Abnormal Spindle Organization Research Articles

PARP12 regulates mouse oocyte meiotic maturation.

Poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification of proteins that involves the transfer of ADP-ribose moieties, and plays important roles in many biological processes including DNA repair, gene expression, RNA processing, ribosome biogenesis, and protein translation. Though it is accepted that PARylation is crucial for oocyte maturation, little is known about how Mono(ADP-ribosyl)ation (MARylation) regulates this process. Here, we report that Parp12,a mon(ADP-ribosyl) transferase of poly(ADP-ribosyl) polymerase (PARP) family, was highly expressed at all stages of oocytes during meiotic maturation. At germinal vesicle (GV) stage, PARP12 was mainly distributed in cytoplasm. Interestingly, PARP12 formed granular aggregation near to spindle poles during metaphase I (MI) and metaphase II (MII). PARP12 depletion results in abnormal spindle organization and chromosome misalignment in mouse oocytes. Chromosome aneuploidy frequency in PARP12 knockdown oocytes was significantly increased. Importantly, PARP12 knockdown triggers activation of spindle assembly checkpoint as shown by active BUBR1 in PARP12-KD MI oocytes. Besides, F actin was significantly attenuated in PARP12-KD MI oocytes which may affect the asymmetric division process. Transcriptomic analysis demonstrated that PARP12 depletion disrupts transcriptome homeostasis. Collectively, our results showed that the maternally expressed mono(ADPribosyl) transferases PARP12 was essential for oocyte meiotic maturation in mouse.

Ovarian endometrioma increases the embryo aneuploid rate: an analysis of 7092 biopsied blastocysts from fertile monogenetic disease carriers

BackgroundEndometriosis affects many reproductive aged patients with fertility decline and poor outcomes of assisted reproductive treatments, mainly by decreased ovarian reserve and lower fertilization and implantation rates. In recent decade, altered oocyte microenvironments and abnormal spindle organization have been reported to be critical to oocyte chromosomal segregation, organization and aneuploid formation. However, clinical evidences are still limited on whether endometriosis influences oocyte and embryo development. We aimed to figure out the impact of endometrioma on embryo aneuploid formation.MethodThis retrospective cohort study included 1,021 patients (7,092 biopsied embryos) from January 2012 to December 2020. Fertile patients without a history of miscarriage who underwent PGT-M treatment with aneuploid screening were included. Patients with ovarian endometrioma were defined as the study group, while patients without endometriosis were defined as the control group. All demographic, controlled ovarian stimulation treatment and aneuploid screening data were recorded and compared.ResultsThe incidence of endometrioma in our study population was 6.5%. There were 7,092 embryos biopsied in total, with 308 embryos in the study group and 6,784 embryos in the control groups. The demographic characteristics were comparable between the two groups except the basal FSH level (6.02 IU/L vs. 5.52 IU/L, p = 0.012). The euploid rate of the study group was significantly lower than that of the control group (52.6% vs. 61.8%, p = 0.012), while the oocyte maturation, fertilization, usable embryo and blastocyst formation rates were comparable. Adjusted for basal FSH level, starting stimulating gonadotropin dosage, total gonadotropin dosage and FSH level on hCG day, euploid rate was still negatively related to endometrioma status.ConclusionsEndometrioma status disturbs oocyte and embryo development. For infertile patients with endometrioma who require assisted reproductive treatment, pre-treatment is necessary to improve treatment outcomes.Trial registrationNot applicable.

GAS6 ameliorates advanced age-associated meiotic defects in mouse oocytes by modulating mitochondrial function.

Previously, we reported that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation by suppressing mitophagy and inducing mitochondrial dysfunction, resulting in fertilization failure. Here, we show that oocyte aging is accompanied by an increase in meiotic defects associated with chromosome misalignment and abnormal spindle organization. Intriguingly, decreased Gas6 mRNA and protein expression were observed in aged oocytes from older females. We further explored the effect of GAS6 on the quality and fertility of aged mouse oocytes using a GAS6 rescue analysis. After treatment with the GAS6 protein, aged oocytes matured normally to the meiosis II (MII) stage. Additionally, maternal age-related meiotic defects were reduced by GAS6 protein microinjection. Restoring GAS6 ameliorated the mitochondrial dysfunction induced by maternal aging. Ultimately, GAS6-rescued MII oocytes exhibited increased ATP levels, reduced ROS levels and elevated glutathione (GSH) levels, collectively indicating improved mitochondrial function in aged oocytes. Thus, the age-associated decrease in oocyte quality was prevented by restoring GAS6. Importantly, GAS6 protein microinjection in aged oocytes also rescued fertility. We conclude that GAS6 improves mitochondrial function to achieve sufficient cytoplasmic maturation and attenuates maternal age-related meiotic errors, thereby efficiently safeguarding oocyte quality and fertility.

