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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.
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Cinesinas , Tubulina (Proteína) , beta Carioferinas/metabolismo , Acetilação , Actinas/metabolismo , Animais , Cinesinas/genética , Mamíferos/metabolismo , Meiose , Camundongos , Oócitos/metabolismo , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
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.
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Cinesinas , Oócitos , Animais , Camundongos , Transporte Biológico , Cinesinas/genética , Meiose , MetáfaseRESUMO
Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon compound that is generated during combustion processes, and is present in various substances such as foods, tobacco smoke, and burning emissions. BaP is extensively acknowledged as a highly carcinogenic substance to induce multiple forms of cancer, such as lung cancer, skin cancer, and stomach cancer. Recently it is shown to adversely affect the reproductive system. Nevertheless, the potential toxicity of BaP on oocyte quality remains unclear. In this study, we established a BaP exposure model via mouse oral gavage and found that BaP exposure resulted in a notable decrease in the ovarian weight, number of GV oocytes in ovarian, and oocyte maturation competence. BaP exposure caused ribosomal dysfunction, characterized by a decrease in the expression of RPS3 and HPG in oocytes. BaP exposure also caused abnormal distribution of the endoplasmic reticulum (ER) and induced ER stress, as indicated by increased expression of GRP78. Besides, the Golgi apparatus exhibited an abnormal localization pattern, which was confirmed by the GM130 localization. Disruption of vesicle transport processes was observed by the abnormal expression and localization of Rab10. Additionally, an enhanced lysosome and LC3 fluorescence intensity indicated the occurrence of protein degradation in oocytes. In summary, our results suggested that BaP exposure disrupted the distribution and functioning of organelles, consequently affecting the developmental competence of mouse oocytes.
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Benzo(a)pireno , Chaperona BiP do Retículo Endoplasmático , Oócitos , Animais , Benzo(a)pireno/toxicidade , Oócitos/efeitos dos fármacos , Feminino , Camundongos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Organelas/efeitos dos fármacos , Camundongos Endogâmicos ICRRESUMO
1,3,6-Trigalloylglucose is a natural compound that can be extracted from the aqueous extracts of ripe fruit of Terminalia chebula Retz, commonly known as "Haritaki". The potential anti-Helicobacter pylori (HP) activity of this compound has not been extensively studied or confirmed in scientific research. This compound was isolated using a semi-preparative liquid chromatography (LC) system and identified through Ultra-high-performance liquid chromatography-MS/MS (UPLC-MS/MS) and Nuclear Magnetic Resonance (NMR). Its role was evaluated using Minimum inhibitory concentration (MIC) assay and minimum bactericidal concentration (MBC) assay, scanning electron microscope (SEM), inhibiting kinetics curves, urea fast test, Cell Counting Kit-8 (CCK-8) assay, Western blot, and Griess Reagent System. Results showed that this compound effectively inhibits the growth of HP strain ATCC 700392, damages the HP structure, and suppresses the Cytotoxin-associated gene A (Cag A) protein, a crucial factor in HP infection. Importantly, it exhibits selective antimicrobial activity without impacting normal epithelial cells GES-1. In vitro studies have revealed that 1,3,6-Trigalloylglucose acts as an anti-adhesive agent, disrupting the adhesion of HP to host cells, a critical step in HP infection. These findings underscore the potential of 1,3,6-Trigalloylglucose as a targeted therapeutic agent against HP infections.
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Helicobacter pylori , Terminalia , Extratos Vegetais/química , Terminalia/química , Cromatografia Líquida , Espectrometria de Massas em Tandem , ÁguaRESUMO
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.
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Cinesinas , Tubulina (Proteína) , Acetilação , Animais , Cinesinas/genética , Pontos de Checagem da Fase M do Ciclo Celular , Meiose , Camundongos , Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
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.
