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After ovulation, senescent oocytes inevitably experience reduced quality and defects in embryonic development. Apigenin (API) is a flavonoid with a wide range of pharmacological effects. Therefore, this study examined the protective effects of API on the quality of porcine oocytes during in-vitro ageing and the underlying mechanisms. The results showed that API treatment could reduce the activation rate after aging for 48 h. In addition, API significantly reduced reactive oxygen species, abnormal distribution of mitochondria, early apoptosis in ageing oocytes, increased glutathione, and mitochondrial adenosine triphosphate levels in ageing oocytes. Importantly, API increased the embryonic development rate in aged oocytes. We also examined molecular changes, finding decreased sirtuin 1 expression in in-vitro postovulatory oocytes, but API reversed this effect. Our results suggest that API attenuates the deterioration of oocyte quality during in-vitro ageing, possibly by reducing oxidative stress through the upregulation of sirtuin 1.
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Apigenina , Sirtuína 1 , Feminino , Animais , Suínos , Sirtuína 1/genética , Sirtuína 1/metabolismo , Apigenina/farmacologia , Apigenina/metabolismo , Regulação para Cima , Senescência Celular/fisiologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Oócitos/fisiologiaRESUMO
Hepatic organoids might provide a golden opportunity for realizing precision medicine in various hepatic diseases. Previously described hepatic organoid protocols from pluripotent stem cells rely on complicated multiple differentiation steps consisting of both 2D and 3D differentiation procedures. Therefore, the spontaneous formation of hepatic organoids from 2D monolayer culture is associated with a low-throughput production, which might hinder the standardization of hepatic organoid production and hamper the translation of this technology to the clinical or industrial setting. Here we describe the stepwise and fully 3D production of hepatic organoids from human pluripotent stem cells. We optimized every differentiation step by screening for optimal concentrations and timing of differentiation signals in each differentiation step. Hepatic organoids are stably expandable without losing their hepatic functionality. Moreover, upon treatment of drugs with known hepatotoxicity, we found hepatic organoids are more sensitive to drug-induced hepatotoxicity compared with 2D hepatocytes differentiated from PSCs, making them highly suitable for in vitro toxicity screening of drug candidates. The standardized fully 3D protocol described in the current study for producing functional hepatic organoids might serve as a novel platform for the industrial and clinical translation of hepatic organoid technology.
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Doença Hepática Induzida por Substâncias e Drogas , Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Diferenciação Celular/genética , OrganoidesRESUMO
Brain organoids have been considered as an advanced platform for in vitro disease modeling and drug screening, but numerous roadblocks exist, such as lack of large-scale production technology and lengthy protocols with multiple manipulation steps, impeding the industrial translation of brain organoid technology. Here, we describe the high-speed and large-scale production of midbrain organoids using a high-throughput screening-compatible platform within 30 days. Micro midbrain organoids (µMOs) exhibit a highly uniform morphology and gene expression pattern with minimal variability. Notably, µMOs show dramatically accelerated maturation, resulting in the generation of functional µMOs within only 30 days of differentiation. Furthermore, individual µMOs display highly consistent responsiveness to neurotoxin, suggesting their usefulness as an in vitro high-throughput drug toxicity screening platform. Collectively, our data indicate that µMO technology could represent an advanced and robust platform for in vitro disease modeling and drug screening for human neuronal diseases.
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Imidacloprid (IMI) is an endogenous neonicotinoid insecticide widely used in agriculture and has attracted researchers' attention because of its risks to the environment and human health. Melatonin (MT) is an antioxidant hormone produced by the pineal gland of the brain. Studies have shown that it has a variety of physiological functions and plays a crucial role in the development of animal germ cells and embryos. The potential protective effects of MT against oocyte damage caused by neonicotinoid pesticide toxicity remain unclear. In this study, we report the toxicity of IMI against, and its effects on the quality of, porcine oocytes and the protective effect of MT on IMI-exposed oocytes. The results show that IMI exposure adversely affected oocyte maturation, while MT supplementation ameliorated its toxic effects. Specifically, IMI exposure increased oxidative stress (OS), endoplasmic reticulum stress (ERS), and apoptosis, which may affect polar body expulsion rates and blastocyst formation. Also, IMI exposure reduced oocyte cleavage rates and the number of cells in blastocysts. However, all of these toxic effects can be restored after a melatonin supplementation treatment. In conclusion, these results suggest that melatonin has a protective effect on IMI-induced defects during porcine oocyte maturation.
