Vinorelbine administration impedes the timely progression of meiotic maturation and induces aneuploidy in mouse oocytes.

Vinorelbine is a commonly used drug to treat various malignancies, such as breast cancer, non-small cell lung cancer, and metastatic pleural mesothelioma. Its side effects include severe neutropenia, local phlebitis, gastrointestinal reactions, and neurotoxicity. In view of the scarcity of research on vinorelbine's reproductive toxicity, this study evaluated the impact of vinorelbine ditartrate, a commonly used form of vinorelbine, on oocyte maturation in vitro. Our investigation revealed that vinorelbine ditartrate had no effect on oocyte meiotic resumption. However, it did reduce the rate of first polar body extrusion, suggesting that it could significantly impede the meiotic maturation of oocytes. Vinorelbine ditartrate exposure was found to disturb the regular spindle assembly and chromosome alignment, leading to the continuous activation of the spindle assembly checkpoint (SAC) and a delayed activation of the anaphase-promoting complex/cyclosome (APC/C), ultimately causing aneuploidy in oocytes. Consequently, the administration of vinorelbine is likely to result in oocyte aneuploidy, which can be helpful in providing a drug reference and fertility guidance in a clinical context.

Effectiveness of non-invasive chromosomal screening for normal karyotype and chromosomal rearrangements.

Purpose: To study the accuracy of non-invasive chromosomal screening (NICS) results, in normal chromosomes and chromosomal rearrangement groups and to investigate whether using trophoblast cell biopsy along with NICS, to choose embryos for transfer can improve the clinical outcomes of assisted pregnancy. Methods: We retrospectively analyzed 101 couples who underwent preimplantation genetic testing at our center from January 2019 to June 2021 and collected 492 blastocysts for trophocyte (TE) biopsy. D3-5 blastocyst culture fluid and blastocyst cavity fluid were collected for the NICS. Amongst them, 278 blastocysts (58 couples) and 214 blastocysts (43 couples) were included in the normal chromosomes and chromosomal rearrangement groups, respectively. Couples undergoing embryo transfer were divided into group A, in which both the NICS and TE biopsy results were euploid (52 embryos), and group B, in which the TE biopsy results were euploid and the NICS results were aneuploid (33 embryos). Results: In the normal karyotype group, concordance for embryo ploidy was 78.1%, sensitivity was 94.9%, specificity was 51.4%, the positive predictive value (PPV) was 75.7%, and the negative predictive value (NPV) was 86.4%. In the chromosomal rearrangement group, concordance for embryo ploidy was 73.1%, sensitivity was 93.3%, specificity was 53.3%, the PPV was 66.3%, and the NPV was 89%. In euploid TE/euploid NICS group, 52 embryos were transferred; the clinical pregnancy rate was 71.2%, miscarriage rate was 5.4%, and ongoing pregnancy rate was 67.3%. In euploid TE/aneuploid NICS group, 33 embryos were transferred; the clinic pregnancy rate was 54.5%, miscarriage rate was 5.6%, and ongoingpregnancy rate was 51.5%. The clinical pregnancy and ongoing pregnancy rates were higher in the TE and NICS euploid group. Conclusion: NICS was similarly effective in assessing both normal and abnormal populations. Identification of euploidy and aneuploidy alone may lead to the wastage of embryos due to high false positives. More suitable reporting methods for NICS and countermeasures for a high number of false positives in NICS are needed. In summary, our results suggest that combining biopsy and NICS results could improve the outcomes of assisted pregnancy.

Transient inhibition of CDK2 activity prevents oocyte meiosis I completion and egg activation in mouse.

