Kinesin KIF3A regulates meiotic progression and spindle assembly in oocyte meiosis.

Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule movement. In this study, we characterized the critical roles of KIF3A during mouse oocyte meiosis. We found that KIF3A associated with microtubules during meiosis and depletion of KIF3A resulted in oocyte maturation defects. LC-MS data indicated that KIF3A associated with cell cycle regulation, cytoskeleton, mitochondrial function and intracellular transport-related molecules. Depletion of KIF3A activated the spindle assembly checkpoint, leading to metaphase I arrest of the first meiosis. In addition, KIF3A depletion caused aberrant spindle pole organization based on its association with KIFC1 to regulate expression and polar localization of NuMA and γ-tubulin; and KIF3A knockdown also reduced microtubule stability due to the altered microtubule deacetylation by histone deacetylase 6 (HDAC6). Exogenous Kif3a mRNA supplementation rescued the maturation defects caused by KIF3A depletion. Moreover, KIF3A was also essential for the distribution and function of mitochondria, Golgi apparatus and endoplasmic reticulum in oocytes. Conditional knockout of epithelial splicing regulatory protein 1 (ESRP1) disrupted the expression and localization of KIF3A in oocytes. Overall, our results suggest that KIF3A regulates cell cycle progression, spindle assembly and organelle distribution during mouse oocyte meiosis.

Kinesin KIFC3 is essential for microtubule stability and cytokinesis in oocyte meiosis.

KIFC3 is a member of Kinesin-14 family motor proteins, which play a variety of roles such as centrosome cohesion, cytokinesis, vesicles transportation and cell proliferation in mitosis. Here, we investigated the functional roles of KIFC3 in meiosis. Our findings demonstrated that KIFC3 exhibited expression and localization at centromeres during metaphase I, followed by translocation to the midbody at telophase I throughout mouse oocyte meiosis. Disruption of KIFC3 activity resulted in defective polar body extrusion. We observed aberrant meiotic spindles and misaligned chromosomes, accompanied by the loss of kinetochore-microtubule attachment, which might be due to the failed recruitment of BubR1/Bub3. Coimmunoprecipitation data revealed that KIFC3 plays a crucial role in maintaining the acetylated tubulin level mediated by Sirt2, thereby influencing microtubule stability. Additionally, our findings demonstrated an interaction between KIFC3 and PRC1 in regulating midbody formation during telophase I, which is involved in cytokinesis regulation. Collectively, these results underscore the essential contribution of KIFC3 to spindle assembly and cytokinesis during mouse oocyte meiosis.

Arf1 GTPase Regulates Golgi-Dependent G2/M Transition and Spindle Organization in Oocyte Meiosis.

ADP-ribosylation factor 1 (Arf1) is a small GTPase belonging to the Arf family. As a molecular switch, Arf1 is found to regulate retrograde and intra-Golgi transport, plasma membrane signaling, and organelle function during mitosis. This study aimed to explore the noncanonical roles of Arf1 in cell cycle regulation and cytoskeleton dynamics in meiosis with a mouse oocyte model. Arf1 accumulated in microtubules during oocyte meiosis, and the depletion of Arf1 led to the failure of polar body extrusion. Unlike mitosis, it finds that Arf1 affected Myt1 activity for cyclin B1/CDK1-based G2/M transition, which disturbed oocyte meiotic resumption. Besides, Arf1 modulated GM130 for the dynamic changes in the Golgi apparatus and Rab35-based vesicle transport during meiosis. Moreover, Arf1 is associated with Ran GTPase for TPX2 expression, further regulating the Aurora A-polo-like kinase 1 pathway for meiotic spindle assembly and microtubule stability in oocytes. Further, exogenous Arf1 mRNA supplementation can significantly rescue these defects. In conclusion, results reported the noncanonical functions of Arf1 in G2/M transition and meiotic spindle organization in mouse oocytes.

Mcrs1 regulates G2/M transition and spindle assembly during mouse oocyte meiosis.

Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.

Exposure to acrylamide induces zygotic genome activation defects of mouse embryos.

