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Maturation of oocytes and early embryo development are regulated precisely by numerous factors at transcriptional and posttranslational levels through precise mechanisms. N6-methyladenosine (m6A) is the most common modification in mRNA which regulates RNA metabolism and gene expression. However, the role of RNA m6A on porcine oocyte maturation and early embryogenesis is largely unknown. Here, we found that oocytes treated with cycloleucine (CL), an RNA m6A inhibitor, express impaired cumulus expansion, increased production of reactive oxygen species (ROS) in the mitochondria, and delayed maturation of oocytes by disrupting spindle organization and chromosome alignment. Also, CL halted the development of embryos at the 4-cell stage and resulted in low-quality blastocysts. Furthermore, CL treatment decreased the RNA m6A, H3K4me3, and H3K9me3 levels, but increased the acetylation level of H4K16 during parthenogenetic embryonic development in pigs. Single-cell RNA-seq (scRNA-seq) analysis further revealed that CL treatment dramatically up-regulated the expression of metabolism-related genes (SLC16A1, and MAIG3 etc.) and maternal related genes, including BTG4, WEE2, and BMP15 among others, at the blastocyst stage. Taken together, inhibition of RNA m6A by CL impaired meiosis of oocytes and early embryonic development of porcine via RNA m6A methylation, histone modifications, and altering the expression of metabolism-related genes in blastocysts.

This study was conducted to elucidate the effects of introducing conjugated linoleic acid (CLA) on meiotic spindle organization of heat stressed (HS) matured oocytes and the resulting blastocysts DNA methylation as well as the expression of the genes involved in DNA methylation (DNMT3a, DNMT3b and DNMT1). Immature bovine oocytes were cultured at 41 °C for the first 12 h and 38.5 °C for the second 12 h of maturation time in the presence of 0 and 50 μM of CLA (HS and HS + CLA groups, respectively). A group of oocytes cultured in medium with no CLA supplementation at normal temperature (38.5 °C for 24 h) was considered as negative control (C). Percentage of normal spindle, and cleavage and blastocyst rates were significantly decreased in the HS group compared to the C group (P 0.05). The global DNA methylation and expression level of DNMT3a gene were increased in HS group compared to the C groups (P 0.05), while the expression level of DNMT3b was decreased. The CLA supplementation improved the percentage of normal microtubules shape in MII oocytes as well as the developmental competence in the HS + CLA group compared to the HS group (P 0.05). However, global DNA methylation and expression level of DNMT3a/b were not ameliorated by CLA supplementation (P 0.05). Based on the obtained results, CLA proved to be capable of improving the oocyte developmental competence as well as decreased the aberrant spindle organization of heat-stressed oocytes and it would not cause epigenetic alteration in the obtained blastocysts.

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.

BACKGROUNDRAB GTPases constitute the largest family of small GTPases and are found in all eukaryotes. RAB GTPases regulate components of the endomembrane system, the nucleus and the plasma membrane, and are involved in intracellular actin/tubulin-dependent vesicle movement, membrane fusion and cell growth in mitosis.OBJECTIVE AND RATIONALERAB GTPases play multiple critical roles during both female and male meiosis. This review summarizes the progress made in our understanding of the role of RAB GTPases in female and male meiosis in different species. We also discuss the potential relationship between RAB GTPases and oocyte/sperm quality, which may help in understanding the mechanisms underlying oogenesis and spermatogenesis and potential genetic causes of infertility.SEARCH METHODSThe PubMed database was searched for articles published between 1991 and 2020 using the following terms: ‘RAB’, ‘RAB oocyte’, ‘RAB sperm’ and ‘RAB meiosis’.OUTCOMESAn analysis of 126 relevant articles indicated that RAB GTPases are present in all eukaryotes, and ten subfamilies (almost 70 members) are expressed in human cells. The roles of 25 RAB proteins and orthologues in female meiosis and 12 in male meiosis have been reported. RAB proteins are essential for the accurate continuity of genetic material, successful fertilization and the normal growth of offspring. Distinct and crucial functions of RAB GTPases in meiosis have been reported. In oocytes, RAB GTPases are involved in spindle organization, kinetochore–microtubule attachment, chromosome alignment, actin filament-mediated spindle migration, cytokinesis, cell cycle and oocyte–embryo transition. RAB GTPases function in mitochondrial processes and Golgi-mediated vesicular transport during female meiosis, and are critical for cortical granule transport during fertilization and oocyte–embryo transition. In sperm, RAB GTPases are vital for cytoskeletal organization and successful cytokinesis, and are associated with Golgi-mediated acrosome formation, membrane trafficking and morphological changes of sperm cells, as well as the exocytosis-related acrosome reaction and zona reaction during fertilization.WIDER IMPLICATIONSAbnormal expression of RAB GTPases disrupts intracellular systems, which may induce diverse diseases. The roles of RAB proteins in female and male reproductive systems, thus, need to be considered. The mechanisms underlying the function of RAB GTPases and the binding specificity of their effectors during oogenesis, spermatogenesis and fertilization remain to be studied. This review should contribute to our understanding of the molecular mechanisms of oogenesis and spermatogenesis and potential genetic causes of infertility.

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.

HDAC11, the sole member of HDAC class IV family, plays vital roles in activating mitosis and apoptosis of tumor cells, but its functions in meiosis are rarely investigated. In the present study, the effect of HDAC11 on meiosis during porcine oocytes maturation was fully studied. The results showed that HDAC11 inhibition by its specific inhibitor JB-3-22 dramatically decreased the porcine oocyte maturation rate by disturbing spindle organization and chromosomes alignment without affecting the cytoplasmic maturation. Further study indicated that HDAC11 inhibition significantly elevated the acetylation levels of α-tubulin and H4K16, which are crucial for spindle organization and chromosomes alignment. Moreover, immunofluorescence staining results showed that HDAC11 inhibition also disturbed other meiosis-related histone modifications, such as increased H3S10pho, H4K5ac and H4K12ac levels and reduced H3T3pho level. Furthermore, RNA-seq analysis results indicated that HDAC11 inhibition disturbed porcine oocytes transcriptome (157 up-regulation, 106 down-regulation). In addition, HDAC11 inhibition compromised oocytes quality and subsequent development after parthenogenetic activation, which may be caused by the aberrant nuclear maturation and transcriptome expression profile during oocytes maturation. Therefore, our results elucidate the function of HDAC11 in porcine oocytes maturation and embryos development through regulating α-tubulin acetylation, meiosis-related histone modifications and transcriptome.