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Proteínas de Ciclo Celular , Meiose , Oócitos , Proteínas Serina-Treonina Quinases , Sumoilação , Animais , Camundongos , Proteínas de Ciclo Celular/metabolismo , Oócitos/metabolismo , Fuso Acromático/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores Etários , Quinase 1 Polo-LikeRESUMO
GBF1 [Golgi brefeldin A (BFA) resistance factor 1] is a member of the guanine nucleotide exchange factors Arf family. GBF1 localizes at the cis-Golgi and endoplasmic reticulum (ER)-Golgi intermediate compartment where it participates in ER-Golgi traffic by assisting in the recruitment of the coat protein COPI. However, the roles of GBF1 in oocyte meiotic maturation are still unknown. In the present study, we investigated the regulatory functions of GBF1 in mouse oocyte organelle dynamics. In our results, GBF1 was stably expressed during oocyte maturation, and GBF1 localized at the spindle periphery during metaphase I. Inhibiting GBF1 activity led to aberrant accumulation of the Golgi apparatus around the spindle. This may be due to the effects of GBF1 on the localization of GM130, as GBF1 co-localized with GM130 and inhibiting GBF1 induced condensation of GM130. Moreover, the loss of GBF1 activity affected the ER distribution and induced ER stress, as shown by increased GRP78 expression. Mitochondrial localization and functions were affected, as the mitochondrial membrane potential was altered. Taken together, these results suggest that GBF1 has wide-ranging effects on the distribution and functions of Golgi apparatus, ER, and mitochondria as well as normal polar body formation in mouse oocytes.
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Fatores de Ribosilação do ADP , Fatores de Troca do Nucleotídeo Guanina , Fatores de Ribosilação do ADP/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Oócitos/metabolismoRESUMO
During aging, oocytes display cytoskeleton dynamics defects and aneuploidy, leading to embryonic aneuploidy, which in turn causes miscarriages, implantation failures, and birth defects. KIF15 (also known as Hklp2), a member of the kinesin-12 superfamily, is a cytoplasmic motor protein reported to be involved in Golgi and vesicle-related transport during mitosis in somatic cells. However, the regulatory mechanisms of KIF15 during meiosis in porcine oocytes and the connection with postovulatory aging remain unclear. In present study, we found that KIF15 is expressed during porcine oocyte maturation, and its localization is dependent on microtubule dynamics. Furthermore, the level of KIF15 expression decreased in postovulatory aged oocytes. The decrease in KIF15 blocked polar body extrusion, thereby hindering oocyte maturation. We demonstrated that KIF15 defects contributed to abnormal spindle morphologies and chromosome misalignment, possibly due to microtubule instability, as evidenced by microtubule depolymerization after cold treatment. Additionally, our data indicated that KIF15 modulates HDAC6 to affect tubulin acetylation in oocytes. Taken together, these results suggest that KIF15 regulates HDAC6-related microtubule stability for spindle organization in porcine oocytes during meiosis, which may contribute to the decline in maturation competence in aged porcine oocytes.
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Desacetilase 6 de Histona , Cinesinas , Microtúbulos , Oócitos , Animais , Oócitos/fisiologia , Oócitos/metabolismo , Microtúbulos/metabolismo , Suínos , Cinesinas/genética , Cinesinas/metabolismo , Desacetilase 6 de Histona/metabolismo , Desacetilase 6 de Histona/genética , Senescência Celular , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterináriaRESUMO
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.
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Fator 1 de Ribosilação do ADP , Fuso Acromático , Camundongos , Animais , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Fuso Acromático/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Meiose , Oócitos/metabolismo , Complexo de Golgi/metabolismoRESUMO
BACKGROUND: The aqueous extract of the dried buds of Syzygium aromaticum (SAAE) have the potential to alleviate Helicobacter pylori infection, but the specific molecular mechanism has not been fully elucidated. PURPOSE: This study aimed to investigate the underlying mechanisms of SAAE on H. pylori pathogenicity. METHODS: The inhibitory kinetics and anti-H. pylori adhesive capacity assays were conducted to examine the effects of SAAE on the growth and adhesive capability of H. pylori. The H. pylori outer membrane vesicles (OMVs) were purified from the culture supernatant through high-speed centrifugation, filtration, and two rounds of ultracentrifugation. Their characteristics and protein composition were then identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and qualitative proteomics study. Subsequently, the effect of SAAE on the pathogenicity of H. pylori OMVs was investigated using the Griess reagent assay, enzyme-linked immunosorbent assay (ELISA), quantitative proteomics study, TEM, and western blotting assay. RESULTS: SAAE exhibited inhibitory effects on H. pylori growth and adhesion. The isolated H. pylori OMVs showed particle size of 27-242 nm and Zeta potential of -9.67 ± 0.53 mV. A total of 599 proteins were identified in the OMVs. Proteomics study indicated that the differential expressed proteins induced by OMVs with or without SAAE commonly enriched in P53 and autophagy pathways. Besides, SAAE counteracted the increased production of pro-inflammatory cytokines and attenuated the induction of cell autophagy caused by H. pylori OMVs. Furthermore, SAAE normalized the abnormal regulation of downstream targets (AIFM2 and IGFBP3) in the P53 signaling pathway caused by H. pylori OMVs. CONCLUSION: SAAE can inhibit the growth and adhesion of H. pylori, reduce the inflammation and autophagy induced by H. pylori OMVs, and combated the abnormal regulation of P53 signaling pathway caused by H. pylori OMVs. These findings may help elucidate the mechanisms through which SAAE reduces the pathogenicity of H. pylori.
