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.

Effects of Regulating Hippo and Wnt on the Development and Fate Differentiation of Bovine Embryo.

The improvement of in vitro embryo development is a gateway to enhance the output of assisted reproductive technologies. The Wnt and Hippo signaling pathways are crucial for the early development of bovine embryos. This study investigated the development of bovine embryos under the influence of a Hippo signaling agonist (LPA) and a Wnt signaling inhibitor (DKK1). In this current study, embryos produced in vitro were cultured in media supplemented with LPA and DKK1. We comprehensively analyzed the impact of LPA and DKK1 on various developmental parameters of the bovine embryo, such as blastocyst formation, differential cell counts, YAP fluorescence intensity and apoptosis rate. Furthermore, single-cell RNA sequencing (scRNA-seq) was employed to elucidate the in vitro embryonic development. Our results revealed that LPA and DKK1 improved the blastocyst developmental potential, total cells, trophectoderm (TE) cells and YAP fluorescence intensity and decreased the apoptosis rate of bovine embryos. A total of 1203 genes exhibited differential expression between the control and LPA/DKK1-treated (LD) groups, with 577 genes upregulated and 626 genes downregulated. KEGG pathway analysis revealed significant enrichment of differentially expressed genes (DEGs) associated with TGF-beta signaling, Wnt signaling, apoptosis, Hippo signaling and other critical developmental pathways. Our study shows the role of LPA and DKK1 in embryonic differentiation and embryo establishment of pregnancy. These findings should be helpful for further unraveling the precise contributions of the Hippo and Wnt pathways in bovine trophoblast formation, thus advancing our comprehension of early bovine embryo development.

Loss of AMPK activity induces organelle dysfunction and oxidative stress during oocyte aging.

BACKGROUND: Oocyte quality is critical for the mammalian reproduction due to its necessity on fertilization and early development. During aging, the declined oocytes showing with organelle dysfunction and oxidative stress lead to infertility. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase which is important for energy homeostasis for metabolism. Little is known about the potential relationship between AMPK with oocyte aging. RESULTS: In present study we reported that AMPK was related with low quality of oocytes under post ovulatory aging and the potential mechanism. We showed the altered AMPK level during aging and inhibition of AMPK activity induced mouse oocyte maturation defect. Further analysis indicated that similar with its upstream regulator PKD1, AMPK could reduce ROS level to avoid oxidative stress in oocytes, and this might be due to its regulation on mitochondria function, since loss of AMPK activity induced abnormal distribution, reduced ATP production and mtDNA copy number of mitochondria. Besides, we also found that the ER and Golgi apparatus distribution was aberrant after AMPK inhibition, and enhanced lysosome function was also observed. CONCLUSIONS: Taken together, these data indicated that AMPK is important for the organelle function to reduce oxidative stress during oocyte meiotic maturation.

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.

NAMPT regulates mitochondria function and lipid metabolism during porcine oocyte maturation.

Oocyte maturation defect can lead to maternal reproduction disorder. NAMPT is a rate-limiting enzyme in mammalian NAD+ biosynthesis pathway, which can regulate a variety of cellular metabolic processes including glucose metabolism and DNA damage repair. However, the function of NAMPT in porcine oocytes remains unknown. In this study, we showed that NAMPT involved into multiple cellular events during oocyte maturation. NAMPT expressed during all stages of porcine oocyte meiosis, and inhibition of NAMPT activity caused the cumulus expansion and polar body extrusion defects. Mitochondrial dysfunction was observed in NAMPT-deficient porcine oocytes, which showed decreased membrane potential, ATP and mitochondrial DNA content, increased oxidative stress level and apoptosis. We also found that NAMPT was essential for spindle organization and chromosome arrangement based on Ac-tubulin. Moreover, lack of NAMPT activity caused the increase of lipid droplet and affected the imbalance of lipogenesis and lipolysis. In conclusion, our study indicated that lack of NAMPT activity affected porcine oocyte maturation through its effects on mitochondria function, spindle assembly and lipid metabolism.

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.