Mammalian oocytes represent impaired quality after undergoing a process of postovulatory aging, which can be alleviated through various effective ways such as reagent treatment. Accumulating evidences have revealed the beneficial effects of astaxanthin (Ax) as a potential antioxidant on reproductive biology. Here, porcine matured oocytes were used as a model to explore whether Ax supplement can protect against oocyte aging in vitro and the underlying mechanism, and therefore they were cultured with or without 2.5 μM Ax for an additional 24 h. Aged oocytes treated with Ax showed improved yield and quality of blastocysts as well as recovered expression of maternal genes. Importantly, oxidative stress in aged oocytes was relieved through Ax treatment, based on reduced reactive oxygen species and enhanced glutathione and antioxidant gene expression. Moreover, inhibition in apoptosis and autophagy of aged oocyte by Ax was confirmed through decreased caspase-3, cathepsin B and autophagic activities. Ax could also maintain spindle organization and actin expression, and rescue functional status of organelles including mitochondria, endoplasmic reticulum, Golgi apparatus and lysosomes according to restored fluorescence intensity. In conclusion, Ax might provide an alternative for ameliorating the oocyte quality following aging in vitro, through the mechanisms mediated by its antioxidant properties.

Monopolar spindle-1 (Mps1) is a critical interphase regulator that also involves into the spindle assembly checkpoint for the cell cycle control in both mitosis and meiosis. However, the functions of Mps1 during mouse early embryo development is still unclear. In this study, we reported the important roles of Mps1 in the first cleavage of mouse embryos. Our data indicated that the loss of Mps1 activity caused precocious cleavage of zygotes to 2-cell embryos; however, prolonged culture disturbed the early embryo development to the blastocyst. We found that the spindle organization was disrupted after Mps1 inhibition, and the chromosomes were misaligned in the first cleavage. Moreover, the kinetochore-microtubule attachment was lost and Aurora B failed to accumulate to the kinetochores, indicating that the spindle assembly checkpoint (SAC) was activated. Furthermore, the inhibition of Mps1 activity resulted in an increase of DNA damage, which further induced oxidative stress, showing with positive γ-H2A.X signal and increased reactive oxygen species level. Ultimately, irreparable DNA damage and oxidative stress-activated apoptosis and autophagy, which was confirmed by the positive Annexin-V signal and increased autophagosomes. Taken together, our data indicated that Mps1 played important roles in the control of SAC and DNA repair during mouse early embryo development.

Accurate chromosome segregation relies on correct chromosome-microtubule interactions within a stable bipolar spindle apparatus. Thus, exposure to spindle disrupting compounds can impair meiotic division and genomic stability in oocytes. The endocrine disrupting activity of bisphenols such as bisphenol A (BPA) is well recognized, yet their damaging effects on spindle microtubules (MTs) is poorly understood. Here, we tested the effect(s) of acute exposure to BPA and bisphenol F (BPF) on assembled spindle stability in ovulated oocytes. Brief (4 h) exposure to increasing concentrations (5, 25, and 50 µg/mL) of BPA or BPF disrupted spindle organization in a dose-dependent manner, resulting in significantly shorter spindles with highly unfocused poles and fragmented pericentrin. The chromosomes remained congressed in an abnormally elongated metaphase-like configuration, yet normal end-on chromosome-MT attachments were reduced in BPF-treated oocytes. Live-cell imaging revealed a rapid onset of bisphenol-mediated spindle MT disruption that was reversed upon compound removal. Moreover, MT stability and regrowth were impaired in BPA-exposed oocytes, with few cold-stable MTs and formation of multipolar spindles upon MT regrowth. MT-associated kinesin-14 motor protein (HSET/KIFC1) labeling along the spindle was also lower in BPA-treated oocytes. Conversely, cold stable MTs and HSET labeling persisted after BPF exposure. Notably, inhibition of Aurora Kinase A limited bisphenol-mediated spindle pole widening, revealing a potential interaction. These results demonstrate rapid MT disrupting activity by bisphenols, which is highly detrimental to meiotic spindle stability and organization. Moreover, we identify an important link between these defects and altered distribution of key spindle associated factors as well as Aurora Kinase A activity.

Formin-like 3 (FMNL3) is a member of the formin-likes (FMNLs), which belong to the formin family. As an F-actin nucleator, FMNL3 is essential for several cellular functions, such as polarity control, invasion, and migration. However, the roles of FMNL3 during oocytes meiosis remain unclear. In this study, we investigated the functions of FMNL3 during mouse oocyte maturation. Our results showed that FMNL3 mainly concentrated in the oocyte cortex and spindle periphery. Depleting FMNL3 led to the failure of polar body extrusion, and we also found large polar bodies in the FMNL3-deleted oocytes, indicating the occurrence of symmetric meiotic division. There was no effect of FMNL3 on spindle organization; however, we observed spindle migration defects at late metaphase I, which might be due to the decreased cytoplasmic actin. Microinjecting Fmnl3-EGFP mRNA into Fmnl3-depleted oocytes significantly rescued these defects. In addition, the results of co-immunoprecipitation and the perturbation of protein expression experiments suggested that FMNL3 interacted with the actin-binding protein FASCIN for the regulation of actin filaments in oocytes. Thus, our results provide the evidence that FMNL3 regulates FASCIN for actin-mediated spindle migration and cytokinesis during mouse oocyte meiosis.

