Kinesin motor KIF16A regulates microtubule stability and actin-dependent spindle migration in mouse oocyte meiosis.

Kif16A, a member of the kinesin-3 family of motor proteins, has been shown to play crucial roles in inducing mitotic arrest, apoptosis, and mitotic cell death. However, its roles during oocyte meiotic maturation have not been fully defined. In this study, we report that Kif16A exhibits unique accumulation on the spindle apparatus and colocalizes with microtubule fibers during mouse oocyte meiotic maturation. Targeted depletion of Kif16A using gene-targeting siRNA disrupts the progression of the meiotic cell cycle. Furthermore, Kif16A depletion leads to aberrant spindle assembly and chromosome misalignment in oocytes. Our findings also indicate that Kif16A depletion reduces tubulin acetylation levels and compromises microtubule resistance to depolymerizing drugs, suggesting its crucial role in microtubule stability maintenance. Notably, we find that the depletion of Kif16A results in a notably elevated incidence of defective kinetochore-microtubule attachments and the absence of BubR1 localization at kinetochores, suggesting a critical role for Kif16A in the activation of the spindle assembly checkpoint (SAC) activity. Additionally, we observe that Kif16A is indispensable for proper actin filament distribution, thereby impacting spindle migration. In summary, our findings demonstrate that Kif16A plays a pivotal role in regulating microtubule and actin dynamics crucial for ensuring both spindle assembly and migration during mouse oocyte meiotic maturation.

Copper oxide nanoparticles impairs oocyte meiosis maturation by inducing mitochondrial dysfunction and oxidative stress.

Copper oxides nanoparticles (CuO NPs) are widely used for a variety of industrial and life science applications. In addition to cause neurotoxicity, hepatotoxicity, immunotoxicity, CuO NPs have also been reported to adversely affect the reproductive system in animals; However, little is known about the effects and potential mechanism of CuO NPs exposure on oocyte quality, especially oocyte maturation. In the present study, we reported that CuO NPs exposure impairs the oocyte maturation by disrupting meiotic spindle assembly and chromosome alignment, as well as kinetochore-microtubule attachment. In addition, CuO NPs exposure also affects the acetylation level of α-tubulin in mice oocyte, which hence impairs microtubule dynamics and organization. Besides, CuO NPs exposure would result in the mis-localization of Juno and Ovastacin, which might be one of the critical factors leading to the failure of oocyte maturation. Finally, CuO NPs exposure impairs the mitochondrial distribution and induced high levels of ROS, which led to the accumulation of DNA damage and occurrence of apoptosis. In summary, our results indicated that CuO NPs exposure had potential toxic effects on female fertility and led to the poor oocyte quality in female mice.

Prothioconazole exposure disrupts oocyte maturation and fertilization by inducing mitochondrial dysfunction and apoptosis in mice.

Prothioconazole (PTC), a novel broad-spectrum triazole fungicide, has attracted widespread concern due to its wide use and toxicological effects on non-target organisms. However, little is known about the impact of PTC on oocyte quality and female fertility, especially on oocyte maturation and fertilization. In the present study, we reported that PTC exposure affects the oocyte developmental competence and oocyte fertilization ability to weaken female fertility. Firstly, PTC compromises oocyte development ability by disrupting spindle morphology and chromosome alignment, as well as decreasing acetylation level of α-tubulin and disrupting kinetochore-microtubule attachments. In addition, PTC compromises oocyte fertilization ability by weakening the sperm binding ability and impairing the dynamics of Juno, Cortical granule and Ovastacin. Finally, single-cell transcriptome analysis revealed that PTC exposure has potentially toxic effects on oocyte development and fertilization, which is caused by the mitochondrial dysfunction and the occurrence of oxidative stress and apoptosis. In summary, our results indicated that PTC exposure had potentially toxic effects on female fertility and led to poor oocyte quality in female mice.

Butylparaben weakens female fertility via causing oocyte meiotic arrest and fertilization failure in mice.