Acrylamide (ACR) is an important chemical raw material for wastewater treatment, paper industry and textile industry, which is widely exposed from occupational, environmental and dietary situation. ACR has neurotoxicity, genotoxicity, potential carcinogenicity and reproductive toxicity. Recent study indicates that ACR affected oocyte maturation quality. In the present study, we reported the effects of ACR exposure on zygotic genome activation (ZGA) in embryos and its related mechanism. Our results showed that ACR treatment caused 2-cell arrest in mouse embryos, indicating the failure of ZGA, which was confirmed by decreased global transcription levels and aberrant expression of ZGA-related and maternal factors. We found that histone modifications such as H3K9me3, H3K27me3 and H3K27ac levels were altered, and this might be due to the occurrence of DNA damage, showing with positive γ-H2A.X signal. Moreover, mitochondrial dysfunction and high levels of ROS were detected in ACR treated embryos, indicating that ACR induced oxidative stress, and this might further cause abnormal distribution of endoplasmic reticulum, Golgi apparatus and lysosomes. In conclusion, our results indicated that ACR exposure disrupted ZGA by inducing mitochondria-based oxidative stress, which further caused DNA damage, aberrant histone modifications and organelles in mouse embryos.

Kinesin KIF15 regulates tubulin acetylation and spindle assembly checkpoint in mouse oocyte meiosis.

Microtubule dynamics ensure multiple cellular events during oocyte meiosis, which is critical for the fertilization and early embryo development. KIF15 (also termed Hklp2) is a member of kinesin-12 family motor proteins, which participates in Eg5-related bipolar spindle formation in mitosis. In present study, we explored the roles of KIF15 in mouse oocyte meiosis. KIF15 expressed during oocyte maturation and localized with microtubules. Depletion or inhibition of KIF15 disturbed meiotic cell cycle progression, and the oocytes which extruded the first polar body showed a high aneuploidy rate. Further analysis showed that disruption of KIF15 did not affect spindle morphology but resulted in chromosome misalignment. This might be due to the reduced stability of the K-fibers, which further induced the loss of kinetochore-microtubule attachment and activated spindle assembly checkpoint, showing with the failed release of Bub3 and BubR1. Based on mass spectroscopy analysis and coimmunoprecipitation data we showed that KIF15 was responsible for recruiting HDAC6, NAT10 and SIRT2 to maintain the acetylated tubulin level, which further affected tubulin acetylation for microtubule stability. Taken together, these results suggested that KIF15 was essential for the microtubule acetylation and cell cycle control during mouse oocyte meiosis.

High-dose zearalenone exposure disturbs G2/M transition during mouse oocyte maturation.

Zearalenone is a mycotoxin produced by fungi of the genus Fusarium, which has severe toxicity on animal and human health including reproduction. Previous study showed that zearalenone exposure inhibited oocyte polar body extrusion, while in present study we found that high dose zearalenone disturbed oocyte meiosis resumption. Our results showed that a high concentration of 100 µM zearalenone reduced the rate of germinal vesicle (GV) breakdown in mouse oocytes. Further analysis indicated that zearalenone caused the decrease of Cyclin B1 and CDK1 expression, indicating MPF activity was affected, which further induced G2/M arrest, and this could be rescued by the inhibition of Wee1 activity. We found that the oocytes under high concentration of zearalenone showed lower γ-H2A.X expression, suggesting that DNA damage repair was disturbed, which further activated of DNA damage checkpoints. This could be confirmed by the altered expression of CHK1 and CHK2 after zearalenone treatment. Moreover, the organelles such as mitochondria, ribosome, endoplasmic reticulum and Golgi apparatus were diffused from germinal vesicle periphery after zearalenone exposure, indicating that zearalenone affected protein synthesis, modification and transport, which further induced the arrest of G2/M transition. Taken together, our results showed that high dose of zearalenone exposure induced G2/M transition defect by affecting organelle function-related CHK1/2-Wee1-MPF pathway.

Kinesin motor KIFC1 is required for tubulin acetylation and actin-dependent spindle migration in mouse oocyte meiosis.

