; iFSP1 treatment, n = 72. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. (C) Representative images of TMRE in the control and iFSP1-treated oocytes. TMRE, violet. Scale bar, 25 μm. (D) Relative fluorescence intensity statistics of TMRE in both groups. Control, n = 60; iFSP1 treatment, n = 48. ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. (E) Relative ATP levels in both groups. Control, n = 75; iFSP1 treatment, n = 75. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. (F) Representative images of DRP1 in the control and iFSP1-treated oocytes. DRP1, violet; α-tubulin, green; DNA, blue. Scale bar, 25 μm.\u003c/p\u003e","description":"","filename":"F7.png","url":"https://assets-eu.researchsquare.com/files/rs-4675534/v1/1bba9039beaf304584d98745.png"},{"id":61429367,"identity":"35d4d2c5-73a2-4c1a-a135-e90597d6e021","added_by":"auto","created_at":"2024-07-30 15:34:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":55764841,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4675534/v1/2d8bc6e1-9794-4c67-adda-a71fb289ee76.pdf"},{"id":61344801,"identity":"ec30744f-6116-4b85-8bec-5348239b996b","added_by":"auto","created_at":"2024-07-29 17:46:25","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14192,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4675534/v1/b43dac29cc1729e95a7f2885.docx"},{"id":61344354,"identity":"91ec54f7-51f2-452e-a9f5-977b522e82c3","added_by":"auto","created_at":"2024-07-29 17:38:25","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12012,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4675534/v1/027ce862da5055ca4defdae9.docx"},{"id":61344360,"identity":"2c784318-bc51-45dd-8305-6bef9a96cd90","added_by":"auto","created_at":"2024-07-29 17:38:25","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12768,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4675534/v1/9ab1c4e4525e33b841dc6708.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ferroptosis suppressor 1 regulates ferroptosis and mitochondrial function during mouse oocyte maturation","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eOocyte quality is critical for female fertility, with compromised quality affecting sperm-egg binding and early embryonic development, influencing pregnancy outcome [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Numerous oocytes form during oogenesis, a complex and discontinuous meiotic process in female mammals. The undeveloped oocyte is halted in the initial meiotic prophase (G2) and exhibits a visible nucleus known as the germinal vesicle (GV). At sexual maturity, hormones stimulate the oocyte to undergo germinal vesicle breakdown (GVBD), marking the G2-M transition and resuming the first meiotic division [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The formation of the spindle, composed of microtubules, and the alignment of homologous chromosomes on the equatorial plate are critical events during the first meiotic metaphase (MI). The oocyte undergoes homologous chromosome segregation during the final phase of the first meiotic division, extrudes the first polar body, and stalls at the second meiotic metaphase (metaphase II, MII), developing into a mature oocyte capable of fertilization. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Proper spindle assembly during meiosis is crucial for fertility as abnormal spindle morphology and chromosome alignment can cause oocyte meiotic defects, resulting in malformed embryos, miscarriages, and birth defects [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, exploring the regulatory mechanisms of oocyte development and identifying potential molecular regulatory targets have considerable theoretical and clinical applications.\u003c/p\u003e \u003cp\u003eFerroptosis represents a form of controlled cellular demise characterized by iron-mediated oxidation of lipids, which is mechanistically and morphologically distinct from apoptosis and alternative modes of regulated cell death [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Polyunsaturated fatty acids, iron, and reactive oxygen species (ROS) are the main positive factors triggering ferroptosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Polyunsaturated fatty acids highly expressed in the cell membrane form polyunsaturated phospholipids via long-chain acyl-CoA synthetase family 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) activity. These phospholipids then utilize free Fe\u003csup\u003e2+\u003c/sup\u003e as a catalyst for the Fenton reaction with O\u003csub\u003e2\u003c/sub\u003e, where electrons are transferred to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate ROS, triggering lipid peroxidation and ferroptosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Ferroptosis assumes a crucial function in the development of female reproductive disorders [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Premature ovarian insufficiency, endometriosis, polycystic ovary syndrome, and trophoblastic dysfunction disorders are characterized by varying degrees of ferroptosis, suggesting it may be a therapeutic target for related diseases. For example, basonuclin zinc finger protein 1 (\u003cem\u003eBNC1\u003c/em\u003e) mutations in humans and mice cause ferroptosis-induced oocyte death and follicular atresia, leading to premature ovarian insufficiency. Inhibiting ferroptosis in mice attenuates \u003cem\u003eBnc1\u003c/em\u003e mutation-induced premature ovarian insufficiency [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Evidence indicates that ferroptosis is strongly associated with impaired oocyte meiotic maturation [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]; however, our knowledge of ferroptosis in oocyte meiotic maturation remains limited.