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Triacylglycerol (TGA) is the primary component of intramuscular fat. Expression of diacylglyceryl transferase (DGAT) determines the polyester differentiation ability of precursor adipocytes. The two DGAT isoforms (DGAT1 and DGAT2) play different roles in TAG metabolism. This study investigates the roles of DGAT1 and DGAT2 in signaling pathways related to differentiation and lipid metabolism in Yanbian bovine preadipocytes. sh-DGAT1 (sh-1), sh-DGAT2 (sh-2), and sh-DGAT1 + sh-DGAT2 (sh-1 + 2) were prepared using short interfering RNA (siRNA) interference technique targeting DGAT1 and DGAT2 genes and infected bovine preadipocytes. Molecular and transcriptomic techniques, including differentially expressed genes (DEGs) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis, were used to investigate the effects on the differentiation of Yanbian bovine preadipocytes. After interference with DGAT1 and DGAT2 genes, the contents of TAG and adiponectin were decreased. The TAG content in the sh-2 and sh-1 + 2 groups was significantly lower than that in the sh-NC group. RNA sequencing (RNA-seq) results showed 2070, 2242, and 2446 DEGs in the sh-1, sh-2, and sh-1 + 2 groups, respectively. The DEGs of the sh-2 group were mainly concentrated in the PPAR, AMPK, and Wnt signaling pathways associated with adipocyte proliferation and differentiation. These results demonstrated that at the mRNA level, DGAT2 plays a more important role in lipid metabolism than DGAT1.
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A Bi2WO6/BiVO4 composite photocatalytic material was synthesized by the hydrothermal method, and achieved the effective degradation of oxytetracycline (OTC) and tetracycline (TC) under visible light. The compositions, structures, chemical states and optoelectronic properties of Bi2WO6, BiVO4 and Bi2WO6/BiVO4 composites were characterized by systematic characterization. The results show that the existence of the heterojunction interface facilitates the separation of photogenerated carriers. Compared with the pure catalyst of Bi2WO6 and BiVO4, the Bi2WO6/BiVO4 composite material significantly improves the degradation efficiency of OTC and TC. The degradation rate is 6.22 and 3.02 times higher than that of Bi2WO6 and BiVO4, respectively. Through the free radical quenching experiments, it is known that photogenerated holes (h+) and superoxide anion free radicals (·O2 -) are the main active substances in the degradation of OTC. By analyzing the process of photocatalytic degradation of OTC, there are mainly six intermediates during the process. Their possible degradation pathways are also inferred in this paper.
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Here we described two human induced pluripotent stem cell (hiPSC) lines from peripheral blood mononuclear cells (PBMCs) of idiopathic autism spectrum disorder (ASD) patients through forced expression of OCT4, SOX2, KLF4, and c-MYC. The hiPSC lines displayed morphology, gene expression patterns, and pluripotential differentiation potentials similar to those of human embryonic stem cells (hESCs). The hiPSC lines from idiopathic ASD patients might be useful to unveil the underlying mechanism of idiopathic ASD and finding its therapeutics.
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Transtorno do Espectro Autista , Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Humanos , Fator 4 Semelhante a Kruppel , Leucócitos MononuclearesRESUMO
Carnosic acid (CA), a natural catechol rosin diterpene, is used as an additive in animal feeds and human foods. However, the effects of CA on mammalian reproductive processes, especially early embryonic development, are unclear. In this study, we added CA to parthenogenetically activated porcine embryos in an in vitro culture medium to explore the influence of CA on apoptosis, proliferation, blastocyst formation, reactive oxygen species (ROS) levels, glutathione (GSH) levels, mitochondrial membrane potential, and embryonic development-related gene expression. The results showed that supplementation with 10 µM CA during in vitro culture significantly improved the cleavage rates, blastocyst formation rates, hatching rates, and total numbers of cells of parthenogenetically activated porcine embryos compared with no supplementation. More importantly, supplementation with CA also improved GSH levels and mitochondrial membrane potential, reduced natural ROS levels in blastomeres, upregulated Nanog, Sox2, Gata4, Cox2, Itga5, and Rictor expression, and downregulated Birc5 and Caspase3 expression. These results suggest that CA can improve early porcine embryonic development by regulating oxidative stress. This study elucidates the effects of CA on early embryonic development and their potential mechanisms, and provides new applications for improving the quality of in vitro-developed embryos.