The antioxidant dieckol reduces damage of oxidative stress-exposed porcine oocytes and enhances subsequent parthenotes embryo development.

This study investigated the effect of the antioxidant dieckol, a component of Ecklonia cava, on maturation and developmental competence of porcine oocytes exposed to oxidative stress in vitro. Oocytes were matured in in vitro maturation (IVM) medium containing various concentrations of dieckol. The blastocyst formation rate was highest in the 0.5 μM dieckol-treated (0.5 DEK) group. The reactive oxygen species level was decreased, and the level of glutathione and expression of antioxidant genes (NFE2L, SOD1, and SOD2) at metaphase II were increased in the 0.5 DEK group. Abnormal spindle organization and chromosome misalignment were prevented in the 0.5 DEK group. Expression of maternal markers (CCNB1 and MOS) and activity of p44/42 mitogen-activated protein kinase were increased in the 0.5 DEK group. After parthenogenetic activation, the total number of cells per blastocyst was increased and the percentage of apoptotic cells was decreased in the 0.5 DEK group. Expression of development-related genes (CX45, CDX2, POU5F1, and NANOG), antiapoptotic genes (BCL2L1 and BIRC5), and a proapoptotic gene (CASP3) were altered in the 0.5 DEK group. These results indicate that the antioxidant dieckol improves IVM and subsequent development of porcine oocytes and can be used to improve the quality of oocytes under peroxidation experimental conditions.

Epigallocatechin-3-gallate (EGCG) protects the oocytes from methyl parathion-induced cytoplasmic deformities by suppressing oxidative and endoplasmic reticulum stress

Methyl parathion (MP) is a commonly used organophosphorus insecticide in commercial farming. It is well known that MP exposure can affect the function of nervous, respiratory, cardiovascular and reproductive systems. In our previous report we have demonstrated that MP exposure results in poor oocyte maturation and defective embryo development which is mainly mediated through oxidative stress. The present investigation was designed to explore whether using a potent free radical scavenger like Epigallocatechin-3-gallate (EGCG) can help in reducing the detrimental effects of MP on the oocytes. For the study, germinal vesicle (GV) stage oocytes collected from the ovaries of adult Swiss albino mice were subjected to in vitro maturation (IVM) in the presence or absence of MP (100 μg/mL) and/or EGCG (0.25 μM). MP significantly reduced the nuclear maturation rate, and resulted in poor cytoplasmic organization which was evident from the altered distribution pattern of mitochondria, endoplasmic reticulum and abnormal spindle organization. These changes were associated with significant elevation in oxidative stress and expression of ER stress markers such as 78 kDa Glucose regulated protein (GRP78) as well as X-box binding protein-1 (XBP1) in the oocytes. Further, the oocytes exposed to MP had lower activation rate and developmental potential. Supplementation of EGCG during IVM not only improved the nuclear maturation rate but also reduced the cytoplasmic abnormalities. These beneficial effects appear to be due to mitigation of oxidative and ER stress in oocytes. In conclusion, results of our study indicate that EGCG can help in alleviating MP-induced oocyte abnormalities.

HDAC11 promotes meiotic apparatus assembly during mouse oocyte maturation via decreasing H4K16 and α-tubulin acetylation

ABSTRACT The smallest histone deacetylase (HDAC) and the solely member of class IV, HDAC11, is reported to regulate mitosis process and tumorigenesis, yet its roles in meiosis process remain unknown. In the present study, we first analyzed the expression of HDAC11 in mouse oocytes. HDAC11 showed gradual lower expression from GV (Germinal Vesicle) to MII (Metaphase II) stage oocytes. Then, the specific inhibitor of HDAC11, JB3-22 was used to explore the role of HDAC11 during mouse oocytes maturation. We found that inhibition of HDAC11 significantly interrupted mouse oocytes meiosis progress, caused abnormal spindle organization and misaligned chromosomes, impaired kinetochore-microtubule attachment and spindle assembly checkpoint (SAC) function. Moreover, HDAC11 inhibition significantly increased the acetylation level of α-tubulin that is associated with microtubule stability, and increased acetylation level of H4K16 that is important for kinetochore function. In conclusion, our study indicates that HDAC11 is an essential factor for oocytes maturation and it promotes meiotic process most likely though decreasing acetylation status of α-tubulin and H4K16.