Mammalian oocyte maturation is a unique asymmetric division, which is mainly because of actin-based spindle migration to the cortex. In the present study, we report that a kinesin motor KIFC1, which is associated with microtubules for the maintenance of spindle poles in mitosis, is also involved in actin dynamics in murine oocyte meiosis, co-localizing with microtubules during mouse oocyte maturation. Depletion of KIFC1 caused the failure of polar body extrusion, and we found that meiotic spindle formation and chromosome alignment were disrupted. This might be because of the effects of KIFC1 on HDAC6 and NAT10-based tubulin acetylation, which further affected microtubule stability. Mass spectroscopy analysis revealed that KIFC1 also associated with several actin nucleation factors and we found that KIFC1 was essential for the distribution of actin filaments, which further affected spindle migration. Depletion of KIFC1 leaded to aberrant expression of formin 2 and the ARP2/3 complex, and endoplasmic reticulum distribution was also disturbed. Exogenous KIFC1 mRNA supplement could rescue these defects. Taken together, as well as its roles in tubulin acetylation, our study reported a previously undescribed role of kinesin KIFC1 on the regulation of actin dynamics for spindle migration in mouse oocytes.

LRRK2 regulates actin assembly for spindle migration and mitochondrial function in mouse oocyte meiosis.

Leucine-rich-repeat kinase 2 (LRRK2) belongs to the Roco GTPase family and is a large multidomain protein harboring both GTPase and kinase activities. LRRK2 plays indispensable roles in many processes, such as autophagy and vesicle trafficking in mitosis. In this study, we showed the critical roles of LRRK2 in mammalian oocyte meiosis. LRRK2 is mainly accumulated at the meiotic spindle periphery during oocyte maturation. Depleting LRRK2 led to the polar body extrusion defects and also induced large polar bodies in mouse oocytes. Mass spectrometry analysis and co-immunoprecipitation results showed that LRRK2 was associated with several actin-regulating factors, such as Fascin and Rho-kinase (ROCK), and depletion of LRRK2 affected the expression of ROCK, phosphorylated cofilin, and Fascin. Further analysis showed that LRRK2 depletion did not affect spindle organization but caused the failure of spindle migration, which was largely due to the decrease of cytoplasmic actin filaments. Moreover, LRRK2 showed a similar localization pattern to mitochondria, and LRRK2 was associated with several mitochondria-related proteins. Indeed, mitochondrial distribution and function were both disrupted in LRRK2-depleted oocytes. In summary, our results indicated the critical roles of LRRK2 in actin assembly for spindle migration and mitochondrial function in mouse oocyte meiosis.

Fumonisin B1 exposure deteriorates oocyte quality by inducing organelle dysfunction and DNA damage in mice.

Oocyte quality is critical for fertilization and early embryo development. Fumonisin B1 (FB1) is a Fusarium mycotoxin and it is commonly found in contaminated food and feedstuff, posing a potential health hazard to both animals and human. FB1 is reported to have hepatotoxicity, neurotoxicity, nephrotoxicity, immunotoxicity and embryotoxicity. However, the effects of FB1 on mouse oocyte quality are still unknown. Here, we explored the toxic effects and potential mechanisms of FB1 on oocyte maturation quality in mice. FB1 exposure inhibited the first polar body extrusion at concentrations of 30 µM and 50 µM, which further induced oocyte meiotic arrest. Besides, disrupted spindle structure was found in oocytes after FB1 exposure. Our results also showed that FB1 exposure impaired mitochondria dysfunction, which further induced oxidative stress and early apoptosis. In addition, we reported that FB1 exposure induced the accumulation of lysosome and occurrence of autophagy. Aberrant ER distribution and ER stress were also found in FB1-exposed oocytes. Moreover, DNA damage was also observed. These results together suggested that FB1 exposure affected oocyte quality by destroying spindle structure, leading to mitochondria, lysosome and ER dysfunction, which further induced oxidative stress, apoptosis, autophagy and DNA damage in mouse oocytes.

Exposure to citrinin induces DNA damage, autophagy, and mitochondria dysfunction during first cleavage of mouse embryos.

Citrinin (CTN) is a mycotoxin, which is isolated from Penicillium citrinum and widely existed in the contaminated feeds. It is reported that CTN is toxic to heart, liver, and reproductive system. Previous studies indicated that CTN induced apoptosis in oocytes and embryos. In this study, we reported the potential causes of CTN on embryo development. Our results showed that 40 µM CTN exposure significantly reduced the first cleavage of mouse embryos, showing with the low rate of 2-cell embryos. We found that CTN induced DNA damage, showing the higher positive γH2A.X signals. Autophagy was occurred since more LC3 positive autophagosomes were found in the cytoplasm. This could be confirmed by the enhanced lysosome function, since higher accumulated lysosome distribution were found and LAMP2 was also increased under CTN exposure. Besides, we showed that mitochondria distribution was disturbed, indicating that CTN could disrupt mitochondria function, which could be the possible reason for the oxidative stress and apoptosis in CTN-exposed embryos. In conclusion, our study showed that CTN exposure had adverse effects on the early embryo development during first cleavage through its effects on the induction of DNA damage, autophagy, and mitochondria dysfunction.

