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.

SRSF2 is required for mRNA splicing during spermatogenesis.

BACKGROUND: RNA splicing plays significant roles in fundamental biological activities. However, our knowledge about the roles of alternative splicing and underlying mechanisms during spermatogenesis is limited. RESULTS: Here, we report that Serine/arginine-rich splicing factor 2 (SRSF2), also known as SC35, plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf2 by Stra8-Cre caused complete infertility and defective spermatogenesis. Further analyses revealed that deletion of Srsf2 disrupted differentiation and meiosis initiation of spermatogonia. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF2 regulatory networks play critical roles in several major events including reproductive development, spermatogenesis, meiotic cell cycle, synapse organization, DNA recombination, chromosome segregation, and male sex differentiation. Furthermore, SRSF2 affected expression and alternative splicing of Stra8, Stag3 and Atr encoding critical factors for spermatogenesis in a direct manner. CONCLUSIONS: Taken together, our results demonstrate that SRSF2 has important functions in spermatogenesis and male fertility by regulating alternative splicing.

SRSF1-mediated alternative splicing is required for spermatogenesis.

Alternative splicing (AS) plays significant roles in a multitude of fundamental biological activities. AS is prevalent in the testis, but the regulations of AS in spermatogenesis is only little explored. Here, we report that Serine/arginine-rich splicing factor 1 (SRSF1) plays critical roles in alternative splicing and male reproduction. Male germ cell-specific deletion of Srsf1 led to complete infertility by affecting spermatogenesis. Mechanistically, by combining RNA-seq data with LACE-seq data, we showed that SRSF1 affected the AS of Stra8 in a direct manner and Dazl, Dmc1, Mre11a, Syce2 and Rif1 in an indirect manner. Our findings demonstrate that SRSF1 has crucial functions in spermatogenesis and male fertility by regulating alternative splicing.

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.

Inhibition of NADPH Oxidase-ROS Signal using Hyaluronic Acid Nanoparticles for Overcoming Radioresistance in Cancer Therapy.

Upregulation of NADPH oxidases (NOXs) in cancer cells leads to chronic increase in intracellular reactive oxygen species (ROS) and adaptation to a high ROS level for cell survival and, thereby, low sensitivity to radiotherapy. To overcome resistance to radiotherapy, we have developed a bioactive and CD44 targeted hyaluronic acid nanoparticle encapsulated with an NOX inhibitor, GKT831 (HANP/GKT831). We found that HANP/GKT831 had stronger inhibitory effects on ROS generation and cell proliferation than that of GKT831 alone in cancer cells. Systemic delivery of HANP/GKT831 led to the targeted accumulation in breast cancer patient derived xenograft (PDX) tumors in nude mice. Importantly, the combination of systemic delivery of HANP/GKT831 with a low dose of local radiotherapy significantly enhanced tumor growth inhibition in breast cancer PDX models. Our results showed that HANP/GKT831 primed tumor cells to radiation-induced DNA damage and cell death by downregulation of DNA repair function and oncogenic signal pathways.

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.

An endometrial receptivity scoring system basing on the endometrial thickness, volume, echo, peristalsis, and blood flow evaluated by ultrasonography.

Background: Establishing a successful pregnancy depends on the endometrium and the embryo. It is estimated that suboptimal endometrial receptivity account for one-third of implantation failures. Despite the indepth understanding of the processes associated with embryo-endometrial cross-talk, little progress has been achieved for diagnosis and treatments for suboptimal endometrial receptivity. Methods: This retrospective study included women undergoing their first frozen-thawed embryo transfer (FET) cycles at our reproductive medicine center from March 2021 to August 2021. Transvaginal three-dimensional (3D) ultrasound was performed in the morning on the day of embryo transfer for all the thawed embryo transfer patients, to evaluate endometrial receptivity, including endometrial thickness, echogenicity, volume, movement and blood flow. Results: A total number of 562 patients of FET with 315 pregnancies (56.0%) was analyzed. It was found that only the echo of the endometrial central line was different between the pregnant group and non-pregnant group. Other parameters, such as endometrial thickness, volume, endometrial peristalsis, or the endometrial blood flow were not statistically different between the two groups. Then, according to the relationship between the different groups and the clinical pregnancy rate, a score of 0 to 2 was respectively scored. The sum of the scores for the six items was the patient's endometrial receptivity score. It showed that the clinical pregnancy rate increased as the endometrial receptivity score increased, and when the receptivity score reaches at least 5, the clinical pregnancy rate is significantly improved (63.7% versus 49.5%, P=0.001). Conclusion: We developed an endometrial receptivity scoring system and demonstrated its validity. It may aid clinicians in choosing the useful marker in clinical practice and for informing further research.

Measurement of Bone Metastatic Tumor Growth by a Tibial Tumorigenesis Assay.