Butylparaben is an ubiquitous environmental endocrine disruptor, that is commonly used in cosmetics and personal care product due to its anti-microbial properties. Butylparaben has been shown to cause developmental toxicity, endocrine and metabolic disorders and immune diseases. However, little is known about the impact on female fertility, especially oocyte quality. In the present study, we reported that butylparaben influenced female fertility by showing the disturbed oocyte meiotic capacity and fertilization potential. Specifically, butylparaben results in the oocyte maturation arrest by impairing spindle/chromosome structure and microtubule stability. Besides, butylparaben results in fertilization failure by impairing the dynamics of Juno and ovastacin and the sperm binding ability. Last, single-cell transcriptome analysis showed that butylparaben-induced oocyte deterioration was caused by mitochondrial dysfunction, which led to the accumulation of ROS and occurrence of apoptosis. Collectively, our study indicates that mitochondrial dysfunction and redox perturbation is the major cause of the weakened female fertility expoesd to butylparaben.

Alpha-lipoic acid supplementation reverses the declining quality of oocytes exposed to cyclophosphamide.

Cyclophosphamide (CY) is a chemotherapeutic drug that is commonly used to treat malignancies of the ovary, breast, and hematology, as well as autoimmune disorders. As a cofactor of mitochondrial multienzyme complexes, alpha lipoic acid (ALA) is well known for its antioxidant characteristics, which operate directly on the scavenging of reactive oxygen species (ROS) and indirectly on the intracellular recycling of other antioxidants. However, the underlying mechanisms through which CY exerts its toxic effects on meiosis and oocyte quality, as well as a viable approach for protecting oocyte quality and preserving fertility, remain unknown. In present study, immunostaining and fluorescence intensity quantification were applied to assess the effects of CY and ALA supplementation on the key processes during the oocyte meiotic maturation. Our results show that supplementing oocytes with ALA, a well-known antioxidant and free radical scavenger, can reverse CY-induced oocyte meiotic maturation failure. Specifically, we found that CY exposure caused oocyte meiotic failure by disrupting meiotic organelle dynamics and arrangement, as well as a prominently impaired cytoskeleton assembly. In addition, CY caused an abnormal distribution of mitochondrion and cortical granules, two indicators of oocyte cytoplasmic maturation. More importantly, we show that ALA supplementation effectively reverses CY-induced meiotic failure and oocyte quality decline by suppressing oxidative stress-induced DNA damage and apoptosis in oocytes. Collectively, our data reveal that ALA supplementation is a feasible approach to protect oocytes from CY-exposed deterioration, providing a better understanding of the mechanisms involved in chemotherapy-induced meiotic failure.

Alpha-lipoic acid supplementation restores the meiotic competency and fertilization capacity of porcine oocytes induced by arsenite.

Arsenite is known as a well-known endocrine disrupting chemicals, and reported to be associated with an increased incidence of negative health effects, including reproductive disorders and dysfunction of the endocrine system. However, it still lacks of the research regarding the beneficial effects of ALA on arsenite exposed oocytes, and the underlying mechanisms have not been determined. Here, we report that supplementation of alpha-lipoic acid (ALA), a strong antioxidant naturally present in all cells of the humans, is able to restore the declined meiotic competency and fertilization capacity of porcine oocytes induced by arsenite. Notably, ALA recovers the defective nuclear and cytoplasmic maturation of porcine oocytes caused by arsenite exposure, including the impaired spindle formation and actin polymerization, the defective mitochondrion integrity and cortical granules distribution. Also, ALA recovers the compromised sperm binding ability to maintain the fertilization potential of arsenite-exposed oocytes. Importantly, ALA suppresses the oxidative stress by reducing the levels of ROS and inhibits the occurrence of DNA damage along with apoptosis. Above all, we provide a new perspective for the application of ALA in effectively preventing the declined oocyte quality induced by environmental EDCs.

Paraquat exposure impairs porcine oocyte meiotic maturation.