Loss of ESRP1 blocks mouse oocyte development and leads to female infertility.

Alternative splicing (AS) contributes to gene diversification, but the AS program during germline development remains largely undefined. Here, we interrupted pre-mRNA splicing events controlled by epithelial splicing regulatory protein 1 (ESRP1) and found that it induced female infertility in mice. Esrp1 deletion perturbed spindle organization, chromosome alignment and metaphase-to-anaphase transformation in oocytes. The first polar body extrusion was blocked during oocyte meiosis owing to abnormal activation of spindle assembly checkpoint and insufficiency of anaphase-promoting complex/cyclosome in Esrp1-knockout oocytes. Esrp1-knockout hampered follicular development and ovulation; eventually, premature ovarian failure occurred in six-month-old Esrp1-knockout mouse. Using single-cell RNA-seq analysis, 528 aberrant AS events of maternal mRNA transcripts were revealed and were preferentially associated with microtubule cytoskeletal organization. Notably, we found that loss of ESRP1 disturbed a comprehensive set of gene-splicing sites - including those within Trb53bp1, Rac1, Bora, Kif2c, Kif23, Ndel1, Kif3a, Cenpa and Lsm14b - that potentially caused abnormal spindle organization. Collectively, our findings provide the first report elucidating the ESRP1-mediated AS program of maternal mRNA transcripts, which may contribute to oocyte meiosis and female fertility in mice.

Exposure to first line anti-tuberculosis drugs in prepubertal age reduces the quality and functional competence of spermatozoa and oocytes in Swiss albino mice

Prepubertal Swiss albino mice of both sex were administered with first-line anti-tuberculosis drugs (ATDs) viz; rifampicin, isoniazid, pyrazinamide, streptomycin and ethambutol intraperitoneally, for 4 weeks. Two weeks after the completion of treatment, male mice were sacrificed to collect caudal spermatozoa and female mice were superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to collect metaphase II (MII) oocytes from oviduct. Administration of ATDs not only decreased the count, motility and, nuclear maturity and also, increased the head abnormalities, mitochondrial damage and DNA damage in epididymal spermatozoa. Reduction in number of ovulated oocytes, increased degeneration rate and altered distribution pattern of cytoplasmic organelles was observed in oocytes of female mice. Presence of ATDs in in vitro maturation (IVM) medium increased abnormalities in meiotic resulted in abnormal spindle organization (except ethambutol) without affecting nuclear maturation. In conclusion, the result of this study indicates that ATDs have considerable adverse effects on the functional competence of male and female gametes, however, with varied degree of toxicity.

Mogroside V protects porcine oocytes from in vitro ageing by reducing oxidative stress through SIRT1 upregulation.

Postovulatory ageing compromises oocyte quality and subsequent development in various manners. We aimed to assay the protective effects of mogroside V on porcine oocyte quality during in vitro ageing and explore the related causes. We observed that mogroside V can effectively maintain normal oocyte morphology and early embryo development competence after prolonged culture for 24 h. Moreover, mogroside V can markedly reduce reactive oxygen species (ROS) levels, alleviate spindle formation and chromosome alignment abnormalities, improve mitochondrial contents, adenosine triphosphate (ATP) levels and the membrane potential (ΔΨm), and reduce early apoptosis in aged oocytes. We examined the molecular changes and found that SIRT1 expression was decreased in in vitro aged oocytes but was maintained by exposure to mogroside V. However, when SIRT1 was successfully inhibited by the specific inhibitor EX-527, mogroside V could not reduce ROS levels or alleviate abnormal spindle organization and chromosome misalignment. In summary, our results demonstrated that mogroside V can alleviate the deterioration of oocyte quality during in vitro ageing, possibly by reducing oxidative stress through SIRT1 upregulation.

Tributyltin oxide exposure impairs mouse oocyte maturation and its possible mechanisms.

