Small Molecule Inhibitor of Formin Homology 2 Domains (SMIFH2) Reveals the Roles of the Formin Family of Proteins in Spindle Assembly and Asymmetric Division in Mouse Oocytes

Dynamic actin reorganization is the main driving force for spindle migration and asymmetric cell division in mammalian oocytes. It has been reported that various actin nucleators including Formin-2 are involved in the polarization of the spindle and in asymmetric cell division. In mammals, the formin family is comprised of 15 proteins. However, their individual roles in spindle migration and/or asymmetric division have not been elucidated yet. In this study, we employed a newly developed inhibitor for formin family proteins, small molecule inhibitor of formin homology 2 domains (SMIFH2), to assess the functions of the formin family in mouse oocyte maturation. Treatment with SMIFH2 during in vitro maturation of mouse oocytes inhibited maturation by decreasing cytoplasmic and cortical actin levels. In addition, treatment with SMIFH2, especially at higher concentrations (500 μM), impaired the proper formation of meiotic spindles, indicating that formins play a role in meiotic spindle formation. Knockdown of the mDia2 formins caused a similar decrease in oocyte maturation and abnormal spindle morphology, mimicking the phenotype of SMIFH2-treated cells. Collectively, these results suggested that besides Formin-2, the other proteins of the formin, including mDia family play a role in asymmetric division and meiotic spindle formation in mammalian oocytes.     

The importance of mitochondrial metabolic activity and mitochondrial DNA replication during oocyte maturation in vitro on oocyte quality and subsequent embryo developmental competence

Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high-dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization.     

Intersectin-Cdc42 interaction is required for orderly meiosis in porcine oocytes

Intersectins (ITSNs) have been shown to act as adaptor proteins that govern multiple cellular events via regulating Cdc42 activity. However, it remains to be determined whether the ITSN-Cdc42 pathway is functional in porcine oocytes. To address this question, we used a small molecule, ZCL278, to selectively disrupt the ITSN2-Cdc42 interaction. In the present study, we find that porcine oocytes exposed to ZCL278 are unable to completely progress through meiosis. Meanwhile, the spindle defects and chromosomal congression failure are frequently detected in these oocytes. In support of this, we observed the accumulated distribution of vesicle-like ITSN2 signals around the chromosome/spindle region during porcine oocyte maturation. In addition, our results also showed that inhibition of the ITSN-Cdc42 interaction impairs the actin polymerization in porcine oocytes. In summary, the findings support a model where ITSNs, through the interaction with Cdc42, modulates the assembly of meiotic apparatus and actin polymerization, consequently ensuring the orderly meiotic progression during porcine oocyte maturation.     

Cover Image,Volume 234, Number 10, October 2019

Cytoskeleton which includes microtubule and actin filaments plays important roles during mammalian oocyte maturation. In the present study, we showed that protein kinase C mu (PKC mu) was one potential key molecule which affected cytoskeleton dynamics in mouse oocytes. Our results showed that PKC mu expressed and localized at the poles of the spindle during oocyte maturation, and PKC mu expression reduced in the oocytes from 6-month-old mice or 24 hr in vitro culture. We knocked down the expression of PKC mu in oocytes using morpholino injection to explore the relationship between PKC mu and subcellular structure defects. The loss of PKC mu reduced oocyte maturation competence, showing with decreased polar body extrusion rate and increased rate of symmetric division. Further analysis indicated that PKC mu decrease caused the spindle organization defects, and this could be confirmed by the decreased tubulin acetylation level. Moreover, we found that PKC mu affected the phosphorylation level of cofilin for actin assembly, which further affected cytoplasmic actin distribution and spindle positioning. In summary, our data indicated that PKC mu is one key factor for oocyte maturation through its roles on the spindle organization and actin filament distribution.     