Paraquat (PQ) is a heterocyclic pesticide that not only damages the testicular development and reduces the quality of semen, but also disturbs the secretion of hormones in the reproductive system. However, the effects of PQ on oocyte maturation and its toxic mechanism have not been yet fully clarified. Here we showed that PQ exposure could have toxic effects on porcine oocyte maturation. PQ exposure with 100 µM inhibited cumulus cell expansion and significantly reduced the rate of first polar body extrusion during oocyte maturation. PQ-exposed oocytes could not develop to the 2-cell and blastocyst stage. PQ exposure with 100 µM significantly increased abnormal spindle rate (65.2% ± 1.0%) and misaligned chromosome rate (63.2% ± 3.4%) compared to the control group (38.3% ± 1.0% and 38.4% ± 1.0%, respectively; P < 0.05). F-actin also exhibited reduced distribution in PQ-exposed oocytes (10.3% ± 1.0%) compared to the control group (14.4% ± 1.0%, P < 0.05). In addition, PQ exposure reduced the active mitochondria levels, but apparently increased the reactive oxygen species (ROS), rH2AX, and LC3 (autophagy marker) levels. qPCR analyses showed that PQ exposure caused the aberrant expression of genes associated with cumulus cell expansion, but did not affect the expression of apoptosis-related genes. Taken together, these results indicate that PQ exposure impaired oocyte nuclear and cytoplasmic maturation probably through oxidative stress.

Insufficiency of melatonin in follicular fluid is a reversible cause for advanced maternal age-related aneuploidy in oocytes.

Age-related decline in female fertility is a common feature that occurs in the fourth decade of women as a result of a reduction in both oocyte quality and quantity [1]. However, strategies to prevent the deterioration of maternal aged oocytes and relevant mechanisms are still underexplored. Here, we find that the reduced abundance of melatonin in the follicular fluid highly correlates with the advanced maternal age-related aneuploidy. Of note, we show that exposure of oocytes from aged mice both in vitro and in vivo to exogenous melatonin not only eliminates the accumulated reactive oxygen species-induced DNA damage and apoptosis, but also suppresses the occurrence of aneuploidy caused by spindle/chromosome defect that is frequently observed in aged oocytes. Importantly, we reveal that melatonin supplementation reverses the defective phenotypes in aged oocytes through a Sirt1/Sod2-dependent mechanism. Inhibition of Sirt1 activity abolishes the melatonin-mediated improvement of aged oocyte quality. Together our findings provide evidence that supplementation of melatonin is a feasible way to protect oocytes from advanced maternal age-related meiotic defects and aneuploidy, demonstrating the potential for improving the quality of oocytes from aged women and the efficiency of assisted reproductive technology.

Coenzyme Q10 ameliorates the quality of postovulatory aged oocytes by suppressing DNA damage and apoptosis.

Postovulatory aging is known to compromise the oocyte quality as well as subsequent embryo development in many different animal models, and becomes one of the most intractable issues that limit the outcome of human assisted reproductive technology (ART). However, the strategies to prevent the deterioration of aged oocytes and relevant mechanisms are still underexplored. Here, we find that supplementation of CoQ10, a natural antioxidant present in human follicular fluids, is able to restore the postovulatory aging-induced fragmentation of oocytes and decline of fertilization. Importantly, we show that CoQ10 supplementation recovers postovulatory aging-caused meiotic defects such as disruption of spindle assembly, misalignment of chromosome, disappearance of actin cap, and abnormal distribution patterns of mitochondria and cortical granules. In addition, CoQ10 protects aged oocytes from premature exocytosis of ovastacin, cleavage of sperm binding site ZP2, and loss of localization of Juno, to maintain the fertilization potential. Notably, CoQ10 suppresses the aging-induced oxidative stress by reducing the levels of superoxide and DNA damage, ultimately inhibiting the apoptosis. Taken together, our findings demonstrate that CoQ10 supplementation is a feasible and effective way to prevent postovulatory aging and preserve the oocyte quality, potentially contributing to improve the successful rate of IVF (in vitro fertilization) and ICSI (intracytoplasmic sperm injection) during human ART.

The cohesion establishment factor Esco1 acetylates α-tubulin to ensure proper spindle assembly in oocyte meiosis.