\u003c/p\u003e \u003cp\u003eFerroptosis suppressor protein 1 (FSP1), or apoptosis inducing factor mitochondria associated (AIFM2), is a highly conserved flavoprotein initially identified as a p53-responsive gene due to its structural similarity with the apoptosis-inducing factor (AIF). Recent evidence indicates that FSP1 functions as a glutathione-independent inhibitor of ferroptosis that participates in ubiquinone (CoQ10) reduction, which prevents lipid oxidation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. N-myristoylated, plasma membrane-localized FSP1 promotes the recruitment of CoQ10 to the cell membrane and its NADPH-catalyzed reduction to ubiquinol (CoQ10H2). Consequently, CoQ10H2 traps free radicals to hinder lipid peroxidation and inhibit cellular ferroptosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The FSP1-CoQ10-NADPH axis, in conjunction with glutathione and glutathione peroxidase 4 (GPX4), acts as an independent parallel system essential for maintaining phospholipid redox homeostasis by inhibiting ferroptosis and phospholipid peroxidation. iFSP1 is a selective FSP1 inhibitor [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] that binds to FSP1 at the plasma membrane, impeding its NADH oxidase activity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Hence, iFSP1 is often used in FSP1-related studies, and its inhibitory effects are well described. Interestingly, a recent study in pigs showed that FSP1 inhibition impairs early embryonic development [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], implying that FSP1 has a vital role in gametogenesis and embryonic development. Nevertheless, the function of FSP1 during oocyte meiosis and the underlying mechanism is still unknown and require a detailed exploration to elucidate our understanding of the events decisive to oocyte quality.\u003c/p\u003e \u003cp\u003eIn our investigation, we examined the function of FSP1 during mouse oocyte meiosis utilizing the iFSP1 inhibitor. We demonstrated that FSP1 was situated in the cytoplasm during mouse oocyte meiosis. Moreover, we found that FSP1 inhibition impaired oocyte maturation in conjunction with ferroptosis, accompanied by mitochondrial dysfunction. Our findings offer initial proof that FSP1 is crucial in mouse oocyte meiosis.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODOLOGIES","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Experimental animals\u003c/h2\u003e \u003cp\u003eThe 3-week-old female, 8-week-old male, and 8-month-old female Kunming (KM) mice were obtained from the Laboratory Animal Center of Anhui Medical University (Hefei, China) and Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China). The mice were housed at the Laboratory Animal Center of Anhui Medical University in a controlled environment with a steady 24\u0026deg;C temperature and a 12-h light-dark cycle. They were granted unlimited access to nourishment and hydration for the duration of the investigation. All procedures involving animals were sanctioned by the Ethical Committee of Anhui Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental reagents and antibodies\u003c/h2\u003e \u003cp\u003eThe primary reagents utilized in this research are detailed in Supplementary Table\u0026nbsp;1. Antibody information is provided in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. In vitro oocyte maturation and iFSP1 treatment\u003c/h2\u003e \u003cp\u003eFollowing adaptation, female mice were euthanized, and their ovaries were extracted and immediately transferred to an M16 medium supplemented with 50 \u0026micro;M 3-isobutyl-1-methylxanthine. The GV-expressing oocytes were collected under a stereomicroscope and relocated to a balanced M16 culture medium. The specimens were maintained in an incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e and complete humidity for 0 h (GV stage), 2 h (GVBD stage), 8 h (MI stage), and 12 h (MII stage). A stock solution of iFSP1 was prepared by dissolving it in dimethylsulfoxide. Treatment cohorts were subjected to the M16 medium with incremental concentrations of iFSP1: 5 \u0026micro;M, 10 \u0026micro;M, and 15 \u0026micro;M, and the control cohort to an equivalent volume of dimethylsulfoxide in the M16 medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eMouse ovarian sections were available from 3-week-old and 8-month-old female KM mice. Sections underwent deparaffinization in xylene and subsequent rehydration via a gradient of diminishing alcohol concentrations (100%-70%). Endogenous peroxidase was inhibited in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 30 min. Antigen retrieval was performed in citrate buffer (pH 6.0) at 100\u0026deg;C for 2 min. The sections were rinsed in phosphate-buffered saline (PBS) and then in 5% BSA for 1 h to reduce nonspecific binding. The sections were exposed to anti-FSP1 as the primary antibody (1:500) at 4\u0026deg;C overnight. The samples underwent washing and were then exposed to the secondary antibody for 1 h. The sections were treated with diaminobenzidine and hematoxylin, dehydrated, and mounted in neutral resin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Immunofluorescence\u003c/h2\u003e \u003cp\u003eThe fertilized oocytes were placed in 4% paraformaldehyde with 0.5% Triton X-100, then incubated for 50 min at ambient temperature. Following this, the oocytes were rinsed utilizing PBS with 2% bovine serum albumin (BSA) for 1 h at ambient temperature. An overnight incubation followed at 4\u0026deg;C with the primary antibodies at a 1:100 dilution ratio. The oocytes were rinsed 3\u0026times; with PBS coupled with 0.1% Tween 20 and 0.01% Triton X-100. Next, they underwent incubation with anti-α-tubulin-FITC (1:100) or Cy3-conjugated goat anti-rabbit IgG (1:100) as the secondary antibody at 37\u0026deg;C for 1 h. After DAPI staining for 10 min, the oocytes were positioned in the middle of a slide with a droplet of antifade mounting medium for fluorescence examination. Fluorescence intensity was analyzed with ImageJ (NIH, Bethesda, MA, USA) or ZEN3.4 (Zeiss, Jena, Germany) software. Mouse ovarian sections underwent dewaxed with xylene and rehydration in an alcohol gradient for immunofluorescence. Fluorescence intensity was investigated utilizing ZEN3.4 (Zeiss).