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Abietanos/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Espécies Reativas de Oxigênio , Animais , Apoptose , Blastocisto/citologia , Proliferação de Células , Meios de Cultura , Técnicas de Cultura Embrionária , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Potencial da Membrana Mitocondrial , Estresse Oxidativo , Partenogênese , Gravidez , Prenhez , SuínosRESUMO
Actin-interacting protein 1 (AIP1), also known as WD repeat-containing protein 1 (WDR1), is ubiquitous in eukaryotic organisms, and it plays critical roles in the dynamic reorganization of the actin cytoskeleton. However, the biological function and mechanism of AIP1 in mammalian oocyte maturation is still largely unclear. In this study, we demonstrated that AIP1 boosts ADF/Cofilin activity in mouse oocytes. AIP1 is primarily distributed around the spindle region during oocyte maturation, and its depletion impairs meiotic spindle migration and asymmetric division. The knockdown of AIP1 resulted in the gathering of a large number of actin-positive patches around the spindle region. This effect was reduced by human AIP1 (hAIP1) or Cofilin (S3A) expression. AIP1 knockdown also reduced the phosphorylation of Cofilin near the spindle, indicating that AIP1 interacts with ADF/Cofilin-decorated actin filaments and enhances filament disassembly. Moreover, the deletion of AIP1 disrupts Cofilin localization in metaphase I (MI) and induces cytokinesis defects in metaphase II (MII). Taken together, our results provide evidence that AIP1 promotes actin dynamics and cytokinesis via Cofilin in the gametes of female mice.
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Fatores de Despolimerização de Actina/metabolismo , Citocinese/fisiologia , Metáfase/fisiologia , Oócitos/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Actinas/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Fosforilação/fisiologia , Fuso Acromático/metabolismoRESUMO
Leonurine (LEO) is pseudoalkaloid that has been isolated from motherwort. It has been found to have various biological activities, including an antioxidant capacity. This study aimed to confirm whether LEO could be used in porcine in vitro culture (IVC) medium for its antioxidant effect and related molecular mechanisms. The results showed that embryos in IVC medium supplemented with 40 µM LEO had an increased blastocyst formation rate, total cell number, and proliferation capacity and a low apoptosis rate. LEO supplementation decreased reactive oxygen species levels and increased glutathione levels. Moreover, LEO-treated embryos exhibited improved intracellular mitochondrial membrane potential and reduced autophagy. In addition, pluripotency related gene was up-regulated while apoptosis and autophagy related genes were down-regulated with LEO supplementation. These results suggest that LEO has a beneficial effect on pre-implantation embryo development by reducing oxidative stress and enhancing mitochondrial function.
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Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Animais , Blastocisto , Técnicas de Cultura Embrionária/veterinária , Ácido Gálico/análogos & derivados , Mitocôndrias , Espécies Reativas de Oxigênio , SuínosRESUMO
Zearalenone (ZEA), which is produced by several fusarium mycotoxins, is found in animal feed and food products, and can exert estrogen-like activity. Melatonin (MT) is emerging as a supplement that can fight the toxic effects of mycotoxins. With a variety of physiological functions that play crucial roles in the development of animal germ cells and embryos, melatonin regulates circadian rhythms and has an anti-inflammatory and anti-oxidative role. This study investigated the protective effects of melatonin against ZEA in porcine early embryonic development. Our results showed that ZEA adversely affected this development, while melatonin supplementation ameliorated the toxic effects. ZEA exposure increased oxidative stress and impaired mitochondrial function, which may affect blastocyst formation. Moreover, we found that ZEA exposure promotes apoptosis, DNA damage, and autophagy in porcine blastocysts. The toxic effects of ZEA on early embryos may be the result of oxidative stress-mediated early apoptosis, while melatonin treatment significantly improved these phenotypes in ZEA-exposed porcine early embryos. Taken together, our results indicate that melatonin has a protective effect on defects caused by ZEA during early porcine embryonic development.
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Blastocisto/efeitos dos fármacos , Melatonina/farmacologia , Suínos/embriologia , Zearalenona/toxicidade , Animais , Apoptose/efeitos dos fármacos , Técnicas de Cultura Embrionária , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Partenogênese , Espécies Reativas de OxigênioRESUMO
Imperatorin (IMP) exhibits a variety of pharmacological properties, including antioxidant, anti-inflammatory, antibacterial, anti-cancer, and anti-hypertension activities. However, its effects on animal reproduction systems, especially oocyte development, maturation, and aging are not yet clear. In this study, the effects of IMP on oocyte development and aging as well as the underlying molecular mechanisms were explored. Oocytes were cultured for an additional 24 h for aging. Results revealed that the blastocyst formation and hatching rates of embryos, which were parthenogenetically activated aged oocytes, were significantly increased with IMP treatment (40 µM). Simultaneously, well-distributed cortical granules but no significant difference in zona pellucida hardness were observed after IMP treatment. During this stage, intracellular reactive oxygen species, apoptosis, and autophagy levels were decreased, while mitochondrial membrane potential, glutathione level, and activity of superoxide dismutase and catalase were increased. IMP-treated aged oocytes also showed significantly higher expression of MOS, CCNB1, BMP15, and GDF9 than non-IMP-treated aged oocytes although their levels were still lower than those in the fresh oocytes. These results suggest that IMP can effectively ameliorate the quality of aged porcine oocytes by reducing oxidative stress and protecting mitochondrial function.