Esco1 has been reported to function as a cohesion establishment factor that mediates chromosome cohesion and segregation in mitotic cells. However, its exact roles in meiosis have not been clearly defined. Here, we document that Esco1 is expressed and localized to both the nucleus and cytoplasm during mouse oocyte meiotic maturation. Depletion of Esco1 by siRNA microinjection causes the meiotic progression arrest with a severe spindle abnormality and chromosome misalignment, which is coupled with a higher incidence of the erroneous kinetochore-microtubule attachments and activation of spindle assembly checkpoint. In addition, depletion of Esco1 leads to the impaired microtubule stability shown by the weakened resistance ability to the microtubule depolymerizing drug nocodazole and the decreased level of acetylated α-tubulin. Conversely, overexpression of Esco1 causes hyperacetylation of α-tubulin and spindle defects. Moreover, we find that Esco1 binds to α-tubulin and is required for its acetylation. The reduced acetylation level of α-tubulin in Esco1-depleted oocytes can be restored by the ectopic expression of exogenous wild-type Esco1 but not enzymatically dead Esco1-G768D. Purified wild-type Esco1 instead of mutant Esco1-G768D acetylates the synthesized peptide of α-tubulin in vitro. Collectively, our data assign a novel function to Esco1 as a microtubule regulator during oocyte meiotic maturation beyond its conventional role in chromosome cohesion.

BaP exposure causes oocyte meiotic arrest and fertilization failure to weaken female fertility.

Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant and carcinogen that is frequently found in particulate matter, with a diameter of ≤2.5 µm (PM2.5). It has been reported to interrupt the normal reproductive system, but the exact molecular basis has not been clearly defined. To understand the underlying mechanisms regarding how BaP exposure disrupts female fertility, we evaluated oocyte quality by assessing the critical regulators and events during oocyte meiotic maturation and fertilization. We found that BaP exposure compromised the mouse oocyte meiotic progression by disrupting normal spindle assembly, chromosome alignment, and kinetochore-microtubule attachment, consequently leading to the generation of aneuploid eggs. In addition, BaP administration significantly decreased the fertilization rate of mouse eggs by reducing the number of sperm binding to the zona pellucida, which was consistent with the premature cleavage of N terminus of zona pellucida sperm-binding protein 2 and precocious exocytosis of ovastacin. Furthermore, BaP exposure interfered with the gamete fusion process by perturbing the localization and protein level of Juno. Notably, we found that BaP exposure induced oxidative stress with an increased level of reactive oxygen species and apoptosis in oocytes and thereby led to the deterioration of critical regulators and events during oocyte meiotic progression and fertilization. Our data document that BaP exposure reduces female fertility via impairing oocyte maturation and fertilization ability induced by oxidative stress and early apoptosis in murine models.-Zhang, M., Miao, Y., Chen, Q., Cai, M., Dong, W., Dai, X., Lu, Y., Zhou, C., Cui, Z., Xiong, B. BaP exposure causes oocyte meiotic arrest and fertilization failure to weaken female fertility.

The protective role of melatonin in porcine oocyte meiotic failure caused by the exposure to benzo(a)pyrene.

STUDY QUESTION: Does melatonin restore the benzo(a)pyrene (BaP)-induced meiotic failure in porcine oocytes? SUMMARY ANSWER: Melatonin effectively inhibits the increased reactive oxygen species (ROS) level and apoptotic rate in BaP-exposed porcine oocytes to recover the meiotic failure. WHAT IS KNOWN ALREADY: BaP, a widespread environmental carcinogen found in particulate matter, 2.5 µm or less (PM2.5), has been shown to have toxicity at the level of the reproductive systems. BaP exposure disrupts the steroid balance, alters the expression of ovarian estrogen receptor and causes premature ovarian failure through the rapid depletion of the primordial follicle pool. In addition, acute exposure to BaP has transient adverse effects on the follicle growth, ovulation and formation of corpora lutea, which results in transient infertility. STUDY DESIGN, SIZE, DURATION: Porcine oocytes were randomly assigned to control, BaP-exposed and melatonin-supplemented groups. BaP was dissolved in dimethylsulphoxide and diluted to a final concentration of 50, 100 or 250 µM with maturation medium, respectively. Melatonin was dissolved in the absolute ethanol and diluted with maturation medium to a final concentration of 1 nM, 100 nM, 10 µM and 1 mM, respectively. The in vitro cultured oocytes from each group after treatment were applied to the subsequent analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Acquisition of oocyte meiotic competence was assessed using immunostaining, fluorescent intensity quantification and/or immunoblotting to analyse the cytoskeleton assembly, mitochondrial integrity, cortical granule dynamics, ovastacin distribution, ROS level and apoptotic rate. Fertilization ability of oocytes was examined by sperm binding assay and IVF. MAIN RESULTS AND THE ROLE OF CHANCE: BaP exposure resulted in the oocyte meiotic failure (P = 0.001) via impairing the meiotic apparatus, showing a prominently defective spindle assembly (P = 0.003), actin dynamics (P < 0.001) and mitochondrion integrity (P < 0.001). In addition, BaP exposure caused the abnormal distribution of cortical granules (P < 0.001) and ovastacin (P = 0.003), which were consistent with the observation that fewer sperm bound to the zona pellucida surrounding the unfertilized BaP-exposed eggs (P < 0.001), contributing to the fertilization failure (P < 0.001). Conversely, melatonin supplementation recovered, at least partially, all the meiotic defects caused by BaP exposure through inhibiting the rise in ROS level (P = 0.015) and apoptotic rate (P = 0.001). LIMITATIONS, REASONS FOR CAUTION: We investigated the negative impact of BaP on the oocyte meiotic maturation in vitro, but not in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our findings not only deeply clarify the potential mechanisms of BaP-induced oocyte meiotic failure, but also extend the understanding about how environmental pollutants influence the reproductive systems in humans. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Natural Science Foundation of China (31571545) and the Natural Science Foundation of Jiangsu Province (BK20150677). The authors have no conflict of interest to disclose.