SIRT3 Regulates Levels of Deacetylated SOD2 to Prevent Oxidative Stress and Mitochondrial Dysfunction During Oocyte Maturation in Pigs.

Mammalian oocyte maturation relies on mitochondrial ATP production, but this can lead to damaging reactive oxygen species (ROS). SIRT3, a mitochondrial sirtuin, plays a critical role in regulating mitochondrial redox balance in mouse oocytes under stress; however, its specific roles in porcine oocytes remain unclear. In this study, we utilized the SIRT3 inhibitor 3-TYP to investigate SIRT3's importance in porcine oocyte maturation. Our findings revealed that SIRT3 is expressed in porcine oocytes and its inhibition leads to maturation failure. This was evident through reduced polar body extrusion, arrested cell cycle, as well as disrupted spindle organization and actin distribution. Furthermore, SIRT3 inhibition resulted in a decrease in mitochondrial DNA copy numbers, disruption of mitochondrial membrane potential, and reduced ATP levels, all indicating impaired mitochondrial function in porcine oocytes. Additionally, the primary source of damaged mitochondria was associated with decreased levels of deacetylated superoxide dismutase 2 (SOD2) after SIRT3 inhibition, which led to ROS accumulation and oxidative stress-induced apoptosis. Taken together, our results suggest that SIRT3 regulates the levels of deacetylated SOD2 to maintain redox balance and preserve mitochondrial function during porcine oocyte maturation, with potential implications for improving pig reproduction.

Toxicity of the Mycotoxin Deoxynivalenol on Early Cleavage of Mouse Embryos by Fluorescence Intensity Analysis.

Deoxynivalenol is a mycotoxin, produced by Fusarium from contaminated corn, wheat, and other grains, that induces multiple effects in humans and animals, including cytotoxic, genotoxic, immunotoxic, and carcinogenic effects. Recent studies show that deoxynivalenol also affects the reproductive system of mammals, including oocyte quality. However, the effects of deoxynivalenol on early embryonic development have not been reported. In this study, fluorescence intensity analysis was used to show that deoxynivalenol disrupted the first cleavage of the zygote. The high deoxynivalenol dose disturbed the movement of the pronucleus after fertilization, while the low deoxynivalenol dose caused aberrant spindle morphology during the metaphase of the first cleavage. Further analysis showed that the reactive oxygen species level increased in the deoxynivalenol-exposed two-cell embryos, indicating oxidative stress. Moreover, deoxynivalenol caused DNA damage in the embryos, as positive γH2A.X signals were detected in the nucleus. These events led to the early apoptosis of mouse embryos, which was confirmed by autophagy. Taken together, our study provides evidence for the toxicity of deoxynivalenol during early embryonic development in the mouse model.

Nivalenol disrupts mitochondria functions during porcine oocyte meiotic maturation.

Oocyte maturation is important for fertility in mammals, since the quality of oocytes directly affects fertilization, embryo attachment and survival. Nivalenol is widely present in nature as a common toxin that contaminates grain and feed, and it has been reported to cause acute toxicity, immunotoxicity, reproductive toxicity and carcinogenic effects. In this study, we explored the impact of nivalenol on the porcine oocyte maturation and its possible mechanisms. The extrusion of the first polar body was significantly inhibited after incubating oocytes with nivalenol. Meanwhile, nivalenol exposure led to the abnormal distribution of mitochondria, aberrant calcium concentration and the reduction of membrane potential caused a significant decrease in the capacity of mitochondria to generate ATP. In addition, nivalenol induced oxidative stress, and the level of ROS was significantly increased in the nivalenol-treated group, which was confirmed by the perturbation of oxidative stress-related genes. We found that nivalenol-treated oocytes showed positive Annexin-V and γH2A.X signals, indicating the occurrence of apoptosis and DNA damage. In all, our data suggest that nivalenol disrupted porcine oocyte maturation through its effects on mitochondria-related oxidative stress, apoptosis and DNA damage.

Aflatoxin B1 impairs porcine oocyte quality via disturbing intracellular membrane system and ATP production.