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Helicobacter pylori , Extratos Vegetais , Syzygium , Helicobacter pylori/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Syzygium/química , Humanos , Aderência Bacteriana/efeitos dos fármacos , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/microbiologia , Proteômica , Proteína Supressora de Tumor p53/metabolismo , Antibacterianos/farmacologia , Autofagia/efeitos dos fármacosRESUMO
Background: Helicobacter pylori (H. pylori) is thought to primarily colonize the human stomach and lead to various gastrointestinal disorders, such as gastritis and gastric cancer. Currently, main eradication treatment is triple or quadruple therapy centered on antibiotics. Due to antibiotic resistance, the eradication rate of H. pylori is decreasing gradually. Therefore, searching for anti-H. pylori drugs from herbal sources has become a strategy for the treatment. Our team proposed a Hezi Qingyou Formula (HZQYF), composed of Chebulae Fructus, Ficus hirta Vahl and Cloves, and studied its anti-H. pylori activity and mechanism. Methods: Chemical components of HZQYF were studied using UHPLC-MS/MS and HPLC. Broth microdilution method and agar dilution method were used to evaluate HZQYF's antibacterial activity. The effects of HZQYF on expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF), and flagellar genes (flaA, flaB) were explored using Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) technology. Effects on morphology and permeability of the extracellular membrane were studied using scanning electron microscopy (SEM) and N-phenylnaphthalen-1-amine (NPN) uptake. Effect on urease activity was studied using a urease kinetics analysis in vitro. Immunofluorescence staining method was used to examine the effect on adhesion. Western blot was used to examine the effect on cagA protein. Results: Minimum inhibitory concentration (MIC) values of the formula against H. pylori clinical strains and standard strains were 80-160 µg/mL, and minimum bactericidal concentration (MBC) values were 160-320 µg/mL. The formula could down-regulate the expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF) and flagellar genes (flaA, flaB), change the morphology of H. pylori, increase its extracellular membrane permeability, and decrease its urease activity. Conclusion: Present studies confirmed that HZQYF had promising in vitro anti-H. pylori activities and demonstrated its possible mechanism of action by down-regulating the bacterial adhesion, urease, and flagellar gene expression, which provided scientific bases for further clinical investigations.