Abnormal lipid metabolism plays crucial roles in the development of cancer. Spindlin 1 (SPIN1) involving the process of spindle organization and chromosomal stability serves as an important player in the carcinogenesis. In this study, we try to identify the new function of SPIN1 in lipid metabolism of liver cancer. Tissue microarray showed that 75% (60/80) of hepatocellular carcinoma (HCC) tissues were positive for SPIN1, which was highly expressed in clinical HCC samples and positively associated with malignancy of HCC. Strikingly, SPIN1 could modulate abnormal lipid metabolism by increasing intracellular triglycerides, cholesterols, and lipid droplets in hepatoma cells, which could remarkably enhance the proliferation of hepatoma cells. Mechanistically, SPIN1 up-regulated FASN in hepatoma cells. SPIN1 co-activated transcriptional factor SREBP1c in the promoter of FASN through interaction with SREBP1c. Moreover, SPIN1 promoted the growth of liver cancer in vitro and in vivo and the levels of intracellular triglycerides, cholesterols and lipid droplets were increased in the tumor tissues from mice. In conclusion, SPIN1 modulates abnormal lipid metabolism and enhances growth of liver cancer through SREBP1c-triggered FASN signaling. Therapeutically, SPIN1 may serve as a novel target for HCC.

Non-functioning pituitary adenomas (NFPAs) are typical pituitary macroadenomas in adults that are associated with increased mortality and morbidity. Although pituitary adenomas are commonly considered slow-growing benign brain tumors, numerous of them possess an invasive nature. Such tumors destroy sella turcica and invade the adjacent tissues such as the cavernous sinus and sphenoid sinus. The most critical obstacle for complete surgical removal in these cases is the high risk of damaging adjacent vital structures. Therefore, the development of novel therapeutic strategies for either early diagnosis through biomarkers or medical therapies to reduce the recurrence rate of NFPAs is imperative. Identification of gene interactions has paved the way for decoding complex molecular mechanisms, including disease-related pathways, and identifying the most momentous genes involved in a specific disease. Currently, our knowledge of the invasion of the pituitary adenoma at the molecular level is not sufficient. The current study aimed to identify critical biomarkers and biological pathways associated with invasiveness in the NFPAs using a three-way interaction model for the first time. In the current study, the Liquid association method was applied to capture the statistically significant triplets that are involved in NFPAs invasiveness. Subsequently, Random Forest analysis was applied to select the most important switch genes. Finally, gene set enrichment (GSE) and gene regulatory network (GRN) analyses were applied to trace the biological relevance of the statistically significant triplets. The results of this study suggest that “mRNA processing” and “spindle organization” biological processes are important in NFAPs invasiveness. Specifically, our results suggest Nkx3-1 and Fech as two switch genes in NFAPs invasiveness that may be a potential biomarkers or target genes, in this pathology.

Alternative polyadenylation (APA) affects the length and composition of 3'-untranslated region (3'-UTR), and regulates mRNA stability or translational activity to affect important biological processes. However, global 3'-UTR landscape and its relationship with gamete maturation remain less studied. Here, we analyzed our previously reported single cell (sc)RNA-seq data of germinal vesicle (GV) and metaphase II (MII) stage oocytes in pigs, to systematically catalogue the 3'-UTR dynamics during oocyte maturation. Two softwares (Dapars and APAtrap) were employed, and identified 110 and 228 mRNAs with significantly different 3'-UTRs (Padj.≤0.05), respectively. Gene enrichment analyses found signaling pathways related with biological processes of female gametophyte production, methyltransferase activity, and mRNA surveillance (Dapars); and cell cycle process, regulation of ERK1 and ERK2 cascade/translation, spindle organization, kinetochore, condensed chromosome, and progesterone-mediated oocyte maturation (APAtrap), respectively. Moreover, 18 of 110 mRNAs (|△PDUI|≥0.25 and |log2 PDUI ratio|≥0.59) and 15 of 228 mRNAs (Perc. Diff.≥0.5) were with more significantly difference of 3'-UTR changes, and Integrative genomics viewer (IGV) analysis further identified 4 (Alg10, Hadhb, Hsd17b4 and Sbds) of 18 mRNAs to be with 3'-UTR length differed ≥150bp and 6 (Gcc1, Hnrnpa2b1, Lsm6, Prpf18, Sfr1 and Ust) of 15 mRNAs to be with 3'-UTR abundance extremely differed. Furthermore, the location, sequences and number of cis-elements were predicted, which were shown to derange cytoplasmic polyadenylation element (CPE), Poly(A) site (PAS) and microRNA binding sites within 3'-UTRs of Alg10, Hadhb, Hsd17b4 and Sbds mRNAs. Taken together, global 3'-UTR landscape changes dynamically with oocyte meiotic maturation, potentially involved in regulating oocyte meiotic process in pigs.

Objectives Histone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood. Materials and methods HDAC8‐targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI‐34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ‐tubulin dynamics and acetylation level of α‐tubulin. Results We observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ‐tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI‐34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α‐tubulin. Conclusions Collectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation., Our findings document that HDAC8 locates to the spindle apparatus in porcine oocytes to take part in the spindle assembly via recruitment of γ‐tubulin to the spindle poles, and hence driving the oocyte meiotic maturation. This function of HDAC8 relies on its enzyme activity and is conserved among species in mammals.

Centrosomes are highly conserved organelles that act as the major microtubule-organizing center (MTOC) in animal somatic cells. Through their MTOC activity, centrosomes play various roles throughout the cell cycle, such as supporting cell migration in interphase and spindle organization and positioning in mitosis. Various approaches for removing centrosomes from somatic cells have been developed and applied over the past few decades to understand the precise roles of centrosomes. Centrinone, a reversible and selective PLK4 (polo-like kinase 4) inhibitor, has recently emerged as an efficient approach to eliminate centrosomes. In this review, we describe the latest findings on centrosome function that have been revealed using various centrosome-eliminating approaches. In addition, we discuss our recent findings on the mechanism of centrosome-independent spindle bipolarization, discovered through the use of centrinone.Key words: centrosome, centrinone, mitotic spindle, bipolarity, NuMA.