银鲫卵母细胞Spindlin基因的表达特征及其功能分析

在卵母细胞成熟过程中,Spindlin与纺锤体微管蛋白相互作用,在配子到胚胎的过程中具有调节细胞周期的作用。前期研究表明,银鲫Spindlin(CagSpin)与微管蛋白相互作用,并与减数分裂的纺锤体共定位。在成熟过程中,CagSpin被磷酸化,在卵母细胞受精和卵胚转换中发挥重要的作用。研究通过对激素诱导后的卵母细胞进行追踪,采用RT-PCR和Western-blot分析,揭示卵母细胞在完成成熟的过程中,CagSpin持续大量表达。采用体外诱导卵母细胞成熟技术和显微注射的方法,揭示过量表达CagSpin导致胚泡(Germinalvesicle,GV)不能破裂,卵母细胞成熟过程被抑制。这些结果表明,CagSpin在卵母细胞成熟过程中发挥着关键的作用,同时为深入研究CagSpin的功能提供依据。     

Simulated Microgravity Compromises Mouse Oocyte Maturation by Disrupting Meiotic Spindle Organization and Inducing Cytoplasmic Blebbing

In the present study, we discovered that mouse oocyte maturation was inhibited by simulated microgravity via disturbing spindle organization. We cultured mouse oocytes under microgravity condition simulated by NASA''s rotary cell culture system, examined the maturation rate and observed the spindle morphology (organization of cytoskeleton) during the mouse oocytes meiotic maturation. While the rate of germinal vesicle breakdown did not differ between 1 g gravity and simulated microgravity, rate of oocyte maturation decreased significantly in simulated microgravity. The rate of maturation was 8.94% in simulated microgravity and was 73.0% in 1 g gravity. The results show that the maturation of mouse oocytes in vitro was inhibited by the simulated microgravity. The spindle morphology observation shows that the microtubules and chromosomes can not form a complete spindle during oocyte meiotic maturation under simulated microgravity. And the disorder of γ-tubulin may partially result in disorganization of microtubules under simulated microgravity. These observations suggest that the meiotic spindle organization is gravity dependent. Although the spindle organization was disrupted by simulated microgravity, the function and organization of microfilaments were not pronouncedly affected by simulated microgravity. And we found that simulated microgravity induced oocytes cytoplasmic blebbing via an unknown mechanism. Transmission electron microscope detection showed that the components of the blebs were identified with the cytoplasm. Collectively, these results indicated that the simulated microgravity inhibits mouse oocyte maturation via disturbing spindle organization and inducing cytoplasmic blebbing.     

Activated proline‐rich tyrosine kinase 2 regulates meiotic spindle assembly in the mouse oocyte

Proline‐rich tyrosine kinase 2 (PYK2), a member of the protein tyrosine kinase family, plays an important role in various cellular processes. PYK2 can be phosphorylated on tyrosine 402 by diverse stimuli at the cell surface, and recent studies have shown that this activated form of PYK2 is enriched in oocytes and required for fertilization. However, the subcellular localization and functions of activated PYK2 in oocytes remain elusive. In this study, we demonstrate that the localization of p‐PYK2 undergoes dynamic changes during in vitro maturation of mouse oocytes. The signal of p‐PYK2 is initially dispersed in the cytoplasm, but begins to decorate organized microtubules after the germinal vesicle breakdown and localizes to spindle poles at metaphase. Our data further show that p‐PYK2 colocalizes with γ‐tubulin from the germinal vesicle stage through the end of meiosis in mouse oocytes. Nocodazole treatment and washout experiments confirm that p‐PYK2 associates with the oocyte spindle and spindle poles. Moreover, pharmacological inhibition of PYK2 activity dramatically alters the morphology of the bipolar spindle and prevents oocyte maturation. Together, these data suggest that activated PYK2 may function as a component of the microtubule organizing center to regulate spindle assembly during the meiotic process of mouse oocytes.     