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Western blotting\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from tissues using RIPA lysis buffer (P0013, Beyotime) supplemented with SDS-PAGE sample loading buffer (P0295, Beyotime). The protein samples were heat-denatured at 95\u0026deg;C for 10 min, electrophoretically separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (IPVH00010, Millipore) with 0.45-\u0026micro;m pores. The membranes underwent a blocking phase in 5% bovine serum albumin for 2 h and a washing process, followed by an incubation with the primary antibodies. Following another wash, the membrane underwent a 2-h incubation at an ambient temperature with secondary antibodies the next day. The detection and densitometric evaluation of the immunoblots was performed using a chemiluminescence detection kit (P0018, Beyotime), and the bands were visually documented using the Tanon 5200 image-capturing system (Tanon).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. RNA isolation and reverse transcription-quantitative PCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eSeventy-five oocytes each from control and iFSP1-treated groups treated for 8 h were collected. Subsequently, total RNA extraction was conducted using the RNAprep Pure Micro Kit as per the instructions, which consisted mainly of cell lysis and elution from the adsorption column. Reverse transcription was performed to obtain cDNA as per the instructions of the PrimeScript\u0026trade; RT reagent Kit (Perfect Real Time). cDNA levels of target genes were measured utilizing the LightCycler 480 SYBR Green I Master. The reaction system consisted of 1 \u0026micro;l cDNA, 10 \u0026micro;l SYBR Green I Master, 2 \u0026micro;l primers, and 7 \u0026micro;l DEPC water. Reactions were performed on a LightCycler\u0026reg; 96 (Roche, Germany) instrument. The relative expression levels of genes to Gapdh were quantified utilizing the 2-ΔΔCT method, with Gapdh serving as a control. The specific information is listed in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.8. Fe\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eand lipid peroxidation level detection\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eFe\u003csup\u003e2+\u003c/sup\u003e levels were identified utilizing the FerroOrange fluorescent probe, and lipid peroxidation levels were determined with the BODIPY 581/591 C11 fluorescent probe. For detection, oocytes were harvested, rinsed, placed in the staining medium, and maintained at 37\u0026deg;C for 25 min. The excess staining solution was removed by rinsing with PBS, and the oocytes were placed into confocal petri dishes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.9. Mitochondrial localization and membrane potential detection\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eMitochondrial localization in oocytes was determined with Mito-Tracker Red. The mitochondrial membrane potential of oocytes was assessed with a TMRE-mitochondrial membrane potential assay kit. In brief, oocytes were harvested, rinsed, placed in the dye medium, and maintained at 37\u0026deg;C for 25 min. The excess staining solution was removed by rinsing with PBS, and the oocytes were placed into confocal petri dishes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.10. Superoxide anion and ROS detection\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eROS levels within mouse oocytes were assessed utilizing DCFH-DA (dichloro-dihydro-fluorescein diacetate), and superoxide anions were assessed utilizing dihydroethidium (DHE). Control and iFSP1-exposed oocytes cultivated for 8 h were gathered and placed in an M16 medium containing 20 \u0026micro;M DCFH-DA or DHE for 25 min at 37\u0026deg;C. The oocytes were rinsed utilizing an M16 medium without DCFH-DA or DHE to remove the indicators that failed to penetrate the cytoplasm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.11. Annexin-V assay\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eInitial stages of programmed cell death in oocytes were identified utilizing an annexin V-FITC apoptosis detection kit. The outer membrane was eliminated employing Tyrode\u0026rsquo;s medium, and the oocytes were placed in an annexin V-FITC binding solution and maintained at 37\u0026deg;C for 25 min. The oocytes were rinsed 3\u0026times; with PBS and placed in confocal petri dishes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.12. Adenosine triphosphate detection\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAdenosine triphosphate (ATP) levels in oocytes were quantified employing an ATP assay kit. A total of 30 oocytes from the control or iFSP1-treated group were gathered, placed into 100 \u0026micro;L of lysis solution, and disrupted on ice for 3 min. Afterward, 100 \u0026micro;L of the cell extract was transferred into a 96-well plate containing the test mixture and blended completely. The chemiluminescence intensity was quantified employing a SpectraMax iD3 multifunctional microplate reader and SoftMax Pro 7 software (Molecular Devices, LLC.