Bone metastasis is a frequent and lethal complication of many cancer types (i.e., prostate cancer, breast cancer, and multiple myeloma), and a cure for bone metastasis remains elusive. To recapitulate the process of bone metastasis and understand how cancer cells metastasize to bone, intracardiac injection and intracaudal arterial animal models were developed. The intratibial injection animal model was established to investigate the communication between cancer cells and the bone microenvironment and to mimic the setting of prostate cancer patients with bone metastasis. Given that detailed protocols of intratibial injection and its quantitative analysis are still insufficient, in this protocol, we provide hands-on procedures for how to prepare cells, perform the tibial injection, monitor tibial tumor growth, and quantitatively evaluate the tibial tumors in pathological samples. This manuscript provides a ready-to-use experimental protocol for investigating cancer cell behaviors in bone and developing novel therapeutic strategies for bone metastatic cancer patients.

Toxic effects of patulin on mouse oocytes and its possible mechanisms.

Patulin is a secondary metabolite mainly secreted by fungi and is the most common mycotoxin found in apples and apple-based products. For the past few years, numerous studies suggested the wide distribution and toxicity of patulin. In this study, we investigated the toxic effect of patulin on mouse oocytes and its possible mechanisms. The results showed that patulin treatment did not affect meiotic resumption, but inhibited oocyte maturation as indicated by failure of first polar body extrusion. Further mechanistic study showed that patulin treatment disturbed normal spindle assembly, chromosome alignment and morphology. We also found increased oxidative stress by testing the level of ROS and decreased mitochondrial membrane potential, indicating mitochondria dysfunction. In summary, our results suggest that patulin treatment causes oocyte meiotic arrest by disturbing normal spindle assembly and chromosome alignment, which may be caused by dysfunctions of mitochondria.

Specific deletion of protein phosphatase 6 catalytic subunit in Sertoli cells leads to disruption of spermatogenesis.

Protein phosphatase 6 (PP6) is a member of the PP2A-like subfamily, which plays significant roles in numerous fundamental biological activities. We found that PPP6C plays important roles in male germ cells recently. Spermatogenesis is supported by the Sertoli cells in the seminiferous epithelium. In this study, we crossed Ppp6cF/F mice with AMH-Cre mice to gain mutant mice with specific depletion of the Ppp6c gene in the Sertoli cells. We discovered that the PPP6C cKO male mice were absolutely infertile and germ cells were largely lost during spermatogenesis. By combing phosphoproteome with bioinformatics analysis, we showed that the phosphorylation status of ß-catenin at S552 (a marker of adherens junctions) was significantly upregulated in mutant mice. Abnormal ß-catenin accumulation resulted in impaired testicular junction integrity, thus led to abnormal structure and functions of BTB. Taken together, our study reveals a novel function for PPP6C in male germ cell survival and differentiation by regulating the cell-cell communication through dephosphorylating ß-catenin at S552.

Cell Division Cycle 5-Like Regulates Metaphase-to-Anaphase Transition in Meiotic Oocyte.

The quality of oocytes is a vital factor for embryo development. Meiotic progression through metaphase I usually takes a relatively long time to ensure correct chromosome separation, a process that is critical for determining oocyte quality. Here, we report that cell division cycle 5-like (Cdc5L) plays a critical role in regulating metaphase-to-anaphase I transition during mouse oocyte meiotic maturation. Knockdown of Cdc5L by small interfering RNA injection did not affect spindle assembly but caused metaphase I arrest and subsequent reduced first polar body extrusion due to insufficient anaphase-promoting complex/cyclosome activity. We further showed that Cdc5L could also directly interact with securin, and Cdc5L knockdown led to a continuous high expression level of securin, causing severely compromised meiotic progression. The metaphase-to-anaphase I arrest caused by Cdc5L knockdown could be rescued by knockdown of endogenous securin. In summary, we reveal a novel role for Cdc5L in regulating mouse oocyte meiotic progression by interacting with securin.

Evaluation of the Genetic Variation Spectrum Related to Corneal Dystrophy in a Large Cohort.