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ETHNOPHARMACOLOGICAL RELEVANCE: Phellodendron chinense C.K.Schneid(P. chinense Schneid) is known in TCM as Huang Bo, is traditionally used to support gastrointestinal function and alleviate stomach-related ailments, including gastric ulcer bleeding and symptoms of gastroesophageal reflux disease. Helicobacter pylori (H. pylori) is classified by the WHO as a Group 1 carcinogen. However, the specific activity and mechanism of action of P. chinense Schneid against H. pylori infection remain unclear. It has been noted that Huangjiu processing may alter the bitter and cold properties of P. chinense Schneid, but its effect on antimicrobial activity requires further investigation. Additionally, it remains uncertain whether berberine is the sole antimicrobial active component of P. chinense Schneid. AIM OF STUDY: This study aims to elucidate the anti-H. pylori infection activity of P. chinense Schneid, along with its mechanism of action and key antimicrobial active components. MATERIALS AND METHODS: Phytochemical analysis was carried out by UPLC-MS/MS. HPLC was employed to quantify the berberine content of the extracts. Antimicrobial activity was assessed using the micro broth dilution method. Morphology was observed using SEM. The impact on urease activity was analyzed through in vitro urease enzyme kinetics. RT-qPCR was employed to detect the expression of virulence genes, including adhesin, flagellum, urease, and cytotoxin-related genes. The adhesion effect was evaluated by immunofluorescence staining and agar culture. RESULTS: P. chinense Schneid exhibited strong antimicrobial activity against both antibiotic-sensitive and resistant H. pylori strains, with MIC ranging from 40 to 160 µg/mL. Combination with amoxicillin, metronidazole, levofloxacin, and clarithromycin did not result in antagonistic effects. P. chinense Schneid induced alterations in bacterial morphology and structure, downregulated the expression of various virulence genes, and inhibited urease enzyme activity. In co-infection systems, P. chinense Schneid significantly attenuated H. pylori adhesion and urease relative content, thereby mitigating cellular damage caused by infection. Huangjiu processing enhanced the anti-H. pylori activity of P. chinense Schneid. Besides berberine, P. chinense Schneid contained seven other components with anti-H. pylori activity, with palmatine exhibiting the strongest activity, followed by jatrorrhizine. CONCLUSIONS: This study sheds light on the potential therapeutic mechanisms of P. chinense Schneid against H. pylori infection, demonstrating its capacity to disrupt bacterial structure, inhibit urease activity, suppress virulence gene transcription, inhibit adhesion, and protect host cells. The anti-H. pylori activity of P. chinense Schneid was potentiated by Huangjiu processing, and additional components beyond berberine were identified as possessing strong anti-H. pylori activity. Notably, jatrorrhizine, a core component of P. chinense Schneid, exhibited significant anti-H. pylori activity, marking a groundbreaking discovery.
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Antibacterianos , Berberina , Helicobacter pylori , Phellodendron , Extratos Vegetais , Helicobacter pylori/efeitos dos fármacos , Phellodendron/química , Antibacterianos/farmacologia , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Berberina/farmacologia , Testes de Sensibilidade Microbiana , Urease/metabolismo , Humanos , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/microbiologiaRESUMO
Sanguisorba officinalis L., a traditional Chinese medicine (TCM) called DiYu (DY) in China, has a strong tradition of utilization as a scorching, blood-cooling, and hemostatic medication, and was used for cancer prevention and treatment due to its potential immune-enhancing and hematological toxicity-reducing effects. Previous studies have reported significant effects of DY on cancers including colorectal cancer (CRC), which is one of the most common malignancies worldwide. The first-line cure 5-fluorouracil (5-FU) plays decisive commerce in the sedative of CRC as a clinically available chemotherapeutic agent. One of the primary causes of cancer treatment failure is the acquisition of chemotherapy drug resistance. In order to successfully combat the emergence of chemoresistance, it is essential to identify herbs or traditional Chinese medicine that have adjuvant therapeutic effects on CRC. Therefore, this study aimed to determine whether DY could improve the sensitivity, conquer the chemoresistance of 5-FU-resistant CRC cells, and investigate its intrinsic mechanism. Materials and methods: MTT, Hoechst 33258 staining, and flow cytometry assays were used to determine the anticancer activity of DY alone or in combination with 5-FU against 5-FU-resistant CRC cells (RKO-R and HCT15-R) and wound healing assays were conducted to detect cell migration. Transcriptomic techniques were carried out to explore the effect and mechanism of DY on drug-resistant CRC cells. Western Blot and RT q-PCR assays were performed to validate the mechanism by which DY overcomes drug-resistant CRC cells. Results: These results indicated that DY alone or in combination with 5-FU significantly inhibited the proliferation and the migration of resistant CRC cells, and potentiated the susceptibility of 5-FU to drug-resistant CRC cells. GO and KEGG enrichment analysis showed that the mechanisms of drug resistance in CRC cells and DY against drug-resistant CRC cells highly overlapped, involved in the modulation of biological processes such as cell migration, positive regulation of protein binding and cytoskeleton, and MAPK (Ras-ERK-MEK), PI3K/Akt, and other signaling pathways. Moreover, DY can mediate the expression of p-R-Ras, p-ERK1/2, p-MEK1/2, p-PI3K, p-AKT, HIF-1A and VEGFA proteins. In addition, DY significantly suppressed the expression of AKT3, NEDD9, BMI-1, and CXCL1 genes in resistant CRC cells. Conclusion: In conclusion, DY could inhibit the proliferation and migration of 5-FU-resistant cells and strengthen the sensitivity of 5-FU to CRC-resistant cells. Furthermore, DY may prevail over chemoresistance through the Ras/MEK/ERK and PI3K/Akt pathways. These findings imply that DY may be a potential drug for clinical treatment or adjuvant treatment of drug-resistant CRC.