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll results were denoted as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and derived from at least 3 separate replicate experiments. Data were assessed and visualized with GraphPad Prism. The variations across the 2 cohorts were determined utilizing the \u003cem\u003et\u003c/em\u003e test, and those across multiple cohorts were assessed utilizing the analysis of variance (ANOVA). The Bonferroni post hoc analysis was carried out utilizing the findings of the chi-square test. Statistical significance was identified when \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Expression and localization of FSP1 in mouse ovaries and oocytes\u003c/h2\u003e \u003cp\u003eUterine, testicular, and ovarian tissues were collected for Western blotting to determine FSP1 expression in mouse reproductive organs. The FSP1 protein was expressed in all 3 tissues, with significantly higher levels in ovaries than in uteruses and testicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Moreover, immunofluorescence analysis of mouse ovarian tissues revealed that FSP1 was expressed across all stages of follicular development: primordial, primary, secondary, and mature follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We also compared FSP1 expression between the ovaries of 3-week-old and 8-month-old female mice to investigate the association between FSP1 and age-related changes. We observed reduced FSP1 expression in the ovaries of 8-month-old female mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E), indicating that FSP1 expression decreases with age. Strikingly, oocytes of 8-month-old mice showed lower FSP1 expression than those of 3-week-old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). Thus, we collected oocytes at the GV, GVBD, MI, and MII phases to delineate FSP1 expression patterns during mouse oocyte meiosis. We demonstrated that FSP1 had consistent expression throughout oocyte maturation, from the GV to MII stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, I). In addition, oocytes immunofluorescently stained with anti-FSP1 antibody exhibited a punctate distribution of FSP1 within the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). These data suggest that FSP1 has high but age-dependent expression in mouse ovaries. Its persistent expression throughout the stages of oocyte meiotic maturation implies that FSP1 has an indispensable role in oocyte maturation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. FSP1 inhibition impairs oocyte cell cycle and meiotic maturation\u003c/h2\u003e \u003cp\u003eWe used the iFSP1 inhibitor to investigate whether FSP1 expression affects oocyte quality regulation during meiotic maturation. We incubated oocytes for 2 h in the M16 medium with escalating iFSP1 levels (0 \u0026micro;M, 5 \u0026micro;M, 10 \u0026micro;M, or 15 \u0026micro;M). We showed that FSP1 inhibition at 5 \u0026micro;M and 10 \u0026micro;M iFSP1 concentrations did not significantly affect oocytes at GVBD and PBE stages compared with those from the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). However, 15 \u0026micro;M iFSP1 significantly impaired the occurrence of the GVBD stage and diminished the frequency of PBE in comparison with oocytes from the control cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). Hence, we selected 15 \u0026micro;M iFSP1 for subsequent experiments to explore how the iFSP1-induced loss of FSP1 activity compromises mouse oocyte GVBD and PBE stages. Following a 12 h treatment with iFSP1, oocytes were collected for cell cycle analysis to gain detailed insights into meiosis under FSP1 inhibition. Most oocytes in the FSP1-inhibited cohort were halted at the GVBD and MI phases, whereas the bulk of oocytes in the control cohort progressed to the MII stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The results suggest that pharmacological FSP1 inhibition impairs oocyte cell cycle progression, provoking meiotic failure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3. FSP1 inhibition impairs oocyte spindle assembly and chromosomal arrangement\u003c/h2\u003e \u003cp\u003eBecause the precise assembly of the spindle and the correct chromosomal arrangement are critical for oocyte maturation, we sought to explore the spindle formation at MI in iFSP1-treated oocytes. Oocytes in the control cohort displayed characteristic barrel-shaped MI spindles with well-aligned chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Conversely, iFSP1-treated oocytes exhibited disorganized spindle morphology and misaligned chromosomes, marked by diminished spindle dimensions and haphazard chromosomal distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A statistical analysis confirmed a notably elevated frequency of irregular spindles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and misaligned chromosomes in the iFSP1-treated cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Additionally, our analysis suggested that the iFSP1-treated oocytes showed a conspicuously decreased spindle length (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) but observed no significant differences in spindle width relative to the control cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Moreover, iFSP1-treated oocytes displayed a markedly elevated chromosome width (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) and the ratio of chromosome width to spindle length versus the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4. FSP1 inhibition disturbs subcellular localization of Pericentrin, p-MAPK, and p-Aurora A\u003c/h2\u003e \u003cp\u003eSince mouse oocytes lack centrosomes, they rely on acentriolar microtubule-organizing centers (MTOCs), which contain essential pericentriolar material, for spindle assembly. Pericentrin, p-MAPK, and p-Aurora A are MTOC-related proteins that play a critical role in the spindle formation during meiosis, so we assessed their expression by immunofluorescence under FSP1 inhibition in oocytes. Whereas control oocytes had Pericentrin and p-MAPK localized to the MI spindle poles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B), their localization in iFSP1-treated oocytes showed an irregular distribution. Likewise, control oocytes displayed