Melatonin protects against defects induced by malathion during porcine oocyte maturation

Malathion (MAL) is a common organophosphorus pesticide and affects both animal and human reproduction. However, the mechanisms regarding how MAL affects the mammalian oocyte quality and how to prevent it have not been fully investigated. In this study, we used porcine oocyte as a model and proved that MAL impaired porcine oocyte quality in a dose-dependent manner during maturation. MAL decreased the first polar body extrusion, disrupted spindle assembly and chromosome alignment, impaired cortical granules (CGs) distribution, and increased reactive oxygen species (ROS) level in oocytes. RNA-seq analysis showed that MAL exposure altered the expression of 2,917 genes in the porcine maturated oocytes and most genes were related to ROS, the lipid droplet process, and the energy supplement. Nevertheless, these defects could be remarkably ameliorated by adding melatonin (MLT) into the oocyte maturation medium. MLT increased oocyte maturation rate and decreased the abnormities of spindle assembly, CGs distribution and ROS accumulation in MAL-exposed porcine oocytes. More important, MLT upregulated the expression of genes related to lipid droplet metabolism (PPARγ and PLIN2), decreased lipid droplet size and lipid peroxidation in MAL-exposed porcine oocytes. Finally, we found that MLT increased the blastocysts formation and the cell numbers of blastocysts in MAL-exposed porcine oocytes after parthenogenetic activation, which was mediated by reduction of ROS levels and maintaining lipid droplet metabolism. Taken together, our results revealed that MLT had a protective action against MAL-induced deterioration of porcine oocyte quality.     

SKAP2 regulates Arp2/3 complex for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes

SKAP2 (Src kinase-associated phosphoprotein 2), a substrate of Src family kinases, has been suggested to be involved in actin-mediated cellular processes. However, little is known about its role in mouse oocyte maturation. In this study, we thus investigated the expression, localization, and functions of SKAP2 during mouse oocyte asymmetric division. SKAP2 protein expression was detected at all developmental stages in mouse oocytes. Immunofluorescent staining showed that SKAP2 was mainly distributed at the cortex of the oocytes during maturation. Treatment with cytochalasin B in oocytes confirmed that SKAP2 was co-localized with actin. Depletion of SKAP2 by injection with specific short interfering RNA caused failure of spindle migration, polar body extrusion, and cytokinesis defects. Meanwhile, the staining of actin filaments at the oocyte membrane and in the cytoplasm was significantly reduced after these treatments. SKAP2 depletion also disrupted actin cap and cortical granule-free domain formation, and arrested a large proportion of oocytes at the telophase stage. Moreover, Arp2/3 complex and WAVE2 expression was decreased after the depletion of SKAP2 activity. Our results indicate that SKAP2 regulates the Arp2/3 complex and is essential for actin-mediated asymmetric cytokinesis by interacting with WAVE2 in mouse oocytes.     

The Microtubule-Associated Protein ASPM Regulates Spindle Assembly and Meiotic Progression in Mouse Oocytes

The microtubule-associated protein ASPM (abnormal spindle-like microcephaly-associated) plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes.     

HDAC3 inhibition disrupts the assembly of meiotic apparatus during porcine oocyte maturation

Histone deacetylases (HDACs) are involved in a wide array of biological processes. However, the role of HDAC3 in porcine oocytes remains unclear. In the current study, we examine the effects of HDAC3 inhibition on porcine oocyte maturation using RGFP966, a selective HDAC3 inhibitor. We find that suppression of HDAC3 activity prevents not only the expansion of cumulus cells but also the meiotic progression of oocytes. It is interesting to note that HDAC3 displays a spindle-like distribution pattern as the porcine oocytes enter meiosis. In line with this, confocal microscopy reveals the high frequency of spindle defects and chromosomal congression failure in metaphase oocytes exposed to RGFP966. Moreover, HDAC3 inhibition results in the hyperacetylation of α-tubulin during oocyte meiosis. These findings indicate that HDAC3 activity might control the microtubule stability via the deacetylation of tubulin, which is critical for maintaining the proper spindle assembly, accurate chromosome separation, and orderly meiotic progression during porcine oocyte maturation.     