SIRT6, a member of the sirtuin family, is a NAD + dependent protein deacetylase and has been implicated in transcriptional regulation of somatic cells and post-transcriptional regulation of oocyte meiosis. However, the function of cumulus cell-derived and maternal SIRT6 in meiotic maturation of porcine oocytes is not yet known. Here, we report that SIRT6 mRNA and protein exists in the oocyte and its surrounding cumulus cells during meiotic maturation. Functional studies using a specific inhibitor in cumulus-enclosed oocytes revealed important roles for SIRT6 in germinal vesicle breakdown (GVBD) and cumulus expansion. Moreover, inhibitor treatment led to a significant reduction in the rate of first polar body (PB1) extrusion and early development of parthenogenetically activated embryos. In contrast, SIRT6 inhibition in cumulus-free oocytes only resulted in a significant reduction in the rate of PB1 extrusion. Furthermore, SIRT6 dysfunction regardless of the origin in both cumulus cells and oocytes severely impaired spindle organization and chromosome alignment at the metaphase stage. Molecularly, SIRT6 inhibition in cumulus cells significantly reduced expression of genes associated with cumulus expansion and gap junctional communication and even expression levels of active phosphorylated CDK1 in oocytes. Importantly, adenylate cyclase inhibition could partially rescue GVBD and PB1 extrusion in SIRT6-inhibited cumulus-enclosed oocytes. Taken together, these results demonstrate that cumulus cell-expressed and maternal SIRT6 differentially regulates porcine oocyte meiotic maturation.

Bipolar spindles are organized by motor proteins that generate microtubule-­dependent forces to separate the two spindle poles. The fission yeast Cut7 (kinesin-5) is a plus-end-directed motor that generates the outward force to separate the two spindle poles, whereas the minus-end-directed motor Pkl1 (kinesin-14) generates the inward force. Balanced forces by these antagonizing kinesins are essential for bipolar spindle organization in mitosis. Here, we demonstrate that chromosomes generate another outward force that contributes to the bipolar spindle assembly. First, it was noted that the cut7 pkl1 double knockout failed to separate spindle poles in meiosis I, although the mutant is known to succeed it in mitosis. It was assumed that this might be because meiotic kinetochores of bivalent chromosomes joined by cross-overs generate weaker tensions in meiosis I than the strong tensions in mitosis generated by tightly tethered sister kinetochores. In line with this idea, when meiotic mono-oriented kinetochores were artificially converted to a mitotic bioriented layout, the cut7 pkl1 mutant successfully separated spindle poles in meiosis I. Therefore, we propose that spindle pole separation is promoted by outward forces transmitted from kinetochores to spindle poles through microtubules.

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.

B chromosomes (Bs) are supernumerary chromosomes, which are often preferentially inherited. When transmission rates of chromosomes are higher than 0.5, not obeying the Mendelian law of equal segregation, the resulting transmission advantage is collectively referred to as 'chromosome drive'. Here we analysed the drive mechanism of Aegilops speltoides Bs. The repeat AesTR-183 of A. speltoides Bs, which also can be detected on the Bs of Aegilops mutica and rye, was used to track Bs during pollen development. Nondisjunction of CENH3-positive, tubulin interacting B sister chromatids and an asymmetric spindle during first pollen grain mitosis are key for the accumulation process. A quantitative flow cytometric approach revealed that, independent of the number of Bs present in the mother plant, Bs accumulate in the generative nuclei to > 93%. Nine out of 11 tested (peri)centromeric repeats were shared by A and B chromosomes. Our findings provide new insights into the process of chromosome drive. Quantitative flow cytometry is a useful and reliable method to study the drive frequency of Bs. Nondisjunction and unequal spindle organization accompany during first pollen mitosis the drive of A. speltoides Bs. The prerequisites for the drive process seems to be common in Poaceae.

Cytoskeleton which includes microtubule and actin filaments plays important roles during mammalian oocyte maturation. In the present study, we showed that protein kinase C mu (PKC mu) was one potential key molecule which affected cytoskeleton dynamics in mouse oocytes. Our results showed that PKC mu expressed and localized at the poles of the spindle during oocyte maturation, and PKC mu expression reduced in the oocytes from 6-month-old mice or 24 hr in vitro culture. We knocked down the expression of PKC mu in oocytes using morpholino injection to explore the relationship between PKC mu and subcellular structure defects. The loss of PKC mu reduced oocyte maturation competence, showing with decreased polar body extrusion rate and increased rate of symmetric division. Further analysis indicated that PKC mu decrease caused the spindle organization defects, and this could be confirmed by the decreased tubulin acetylation level. Moreover, we found that PKC mu affected the phosphorylation level of cofilin for actin assembly, which further affected cytoplasmic actin distribution and spindle positioning. In summary, our data indicated that PKC mu is one key factor for oocyte maturation through its roles on the spindle organization and actin filament distribution.

Objectives RAB14 is a member of small GTPase RAB family which localizes at the endoplasmic reticulum (ER), Golgi apparatus and endosomal compartments. RAB14 acts as molecular switches that shift between a GDP‐bound inactive state and a GTP‐bound active state and regulates circulation of vesicles between the Golgi and endosomal compartments. In present study, we investigated the roles of RAB14 during oocyte meiotic maturation. Materials and methods Microinjection with siRNA and exogenous mRNA for knock down and rescue, and immunofluorescence staining, Western blot and real‐time RT‐PCR were utilized for the study. Results Our results showed that RAB14 localized in the cytoplasm and accumulated at the cortex during mouse oocyte maturation, and it was also enriched at the spindle periphery. Depletion of RAB14 did not affect polar body extrusion but caused large polar bodies, indicating the failure of asymmetric division. We found that absence of RAB14 did not affect spindle organization but caused the spindle migration defects, and this might be due to the regulation on cytoplasmic actin assembly via the ROCK‐cofilin signalling pathway. We also found that RAB14 depletion led to aberrant Golgi apparatus distribution. Exogenous Myc‐Rab14 mRNA supplement could significantly rescue these defects caused by Rab14 siRNA injection. Conclusions Taken together, our results suggest that RAB14 affects ROCK‐cofilin pathway for actin‐based spindle migration and Golgi apparatus distribution during mouse oocyte meiotic maturation., RAB14 localized in the cytoplasm and accumulated at the cortex during mouse oocyte maturation, and it was also enriched at the spindle periphery. Depletion of RAB14 caused large polar bodies, indicating the failure of asymmetric division. Absence of RAB14 did not affect spindle organization but caused the spindle migration defects, and this might be due to the regulation on cytoplasmic actin assembly via the ROCK‐cofilin signalling pathway. We also found that RAB14 depletion led to aberrant Golgi apparatus distribution. Exogenous Myc‐Rab14 mRNA supplement could significantly rescue these defects caused by Rab14 siRNA injection. Our results suggest that RAB14 affects ROCK‐cofilin pathway for actin‐based spindle migration and Golgi apparatus distribution during mouse oocyte meiotic maturation.