Postovulatory aging causes the deterioration of porcine oocytes via induction of oxidative stress.

Negative effects of postovulatory aging on fertilization ability and subsequent embryo development have been reported in rodents; however, the molecular and cellular changes during this process have not been fully defined. Here, we used porcine oocytes, a model that is physiologically and developmentally similar to humans, to explore the molecular mechanisms that underlie how postovulatory aging affects oocyte quality and fertilization capacity. We found that postovulatory aging caused the morphologic change of porcine oocytes by exhibiting the incompact expansion of cumulus cells and an increased occurrence of fragmentation. Aging also impaired oocyte quality by disrupting organelle structures, including the spindle assembly, actin polymerization, and mitochondrial integrity. Moreover, postovulatory aging led to the abnormal distribution of cortical granules and ovastacin, which, in turn, resulted in defective sperm binding and consequently compromised fertilization potential. Of note, we observed that postovulatory aging induced oxidative stress with a high level of reactive oxygen species and apoptotic rate in oocytes, thereby resulting in the deterioration of critical factors in the maintenance of oocyte quality and fertilization capacity. Taken together, our findings demonstrate that postovulatory aging perturbs a variety of molecular and cellular changes in porcine oocytes by inducing oxidative stress.-Miao, Y., Zhou, C., Cui, Z., Zhang, M., ShiYang, X., Lu, Y., Xiong, B. Postovulatory aging causes the deterioration of porcine oocytes via induction of oxidative stress.

Dynein promotes porcine oocyte meiotic progression by maintaining cytoskeletal structures and cortical granule arrangement.

Cytoplasmic dynein is a family of cytoskeletal motor proteins that move towards the minus-end of the microtubules to perform functions in a variety of mitotic processes such as cargo transport, organelle positioning, chromosome movement and centrosome assembly. However, its specific roles during mammalian oocyte meiosis have not been fully defined. Herein, we investigated the critical events during porcine oocyte meiotic maturation after inhibition of dynein by Ciliobrevin D treatment. We found that oocyte meiotic progression was arrested when inhibited of dynein by showing the poor expansion of cumulus cells and decreased rate of polar body extrusion. Meanwhile, the spindle assembly and chromosome alignment were disrupted, accompanied by the reduced level of acetylated α-tubulin, indicative of weakened microtubule stability. Defective actin polymerization on the plasma membrane was also observed in dynein-inhibited oocytes. In addition, inhibition of dynein caused the abnormal distribution of cortical granules and precocious exocytosis of ovastacin, a cortical granule component, which predicts that ZP2, the sperm binding site in the zona pellucida, might be prematurely cleaved in the unfertilized dynein-inhibited oocytes, potentially leading to the fertilization failure. Collectively, our findings reveal that dynein plays a part in porcine oocyte meiotic progression by regulating the cytoskeleton dynamics including microtubule stability, spindle assembly, chromosome alignment and actin polymerization. We also find that dynein mediates the normal cortical granule distribution and exocytosis timing of ovastacin in unfertilized eggs which are the essential for the successful fertilization.