AIMS: To characterize the genetic landscape and mutation spectrum of patients with corneal dystrophies (CDs) in a large Han ethnic Chinese Cohort with inherited eye diseases (IEDs). METHODS: Retrospective study. A large IED cohort was recruited in this study, including 69 clinically diagnosed CD patients, as well as other types of eye diseases patients and healthy family members as controls. The 792 genes on the Target_Eye_792_V2 chip were used to screen all common IEDs in our studies, including 22 CD-related genes. RESULTS: We identified 2334 distinct high-quality variants on 22 CD-related genes in a large IEDs cohort. A total of 21 distinct pathogenic or likely pathogenic mutations were identified, and the remaining 2313 variants in our IED cohort had no evidence of CD-related pathogenicity. Overall, 81.16% (n = 56/69) of CD patients received definite molecular diagnoses, and transforming growth factor-beta-induced protein (TGFBI), CHTS6, and SLC4A11 genes covered 91.07, 7.14, and 1.79% of the diagnosed cases, respectively. Twelve distinct disease-associated mutations in the TGFBI gene were identified, 11 of which were previously reported and one is novel. Four of these TGFBI mutations (p.D123H, p.M502V, p.P501T, and p.P501A) were redefined as likely benign in our Han ethnic Chinese IED cohort after performing clinical variant interpretation. These four TGFBI mutations were detected in asymptomatic individuals but not in CD patients, especially the previously reported disease-causing mutation p.P501T. Among 56 CD patients with positive detected mutations, the recurrent TGFBI mutations were p.R124H, p.R555W, p.R124C, p.R555Q, and p.R124L, and the proportions were 32.14, 19.64, 14.29, 10.71, and 3.57%, respectively. Twelve distinct pathogenic or likely pathogenic mutations of CHTS6 were detected in 28 individuals. The recurrent mutations were p.Y358H, p.R140X, and p.R205W, and the proportions were 25.00, 21.43, and 14.29%, respectively. All individuals associated with TGFBI were missense mutations; 74.19% associated with CHTS6 mutations were missense mutations, and 25.81% were non-sense mutations. Hot regions were located in exons 4 and 12 of TGFBI individuals and located in exon 3 of CHTS6 individuals. No de novo mutations were identified. CONCLUSION: For the first time, our large cohort study systematically described the variation spectrum of 22 CD-related genes and evaluated the frequency and pathogenicity of all 2334 distinct high-quality variants in our IED cohort. Our research will provide East Asia and other populations with baseline data from a Han ethnic population-specific level.

The Cyclin B2/CDK1 Complex Conservatively Inhibits Separase Activity in Oocyte Meiosis II.

Recently, we have reported that the cyclin B2/CDK1 complex regulates homologous chromosome segregation through inhibiting separase activity in oocyte meiosis I, which further elucidates the compensation of cyclin B2 on cyclin B1's function in meiosis I. However, whether cyclin B2/CDK1 complex also negatively regulates separase activity during oocyte meiosis II remains unknown. In the present study, we investigated the function of cyclin B2 in meiosis II of oocyte. We found that stable cyclin B2 expression impeded segregation of sister chromatids after oocyte parthenogenetic activation. Consistently, stable cyclin B2 inhibited separase activation, while introduction of non-phosphorylatable separase mutant rescued chromatid separation in the stable cyclin B2-expressed oocytes. Therefore, the cyclin B2/CDK1 complex conservatively regulates separase activity via inhibitory phosphorylation of separase in both meiosis I and meiosis II of mouse oocyte.

Critical Functions of PP2A-Like Protein Phosphotases in Regulating Meiotic Progression.

Meiosis is essential to the continuity of life in sexually-reproducing organisms through the formation of haploid gametes. Unlike somatic cells, the germ cells undergo two successive rounds of meiotic divisions after a single cycle of DNA replication, resulting in the decrease in ploidy. In humans, errors in meiotic progression can cause infertility and birth defects. Post-translational modifications, such as phosphorylation, ubiquitylation and sumoylation have emerged as important regulatory events in meiosis. There are dynamic equilibrium of protein phosphorylation and protein dephosphorylation in meiotic cell cycle process, regulated by a conservative series of protein kinases and protein phosphatases. Among these protein phosphatases, PP2A, PP4, and PP6 constitute the PP2A-like subfamily within the serine/threonine protein phosphatase family. Herein, we review recent discoveries and explore the role of PP2A-like protein phosphatases during meiotic progression.

Acetylation of KLF5 maintains EMT and tumorigenicity to cause chemoresistant bone metastasis in prostate cancer.

Advanced prostate cancer (PCa) often develops bone metastasis, for which therapies are very limited and the underlying mechanisms are poorly understood. We report that bone-borne TGF-ß induces the acetylation of transcription factor KLF5 in PCa bone metastases, and acetylated KLF5 (Ac-KLF5) causes osteoclastogenesis and bone metastatic lesions by activating CXCR4, which leads to IL-11 secretion, and stimulating SHH/IL-6 paracrine signaling. While essential for maintaining the mesenchymal phenotype and tumorigenicity, Ac-KLF5 also causes resistance to docetaxel in tumors and bone metastases, which is overcome by targeting CXCR4 with FDA-approved plerixafor. Establishing a mechanism for bone metastasis and chemoresistance in PCa, these findings provide a rationale for treating chemoresistant bone metastasis of PCa with inhibitors of Ac-KLF5/CXCR4 signaling.