Huang et al. reveal that the microcephaly-related protein WDR62 possesses an intrinsic affinity for curved microtubules and regulates spindle dynamics by functioning as an adaptor protein between its recruiting factor TPX2/Aurora A and the effector katanin., WDR62 is a microcephaly-related, microtubule (MT)-associated protein (MAP) that localizes to the spindle pole and regulates spindle organization, but the underlying mechanisms remain elusive. Here, we show that WDR62 regulates spindle dynamics by recruiting katanin to the spindle pole and further reveal a TPX2–Aurora A–WDR62–katanin axis in cells. By combining cellular and in vitro experiments, we demonstrate that WDR62 shows preference for curved segments of dynamic GDP-MTs, as well as GMPCPP- and paclitaxel-stabilized MTs, suggesting that it recognizes extended MT lattice. Consistent with this property, WDR62 alone is inefficient in recruiting katanin to GDP-MTs, while WDR62 complexed with TPX2/Aurora A can potently promote katanin-mediated severing of GDP-MTs in vitro. In addition, the MT-binding affinity of WDR62 is autoinhibited through JNK phosphorylation-induced intramolecular interaction. We propose that WDR62 is an atypical MAP and functions as an adaptor protein between its recruiting factor TPX2/Aurora A and the effector katanin to orchestrate the regulation of spindle dynamics.

Microtubule-severing protein (MTSP) is critical for the survival of both mitotic and postmitotic cells. However, the study of MTSP during meiosis of mammalian oocytes has not been reported. We found that spastin, a member of the MTSP family, was highly expressed in oocytes and aggregated in spindle microtubules. After knocking down spastin by specific siRNA, the spindle microtubule density of meiotic oocytes decreased significantly. When the oocytes were cultured in vitro, the oocytes lacking spastin showed an obvious maturation disorder. Considering the microtubule-severing activity of spastin, we speculate that spastin on spindles may increase the number of microtubule broken ends by severing the microtubules, therefore playing a nucleating role, promoting spindle assembly and ensuring normal meiosis. In addition, we found the colocalization and interaction of collapsin response mediator protein 5 (CRMP5) and spastin in oocytes. CRMP5 can provide structural support and promote microtubule aggregation, creating transportation routes, and can interact with spastin in the microtubule activity of nerve cells (30). Knocking down CRMP5 may lead to spindle abnormalities and developmental disorders in oocytes. Overexpression of spastin may reverse the abnormal phenotype caused by the deletion of CRMP5. In summary, our data support a model in which the interaction between spastin and CRMP5 promotes the assembly of spindle microtubules in oocytes by controlling microtubule dynamics, therefore ensuring normal meiosis.

During spermatogenesis, the Golgi apparatus serves important roles including the formation of the acrosome, which is a sperm-specific organelle essential for fertilization. We have previously demonstrated that D. melanogaster ATP-dependent Citrate Lyase (ATPCL) is required for spindle organization, cytokinesis, and fusome assembly during male meiosis, mainly due to is activity on fatty acid biosynthesis. Here, we show that depletion of DmATPCL also affects the organization of acrosome and suggest a role for this enzyme in the assembly of Golgi-derived structures during Drosophila spermatogenesis.

Perfluorooctane sulfonate (PFOS) is a widely used artificial surfactant with potential toxicity to humans and animals. However, little is known about the impact of PFOS on the female germ cell development. Here, we report that PFOS exposure weakens oocyte quality by disturbing oocyte meiotic competency and fertilization ability. Specifically, PFOS exposure impaired cytoskeleton assembly including spindle organization and actin polymerization to cause the oocyte maturation arrest. In addition, PFOS exposure also impaired the mitochondrial dynamics and function, resulting in the increased levels of reactive oxygen species (ROS) and DNA damage as well as generation of apoptosis. Lastly, PFOS exposure compromised the distribution of cortical granules (CGs) and their component ovastacin, leading to the failure of sperm binding and fertilization. Altogether, our study illustrates that oxidative stress-induced apoptosis is a major cause for the deteriorated quality of porcine oocytes exposed to PFOS.

