Extracellular signal-regulated protein kinase 5 modulates the spindle assembly to coordinate the oocyte meiotic maturation.

Extracellular signal-regulated protein kinase 5 (Erk5), a member of the mitogen-activated protein kinase (MAPK) family, is ubiquitously expressed in all eukaryotic cells and is implicated in the various mitotic processes such as cell survival, proliferation, migration, and differentiation. However, the potential functional roles of Erk5 in oocyte meiosis have not been fully determined. In this study, we document that ERK5 participates in the meiotic maturation of mouse oocytes by regulating the spindle assembly to ensure the meiotic progression. We unexpectedly found that phosphorylated ERK5 was localized in the spindle pole region at metaphase I and II stages by immunostaining analysis. Inhibition of ERK5 activity using its specific inhibitor XMD8-92 dramatically reduced the incidence of first polar body extrusion. In addition, inhibition of ERK5 evoked the spindle assembly checkpoint to arrest oocytes at metaphase I stage by impairing the spindle assembly, chromosome alignment and kinetochore-microtubule attachment. Mechanically, over-strengthened microtubule stability was shown to disrupt the microtubule dynamics and thus compromise the spindle assembly in ERK5-inhibited oocytes. Conversely, overexpression of ERK5 caused decreased level of acetylated α-tubulin and spindle defects. Collectively, we conclude that ERK5 plays an important role in the oocyte meiotic maturation by regulating microtubule dynamics and spindle assembly.

The cohesin release factor Wapl interacts with Bub3 to govern SAC activity in female meiosis I.

During mitotic prophase, cohesins are removed from chromosome arms by Wapl to ensure faithful sister chromatid separation. However, during female meiosis I, the resolution of chiasmata requires the proteolytic cleavage of cohesin subunit Rec8 along chromosome arms by Separase to separate homologs, and thus the role of Wapl remained unknown. Here, we report that Wapl functions as a regulator of spindle assembly checkpoint (SAC) to prevent aneuploidy in meiosis I. Depletion of Wapl accelerates meiotic progression, inactivates SAC, and causes meiotic defects such as aberrant spindle/chromosome structure and incorrect kinetochore-microtubule (K-MT) attachment, consequently leading to aneuploid eggs. Notably, we identify Bub3 as a binding partner of Wapl by immunoprecipitation and mass spectrometry analysis. We further determine that Wapl controls the SAC activity by maintaining Bub3 protein level and document that exogenous Bub3 restores the normal meiosis in Wapl-depleted oocytes. Together, our findings uncover unique, noncanonical roles for Wapl in mediating control of the SAC in female meiosis I.

Casein kinase 2 modulates the spindle assembly checkpoint to orchestrate porcine oocyte meiotic progression.

BACKGROUND: CK2 (casein kinase 2) is a serine/threonine-selective protein kinase that has been involved in a variety of cellular processes such as DNA repair, cell cycle control and circadian rhythm regulation. However, its functional roles in oocyte meiosis have not been fully determined. RESULTS: We report that CK2 is essential for porcine oocyte meiotic maturation by regulating spindle assembly checkpoint (SAC). Immunostaining and immunoblotting analysis showed that CK2 was constantly expressed and located on the chromosomes during the entire oocyte meiotic maturation. Inhibition of CK2 activity by its selective inhibitor CX-4945 impaired the first polar body extrusion and arrested oocytes at M I stage, accompanied by the presence of BubR1 at kinetochores, indicative of activated SAC. In addition, we found that spindle/chromosome structure was disrupted in CK2-inhibited oocytes due to the weakened microtubule stability, which is a major cause resulting in the activation of SAC. Last, we found that the level DNA damage as assessed by γH2A.X staining was considerably elevated when CK2 was inhibited, suggesting that DNA damage might be another critical factor leading to the SAC activation and meiotic failure of oocytes. CONCLUSIONS: Our findings demonstrate that CK2 promotes the porcine oocyte maturation by ensuring normal spindle assembly and DNA damage repair.