Gefitinib reduces oocyte quality by disturbing meiotic progression.

Gefitinib is a first-line anti-cancer drug for the treatment of advanced non-small cell lung cancer (NSCLC). It has been reported that gefitinib can generate several drug-related adverse effects, including nausea, peripheral edema, decreased appetite and rash. However, the reproductive toxicity of gefitinib has not been clearly defined until now. Here we assessed the effects of gefitinib on oocyte quality by examining the critical events and molecular changes of oocyte maturation. Gefitinib at 1, 2, 5 or 10 µM concentration was added to culture medium (M2). We found that gefitinib at its median peak concentration of 1 µM did not affect oocyte maturation, but 5 µM gefitinib severely blocked oocyte meiotic progression as indicated by decreased rates of germinal vesicle breakdown (GVBD) and polar body extrusion (PBE). We further showed that gefitinib treatment increased phosphorylation of CDK1 at the site of Try15, inhibited cyclin B1 entry into the nucleus, and disrupted normal spindle assembly, chromosome alignment and mitochondria dynamics, finally leading to the generation of aneuploidy and early apoptosis of oocytes. Our study reported here provides valuable evidence for reproductive toxicity of gefitinib administration employed for the treatment of cancer patients.

Ultra-low dose rituximab as add-on therapy in anti-MDA5-positive patients with polymyositis /dermatomyositis associated ILD.

OBJECTIVES: To evaluate the efficacy and safety of ultra-low dose (100 mg) rituximab (RTX) administration in anti-melanoma differentiation-associated gene 5 (MDA5) positive patients with polymyositis/dermatomyositis (PM/DM) associated interstitial lung disease. METHODS: This retrospective study included anti-MDA5 antibody positive ILD subjects in the First Affiliated Hospital of Guangzhou Medical University from November 2017 to March 2019. Independent predictors for 180-day mortality were measured by Cox regression analysis. Patients were divided into 3 groups: Group 1 (non-cyclophosphamide (CTX)/RTX) (n = 10), Group 2 (CTX only) (n = 19) and Group 3 (RTX with/without CTX) (n = 11). The 180-day mortality was compared among 3 groups with Kaplan-Meier analysis. Post-RTX serological parameters as well as adverse events were evaluated. RESULTS: Forty patients were included with the mean age of 51.3 years. Elevated IL-10 level and CD4+/8+ ratio were considered as risk factors of 180-day mortality. Kaplan-Meier analysis showed a trend toward decrease, albeit non-significant, in 180-day mortality in Group 3 (P = 0.26). The administration of 100 mg RTX brought down B cell within 7 days that lasted for 180 days. There were 7 and 6 infection events observed within 2 months of CTX/RTX treatment in Group 2 and 3, with 5 and 2 fatal cases respectively. Cytomegalovirus infection accounted for half infection events in Group 3. CONCLUSION: We found a pronounced and prolonged B cell depletion following 100 mg RTX infusion and RTX add-on may be effective in anti-MDA5 positive ILD patients. However, infection, especially opportunistic infection, should be concerned during the treatment.

Treatment of Infertile Women with Unilateral Tubal Occlusion Diagnosed by Hysterosalpingography: The Role of Intrauterine Insemination.

The optimal assisted reproductive treatment strategy for infertile women with unilateral tubal obstruction remains uncertain. To investigate the role of intrauterine insemination (IUI) in the treatment of infertile women with unilateral tubal occlusion, the data of 148 couples were retrospectively collected and analyzed. Seventy-eight infertile women with unilateral occlusion diagnosed by hysterosalpingography (HSG) were categorized as the study group and 70 others with unexplained infertility as the control group. The study group was divided into a proximal occlusion subgroup and a mid-distal occlusion subgroup for further analysis. The main outcomes, namely the clinical pregnancy rate (CPR), ongoing pregnancy rate (OPR), and live birth rate (LBR) per cycle, were analyzed. Our results showed a tendency of lower CPR, OPR, and LBR in the study group than in the control group, without statistical significance. Further investigations revealed that the unilateral proximal occlusion subgroup had similar CPR, OPR, and LBR as the control group, while the unilateral mid-distal occlusion subgroup had significantly lower CPR (5.1% vs. 20.0%, P=0.035), OPR (5.1% vs. 20.0%, P=0.035), and LBR (5.1% vs. 20.0%, P=0.035) than the control group. In conclusion, the clinical outcomes of IUI were worse in patients with unilateral tubal occlusion than in those with unexplained infertility. This might be primarily caused by the worse outcome of patients with unilateral mid-distal tubal occlusion instead of proximal occlusion.

Overexpression of cyclin A1 promotes meiotic resumption but induces premature chromosome separation in mouse oocyte.

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation.

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.