Tributyltin oxide (TBTO) has been widely used as marine antifouling composition, preservative, biocide, and a stabilizer in plastic industry. Previous studies have indicated that TBTO can cause immunotoxicity as an environmental pollutant. However, little is known about its reproductive toxicity, especially on female oocyte maturation and the underlying mechanisms. In this study, mouse oocytes were cultured with different concentrations of TBTO in vitro, and several crucial events during meiotic maturation were evaluated. We found that the first polar body extrusion rate was significantly reduced, which reflected the disruption of meiotic maturation. The rate of abnormal spindle organization increased significantly, accompanied with a higher rate of chromosome misalignment. In addition, TBTO treatment increased reactive oxygen speciesgeneration markedly, which also accelerated the early-stage apoptosis. Moreover, heterogeneous mitochondrial distribution, mitochondrial dysfunction, and higher rate of aneuploidy were detected, which consequently disrupted in vitro fertilization. In conclusion, our results indicated that TBTO exposure could impair mouse oocyte maturation by affecting spindle organization, chromosome alignment, mitochondria functions, oxidative stress, and apoptosis.

Maytansinoid-antibody conjugates induce mitotic arrest by suppressing microtubule dynamic instability.

Maytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoids (DM1 and DM4) attached to tumor-specific antibodies have shown promising clinical results. To determine the mechanism by which the antibody-DM1 conjugates inhibit cell proliferation, we examined the effects of the cleavable anti-EpCAM-SPP-DM1 and uncleavable anti-EpCAM-SMCC-DM1 conjugates on MCF7 human breast tumor cells. We also examined the effects of the free maytansinoids, maytansine and S-methyl DM1 (a version of DM1 that is stable in cell culture medium), for comparison. Both the conjugates and free maytansinoids potently inhibited MCF7 cell proliferation at nanomolar and subnanomolar concentrations, respectively, by arresting the cells in mitotic prometaphase/metaphase. Arrest occurred in concert with the internalization and intracellular processing of both conjugates under conditions that induced abnormal spindle organization and suppressed microtubule dynamic instability. Microtubule depolymerization occurred only at significantly higher drug concentrations. The results indicate that free maytansinoids, antibody-maytansinoid conjugates, and their metabolites exert their potent antimitotic effects through a common mechanism involving suppression of microtubule dynamic instability.

5-aminosalicylic acid interferes in the cell cycle of colorectal cancer cells and induces cell death modes

Epidemiological data suggests that 5-aminosalicylic acid (5-ASA), a nonsteroidal antiinflammatory drug used in the treatment of inflammatory bowel diseases, prevents colorectal cancer development in these patients, although the mechanisms remain incompletely understood. Here we report that 5-ASA prevents growth of several colorectal cancer cell lines by interfering in the cell cycle, i.e., an S-phase and G2/M phase arrest, dependent on 5-ASA dosage and concentration, together with an increased mitotic index. In addition, prolonged cell cycle arrest by repeated 5-ASA treatment induced apoptosis and caused abnormal spindle organization leading to mitotic catastrophe, another form of cell death. These observations illustrate that 5-ASA has chemopreventive and chemotherapeutic properties.

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.

Dynamic changes in NuMA and microtubules in monkey-rabbit nuclear transfer embryos

Previous reports have indicated that failure in cloning monkey is attributed to the removal of nuclear mitotic apparatus (NuMA) during enucleation and subsequent abnormal organization of mitotic apparatus. This study investigated the transformation and assembly of tubulin and NuMA protein during the first cell cycle of cloned monkey embryos reconstructed by using enucleated rabbit oocytes as recipients. After the oocyte fused with a fibroblast, extensive microtubule organization was observed around the introduced nucleus in most reconstructed embryos, suggesting the introduction of a somatic cell centrosome. A high proportion of fibroblast nuclei transferred into non-activated oocytes underwent premature chromosome condensation (PCC), transient spindle organization and chromosomes separation, followed by the formation of two pronucleus-like structures. In contrast, fibroblast nuclei in pre-activated ooplasm rarely underwent PCC, but formed a swollen pronucleus-like structure. Normal spindles were observed in about one third of the cloned embryos reconstructed by both methods. After transferring monkey fibroblasts into NuMA-removed enucleated rabbit oocytes, NuMA was localized in pseudo-pronuclei and gradually moved to mitotic spindle poles at the first mitotic spindle poles. NuMA antibody microinjection resulted in spindle disorganization and chromosome misalignment, but did not significantly affect early cleavage. Our findings indicate that: 1. NuMA in donor monkey fibroblast may contribute to form a normal spindle in enucleated rabbit oocyte; 2. when non-activated cytoplasts and pre-activated cytoplasts are used as recipients, the donor nuclei undergo different morphological changes, but yield similar early embryo development; 3. although abnormal spindle organization and chromosome alignment may cause low efficiency of animal cloning, these abnormalities do not significantly affect early cleavage.