MEK1/2 is a critical regulator of microtubule assembly and spindle organization during rat oocyte meiotic maturation

MEK (MAPK kinase) is an upstream protein kinase of MAPK in the MOS/MEK/MAPK/p90rsk signaling pathway. We previously reported the function and regulation of MAPK during rat oocyte maturation. In this study, we further investigated the localization and possible roles of MEK1/2. First, immunofluorescent staining revealed that p-MEK1/2 was restricted to the germinal vesicle (GV). After germinal vesicle breakdown (GVBD), p-MEK1/2 condensed in the vicinity of chromosomes and then translocated to the spindle poles at metaphase I, while spindle microtubules stained faintly. When the oocyte went through anaphase I and telophase I, p-MEK1/2 disappeared from spindle poles and became associated with the midbody. By metaphase II, p-MEK1/2 was again localized to the spindle poles. Second, p-MEK1/2 was localized to the centers of cytoplasmic microtubule asters induced by taxol. Third, p-MEK1/2 co-localized with gamma-tubulin in microtubule-organizing centers (MTOCs). Forth, treatment with U0126, a non-competitive MEK1/2 inhibitor, did not affect germinal vesicle breakdown, but caused chromosome mis-alignment in all MI oocytes examined and abnormal spindle organization as well as small cytoplasmic spindle-like structure formation in MII oocytes. Finally, U0126 reduced the number of cytoplasmic asters induced by taxol. Our data suggest that MEK1/2 has regulatory functions in microtubule assembly and spindle organization during rat oocyte meiotic maturation.     

NMNAT2‐mediated NAD+ generation is essential for quality control of aged oocytes

Advanced maternal age has been reported to impair oocyte quality; however, the underlying mechanisms remain to be explored. In the present study, we identified the lowered NAD⁺ content and decreased expression of NMNAT2 protein in oocytes from old mice. Specific depletion of NMNAT2 in mouse oocytes disturbs the meiotic apparatus assembly and metabolic activity. Of note, nicotinic acid supplementation during in vitro culture or forced expression of NMNAT2 in aged oocytes was capable of reducing the reactive oxygen species (ROS) production and incidence of spindle/chromosome defects. Moreover, we revealed that activation or overexpression of SIRT1 not only partly prevents the deficient phenotypes of aged oocytes but also ameliorates the meiotic anomalies and oxidative stress in NMNAT2‐depleted oocytes. To sum up, our data indicate a role for NMNAT2 in controlling redox homeostasis during oocyte maturation and uncover that NMNAT2‐ NAD⁺‐SIRT1 is an important pathway mediating the effects of maternal age on oocyte developmental competence.     

Dynamic distribution of spindlin in nucleoli, nucleoplasm and spindle from primary oocytes to mature eggs and its critical function for oocyte-to-embryo transition in gibel carp

Spindlin (Spin) was thought as a maternal-effect factor associated with meiotic spindle. Its role for the oocyte-to-embryo transition was suggested in mouse, but its direct evidence for the function had been not obtained in other vertebrates. In this study, we used the CagSpin-specific antibody to investigate CagSpin expression pattern and distribution during oogenesis of gibel carp (Carassius auratus gibelio). First, the oocyte-specific expression pattern and dynamic distribution was revealed in nucleoli, nucleoplasm, and spindle from primary oocytes to mature eggs by immunofluorescence localization. In primary oocytes and growth stage oocytes, CagSpin accumulates in nucleoli in increasing numbers along with the oocyte growth, and its disassembly occurs in vitellogenic oocytes, which implicates that CagSpin may be a major component of a large number of nucleoli in fish growth oocytes. Then, co-localization of CagSpin and β-tubulin was revealed in meiotic spindle of mature egg, indicating that CagSpin is one spindle-associated factor. Moreover, microinjection of CagSpin-specific antibody into the fertilized eggs blocked the first cleavage, and found that the CagSpin depletion resulted in spindle assembly disturbance. Thereby, our study provided the first direct evidence for the critical oocyte-to-embryo transition function of Spin in vertebrates, and confirmed that Spin is one important maternal-effect factor that participates in oocyte growth, oocyte maturation, and oocyte-to-embryo transition.     