a distinct positive signal for p-Aurora A protein at spindle poles, while iFSP1-treated oocytes exhibited a scattered and irregularly distributed p-Aurora A signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In summary, FSP1 inhibition disorganizes the localization of various MTOC-associated proteins in mouse oocytes, indicating protein dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5. FSP1 inhibition persistently activates the spindle assembly checkpoint\u003c/h2\u003e \u003cp\u003eAberrant spindle phenotype and the altered chromosome arrangement in oocytes after FSP1 inhibition suggest that the spindle assembly checkpoint (SAC) is activated to arrest meiotic progression. To validate our assumption, we investigated iFSP1-treated mouse oocytes for the localization of mitotic checkpoint Bub1-related kinase or MAD3/Bub1b (BubR1), an essential element of the SAC. We observed bright BubR1 signals in control and iFSP1-treated oocytes after 4.5 h culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Moreover, after 10.5 h culture, no positive BubR1 signal was detected in control oocytes, only segregated homologous chromosomes. By contrast, BubR1 was apparent as a bright fluorescence signal on the chromosomes in iFSP1-treated oocytes, and the chromosomes were unsegregated. Indeed, statistical data confirmed that the fraction of activated BubR1 in the iFSP1-administered cohort was notably elevated relative to the control cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These results indicate that repressed FSP1 activity in mouse oocytes sustains SAC activation, blocking oocyte meiotic progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.6. FSP1 inhibition induces ferroptosis in mouse oocytes\u003c/h2\u003e \u003cp\u003eThe FSP1 protein is a suppressor of ferroptosis; therefore, we investigated whether ferroptosis contributes to oocyte maturation failure in the absence of FSP1 activity. Considering the importance of Fe\u003csup\u003e2+\u003c/sup\u003e in lipid peroxidation and ferroptosis, we first utilized the Fe\u003csup\u003e2+\u003c/sup\u003e-selective fluorescent probe FerroOrange to determine Fe\u003csup\u003e2+\u003c/sup\u003e levels in oocytes. The iFSP1-treated cohort showed markedly elevated Fe\u003csup\u003e2+\u003c/sup\u003e levels relative to the control cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). In addition, iFSP1-treated oocytes had significantly elevated ROS and DHE levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-F), implying that oocytes experience excessive oxidative stress under FSP1 inhibition. We also detected increased levels of oxidized lipids in iFSP1-treated oocytes, indicated by higher fluorescence of oxidized (green) BODIPY 581/591 C11 reporter in treated oocytes compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Concurrently, a marked reduction in non-oxidized (violet) BODIPY 581/591 C11 fluorescence was revealed in the iFSP1-treated group, suggesting enhanced lipid peroxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). The ratio of oxidized lipids to reduced lipids markedly elevated in the iFSP1-treated oocytes, further substantiating ferroptosis induction under FSP1 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Moreover, iFSP1-treated oocytes showed aberrant expression of ferroptosis marker genes: prostaglandin-endoperoxide synthase 2 (\u003cem\u003ePtgs2\u003c/em\u003e), lysophosphatidylcholine acyltransferase 3 (\u003cem\u003eLpcat3\u003c/em\u003e), acyl-CoA synthetase long-chain family member 4 (\u003cem\u003eAcs4\u003c/em\u003e), solute carrier family 7 (\u003cem\u003eSlc7a11\u003c/em\u003e), and cytochrome p450 oxidoreductase (\u003cem\u003ePor\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). Finally, to distinguish ferroptosis from apoptosis, we also assessed apoptosis levels by staining oocytes with annexin V (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). The absence of an annexin V signal confirmed that FSP1 inhibition did not cause apoptosis in iFSP1-treated oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). In conclusion, these findings suggest that FSP1 inhibition affects mouse oocyte development by inducing ferroptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Inhibited FSP1 protein causes mitochondria dysfunction in oocytes\u003c/h2\u003e \u003cp\u003eSince ferroptosis is typically accompanied by abnormalities in mitochondrial function, we investigated the impact of FSP1 inhibition on mitochondrial performance in oocytes. We discovered that mitochondria were uniformly distributed in the cytoplasm of the control group, whereas a notable aggregation of mitochondria was evident in the iFSP1-treated cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Statistically, the treated cohort displayed a marked elevate in the proportion of abnormal mitochondrial distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Mitochondrial function under FSP1 inhibition was further assessed by measuring mitochondrial membrane potential with the TMRE probe. Oocytes treated with iFSP1 showed a significant increase in the membrane potential versus control oocytes, indicating impaired mitochondrial function (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Indeed, significantly reduced ATP content in iFSP1-treated oocytes confirmed enhanced mitochondrial stress under suppressed FSP1 activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). We also investigated the expression of dynamin-related protein 1 (DRP1), an essential mediator of mitochondrial fission. The DRP1 protein had even cytosolic distribution in control oocytes but became concentrated around the outer mitochondrial membrane in iFSP1-treated oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF), corroborating elevated mitochondrial stress. These findings demonstrate that FSP1 inhibition promotes ferroptosis and mitochondrial dysfunction in mouse oocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eFemale fertility experiences a notable decline with increasing age, particularly beyond 35 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Assisted reproductive technologies can improve reproductive outcomes to some extent, but their success rates also decline [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] largely due to age-related decrease in ovarian reserve and declining oocyte quality [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eThe FSP1 protein reduces CoQ10 to prevent lipid oxidation independently of the GPX4 pathway, inhibiting ferroptosis via an unknown regulatory mechanism [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. 