Cohesin acetyltransferase Esco2 regulates SAC and kinetochore functions via maintaining H4K16 acetylation during mouse oocyte meiosis.

Sister chromatid cohesion, mediated by cohesin complex and established by the acetyltransferases Esco1 and Esco2, is essential for faithful chromosome segregation. Mutations in Esco2 cause Roberts syndrome, a developmental disease characterized by severe prenatal retardation as well as limb and facial abnormalities. However, its exact roles during oocyte meiosis have not clearly defined. Here, we report that Esco2 localizes to the chromosomes during oocyte meiotic maturation. Depletion of Esco2 by morpholino microinjection leads to the precocious polar body extrusion, the escape of metaphase I arrest induced by nocodazole treatment and the loss of BubR1 from kinetochores, indicative of inactivated SAC. Furthermore, depletion of Esco2 causes a severely impaired spindle assembly and chromosome alignment, accompanied by the remarkably elevated incidence of defective kinetochore-microtubule attachments which consequently lead to the generation of aneuploid eggs. Notably, we find that the involvement of Esco2 in SAC and kinetochore functions is mediated by its binding to histone H4 and acetylation of H4K16 both in vivo and in vitro. Thus, our data assign a novel meiotic function to Esco2 beyond its role in the cohesion establishment during mouse oocyte meiosis.

Melatonin protects oocyte quality from Bisphenol A-induced deterioration in the mouse.

Bisphenol A (BPA) has been reported to adversely affect the mammalian reproductive system in both sexes. However, the underlying mechanisms regarding how BPA disrupts the mammalian oocyte quality and how to prevent it have not been fully defined. Here, we document that BPA weakens oocyte quality by impairing both oocyte meiotic maturation and fertilization ability. We find that oral administration of BPA (100 µg/kg body weight per day for 7 days) compromises the first polar body extrusion (78.0% vs 57.0%, P<.05) by disrupting normal spindle assembly, chromosome alignment, and kinetochore-microtubule attachment. This defect could be remarkably ameliorated (76.7%, P<.05) by concurrent oral administration of melatonin (30 mg/kg body weight per day for 7 days). In addition, BPA administration significantly decreases the fertilization rate of oocytes (87.2% vs 41.1%, P<.05) by reducing the number of sperm binding to the zona pellucida, which is consistent with the premature cleavage of ZP2 as well as the mis-localization and decreased protein level of ovastacin. Also, the localization and protein level of Juno, the sperm receptor on the egg membrane, are strikingly impaired in BPA-administered oocytes. Finally, we show that melatonin administration substantially elevates the in vitro fertilization rate (63.0%, P<.05) by restoring above defects of fertilization proteins and events, which might be mediated by the improvement of oocyte quality via reduction of ROS levels and inhibition of apoptosis. Collectively, our data reveal that melatonin has a protective action against BPA-induced deterioration of oocyte quality in mice.

Melatonin improves the fertilization ability of post-ovulatory aged mouse oocytes by stabilizing ovastacin and Juno to promote sperm binding and fusion.

STUDY QUESTION: What are the underlying mechanisms of the decline in the fertilization ability of post-ovulatory aged oocytes? SUMMARY ANSWER: Melatonin improves the fertilization ability of post-ovulatory aged oocytes by reducing aging-induced reactive oxygen species (ROS) levels and inhibiting apoptosis and by maintaining the levels and localization of the fertilization proteins, ovastacin and Juno. WHAT IS KNOWN ALREADY: Following ovulation, the quality of mammalian metaphase II oocytes irreversibly deteriorates over time with a concomitant loss of fertilization ability. Melatonin has been found to prevent post-ovulatory oocyte aging and extend the window for optimal fertilization in mice. STUDY DESIGN, SIZE, DURATION: Mouse oocytes were randomly assigned to three groups and aged in vitro for 0, 6, 12 and 24 h, respectively. Increasing concentrations of melatonin (10-9 M, 10-7 M, 10-5 M and 10-3 M) were added to the 24 h aging group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm binding assays, in-vitro fertilization, immunofluorescent staining and western blotting were performed to investigate key regulators and events during fertilization of post-ovulatory aged mouse oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the actin cap which promotes a cortical granule (CG) free domain is disrupted with a re-distribution of CGs in the subcortex of aged oocytes. Ovastacin, a CG metalloendoprotease, is mis-located and prematurely exocytosed in aged oocytes with subsequent cleavage of the zona pellucida protein ZP2. This disrupts the sperm recognition domain and dramatically reduces the number of sperm binding to the zona pellucida. The abundance of Juno, the sperm receptor on the oocyte membrane, also is reduced in aged oocytes. Exposure of aged oocytes to melatonin significantly elevates in-vitro fertilization rates potentially by rescuing the above age-associated defects of fertilization, and reducing ROS and inhibiting apoptosis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We explored the mechanisms of the decline in fertilization ability decline in aged mouse oocytes, in vitro but not in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our findings may contribute to the development a more efficient method, involving melatonin, for improving IVF success rates. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation (31571545) and the Natural Science Foundation of Jiangsu Province (BK20150677). The authors have no conflict of interest to disclose.