Continuous exposure to bisphenol A during in vitro follicular development induces meiotic abnormalities

Bisphenol A (BPA), a widely used environmental contaminant, may exert weak estrogenic, anti-androgenic and anti-thyroidic activities. BPA is suspected to possess aneugenic properties that may affect somatic cells and mammalian oocytes. Oocyte growth and maturation depend upon a complex bi-directional signaling between the oocyte and its companion somatic cells. Consequently, disturbances in oocyte maturation may originate either from direct effects of BPA at the level of the oocyte or from indirect influences at the follicular level, such as alterations in hormonal homeostasis. This study aimed to analyze the effects of chronic BPA exposure (3 nM to 30 microM) on follicle-enclosed growth and maturation of mouse oocytes in vitro. Oocytes were cultured and their spindle and chromosomes were stained by alpha-tubulin immunofluorescence and ethidium homodimer-2, respectively. Confocal microscopy was utilized for subsequent analysis. Only follicles that were exposed to 30 microM BPA during follicular development showed a slightly reduced granulosa cell proliferation and a lower total estrogen production, but they still developed and formed antral-like cavities. However, 18% of oocytes were unable to resume meiosis after stimulation of oocyte maturation, and 37% arrested after germinal vesicle breakdown, significantly different from controls (p<0.05). Only 45% of the oocytes extruded a first polar body (p < 0.05). 30 microM BPA led also to a significant increase in meiosis I-arrested oocytes with unaligned chromosomes and spindle aberrations. Oocytes that were able to progress beyond meiosis I, frequently arrested at an abnormal telophase I. Additionally, in many oocytes exposed to low chronic BPA that matured to meiosis II chromosomes failed to congress at the spindle equator. In conclusion, mouse follicle culture reveals non-linear dose-dependent effects of BPA on the meiotic spindle in mouse oocytes when exposure was chronic throughout oocyte growth and maturation.     

Chk2 Regulates Cell Cycle Progression during Mouse Oocyte Maturation and Early Embryo Development

As a tumor suppressor homologue during mitosis, Chk2 is involved in replication checkpoints, DNA repair, and cell cycle arrest, although its functions during mouse oocyte meiosis and early embryo development remain uncertain. We investigated the functions of Chk2 during mouse oocyte maturation and early embryo development. Chk2 exhibited a dynamic localization pattern; Chk2 expression was restricted to germinal vesicles at the germinal vesicle (GV) stage, was associated with centromeres at pro-metaphase I (Pro-MI), and localized to spindle poles at metaphase I (MI). Disrupting Chk2 activity resulted in cell cycle progression defects. First, inhibitor-treated oocytes were arrested at the GV stage and failed to undergo germinal vesicle breakdown (GVBD); this could be rescued after Chk2 inhibition release. Second, Chk2 inhibition after oocyte GVBD caused MI arrest. Third, the first cleavage of early embryo development was disrupted by Chk2 inhibition. Additionally, in inhibitor-treated oocytes, checkpoint protein Bub3 expression was consistently localized at centromeres at the MI stage, which indicated that the spindle assembly checkpoint (SAC) was activated. Moreover, disrupting Chk2 activity in oocytes caused severe chromosome misalignments and spindle disruption. In inhibitor-treated oocytes, centrosome protein γ-tubulin and Polo-like kinase 1 (Plk1) were dissociated from spindle poles. These results indicated that Chk2 regulated cell cycle progression and spindle assembly during mouse oocyte maturation and early embryo development.     