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 [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eThe 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 [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. 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 [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] 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.\u003c/p\u003e \u003cp\u003eSince FSP1 inhibits ferroptosis through CoQ10 action, we asked whether the impaired oocyte maturation caused by FSP1 inhibition is associated with ferroptosis. Elevated Fe\u003csup\u003e2+\u003c/sup\u003e 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 Fe\u003csup\u003e2+\u003c/sup\u003e levels are found in oocytes of aging mice, and these oocytes have substantially reduced quality[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. 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 Fe\u003csup\u003e2+\u003c/sup\u003e 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.\u003c/p\u003e \u003cp\u003eMitochondria occupy a crucial position in iron metabolism [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and undergo morphological changes during ferroptosis, encompassing enhanced membrane density and diminished or absent mitochondrial cristae [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Iron overload causes mitochondrial dysfunction, evidenced by reduced mitochondrial respiration, elevated mitochondrial ROS levels, depolarization of the mitochondrial membrane potential, and mitochondrial swelling [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Excess ROS damages lipids, nucleic acids, and proteins, promoting DNA damage and protein dysfunction [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. An inequity among ROS levels and antioxidant defenses induces oxidative stress, and accumulated ROS triggers ferroptosis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Correct mitochondrial function is essential for proper maturation and oocyte competence [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. During oocyte maturation, the mtDNA copy number increases dramatically, and the distribution of mitochondria changes considerably to produce enough energy for meiosis [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Abnormal mitochondrial function leads to abnormal spindle assembly and failed polar body extrusion [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This evidence confirms that oocyte maturation defects observed under FSP1 inhibition are due to mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHongzhen Ruan: Writing \u0026ndash; original draft, Visualization, Formal analysis, Data curation, Conceptualization. Huifen Xiang: Validation, Data curation. Huilei Chen: Visualization, Funding acquisition.Yajing Liu: Visualization, Formal analysis, Data curation. Yunxia Cao: Supervision, Project administration, Funding acquisition. Zhiming Ding: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Supervision, Project administration, Conceptualization. Dan Liang: Supervision,Resources, Funding acquisition.Peiwen Wang : Visualization, Software, Formal analysis.Liuliu Dong: Validation, Methodology. Yaxin Chen and Yingying Zhang and Cong Ma : Validation, Formal analysis. Mengyao Wang and Caiyun Wu: Visualization, Validation.Hongzhen Ruan,Huifen Xiang and Yajing Liu contribute equally and should be considered as co-first authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMiao Y, Cui Z, Gao Q, Rui R, Xiong B. Nicotinamide Mononucleotide Supplementation Reverses the Declining Quality of Maternally Aged Oocytes. CELL REP. 2020;32(5):107987.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReader KL, Stanton JL, Juengel JL. The Role of Oocyte Organelles in Determining Developmental Competence. BIOLOGY-BASEL 2017, 6(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhattacharya S, Basu D, Ak N, Priyadarshini A. Molecular mechanism of oocyte maturation. Soc Reprod Fertil Suppl. 2007;63:45\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Qian WP, Sun QY. Cyclins regulating oocyte meiotic cell cycle progression\u0026dagger;. BIOL REPROD. 2019;101(5):878\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMikwar M, MacFarlane AJ, Marchetti F. Mechanisms of oocyte aneuploidy associated with advanced maternal age. MUTAT RES-REV MUTAT. 2020;785:108320.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang FL, Li WD, Zhu KX, Zhou X, Li L, Lee TL, Shen W. Aging-related aneuploidy is associated with mitochondrial imbalance and failure of spindle assembly. CELL DEATH DISCOV. 2023;9(1):235.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStockwell BR, Friedmann AJ, Bayir H, Bush AI, Conrad M, Dixon SJ, Fulda S, Gasc\u0026oacute;n S, Hatzios SK, Kagan VE et al. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. \u003cem\u003eCELL\u003c/em\u003e 2017, 171(2):273\u0026ndash;285.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoindessous V, Lazareth H, Crambert G, Cheval L, Sampaio JL, Pallet N. STAT3 drives the expression of ACSL4 in acute kidney injury. ISCIENCE. 