RESUMO
BACKGROUND: Drug resistance is an important factor in the fight against influenza A virus (IAV). Natural products offer a rich source of lead compounds for the discovery of novel antiviral drugs. In a previous study, we isolated the sorbicillinoid polyketide HSL-2 from the mycelium of fungus Trichoderma sp. T-4-1. Here, we show that this compound exerts strong antiviral activity against a panel of IAVs. METHODS: The immunofluorescence and qRT-PCR assays were used to detect the inhibitory effect of HSL-2 toward the replication of influenza virus and IAV-induced expression of the pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1ß. RESULTS: The results indicated that HSL-2 inhibited influenza virus replication, and it significantly inhibited IAV-induced overexpression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß through modulating the PPAR-γ/NF-κB pathway. Notably, this effect was decreased when cells were transfected with PPAR-γ siRNA or treated with the PPAR-γ inhibitor T0070907. In addition, HSL-2 was able to attenuate lung inflammatory responses and to improve lung lesions in a mouse model of IAV infection. CONCLUSIONS: In this paper, we identified a microbial secondary metabolite, HSL-2, with anti-influenza virus activity. This report is the first to describe the antiviral activity and mechanism of action of HSL-2, and it provides a new strategy for the development of novel anti-influenza virus drugs from natural sources.
RESUMO
Heat stress (HS) is a stressor that negatively affect female reproduction. Specially, oocytes are very sensitive to HS. It has been demonstrated that some active compounds can protect oocyte from HS. We previously found that Mogroside V (MV), extracted from Siraitia grosvenorii (Luo Han Guo), can protect oocyte from many kinds of stresses. However, how MV alleviates HS-induced disruption of oocyte maturation remains unknown. In this study, we treated the HS-induced porcine oocytes with MV to examine their maturation and quality. Our findings demonstrate that MV can effectively alleviate HS-induced porcine oocyte abnormal cumulus cell expansion, decrease of first polar body extrusion rate, spindle assembly and chromosome separation abnormalities, indicating MV attenuates oocyte mature defects. We further observed that MV can effectively alleviate HS-induced cortical granule distribution abnormality and decrease of blastocyst formation rate after parthenogenesis activation. In addition, MV treatment reversed mitochondrial dysfunction and lipid droplet content decrease, reduced reactive oxygen species levels, early apoptosis and DNA damage in porcine oocytes after HS. Collectively, this study suggests that MV can effectively protect porcine oocytes from HS.
Assuntos
Técnicas de Maturação in Vitro de Oócitos , Oócitos , Triterpenos , Suínos , Feminino , Animais , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oogênese , Espécies Reativas de Oxigênio/farmacologia , Resposta ao Choque TérmicoRESUMO
Influenza A virus (IAV) is one of the leading causes of respiratory illness and continues to cause pandemics around the world. Against this backdrop, drug resistance poses a challenge to existing antiviral drugs, and hence, there is an urgent need for developing new antiviral drugs. In this study, we obtained a phenolic compound SG-7, a derivative of natural compound 2-hydroxymethyl-1,4-hydroquinone, which exhibits inhibitory activity toward a panel of influenza viruses and has low cellular toxicity. Mechanistic studies have shown that SG-7 exerts its anti-IAV properties by acting on the virus itself and modulating host signaling pathways. Namely, SG-7 targets the HA2 subunit of hemagglutinin (HA) to block the fusion of viral-cellular membranes and inhibits IAV-induced oxidative stress and overexpression of pro-inflammatory factors by activating the Nrf2/HO-1 pathway and reducing NF-κB activation. In addition, SG-7 can enhance type I IFN antiviral response by inducing Nrf2 expression. Importantly, SG-7 showed the ability to inhibit viral replication in the lungs of IAV-infected mice and reduce their mortality. Therefore, SG-7 may be a promising lead compound for anti-influenza drug development.