Functional involvement of Xenopus LIM kinases in progression of oocyte maturation

LIM kinases (LIMK), including LIMK1 and LIMK2, are unique LIM-family proteins containing a catalytic (kinase) domain. These kinases phosphorylate an actin-depolymerizing factor, cofilin, involved in the regulation of actin-filament dynamics. An unanswered question is the in vivo function of LIMK and how they contribute to development. When we cloned Xenopus homologues of mammalian LIMK, Xlimk1 and Xlimk2, we found that their mRNA and products were abundantly expressed in oocytes. In addition, we obtained evidence for the functional involvement of Xlimk1/2 during oocyte maturation. The microinjection of Xlimk1/2 mRNA into progesterone-treated oocytes significantly inhibited the appearance of a white maturation spot (WMS), an indicator of entry into meiosis. In oocytes lacking a WMS, the organization and/or migration of the microtubule-derived precursor of the meiotic spindle was predominantly affected. We also found that the ectopic expression of Xlimk1/2 clearly prevented dephosphorylation (activation) of Xenopus cofilin (XAC) during oocyte maturation. Furthermore, co-injection of Xlimk1/2 with the constitutively active type of XAC overcame the inhibitory effects by Xlimk1/2, suggesting that XLIMK-induced abnormality in oocyte maturation was mediated by XAC inactivation. Based on these findings, we propose that XLIMK is a putative regulator of cytoskeletal rearrangements during oocyte maturation, and the interaction between XLIMK activity and microtubule dynamics seems highly likely.     

Leptin accelerates pronuclear formation following intracytoplasmic sperm injection of porcine oocytes: Possible role for MAP kinase inactivation

Leptin, a multifunctional hormone, is present in mammalian oocytes and follicular fluids and cumulus cells. While leptin modulates oocyte maturation in vitro which seems to result in enhancement of embryo development, it is unclear whether leptin treatment of oocytes affects cytoplasmic maturation and fertilization processes. In order to gain a better understanding of the role of leptin during oocyte maturation, we examined microtubule and microfilament assembly following oocyte maturation and blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) in leptin-treated oocytes. Addition of 10 or 100 ng/ml leptin during oocyte maturation did not increase the proportion of metaphase II oocytes, but enhanced development to blastocyst stage by day 7 (P < 0.01) after parthenogenetic activation (PA), accompanied by increased cell number. However there was no effect on the number of apoptotic cells in blastocysts. Following maturation in the presence of leptin, there were more oocytes with normal spindle formation. MAPK activity decreased more rapidly, and pronuclear formation was accelerated after parthenogenetic activation or ICSI of leptin-treated oocytes. These results suggested that exogeneous leptin enhanced spindle assembly and accelerated pronuclear formation following fertilization, possibly via the MAPK pathway.     

Melatonin alleviates the deterioration of oocytes from mice subjected to repeated superovulation

Induction of repeated superovulation with exogenous hormones is widely used in assisted reproductive technology (ART). Though it is generally safe, emerging evidence has indicated that repeated superovulation may compromise oocyte quality. However, few studies have explored how to ameliorate such impairment. Because melatonin has beneficial influences on oocytes in various detrimental environments, we aimed to explore whether melatonin could protect mouse oocytes after repeated superovulation. We found that repeated superovulation markedly reduced meiotic maturation and disrupted spindle organization and chromosome alignment. Furthermore, we observed reduced mitochondrial content and enhanced early apoptosis in oocytes from mice subjected to repeated superovulation. In addition, 5-methylcytosine (5mc) fluorescence intensity was lower in oocytes from experimental mice than in those from control mice, indicating that repeated superovulation disrupts genomic DNA methylation, and elevations in reactive oxygen species levels indicated that repeated superovulation also induces oxidative stress. Conversely, melatonin administration improved oocyte maturation and attenuated the observed defects. Interestingly, supplementation with melatonin during in vitro maturation had the same protective effects on oocytes as in vivo melatonin administration. In summary, our results show that melatonin can improve oocyte quality after repeated superovulation and thus provide a potential strategy to improve ART efficiency.