RAB14 GTPase is essential for actin-based asymmetric division during mouse oocyte maturation.

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.

Increased Environment-Related Metabolism and Genetic Expression in the In Vitro Matured Mouse Oocytes by Transcriptome Analysis.

Infertility in humans at their reproductive age is a world-wide problem. Oocyte in vitro maturation (IVM) is generally used in such cases to acquire the embryo in assisted reproductive technology (ART). However, the differences between an in vivo (IVO) and IVM culture environment in the RNA expression profile in oocytes, remains unclear. In this study, we compared the global RNA transcription pattern of oocytes from in vitro and in vivo maturation. Our results showed that 1,864 genes differentially expressed between the IVO and IVM oocytes. Among these, 1,638 genes were up-regulated, and 226 genes were down-regulated, and these changes were mainly divided into environmental adaption, metabolism, and genetic expression. Our detailed analysis showed that the expression of genes that belonged to metabolism-related processes such as energy metabolism, nucleotide metabolism, and carbohydrate metabolism was changed; and these genes also belonged to organismal systems including environmental adaptation and the circulatory system; moreover, we also found that the relative gene expression of genetic expression processes, such as protein synthesis, modification, and DNA replication and repair were also altered. In conclusion, our data suggests that in vitro maturation of mouse oocyte resulted in metabolism and genetic expression changes due to environmental changes compared with in vivo matured oocytes.

Single-cell transcriptome analysis reveals that maternal obesity affects DNA repair, histone methylation, and autophagy level in mouse embryos.

Obesity causes many reproductive dysfunctions such as reduced conception, infertility, and early pregnancy loss, and this is largely due to the negative effects of obesity on oocyte and embryo quality. In the present study, we employed single-cell RNA transcriptome sequencing to investigate the potential causes for the maternal obesity effects on mouse embryos. Our results showed that the 4-cell and morula/blastocyst rates were all significantly decreased during embryo development in obese mice. Genome-wide analysis indicated that obesity altered the expression of more than 1100 genes in 2-cell embryos, including the genes which were related to the p53 signaling pathway and apoptosis. Further analysis showed that the expression of 47 genes related to DNA damage was changed, and a positive γH2A signal and the altered expression of Rad51 and Tex15 were observed in the obese embryos. Obesity also affected histone methylation, shown by the decrease of the H3K4-me2 level. Besides this, we observed the occurrence of autophagy and apoptosis in the embryos of obese mice. There were 42 genes that were related to autophagy/apoptosis that showed aberrant expression, and the positive LC3 signal and the decrease of Clec16a, Rraga, and Atg10 level were also observed. In summary, our study suggested that obesity affected early embryonic development by inducing DNA damage, aberrant histone methylation, and autophagy levels in mice.

Modified hydrated sodium calcium aluminosilicate-supplemented diet protects porcine oocyte quality from zearalenone toxicity.

Zearalenone (ZEN) is one of the most common mycotoxins produced by fungus in contaminated feed. ZEN has multiple toxicities, including reproductive toxicity of domestic animals, particularly pigs. However, studies on the effects of ZEN on ovary/oocytes have been primarily based on in vitro experiments, and there is still no evidence from porcine in vivo models due to multiple limitations. Moreover, no report has investigated the effect of hydrated sodium calcium aluminosilicate (HSCAS) as a supplement on pig oocyte quality. In the present study, we fed pigs a 1.0 mg/kg ZEN-contaminated diet for 10 days. The results showed that pigs fed ZEN presented reduced oocyte-cumulus cell interactions, an increase in the number of denuded oocytes in ovaries, a decrease in the number of oocytes in each ovary, and an increase in the oocyte death rate. Oocytes from ZEN-exposed pigs exhibited a delayed cell cycle and abnormal cytoskeletal dynamics during meiotic maturation, which could be due to oxidative stress-induced autophagy. Moreover, we also show that supplementing the ZEN-contaminated diet with modified HSCAS effectively protected porcine oocyte quality. Taken together, our study provides in vivo data demonstrating the protective effects of HSCAS against ZEN toxicity in porcine oocytes.