Jie Jiang,1 Bin Liu,2 Ruilin Liu,3 Wenzhuo Yang1 1Department of Gastroenterology and Hepatology, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of China; 2Department of Gastroenterology, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, People’s Republic of China; 3Department of Pulmonary, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of ChinaCorrespondence: Ruilin LiuDepartment of Pulmonary, Shanghai Tongji Hospital, Tongji University School of Medicine, 389 Xincun Road, Shanghai, 200065, People’s Republic of ChinaEmail 18721881628@163.comWenzhuo YangDepartment of Gastroenterology and Hepatology, Shanghai Tongji Hospital, Tongji University School of Medicine, 389 Xincun Road, Shanghai, 200065, People’s Republic of ChinaEmail yangwenzhuo2002@163.comBackground: Taspase 1 (TASP1) is a highly conserved protease involved in site-specific proteolysis. Existing researches have revealed a link between TASP1 expression and carcinogenesis. However, limited data are available regarding the prognosis and functions of TASP1 in hepatocellular carcinoma (HCC).Methods: Western Blotting and qRT-PCR were employed to evaluate the level of TASP1 in HCC cell lines and clinical specimens. TASP1 expression was further calculated in clinical specimens by immunohistochemistry and the mRNA level of TASP1 in HCC was analyzed using Oncomine and UALCAN databases. The TASP1 promoter methylation modification was shown via MEXPRESS and UALCAN. The association between TASP1 expression and postoperative prognosis was evaluated using Kaplan–Meier and Cox regression analysis in clinical patients. The effect of TASP1 on HCC prognosis was analyzed via Kaplan-Meier plotter, GEPIA and UALCAN. Additionally, the regulators, kinases, miRNA and transcription factor targets of TASP1 were identified using LinkedOmics. Moreover, cBioPortal was used to detect the genetic alteration of TASP1. Finally, TIMER was utilized to assess the relation between TASP1 and the immune cell infiltration, whereas the correlation of TASP1 with three immune factors was detected through TISIDB.Results: TASP1 expression was increased in HCC cell lines and HCC tissues. CNV and DNA methylation of TASP1 were changed. Survival analysis revealed that high TASP1 expression was correlated with overall survival (OS). Functional network analysis about TASP1 in HCC showed that the double-strand break repair, peptidyl-threonine modification, spindle organization, peptidyl-lysine modification and microtubule-based movement were modulated. Furthermore, TASP1 expression revealed puissant relation to the infiltration of immune cells and three immune factors in HCC.Conclusion: These data indicate that TASP1 may act as a potential prognostic marker in HCC and regulate HCC via multiple mechanisms.Keywords: hepatocellular carcinoma, TASP1, prognosis, biological functional networks

BackgroundExercise has a positive impact on patients with osteosarcoma, improving function, reducing disability, maintaining independence and quality of life. Exercise may also directly affect the effectiveness of cancer treatment. Cell division cycle-associated protein 4 (CDCA4) is reported to function importantly during numerous human cancers development. Nevertheless, the details toward CDCA4 function are still to be investigated.MethodsThis study comprehensively analyzed the GSE74194 database and obtained aerobic exercise-related genes. Protein-protein interaction network (PPI) and Gene Ontology (GO) analysis were performed on the differentially expressed genes (DEGs). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and tumor genome atlas (TCGA) data mining were applied to measure aerobic exercise-related gene CDCA4 level in osteosarcoma tissue. We conducted lots of functional experiments to uncover CDCA4 function and its corresponding mechanism in osteosarcoma.ResultsWe screened a total of 547 DEGs related to aerobic exercise, of which 373 were up-regulated and 174 were down-regulated. PPI analysis revealed 90 genes that might play key roles. GO analysis showed that aerobic exercise-related DEGs were significantly enriched during the mitotic cell cycle, cell division, mitotic nuclear division and sister chromatid segregation, nuclear division, microtubule cytoskeleton organization involved protein, microtubule-based process, spindle organization, G2/M transition of mitotic cell cycle. Our results indicated that CDCA4 was increased in osteosarcoma tissues and cell lines, and its level had association with high mortality of osteosarcoma patients. Further studies revealed that absence of CDCA4 largely hindered osteosarcoma cancer cell proliferation, invasion, and migration.ConclusionComprehensive bioinformatics analysis improves our understanding of the underlying molecular mechanisms of aerobic exercise on osteosarcoma. This provides evidence for the effect of aerobic exercise on CDCA4 expression. Our data suggested that CDCA4 could facilitate osteosarcoma development, and gave a hint that CDCA4 was a candidate target in the treatment of osteosarcoma, aerobic exercise might help the treatment and prognosis of patients with osteosarcoma.

Cellular organelles such as the mitotic spindle adjust their size to the dimensions of the cell. It is widely understood that spindle scaling is governed by regulation of microtubule polymerization. Here, we use quantitative microscopy in living zebrafish embryos and Xenopus egg extracts in combination with theory to show that microtubule polymerization dynamics are insufficient to scale spindles and only contribute below a critical cell size. In contrast, microtubule nucleation governs spindle scaling for all cell sizes. We show that this hierarchical regulation arises from the partitioning of a nucleation inhibitor to the cell membrane. Our results reveal that cells differentially regulate microtubule number and length using distinct geometric cues to maintain a functional spindle architecture over a large range of cell sizes.

International audience; The spindle is crucial for cell division by allowing the faithful segregation of replicated chromosomes to daughter cells. Proper segregation is ensured only if microtubules and hundreds of other associated factors interact to assemble this complex structure with the appropriate architecture and size. In this review, we describe the latest view of spindle organization as well as the molecular gradients and mechanisms underlying microtubule nucleation and spindle assembly. We then discuss the overlapping physical and molecular constraints that dictate spindle morphology, concluding with a focus on spindle size regulation.

Kornakov et al. investigate mechanisms of bipolar spindle assembly, a critical prerequisite for accurate chromosome segregation in mitosis. They define the molecular organization of an interaction interface between the conserved microtubule plus-end tracking protein EB1 and the minus-end motor Kinesin-14 and demonstrate with specific mutations that this complex is required for proper organization of the metaphase spindle., Kinesin-14s are conserved molecular motors required for high-fidelity chromosome segregation, but their specific contributions to spindle function have not been fully defined. Here, we show that key functions of budding yeast Kinesin-14 Cik1-Kar3 are accomplished in a complex with Bim1 (yeast EB1). Genetic complementation of mitotic phenotypes identifies a novel KLTF peptide motif in the Cik1 N-terminus. We show that this motif is one element of a tripartite binding interface required to form a high-affinity Bim1–Cik1-Kar3 complex. Lack of Bim1-binding by Cik1-Kar3 delays cells in mitosis and impairs microtubule bundle organization and dynamics. Conversely, constitutive targeting of Cik1-Kar3 to microtubule plus ends induces the formation of nuclear microtubule bundles. Cells lacking the Bim1–Cik1-Kar3 complex rely on the conserved microtubule bundler Ase1/PRC1 for metaphase spindle organization, and simultaneous loss of plus-end targeted Kar3 and Ase1 is lethal. Our results reveal the contributions of an EB1–Kinesin-14 complex for spindle formation as a prerequisite for efficient kinetochore clustering and bi-orientation.