2024;27(6):109737.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Su T, Fan Y, Cheng C, Xu L. LiTian: Spotlight on iron overload and ferroptosis: Research progress in female infertility. LIFE SCI. 2024;340:122370.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang F, Liu Y, Ni F, Jin J, Wu Y, Huang Y, Ye X, Shen X, Ying Y, Chen J, et al. BNC1 deficiency-triggered ferroptosis through the NF2-YAP pathway induces primary ovarian insufficiency. NAT COMMUN. 2022;13(1):5871.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu W, Zhang Y, Wang D, Yang T, Qi J, Zhang Y, Jiang H, Zhang J, Sun B, Liang S. Iron Overload-Induced Ferroptosis Impairs Porcine Oocyte Maturation and Subsequent Embryonic Developmental Competence in vitro. FRONT CELL DEV BIOL. 2021;9:673291.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang J, Fan H, Li C, Yang K, Xiong C, Xiong S, Feng S, Chen S, Wang B, Su Y, et al. Dysregulation of ferroptosis-related genes in granulosa cells associates with impaired oocyte quality in polycystic ovary syndrome. FRONT ENDOCRINOL. 2024;15:1346842.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Wu K, Wu Y. The emerging role of ferroptosis in female reproductive disorders. BIOMED PHARMACOTHER. 2023;166:115415.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi Z, Li Y, Song D, Ding J, Mei S, Sun S, Cheng W, Yu J, Zhou L, Kuang Y, et al. Iron-overloaded follicular fluid increases the risk of endometriosis-related infertility by triggering granulosa cell ferroptosis and oocyte dysmaturity. CELL DEATH DIS. 2022;13(7):579.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBersuker K, Hendricks JM, Li Z, Magtanong L, Ford B, Tang PH, Roberts MA, Tong B, Maimone TJ, Zoncu R, et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature. 2019;575(7784):688\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoll S, Freitas FP, Shah R, Aldrovandi M, Da SM, Ingold I, Goya GA, Xavier DST, Panzilius E, Scheel CH, et al. FSP1 is a glutathione-independent ferroptosis suppressor. Nature. 2019;575(7784):693\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi W, Liang L, Liu S, Yi H, Zhou Y. FSP1: a key regulator of ferroptosis. TRENDS MOL MED. 2023;29(9):753\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Gou S, Zhang Q, Yong X, Gan B, Jia D. FSP1 oxidizes NADPH to suppress ferroptosis. CELL RES. 2023;33(12):967\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXavier DST, Schulte C, Alves AN, Maric HM, Friedmann AJ. Molecular characterization of AIFM2/FSP1 inhibition by iFSP1-like molecules. CELL DEATH DIS. 2023;14(4):281.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheu JW, Lee D, Li Q, Goh CC, Bao MH, Yuen VW, Zhang MS, Yang C, Chan CY, Tse AP, et al. Ferroptosis Suppressor Protein 1 Inhibition Promotes Tumor Ferroptosis and Anti-tumor Immune Responses in Liver Cancer. CELL MOL GASTROENTER. 2023;16(1):133\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang YQ, Qu HX, Dong YW, Qi JJ, Wei HK, Sun H, Jiang H, Zhang JB, Sun BX, Liang S. Inhibition of FSP1 impairs early embryo developmental competence in pigs. Theriogenology. 2024;214:257\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWasielak-Politowska M, Kordowitzki P. Chromosome Segregation in the Oocyte: What Goes Wrong during Aging. INT J MOL SCI 2022, 23(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArtini PG, Obino ME, Vergine F, Sergiampietri C, Papini F, Cela V. Assisted reproductive technique in women of advanced fertility age. MINERVA GINECOL. 2018;70(6):738\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeshadri S, Morris G, Serhal P, Saab W. Assisted conception in women of advanced maternal age. BEST PRACT RES CL OB. 2021;70:10\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmstrong DT. Effects of maternal age on oocyte developmental competence. Theriogenology. 2001;55(6):1303\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuncan FE, Hornick JE, Lampson MA, Schultz RM, Shea LD, Woodruff TK. Chromosome cohesion decreases in human eggs with advanced maternal age. Aging Cell. 2012;11(6):1121\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerreira AF, Soares M, Almeida-Santos T, Ramalho-Santos J, Sousa AP. Aging and oocyte competence: A molecular cell perspective. WIRES MECH DIS. 2023;15(5):e1613.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmed TA, Ahmed SM, El-Gammal Z, Shouman S, Ahmed A, Mansour R, El-Badri N. Oocyte Aging: The Role of Cellular and Environmental Factors and Impact on Female Fertility. ADV EXP MED BIOL. 2020;1247:109\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVollenhoven B, Hunt S. Ovarian ageing and the impact on female fertility. \u003cem\u003eF1000Res\u003c/em\u003e 2018, 7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Wang L, Xiang W. Mechanisms of ovarian aging in women: a review. J OVARIAN RES. 2023;16(1):67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLabit H, Fujimitsu K, Bayin NS, Takaki T, Gannon J, Yamano H. Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C. EMBO J. 2012;31(15):3351\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo S, Tong L. Structural biology of the separase-securin complex with crucial roles in chromosome segregation. CURR OPIN STRUC BIOL. 2018;49:114\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVogt E, Kirsch-Volders M, Parry J, Eichenlaub-Ritter U. Spindle formation, chromosome segregation and the spindle checkpoint in mammalian oocytes and susceptibility to meiotic error. MUTAT RES-FUND MOL M. 2008;651(1\u0026ndash;2):14\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSo C, Menelaou K, Uraji J, Harasimov K, Steyer AM, Seres KB, Bucevičius J, Lukinavičius G, M\u0026ouml;bius W, Sibold C, et al. Mechanism of spindle pole organization and instability in human oocytes. Science. 