Mps1 controls spindle assembly, SAC, and DNA repair in the first cleavage of mouse early embryos.

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.

PRC1 is a critical regulator for anaphase spindle midzone assembly and cytokinesis in mouse oocyte meiosis.

Protein regulator of cytokinesis 1 (PRC1) is a microtubule bundling protein that is involved in the regulation of the central spindle bundle and spindle orientation during mitosis. However, the functions of PRC1 during meiosis have rarely been studied. In this study, we explored the roles of PRC1 during meiosis using an oocyte model. Our results found that PRC1 was expressed at all stages of mouse oocyte meiosis, and PRC1 accumulated in the midzone/midbody during anaphase/telophase I. Moreover, depleting PRC1 caused defects in polar body extrusion during mouse oocyte maturation. Further analysis found that PRC1 knockdown did not affect meiotic spindle formation or chromosome segregation; however, deleting PRC1 prevented formation of the midzone and midbody at the anaphase/telophase stage of meiosis I, which caused cytokinesis defects and further induced the formation of two spindles in the oocytes. PRC1 knockdown increased the level of tubulin acetylation, indicating that microtubule stability was affected. Furthermore, KIF4A and PRC1 showed similar localization in the midzone/midbody of oocytes at anaphase/telophase I, while the depletion of KIF4A affected the expression and localization of PRC1. The PRC1 mRNA injection rescued the defects caused by PRC1 knockdown in oocytes. In summary, our results suggest that PRC1 is critical for midzone/midbody formation and cytokinesis under regulation of KIF4A in mouse oocytes.

CHK1 monitors spindle assembly checkpoint and DNA damage repair during the first cleavage of mouse early embryos.

OBJECTIVES: DNA damage and errors of accurate chromosome segregation lead to aneuploidy and foetal defects. DNA repair and the spindle assembly checkpoint (SAC) are the mechanisms developed to protect from these defects. Checkpoint kinase 1 (CHK1) is reported to be an important DNA damage response protein in multiple models, but its functions remain unclear in early mouse embryos. MATERIALS AND METHODS: Immunofluorescence staining, immunoblotting and real-time reverse transcription polymerase chain reaction were used to perform the analyses. Reactive oxygen species levels and Annexin-V were also detected. RESULTS: Loss of CHK1 activity accelerated progress of the cell cycle at the first cleavage; however, it disturbed the development of early embryos to the morula/blastocyst stages. Further analysis indicated that CHK1 participated in spindle assembly and chromosome alignment, possibly due to its regulation of kinetochore-microtubule attachment and recruitment of BubR1 and p-Aurora B to the kinetochores, indicating its role in SAC activity. Loss of CHK1 activity led to embryonic DNA damage and oxidative stress, which further induced early apoptosis and autophagy, indicating that CHK1 is responsible for interphase DNA damage repair. CONCLUSIONS: Our results indicate that CHK1 is a key regulator of the SAC and DNA damage repair during early embryonic development in mice.

Nonylphenol exposure affects mouse oocyte quality by inducing spindle defects and mitochondria dysfunction.

Nonylphenol (NP) is a chemical raw material and intermediate which is mainly used in the production of surfactants, lubricating oil additives and pesticide emulsifiers. NP is reported to be toxic on the immune system, nervous system and reproductive system due to its binding to estrogen receptors. However, the toxicity of NP on mammalian oocyte quality remains unclear. In present study, we explored the effects of NP exposure on mouse oocyte maturation. Our results showed that 4 weeks of NP exposure increased the number of atresia follicles and decreased oocyte developmental competence. Transcriptomic analysis indicated that NP exposure altered the expression of more than 800 genes in oocytes, including multiple biological pathways. Subcellular structure examination indicated that NP exposure disrupted meiotic spindle organization and caused chromosome misalignment. Moreover, aberrant mitochondrial distribution and decreased membrane potential were also observed, indicating that NP exposure caused mitochondria dysfunction. Further analysis showed that NP exposure resulted in the accumulation of reactive oxygen species (ROS), which causes oxidative stress; and the NP-exposed oocytes showed positive Annexin-V signal, indicating the occurrence of early apoptosis. In summary, our results indicated that NP exposure reduced oocyte quality by affecting cytoskeletal dynamics and mitochondrial function, which further induced oxidative stress and apoptosis in mice.