Aflatoxin is the most common type of mycotoxins in contaminated corn, peanuts and rice, which affects the livestock and ultimately endangers human health. Aflatoxin is reported to have carcinogenicity, mutation, growth retardation, immunosuppression and reproductive toxicity. In present study we reported the causes for the declined porcine oocyte quality under aflatoxin exposure. We set up an in vitro exposure model and showed that aflatoxin B1 disturbed cumulus cell expansion and oocyte polar body extrusion. We found that aflatoxin B1 exposure disrupted ER distribution and elevated the expression of GRP78, indicating the occurrence of ER stress, and the increased calcium storage also confirmed this. Besides, the structure of cis-Golgi apparatus, another intracellular membrane system was also affected, showing with decreased GM130 expression. The oocytes under aflatoxin B1 exposure showed aberrant lysosome accumulation and higher LAMP2 expression, a marker for lysosome membrane protection, and this might be due to the aberrant mitochondria function with low ATP production and the increase of apoptosis, since we found that BAX expression increased, and ribosomal protein which is also an apoptosis-related factor RPS3 decreased. Taken together, our study revealed that aflatoxin B1 impairs intracellular membrane system ER, Golgi apparatus, lysosome and mitochondria function to affect porcine oocyte maturation quality.

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.

Age-related SUMOylation of PLK1 is essential to meiosis progression in mouse oocytes.

Polo like kinase 1 (PLK1) is a protein kinase involved in regulating the spindle assembly and cell cycle control in mammalian oocytes. SUMOylation, one way of post-translational modification, regulates oocyte meiosis by controlling several substrates. However, the relation between PLK1 and SUMOylation in oocytes is still unknown. In this study, we investigated that whether PLK1 was modified by SUMOylation in oocytes and its potential relationship with age-related meiotic abnormalities. We showed that PLK1 had colocalization and protein interaction with Small Ubiquitin-Like Modifier (SUMO)-1 and SUMO-2/3 in mouse oocytes, indicating that PLK1 could be modified by SUMO-1 and SUMO-2/3. Overexpression of PLK1 SUMOylation site mutants PLK1K178R and PLK1K191R caused the increase of the abnormal spindle rate of oocytes and the decline of the first polar body extrusion rate with the abnormal localization of PLK1, suggesting that the SUMOylation modification of PLK1 is essential for normal meiosis in oocytes. Compared with young mice, the expression of PLK1 protein increased and the expression of SUMO-1 and SUMO-2/3 protein decreased in the oocytes of aged mice, indicating that the SUMOylation of PLK1 might be related to the mouse aging. Therefore, our data suggested that PLK1 could be SUMOylated by SUMO-1 and SUMO-2/3 in mouse oocytes and SUMOylation of PLK1 regulated the meiosis progression of oocytes which was related with aging.

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.

Nivalenol affects Cyclin B1 level and activates SAC for cell cycle progression in mouse oocyte meiosis.

OBJECTIVES: Nivalenol (NIV) is a secondary metabolite of type B trichothecene mycotoxin produced by Fusarium genera, which is widely found in contaminated food and crops such as corn, wheat and peanuts. NIV is reported to have hepatotoxicity, immunotoxicity, genotoxicity, and reproductive toxicity. Previous studies indicate that NIV disturbs mammalian oocyte maturation. Here, we reported that delayed cell cycle progression might be the reason for oocyte maturation defect caused by NIV exposure. METHODS AND RESULTS: We set up a NIV exposure model and showed that NIV did not affect G2/M transition for meiosis resumption, but disrupted the polar body extrusion of oocytes. Further analysis revealed that oocytes were arrested at metaphase I, which might be due to the lower expression of Cyclin B1 after NIV exposure. After cold treatment, the microtubules were disassembled in the NIV-exposed oocytes, indicating that NIV disrupted microtubule stability. Moreover, NIV affected the attachment between kinetochore and microtubules, which further induced the activation of MAD2/BUBR1 at the kinetochores, suggesting that spindle assemble checkpoint (SAC) was continuously activated during oocyte meiotic maturation. CONCLUSIONS: Taken together, our study demonstrated that exposure to NIV affected Cyclin B1 expression and activated microtubule stability-dependent SAC to ultimately disturb cell cycle progression in mouse oocyte meiosis.