2022;375(6581):j3944.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu T, Dong J, Fu J, Kuang Y, Chen B, Gu H, Luo Y, Gu R, Zhang M, Li W, et al. The mechanism of acentrosomal spindle assembly in human oocytes. Science. 2022;378(6621):q7361.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaumann C, Wang X, Yang L, Viveiros MM. Error-prone meiotic division and subfertility in mice with oocyte-conditional knockdown of pericentrin. J CELL SCI. 2017;130(7):1251\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim S, Leem J, Oh JS, Kim JS. Cytotoxicity of 9,10-Phenanthrenequinone Impairs Mitotic Progression and Spindle Assembly Independent of ROS Production in HeLa Cells. \u003cem\u003eTOXICS\u003c/em\u003e 2022, 10(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOu XH, Li S, Xu BZ, Wang ZB, Quan S, Li M, Zhang QH, Ouyang YC, Schatten H, Xing FQ, et al. p38α MAPK is a MTOC-associated protein regulating spindle assembly, spindle length and accurate chromosome segregation during mouse oocyte meiotic maturation. Cell Cycle. 2010;9(20):4130\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng L, He Y, Wang W, Chu Y, Lin Q, Rui R, Li Q, Ju S. PAK1 Is Involved in the Spindle Assembly during the First Meiotic Division in Porcine Oocytes. INT J MOL SCI 2023, 24(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Zhang J, Tian Y, Xu X, Wang B, Huang Z, Lou S, Kang J, Zhang N, Weng J, et al. Iron accumulation in ovarian microenvironment damages the local redox balance and oocyte quality in aging mice. REDOX BIOL. 2024;73:103195.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu C, Cao N, Zeng S, Zhu W, Fu X, Liu W, Fan S. Role of mitochondria in the regulation of ferroptosis and disease. FRONT MED-LAUSANNE. 2023;10:1301822.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan L, Pei J, Tao H, Guo X, Wei Y, Yang Z, Zhang H. The potential role of ferroptosis in the physiopathology of deep tissue injuries. INT WOUND J 2023, 21(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdegboro AG, Afolabi IS. Molecular mechanisms of mitochondria-mediated ferroptosis: a potential target for antimalarial interventions. FRONT CELL DEV BIOL. 2024;12:1374735.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfsar A, Zhang L. Putative Molecular Mechanisms Underpinning the Inverse Roles of Mitochondrial Respiration and Heme Function in Lung Cancer and Alzheimer's Disease. BIOLOGY-BASEL 2024, 13(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePfeifer GP. DNA Damage and Parkinson's Disease. INT J MOL SCI 2024, 25(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang J, Yan Z, Song Y, Chen T. Nanodrug Delivery Systems for Myasthenia Gravis: Advances and Perspectives. \u003cem\u003ePHARMACEUTICS\u003c/em\u003e 2024, 16(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBahety D, B\u0026ouml;ke E, Rodr\u0026iacute;guez-Nuevo A. Mitochondrial morphology, distribution and activity during oocyte development. TRENDS ENDOCRIN MET; 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang X, Yan L, Wang J. Spatiotemporal Distribution and Function of Mitochondria in Oocytes. REPROD SCI. 2024;31(2):332\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao B, Qin J, Pan B, Qazi IH, Ye J, Fang Y, Zhou G. Oxidative Stress and Oocyte Cryopreservation: Recent Advances in Mitigation Strategies Involving Antioxidants. \u003cem\u003eCELLS-BASEL\u003c/em\u003e 2022, 11(22).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"FSP1, oocyte, meiosis, ferroptosis, mitochondria","lastPublishedDoi":"10.21203/rs.3.rs-4675534/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4675534/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOocyte quality is critical for fertilization and embryo development. Recent studies have shown that ferroptosis may compromise oocyte quality. Ferroptosis suppressor protein 1 (FSP1) is a ferroptosis inhibitor with an undefined role in oocyte quality regulation during meiotic maturation. Here, we found that FSP1 is expressed throughout all stages of meiotic maturation and localizes to the cytoplasm of mouse oocytes. A decline in FSP1 expression was observed in the ovaries and oocytes of aged mice. Pharmacological inhibition of FSP1 caused a failure in germinal vesicle breakdown and polar body emission, accompanied by spindle abnormalities and chromosome misalignment. Moreover, FSP1 inhibition consistently activated the spindle assembly checkpoint, inducing meiotic arrest. Mechanistically, FSP1 inhibition increased Fe\u003csup\u003e2+\u003c/sup\u003e content, elevated dihydroethidium levels, promoted reactive oxygen species buildup, and heightened lipid peroxidation. Additionally, it dysregulated the expression of ferroptosis-related genes, suggesting that oocytes underwent ferroptosis. Furthermore, FSP1 inhibition provoked mitochondrial dysfunction, characterized by abnormal mitochondrial localization, reduced ATP levels, and elevated mitochondrial membrane potential. In summary, our findings demonstrate that FSP1 participates in oocyte meiotic maturation through its involvement in iron homeostasis and mitochondrial activity, and FSP1 inhibition results in ferroptosis-dependent meiotic failure.\u003c/p\u003e","manuscriptTitle":"Ferroptosis suppressor 1 regulates ferroptosis and mitochondrial function during mouse oocyte maturation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 17:38:20","doi":"10.21203/rs.3.rs-4675534/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b81b1023-8bff-4399-bc92-47efc89a0097","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-30T15:25:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-29 17:38:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4675534","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4675534","identity":"rs-4675534","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}