Mammalian oocytes are arrested at the diplotene stage of prophase I during fetal or postnatal development. It was reported that cyclin-dependent kinases (CDK1) was the sole CDK to drive the resumption of meiosis and CDK2 was dispensable for meiosis progression in mouse oocytes according to the conditional knockout studies. However, a recent study showed that CDK2 activity is essential for meiotic division and gametogenesis by means of gene-directed mutagenesis, which avoids the compensatory activation of other CDKs. Taken the compensatory effect between CDKs after gene knockout, the physiological function of CDK2 activity in oocyte maturation remains unclear. To address this issue, we applied a specific small-molecule inhibitor to restrain CDK2 activity transiently during oocyte meiotic maturation. Surprisingly, transient inhibition of CDK2 activity severely prevented the meiosis I completion although the meiotic resumption was not affected. Then we found that CDK2 activity was required for establishment of normal spindle and chromosome dynamics. Notably, CDK2 inhibition interrupted the anaphase-promoting complex/cyclosome (APC/C)-dependent degradation pathway by maintaining the activation of spindle assembly checkpoint (SAC). Interestingly, CDK2 inhibition prevented the egg activation as well. Overall, our data demonstrate that CDK2 kinase activity is required for proper dynamics of spindle and chromosomes, whose disturbance induces the continuous SAC activation and subsequent inactivation of APC/C activity in oocyte meiosis.

CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes.

Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.

NEK5 regulates cell cycle progression during mouse oocyte maturation and preimplantation embryonic development.

NEK5, a member of never in mitosis-gene A-related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA-mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin-dependent kinase 1 in oocytes, resulting in a decrease of maturation-promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1-cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.

PKCß1 regulates meiotic cell cycle in mouse oocyte.

PKCßI, a member of the classical protein kinase C family, plays key roles in regulating cell cycle transition. Here, we report the expression, localization and functions of PKCßI in mouse oocyte meiotic maturation. PKCßI and p-PKCßI (phosphor-PKCßI) were expressed from germinal vesicle (GV) stage to metaphase II (MII) stage. Confocal microscopy revealed that PKCßI was localized in the GV and evenly distributed in the cytoplasm after GV breakdown (GVBD), and it was concentrated at the midbody at telophase in meiotic oocytes. While, p-PKCßI was concentrated at the spindle poles at the metaphase stages and associated with midbody at telophase. Depletion of PKCßI by specific siRNA injection resulted in defective spindles, accompanied with spindle assembly checkpoint activation, metaphase I arrest and failure of first polar body (PB1) extrusion. Live cell imaging analysis also revealed that knockdown of PKCßI resulted in abnormal spindles, misaligned chromosomes, and meiotic arrest of oocytes arrest at the Pro-MI/MI stage. PKCßI depletion did not affect the G2/M transition, but its overexpression delayed the G2/M transition through regulating Cyclin B1 level and Cdc2 activity. Our findings reveal that PKCßI is a critical regulator of meiotic cell cycle progression in oocytes. Abbreviations: PKC, protein kinase C; COC, cumulus-oocyte complexes; GV, germinal vesicle; GVBD, germinal vesicle breakdown; Pro-MI, first pro-metaphase; MI, first metaphase; Tel I, telophase I; MII, second metaphase; PB1, first polar body; SAC, spindle assembly checkpoint.

Kif2a regulates spindle organization and cell cycle progression in meiotic oocytes.

Kif2a is a member of the Kinesin-13 microtubule depolymerases. Here, we report the expression, subcellular localization and functions of Kif2a during mouse oocyte meiotic maturation. Immunoblotting analysis showed that Kif2a was gradually increased form GV to the M I stages, and then decreased slightly at the M II stage. Confocal microscopy identified that Kif2a localized to the meiotic spindle, especially concentrated at the spindle poles and inner centromeres in metaphase and translocated to the midbody at telophase. Kif2a depletion by siRNA microinjection generated severely defective spindles and misaligned chromosomes, reduced microtubule depolymerization, which led to significant pro-M I/M Iarrest and failure of first polar body (PB1) extrusion. Kif2a-depleted oocytes were also defective in spindle pole localization of γ-tubulin and showed spindle assembly checkpoint (SAC) protein Bub3 at the kinetochores even after 10 hr extended culture. These results demonstrate that Kif2a may act as a microtubule depolymerase, regulating microtubule dynamics, spindle assembly and chromosome congression, and thus cell cycle progression during mouse oocyte meiotic maturation.