Aflatoxin B1 exposure disrupts organelle distribution in mouse oocytes.

Aflatoxin B1 (AFB1) is a secondary metabolite produced by the fungus Aspergillus, which is ubiquitous in moldy grain products. Aflatoxin B1 has been reported to possess hepatotoxicity, renal toxicity, and reproductive toxicity. Previous studies have shown that AFB1 is toxic to mammalian oocytes. However, the potential toxicity of AFB1 on the organelles of mouse oocytes is unknown. In this study, we found that exposure to AFB1 significantly reduced mouse oocyte development capacity. Further analysis showed that the endoplasmic reticulum (ER) failed to accumulate around the spindle, and scattered in the cytoplasm under AFB1 exposure. Similar to the ER, the Golgi apparatus showed a uniform localization pattern following AFB1 treatment. In addition, we found that AFB1 exposure caused the condensation of lysosomes in the cytoplasm, presenting as a clustered or spindle peripheral-localization pattern, which indicated that protein modification, transport, and degradation were affected. Mitochondrial distribution was also altered by AFB1 treatment. In summary, our study showed that AFB1 exposure had toxic effects on the distribution of mouse oocyte organelles, which further led to a decline in oocyte quality.

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.

Nonylphenol exposure-induced oocyte quality deterioration could be reversed by melatonin supplementation in mice.

Nonylphenol (NP) belongs to the metabolites of commercial detergents, which acts as an environmental endocrine disruptor. NP is reported to have multiple toxicity including reproductive toxicity. In present study, we reported the protective effects of melatonin on the NP-exposed oocyte quality. We set up a mouse in vivo model of NP exposure (500 µg/L), by daily drinking and continued feeding for 4 weeks; and we gave a daily dose of melatonin (30 mg/kg) to the NP-exposed mice. Melatonin supplementation restores the development ability of oocytes exposed to NP, and this was due to the reduction of ROS level and DNA damage by melatonin. Melatonin could rescue aberrant mitochondria distribution, mitochondria membrane potential, which also was reflected by ATP content and mtDNA copy number. Moreover, melatonin could restore the RPS3 expression to ensure the ribosome function for protein synthesis, and reduced GRP78 protein level to protect against ER stress and ER distribution defects. We also found that vesicle protein Rab11 from Golgi apparatus was protected by melatonin at the spindle periphery of oocytes of NP-exposed mice, which further moderated LAMP2 for lysosome function. Our results indicate that melatonin protects oocytes from NP exposure through its effects on the reduction of oxidative stress and DNA damage, which might be through its amelioration on the organelles in mice.

Acrylamide Exposure Destroys the Distribution and Functions of Organelles in Mouse Oocytes.

Acrylamide (ACR) is a common industrial ingredient which is also found in foods that are cooked at high temperatures. ACR has been shown to have multiple toxicities including reproductive toxicity. Previous studies reported that ACR caused oocyte maturation defects through the induction of apoptosis and oxidative stress. In the present study, we showed that ACR exposure affected oocyte organelle functions, which might be the reason for oocyte toxicity. We found that exposure to 5 mM ACR reduced oocyte maturation. ACR caused abnormal mitochondrial distribution away from spindle periphery and reduced mitochondrial membrane potential. Further analysis showed that ACR exposure reduced the fluorescence intensity of Rps3 and abnormal distribution of the endoplasmic reticulum, indicating that ACR affected protein synthesis and modification in mouse oocytes. We found the negative effects of ACR on the distribution of the Golgi apparatus; in addition, fluorescence intensity of vesicle transporter Rab8A decreased, suggesting the decrease in protein transport capacity of oocytes. Furthermore, the simultaneous increase in lysosomes and LAMP2 fluorescence intensity was also observed, suggesting that ACR affected protein degradation in oocytes. In conclusion, our results indicated that ACR exposure disrupted the distribution and functions of organelles, which further affected oocyte developmental competence in mice.