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L-carnitine (LC) is well known for its antioxidant activity. In this study, we explored the potential mechanistic effects of LC supplementation on aged bovine oocytes in vitro. We showed that in-vitro maturation could enhance the subsequent developmental capacity of aging oocytes, when supplemented with LC. After in vitro fertilization, the blastocyst formation rate in the aged oocytes post-LC treatment significantly increased compared to that in untreated aged oocytes (29.23 ± 2.20% vs. 20.90 ± 3.05%). Furthermore, after LC treatment, the level of intracellular reactive oxygen species in aged oocytes significantly decreased, and glutathione levels significantly increased, compared to those in untreated aged oocytes. Mitochondrial membrane potential, the percentage of early apoptotic oocytes, and caspase-3 activity were significantly reduced in LC-treated aged oocytes compared to those in untreated aged oocytes. Furthermore, during in vitro aging, the mRNA levels of the anti-apoptotic genes, Bcl-xl and survivin in LC-treated aged oocytes were significantly higher than those in untreated aged oocytes. Overall, these results indicate that at least in in vitro conditions, LC can prevent the aging of bovine oocytes and improve the developmental capacity of bovine embryo.
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Bovinos , Senescência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Animais , Carnitina/farmacologia , Bovinos/embriologia , Bovinos/fisiologia , Células Cultivadas , Senescência Celular/genética , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oócitos/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Kaempferol (KAE) is a natural flavonoid present in different plant species and exhibits anti-inflammatory, antioxidant, and anticancer therapeutic properties. In the present study, we investigated the influence and underlying mechanisms of KAE supplementation on porcine oocytes during in vitro aging. The results show that KAE treatment can alleviate the aging-related reduction of developmental competence. We observed that the blastocyst production rate in aged oocytes treated with 0.1 µM KAE was significantly higher than in untreated aging oocytes (36.78 ± 0.86% vs. 27.55 ± 2.60%, respectively, p < .05). The KAE-treated aging oocytes had significantly reduced levels of reactive oxygen species (p < .05). Furthermore, the mRNA levels of the embryonic pluripotency-related genes Oct4, NANOG, and ITGA5 were significantly increased in blastocysts derived from KAE-treated oocytes (p < .05). During excessive oocyte culture, KAE treatment maintained the mitochondrial membrane potential and reduced apoptosis; however, this was not observed in untreated aging oocytes. In conclusion, our results suggest that KAE treatment can alleviate the aging of porcine oocytes by reducing oxidative stress and improving mitochondrial function.
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Senescência Celular , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Quempferóis/farmacologia , Oócitos/fisiologia , Suínos/embriologia , Animais , Blastocisto/metabolismo , Técnicas de Cultura Embrionária , Feminino , Integrinas/genética , Integrinas/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Measurements of the three-dimensional (3D) structure of spermatozoon are crucial for the study of developmental biology and for the evaluation of in vitro fertilization. Here, we present 3D label-free imaging of individual spermatozoon and perform quantitative analysis of bovine, porcine, and mouse spermatozoa morphologies using refractive index tomography. Various morphological and biophysical properties were determined, including the internal structure, volume, surface area, concentration, and dry matter mass of individual spermatozoon. Furthermore, Holstein cows and Korean native cattle spermatozoa were systematically analyzed and revealed significant differences in spermatozoa head length, head width, midpiece length, and tail length between the two breeds. This label-free imaging approach provides a new technique for understanding the physiology of spermatozoa.
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Imageamento Tridimensional , Espermatozoides/citologia , Animais , Bovinos , Masculino , Refratometria , Especificidade da Espécie , Espermatozoides/metabolismoRESUMO
Kaempferol (3,4',5,7-tetrahydroxyflavone, KAE) is one of the most commonly occurring dietary flavonols. The biological and pharmacological effects of kaempferol depend upon whether it acts as an antioxidant, anti-inflammatory, or anticancer agent. The present study explored the influence of KAE supplementation on in vitro damage to porcine oocytes and its underlying mechanisms. Different concentrations of KAE (0.05, 0.1, 0.5, 1⯵M) were added to porcine zygote medium 5 during in vitro culture. The results showed that supplementation with 0.1⯵M KAE significantly increased the blastocyst formation rate. Blastocyst formation and quality were significantly increased in the 200⯵Mâ¯H2O2 treatment group following addition of 0.1⯵M KAE. KAE prevented the H2O2-induced compromise of mitochondrial membrane potential and reactive oxygen species generation. Furthermore, the extent of autophagy and DNA damage in the blastocysts was attenuated by supplementation with KAE in the H2O2 induced oxidative injury group compared to that observed in controls. These results suggest that KAE has beneficial effects on the development of porcine parthenotes by attenuating oxidative stress and increasing mitochondrial function.