Background: Microcystin-leucine arginine (MC-LR) is the most toxic cyanotoxin found in water bodies. Microcystins are produced as secondary products of cyanobacteria metabolism. They have a stable structure, and can bioaccumulate in living organisms. Humans and livestock who drink fresh water containing MC-LR can be poisoned. However, few studies have reported the effects of MC-LR exposure on livestock or human reproduction.Results: We used porcine oocytes as the model to explore the effects of MC-LR on oocyte maturation, and studied the impact of vitamin C (VC) administration on MC-LR‐induced meiosis defects. Exposure to MC-LR significantly restricted cumulus cell expansion and decreased first polar body extrusion. Further studies showed that MC-LR exposure led to meiosis arrest by disturbing cytoskeleton dynamics with MC-LR exposed oocytes displaying aberrant spindle organization, low levels of acetylate α‐tubulin, and disturbed actin polymerization. Additionally, MC-LR exposure impaired cytoplasmic maturation by disturbing mitochondria distribution. Moreover, MC-LR also produced abnormal epigenetic modifications, and induced high levels of oxidative stress and DNA damage. The administration of VC provided partial protection from all of the defects observed in oocytes exposed to MC-LR. Conclusions: These results demonstrate that MC-LR has a toxic effect on oocyte meiosis through the generation of excessive ROS levels and DNA damage. Supplementation of VC was able to protect against MC-LR-induced oocyte damage and represents a potential therapeutic strategy to improve the quality of MC-LR-exposed oocytes.

The early embryonic development is important for the subsequent embryo implantation, and any defects in this process can lead to embryonic aneuploidy, which causes miscarriage and birth defects. Survivin is the member of inhibitor of apoptosis protein (IAP) family, and it is also an essential subunit of chromosomal passenger complex (CPC), which regulates both apoptosis and cell cycle control in many models. However, the roles of survivin in mouse early embryos remain unclear. In the present study, we showed that survivin activity was essential for mouse early embryo development. Our results showed that survivin mainly accumulated at chromosomes at metaphase stage and located at the spindle midzone at anaphase and telophase stages during the first cleavage. Loss of survivin activity led to the failure of cleavage in early mouse embryos. Further analysis indicated that survivin involved into spindle organization and chromosome alignment. Moreover, inhibition of survivin induced oxidative stress and DNA damage, showing with the increase of ROS level, the positive γH2A signal, and the increase of Rad51 level. We also observed the occurrence of autophagy and apoptosis in the survivin-inhibited embryos. In summary, our study suggested that survivin was a critical regulator for early embryo development through its regulation on spindle organization, chromosome alignment, and DNA damage.

Alternative splicing (AS) contributes to gene diversification in cells, but the importance of AS 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. Germline-specific knockout of Esrp1 perturbed spindle organization, chromosome alignment, and metaphase-to-anaphase transformation in oocytes. The first polar body extrusion (PBE) was blocked during oocyte meiosis and was found to be due to abnormal activation of spindle assembly checkpoint (SAC) and insufficiency of anaphase-promoting complex/cyclosome (APC/C) in Esrp1-knockout oocytes. Esrp1-knockout in oocytes hampered follicular development and ovulation; eventually, premature ovarian failure (POF) occurred in six-month-old Esrp1-knockout mouse. Using single-cell RNA sequencing analysis, 528 aberrant AS events of maternal mRNA transcripts were revealed and were preferentially associated with microtubule cytoskeletal organization in Esrp1-knockout oocytes. 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 ultimately caused abnormal spindle organization. Taken together, our findings provide the first report elucidating the AS program of maternal mRNA transcripts, mediated by the splicing factor, ESRP1, that is required for oocyte meiosis and female fertility in mice.

At the onset of mitosis, centrosomes expand the pericentriolar material (PCM) to maximize their microtubule-organizing activity. This step, termed centrosome maturation, ensures proper spindle organization and faithful chromosome segregation. However, as the centrosome expands, how PCM proteins are recruited and held together without membrane enclosure remains elusive. We found that endogenously expressed pericentrin (PCNT), a conserved PCM scaffold protein, condenses into dynamic granules during late G2/early mitosis before incorporating into mitotic centrosomes. Furthermore, the N-terminal portion of PCNT, enriched with conserved coiled-coils (CCs) and low-complexity regions (LCRs), phase separates into dynamic condensates that selectively recruit PCM proteins and nucleate microtubules in cells. We propose that CCs and LCRs, two prevalent sequence features in the centrosomal proteome, are preserved under evolutionary pressure in part to mediate liquid-liquid phase separation, a process that bestows upon the centrosome distinct properties critical for its assembly and functions., Summary: In situ-tagged PCNT condenses into dynamic granules, and its coiled-coil-rich regions phase separate into centrosome-like condensates in human cells, shedding new light on centrosome assembly.

NuMA is an abundant long-coiled-coil protein that plays a prominent role in spindle organization during mitosis. In interphase, NuMA is localized to the nucleus and hypothesized to control gene expression and chromatin organization. However, because of the prominent mitotic phenotype upon NuMA loss, its precise function in the interphase nucleus remains elusive. Here, we report that NuMA is associated with chromatin in interphase and prophase but released upon nuclear envelope breakdown (NEBD) by the action of Cdk1. We uncovered that NuMA directly interacts with DNA via evolutionarily conserved sequences in its C-terminus. Notably, the expression of the DNA-binding mutant of NuMA affects chromatin decondensation at the mitotic exit, and nuclear shape in interphase. The impact on nuclear shape by mutant NuMA expression is due to its potential to polymerize into high-order fibrillar structures. This study links the chromatin binding ability of NuMA with the maintenance of nuclear shape and architecture, which has a well-studied role in regulating gene expression during development and diseases.