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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.
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Acrilamida , Zigoto , Camundongos , Animais , Acrilamida/metabolismo , Zigoto/metabolismo , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Dano ao DNARESUMO
BACKGROUND: Recently, defects in mammalian oocytes maturation induced by environmental pollution results in the decreasing animal reproduction. Animal exposed to glyphosate is largely unavoidable because glyphosate is one of the most widely used herbicide worldwide due to its high-efficiency and broad-spectrum effects, which causes glyphosate an environmental contaminant found in soil, water and food. During the last few years, the growing and wider use of glyphosate has raised great concerns about its effects of reproductive toxicity. In this study, using porcine models, we investigated effects of glyphosate on organelle functions during oocyte meiosis. RESULTS: The results showed glyphosate exposure disrupted porcine oocyte maturation. Expression levels of cumulus expansion-related genes were interfered, further indicating the meiotic defects. The damaging effects were mediated by destruction of mitochondrial distribution and functions, which induced ROS accumulation and oxidative stress, also indicated by the decreased mRNA expression of related antioxidant enzyme genes. We also found an interference of endoplasmic reticulum (ER) distribution, disturbance of Ca2+ homeostasis, as well as fluctuation of ER stress, showing with the reduced ER stress-related mRNA or protein expression, which could indicate the dysfunction of ER for protein processing and signal transduction in glyphosate-exposed oocytes. Moreover, glyphosate exposure induced the disruption of lysosome function for autophagy, showing with the decrease of LAMP2 expression and autophagy-related genes mRNA expression. Additionally, our data showed the distribution of Golgi apparatus and the functions of ribosome were disturbed after glyphosate exposure, which might affect protein synthesis and transport. CONCLUSIONS: Collectively, our study showed that exposed to glyphosate could affect animal reproduction by compromising the quality of oocytes through its wide toxic effects on organelle functions.
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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.
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Aflatoxina B1 , Oócitos , Camundongos , Animais , Aflatoxina B1/toxicidade , Oócitos/metabolismo , Oogênese , Retículo Endoplasmático , Lisossomos , MamíferosRESUMO
Background: Colorectal cancer (CRC) is the most common malignant cancer worldwide. Sanguisorba officinalis has been shown to have anti-inflammatory, anti-bacterial, antioxidant, and anti-tumor effects, while its molecular mechanism against CRC remains unclear. The aim of this study is to explore the underlying mechanism of S. officinalis against CRC cell lines using network pharmacology and transcriptomic sequencing methods. Method: Firstly, the active ingredients and potential targets of S. officinalis against CRC were screened from databases. Secondly, the networks of ingredient-target, ingredient-target-CRC and protein-protein interaction were constructed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of network pharmacology and transcriptomic sequencing were performed. Finally, the effect of S. officinalis against CRC was verified by in vitro experiments. Results: In total, 14 active ingredients and 273 potential targets against CRC were identified in S. officinalis by network pharmacology. PI3K-Akt, HIF-1, and MAPK signaling pathways related to cell proliferation were regulated by S. officinalis in enrichment analyses and transcriptomic sequencing. In vitro, S. officinalis inhibited the proliferation and migration of CRC cells and arrested the cell cycle at the G0-G1 phase. The western blot showed that S. officinalis downregulated the expression of p-PI3K, p-Akt, HIF-1A, VEGFA, cyclin D1, c-Myc, and p-MAPK proteins in CRC cells. Conclusion: In conclusion, network pharmacology and transcriptomic sequencing analyses, in combination with in vitro studies, have been successfully applied to study the underlying mechanism of S. officinalis against CRC cells. Our results demonstrate that S. officinalis suppresses the proliferation, survival, and migration of CRC cells through regulating the PI3K-Akt, HIF-1, and MAPK signaling pathways.
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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.