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Embrião de Mamíferos/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Quempferóis/farmacologia , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Suínos/embriologia , Animais , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , PartenogêneseRESUMO
Laminarin (LAM) is a ß-glucan oligomer known to possess biological activities such as anticancer and antioxidant effects. This study explored the influence of LAM supplementation on in vitro aged porcine oocytes and the underlying mechanisms behind this influence. We found that LAM delayed the aging process and improved the quality of aged oocytes. LAM supplementation enhanced the subsequent developmental competence of aged oocytes during the in vitro aging process. The blastocyst formation rate was significantly increased in aged oocytes treated with 20 µg/ml LAM compared to non-treated aged oocytes (45.3% vs. 28.7%, P < 0.01). The mRNA levels of apoptosis-related genes, B cell lymphoma-2-associated X protein (Bax) and Caspase-3, were signiï¬cantly lower in blastocysts derived from the LAM-treated aged oocytes during the in vitro aging process. Furthermore, the level of intracellular reactive oxygen species was significantly decreased and that of glutathione was significantly increased in aged oocytes following LAM treatment. Mitochondrial membrane potential was increased, and the activities of caspase-3 and cathepsin B were significantly reduced in the LAM-treated aged oocytes compared with the non-treated aged oocytes. Taken together, these results suggest that LAM is beneficial for delaying the aging process in porcine oocytes.
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Antioxidantes/farmacologia , Glucanos/farmacologia , Oócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Envelhecimento/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , SuínosRESUMO
Laminarin (LMA), a ß-glucan mixture with good biocompatibility, improves the growth performance and immune response when used as food additives and nutraceuticals. The aim of the present research was to explore the effects of LMA on porcine early stage embryo development, as well as the underlying mechanisms. The results showed that the developmental competence of porcine early stage embryos was dramatically improved after LMA supplementation during the in vitro culture period. The presence of 20⯵g/mL LMA during the in vitro culture period significantly improved cleavage rate, blastocyst formation rates, hatching rate, and total cell number in the blastocyst compared to that in the control group. Notably, LMA attenuated the intracellular reactive oxygen species generation induced by H2O2. Furthermore, LMA not only increased intracellular glutathione levels, but also ameliorated mitochondrial membrane potential. In addition, the expression of a zygotic genome activation related gene (YAP1), pluripotency-related genes (OCT4, NANOG, and SOX2), and hatching-related genes (COX2, GATA4, and ITGA5) were up-regulated following LMA supplementation during porcine early stage embryo development. These results demonstrate that LMA has beneficial effects on the development of porcine early stage embryos via regulation of oxidative stress. This evidence provides a novel method for embryo development improvement associated with exposure to LMA.
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Desenvolvimento Embrionário/efeitos dos fármacos , Glucanos/farmacologia , Sus scrofa/embriologia , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Técnicas de Cultura Embrionária/métodos , Técnicas de Cultura Embrionária/veterinária , Feminino , Expressão Gênica/efeitos dos fármacos , Glutationa/análise , Peróxido de Hidrogênio/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Calcium and integrin-binding protein 2 (CIB2) belongs to a protein family with four known members, CIB1 through CIB4, which are characterized by multiple calcium-binding EF-hand domains. Among the family members, the Cib1 and Cib2 genes are expressed in mouse cochlear hair cells, and mutations in the human CIB2 gene have been associated with nonsyndromic deafness DFNB48 and syndromic deafness USH1J. To further explore the function of CIB1 and CIB2 in hearing, we established Cib1 and Cib2 knockout mice using the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease (CRISPR/Cas9) genome editing technique. We found that loss of CIB1 protein does not affect auditory function, whereas loss of CIB2 protein causes profound hearing loss in mice. Further investigation revealed that hair cell stereocilia development is affected in Cib2 knockout mice. Noticeably, loss of CIB2 abolishes mechanoelectrical transduction (MET) currents in auditory hair cells. In conclusion, we show here that although both CIB1 and CIB2 are readily detected in the cochlea, only loss of CIB2 results in profound hearing loss, and that CIB2 is essential for auditory hair cell MET.