Assuntos
Vírus da Influenza A , Influenza Humana , Animais , Camundongos , Humanos , Vírus da Influenza A/fisiologia , Fator 2 Relacionado a NF-E2 , Antivirais/farmacologia , Antivirais/uso terapêutico , Influenza Humana/tratamento farmacológico , Replicação ViralRESUMO
Lipopolysaccharide (LPS), a significant virulence factor of gram-negative bacteria, adversely affects female reproduction, especially the maturation and early embryonic development of oocytes, through inducing of inflammatory and oxidative stress-associated toxic responses. Resveratrol (Res), a potent antioxidant, exhibits many beneficial effects on the maturation and developmental competence of oocytes. However, it is unclear whether Res can restore LPS-induced defects in the maturation of oocytes during meiosis. In this study, we used porcine oocytes to explore the protective effects of Res and its underlying mechanism against the toxic impacts of LPS exposure on meiotic maturation and developmental competence of oocytes during meiosis. The oocytes were randomly assigned to a control, LPS-exposed or Res-supplemented group. Nuclear and cytoplasmic maturation was assessed after 26 h (MI) or 44 h (MII) of in vitro maturation (IVM). Our results showed that 10 µM Res significantly improved the rates of oocyte maturation and blastocyst formation after exposure to 15 µg/mL LPS. In addition, Res preserved the normal spindle/chromosome structure and maintained acetylated tubulin levels, actin polymerization and cortical granules (CGs) distribution. Additionally, Res protected mitochondrial content and function, scavenges reactive oxygen species (ROS), and reduced DNA damage and apoptosis in LPS-exposed oocytes. Furthermore, inhibition of SIRT1 by its specific inhibitor EX527 suppressed the recovery of ROS levels, mitochondrial content, and spindle/chromosome structure by Res supplementation. In summary, this study shows that Res can alleviate the impacts of LPS-induced toxicity on meiosis in porcine oocytes by upregulating SIRT1, which ameliorates oxidative stress and increasing mitochondrial content.
Assuntos
Lipopolissacarídeos , Sirtuína 1 , Gravidez , Suínos , Feminino , Animais , Lipopolissacarídeos/toxicidade , Resveratrol/farmacologia , Espécies Reativas de Oxigênio , Oócitos , Técnicas de Maturação in Vitro de OócitosRESUMO
Sixteen secondary metabolites, including one new sesquiterpene (1, named isocyclonerodiol oxide), seven known sesquiterpenes (2-8), two sorbicillinoid polyketides (15, 16), and six known other compounds (9-14) were isolated from the fermentation broth of Trichoderma sp. T-4-1. The structure of 1 was determined by 1 D, 2 D NMR (HMBC, HSQC, 1H-1H COSY, NOESY), and HRESIMS spectra. In addition, sesquiterpenes and sorbicillinoid polyketides showed significant antiviral activities.
RESUMO
Five metabolites (1-5), including two new sesquiterpenoids, designated ganodermanol L (1) and 4α,15-epoxyeudesmane-1ß,6α,11-triol (2), together with three known structurally related compounds (3-5), have been isolated from the cultures of Streptomyces sp. XM17, a bacteria residing in the fresh feces of the giant panda Ailuropoda melanoleuca. The structures of 1-2 were established on the basis of extensive spectroscopic analyses, including 1D- and 2D-NMR (1H-1H COSY, HMQC, HMBC and NOESY) experiments. Furthermore, the absolute configuration of 1 was established by single-crystal X-ray crystallographic analyses. Of noted, these compounds were found to possessed antiviral activities using the 'pretreatment of virus' approach with IC50 values ranging from 4 to 30 nM, indicating that these sesquiterpenoids were potent in inhibiting the entry of influenza A virus.
Assuntos
Vírus da Influenza A , Sesquiterpenos , Streptomyces , Ursidae , Animais , Streptomyces/química , Sesquiterpenos/química , Fezes , Antivirais/farmacologia , Estrutura MolecularRESUMO
Three novel norsesquiterpenoids, (2R,4S,8aR)-8,8a,1,2,3,4-hexahydro-2-hydroxy-4,8a-dimethyl-2(2H)-naphthalenone (1), (1S,3S,4S,4aS,8aR)-4,8a-dimethyloctahydronaphthalene-1,3,4a(3H)-triol(2), (4S,4aS,8aS)-octahydro-4a-hydroxy-4, 8a-dimethyl-1(2H)-naphthalenone (3), as well as six other known analogues (4-9), were isolated from the culture broth of Streptomyces sp. XM17, an actinobacterial strain inhabiting the fresh feces of the giant panda Ailuropoda melanoleuca. The chemical structures of 1-3 were elucidated comprehensively by NMR spectroscopic and MS analyses, furthermore, the stereochemical configurations were resolved by NOESY experiments, along with ECD spectral and single-crystal X-ray crystallographic analyses. These compounds were then tested for their antiviral activities using the "pretreatment of virus" approach, which showed that most of these compounds were potent in inhibiting the entry of influenza A virus, with IC50 values ranging from 5 to 49 nM and selectivity indices all above 500.