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.

Vitamin C protects carboplatin-exposed oocytes from meiotic failure.

CBP (carboplatin) is a second-generation chemotherapeutic drug of platinum compound commonly applied in the treatment of sarcomas and germ cell tumours. Although it is developed to replace cisplatin, which has been proven to have a variety of side effects during cancer treatment, CBP still exhibits a certain degree of toxicity including neurotoxicity, nephrotoxicity, hematotoxicity and myelosuppression. However, the underlying mechanisms regarding how CBP influences the female reproductive system especially oocyte quality have not yet been fully determined. Here, we report that CBP exposure led to the oocyte meiotic defects by impairing the dynamics of the meiotic apparatus, leading to a remarkably aberrant spindle organisation, actin polymerisation and mitochondrial integrity. Additionally, CBP exposure caused compromised sperm binding and fertilisation potential of oocytes by due to an abnormal distribution of cortical granules and its component ovastacin. More importantly, we demonstrated that vitamin C supplementation prevented meiotic failure induced by CBP exposure and inhibited the increase in ROS levels, DNA damage accumulation and apoptotic incidence. Taken together, our findings demonstrate the toxic effects of CBP exposure on oocyte development and provide a potential effective way to improve the quality of CBP-exposed oocytes in vitro.

Tea polyphenol protects against cisplatin-induced meiotic defects in porcine oocytes.

DDP (cisplatin), a DNA cross-linking agent, is one of the most common chemotherapeutic drugs that have been widely used in the treatment of sarcomas and germ cell tumors. DDP treatment exhibits severe side effects including renal toxicity, ototoxicity and embryo-toxicity. Women of reproductive age treated with DDP may lead to loss of primordial follicles, resulting in the depletion of the ovarian reserve and consequent premature ovarian failure. However, the influence of DDP on the oocyte quality and the strategy to prevent it has not yet fully clarified. Here, we report that DDP exposure resulted in the oocyte meiotic failure via disrupting the meiotic organelle dynamics and arrangement, exhibiting a prominently impaired cytoskeleton assembly, including spindle formation and actin polymerization. In addition, exposure to DDP led to the abnormal distribution of mitochondrion and cortical granules, two indicators of cytoplasmic maturation of oocytes. Conversely, TP (tea polyphenols) supplementation partially restored all of the meiotic defects resulted from DDP exposure through suppressing the increase of ROS level and the occurrence of DNA damage as well as apoptosis.

Exposure to aristolochic acid I compromises the maturational competency of porcine oocytes via oxidative stress-induced DNA damage.

Aristolochic acid (AA) is a class of carcinogenic and nephrotoxic nitrophenanthrene carboxylic acids naturally found in Aristolochia plants. These plants have been widely used as herbal medicines and also enter the human food chain as the persistent soil pollutants. It has been known that AA exposure is implicated in multiple cancer types, kidney failure and ovarian dysfunction. However, whether AA exposure would influence the oocyte quality has not yet determined. Here, we document that AAI has the negative effects on the competency of oocyte maturation and fertilization. We show that AAI exposure leads to the oocyte meiotic failure via impairing the meiotic apparatus, displaying a prominently defective spindle assembly, actin dynamics and mitochondrial integrity. AAI exposure also causes the abnormal distribution of cortical granules and ovastacin, which is consistent with the observation that fewer sperm bound to the zona pellucida surrounding the unfertilized AAI-exposed eggs, contributing to the fertilization failure. In addition, AAI exposure induces the increased levels of ROS, DNA damage and early apoptosis in porcine oocytes. Collectively, we demonstrate that AAI exposure perturbs the oocyte meiotic progression and fertilization capacity via disruption of both nuclear maturation and cytoplasmic maturation of oocyte, which might be caused by the excessive oxidative stress-induced DNA damage and apoptosis.

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.