Induction of repeated superovulation with exogenous hormones is widely used in assisted reproductive technology (ART). Though it is generally safe, emerging evidence has indicated that repeated superovulation may compromise oocyte quality. However, few studies have explored how to ameliorate such impairment. Because melatonin has beneficial influences on oocytes in various detrimental environments, we aimed to explore whether melatonin could protect mouse oocytes after repeated superovulation. We found that repeated superovulation markedly reduced meiotic maturation and disrupted spindle organization and chromosome alignment. Furthermore, we observed reduced mitochondrial content and enhanced early apoptosis in oocytes from mice subjected to repeated superovulation. In addition, 5-methylcytosine (5mc) fluorescence intensity was lower in oocytes from experimental mice than in those from control mice, indicating that repeated superovulation disrupts genomic DNA methylation, and elevations in reactive oxygen species levels indicated that repeated superovulation also induces oxidative stress. Conversely, melatonin administration improved oocyte maturation and attenuated the observed defects. Interestingly, supplementation with melatonin during in vitro maturation had the same protective effects on oocytes as in vivo melatonin administration. In summary, our results show that melatonin can improve oocyte quality after repeated superovulation and thus provide a potential strategy to improve ART efficiency.

Enhancer of zeste homolog 2 (EZH2) has been extensively investigated to participate in diverse biological processes, including carcinogenesis, the cell cycle, X-chromosome inactivation, and early embryonic development. However, the functions of this protein during mammalian oocyte meiotic maturation remain largely unexplored. Here, combined with RNA-Seq, we provided evidence that EZH2 is essential for oocyte meiotic maturation in pigs. First, EZH2 protein expression increased with oocyte progression from GV to MII stage. Second, the siRNA-mediated depletion of EZH2 led to accelerated GVBD and early occurrence of the first polar body extrusion. Third, EZH2 knockdown resulted in defective spindle assembly, abnormal SAC activity, and unstable K-MT attachment, which was concomitant with the increased rate of aneuploidy. Finally, EZH2 silencing exacerbated oxidative stress by increasing ROS levels and disrupting the distribution of active mitochondria in porcine oocytes. Furthermore, parthenogenetic embryonic development was impaired following the depletion of EZH2 at GV stage. Taken together, we concluded that EZH2 is necessary for porcine oocyte meiotic progression through regulating spindle organization, maintaining chromosomal integrity, and mitochondrial function.

Protein kinase D (PKD) subfamily which includes PKD1, PKD2 and PKD3 is a novel family of serine/threonine kinases. PKD has been widely implicated in the regulation of multiple physiological effects including immune responses, apoptosis and cell proliferation. However, the roles of PKD in oocytes have not been fully clarified. In this study we investigated the regulatory functions of PKD during porcine oocyte maturation. Our results indicated that PKD expressed in porcine oocytes and the inhibition of PKD family activity led to the failure of meiosis resumption and the first polar body extrusion. Further analysis indicated that the spindle assembly and chromosome alignment were disrupted after PKD family inhibition, and this might be through its regulatory role on MAPK phosphorylation. We also found that PKD phosphorylated cofilin for actin assembly, which further affected cortical actin distribution, indicating the roles of PKD family on cytoskeleton. In addition, a decreased expression of PKD in postovulatory aging porcine oocytes was observed, which might connect PKD with cytoskeleton defects in aged oocytes. Taken together, these results suggest that PKD possesses important functions in porcine oocyte maturation by regulating spindle organization and actin assembly.

The citrus flavonoid hesperetin has a variety of pharmacological actions, including antioxidant, antiinflammatory, and anticancer activities. This study investigated whether hesperetin prevents aging of oocytes in vitro in which it determined the maturation of nuclear and cytoplasm and the developmental capacity of embryo by modulating the reactive oxygen species (ROS) level. Porcine oocytes were matured in vitro for 44 hr (control) and for an additional 24 hr in the presence of 0, 1, 10, 100, and 250 μM hesperetin (aging, H-1, H-10, H-100, and H-250, respectively). Although there was no difference in the rate of maturation among all the groups, both the control and H-100 groups significantly increased in the rate of cleavage and blastocyst formation compared to the aging group. The H-100 group significantly decreased ROS activity and increases the level of glutathione (GSH) and expression of the antioxidant genes (PRDX5, NFE2L, SOD1, and SOD2) compared with the aging group. The H-100 groups prevented aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase and increased the messenger RNA expression of cytoplasmic maturation factor genes (GDF9, CCNB1, BMP15, and MOS). Subsequently, both the control and H-100 groups significantly increased the total cell number and decreased the apoptosis cells at the blastocyst stage compared with aging group. The results indicate that hesperetin improves the quality of porcine oocytes by protecting them against oxidative stress during aging in vitro.

Oocyte meiosis is a complex process coordinated by multiple endocrinal and molecular circuits. Recently, N6-methyladenosine (m6A) epigenetic modification on RNA is revealed to be important for meiotic maturation. However, the molecular mechanism of how m6A modification exerts its effect on oocyte maturation is largely unknown. Here, we showed that endogenous m6A writers (Mettl3 and Wtap) and eraser (Fto) elevated their transcript levels during meiotic maturation of pig oocytes. From germinal vesicle (GV) to metaphase II (MII) stages, global m6A level significantly increased, and existed mostly in ooplasm. Methyl donor (betaine, 16 mM) treatment of porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) significantly boosted nucleic acid m6A level within oocytes, but unchanged meiotic process and oocyte subsequent development. By contrast, methylation inhibitor (cycloleucine, 20 mM) reduced nucleic acid m6A level, and significantly decreased the germinal vesicle breakdown (GVBD) rate, the extrusion rate of the first polar body, and the cleavage and blastocyst rates of parthenotes. In addition, in cycloleucine-treated oocytes Wtap increased but Lin28 decreased their abundances significantly, along with the higher incidence of spindle defects and chromosome misalignment. Furthermore, pT161-CDK1 protein level in pig oocytes was confirmed to be decreased after cycloleucine treatment for 24 h. Taken together, chemical induced reduction of nucleic acid m6A methylation during pig oocyte meiosis could impair meiotic maturation and subsequent development potency, possibly through down-regulating pluripotency marker Lin28 mRNA abundance and disturbing MPF-regulated chromosome/spindle organization.