Smc1ß is required for activation of SAC during mouse oocyte meiosis.

Smc1ß is a meiosis-specific cohesin subunit that is essential for sister chromatid cohesion and DNA recombination. Previous studies have shown that Smc1ß-deficient mice in both sexes are sterile. Ablation of Smc1ß during male meiosis leads to the blockage of spermatogenesis in pachytene stage, and ablation of Smc1ß during female meiosis generates a highly error-prone oocyte although it could develop to metaphase II stage. However, the underlying mechanisms regarding how Smc1ß maintains the correct meiotic progression in mouse oocytes have not been clearly defined. Here, we find that GFP-fused Smc1ß is expressed and localized to the chromosomes from GV to MII stages during mouse oocyte meiotic maturation. Knockdown of Smc1ß by microinjection of gene-specific morpholino causes the impaired spindle apparatus and chromosome alignment which are highly correlated with the defective kinetochore-microtubule attachments, consequently resulting in a prominently higher incidence of aneuploid eggs. In addition, the premature extrusion of polar bodies and escape of metaphase I arrest induced by low dose of nocodazole treatment in Smc1ß-depleted oocytes indicates that Smc1ß is essential for activation of spindle assembly checkpoint (SAC) activity. Collectively, we identify a novel function of Smc1ß as a SAC participant beyond its role in chromosome cohesion during mouse oocyte meiosis.

Stag3 regulates microtubule stability to maintain euploidy during mouse oocyte meiotic maturation.

Stag3, a meiosis-specific subunit of cohesin complex, has been demonstrated to function in both male and female reproductive systems in mammals. However, its roles during oocyte meiotic maturation have not been fully defined. In the present study, we report that Stag3 uniquely accumulates on the spindle apparatus and colocalizes with microtubule fibers during mouse oocyte meiotic maturation. Depletion of Stag3 by gene-targeting morpholino disrupts normal spindle assembly and chromosome alignment in oocytes. We also find that depletion of Stag3 reduces the acetylated level of tubulin and microtubule resistance to microtubule depolymerizing drug, suggesting that Stag3 is required for microtubule stability. Consistent with these observations, kinetochore-microtubule attachment, an important mechanism controlling chromosome alignment, is severely impaired in Stag3-depleted oocytes, resultantly causing the significantly increased incidence of aneuploid eggs. Collectively, our data reveal that Stag3 is a novel regulator of microtubule dynamics to ensure euploidy during moue oocyte meiotic maturation.

Cullin9 protects mouse eggs from aneuploidy by controlling microtubule dynamics via Survivin.

The Cullin9 gene encodes a putative E3 ligase that serves a wide variety of biological functions in mitosis, whereas its roles in meiosis have not yet clearly defined. Here, we report that Cullin9 accumulates on the spindle apparatus and colocalizes with the microtubule fibers during mouse oocyte meiotic maturation. Depletion of Cullin9 by morpholino microinjection results in a remarkably higher rate of disorganized spindles and misaligned chromosomes in oocytes, which is coupled with the impaired kinetochore-microtubule attachments. Resultantly, the incidence of aneuploid eggs significantly increases in Cullin9-depleted oocytes. Moreover, we show that Cullin9 controls Survivin's protein level during meiotic maturation, and thus regulates microtubule stability in oocytes. Thus, our study assigns a new meiotic function to Cullin9 and reveals that it prevents mouse eggs from aneuploidy by regulating microtubule dynamics via Survivin.