Assuntos
Antivirais/isolamento & purificação , Fezes/microbiologia , Vírus da Influenza A/efeitos dos fármacos , Sesquiterpenos/isolamento & purificação , Streptomyces/química , Animais , Antivirais/química , Antivirais/farmacologia , Antivirais/toxicidade , Embrião de Galinha , Dicroísmo Circular , Cristalografia por Raios X , Cães , Concentração Inibidora 50 , Células Madin Darby de Rim Canino , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Sesquiterpenos/toxicidade , UrsidaeRESUMO
A new aliphatic acid, compound 1, together with six known metabolites, including nonactic acid (2), homononactic acid (3), ethyl homononactate (4), homononactylhomononactate (5), valinomycin (6), and cyclo-(Pro-Leu) (7), was isolated from the culture broth of Streptomyces sp. BM-8, an actinobacterial strain isolated from the feces of Equus quagga. The structures of these compounds were established by analyses of spectroscopic data, including 1D and 2D nuclear magnetic resonance spectra (NMR), as well as by HR-ESI-MS spectrometry and chemical derivative analyses. Additionally, a serial analogue of nonactic acid and homononacticacid (8-21) was synthesized. The cytotoxicity of 1-21 wastested against a panel of cancer cell lines, such as HT-29, MCF-7, A375 and K562, with MTT assay. In addition, the cytotoxicity tests revealed that 1 was less cytotoxic toward a panel of cancerous cells, as compared with valinomycin (6).
Assuntos
Antineoplásicos , Citotoxinas , Equidae/microbiologia , Fezes/microbiologia , Neoplasias/tratamento farmacológico , Streptomyces , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Citotoxinas/química , Citotoxinas/farmacologia , Células HT29 , Humanos , Células K562 , Células MCF-7 , Neoplasias/metabolismo , Streptomyces/química , Streptomyces/crescimento & desenvolvimento , Streptomyces/isolamento & purificaçãoRESUMO
Parathyroid hormone-related protein (PTHrP), the main cause of humoral hypercalcemia in malignancies, promotes cell proliferation and delays terminal cell maturation during embryonic development. Our previous study reported that PTHrP plays important roles in blastocyst formation, pluripotency gene expression, and histone acetylation during mouse preimplantation embryonic development. In this study, we further investigated the mechanism of preimplantation embryonic development regulated by PTHrP. Our results showed that Pthrp depletion decreased both the developmental rate of embryos at the cleavage stage and the cell number of morula-stage embryos. Pthrp-depleted embryos had significantly decreased levels of cyclin D1, phospho (p)-AKT (Thr308) and E2F1. However, Pthrp depletion did not cause significant changes in CDK4, ß-catenin or RUNX2 expression. In addition, our results indicated that Pthrp depletion promoted HDAC4 translocation from the cytoplasm to the nucleus in cleavage-stage embryos by stimulating the activity of protein phosphatase 2A (PP2A), which resulted in dephosphorylation of HDAC4. Taken together, these results suggest that PTHrP regulates cleavage division progression and blastocyst formation through the AKT/cyclin D1 pathway and that PTHrP modulates histone acetylation patterns through nuclear translocation of HDAC4 via PP2A-dependent HDAC4 dephosphorylation during preimplantation embryonic development in mice.
Assuntos
Blastocisto/metabolismo , Ciclina D1/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Acetilação , Transporte Ativo do Núcleo Celular , Animais , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilases/genética , Camundongos , Proteína Relacionada ao Hormônio Paratireóideo/genética , Fosforilação , Proteína Fosfatase 2/metabolismo , Transdução de SinaisRESUMO
A new bafilomycin derivative (1) and another seven known bafilomycins (2-8) were isolated from feces-derived Streptomyces sp. HTL16. The structure of 1 was elucidated by 1D and 2D NMR spectroscopic analysis. Biological testing demonstrated that these bafilomycins exhibited potent antiviral activities against the influenza A and SARS-CoV-2 viruses, with IC50 values in the nanomolar range, by inhibiting the activity of endosomal ATP-driven proton pumps.
Assuntos
Antivirais/farmacologia , Fezes/microbiologia , Macrolídeos/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Streptomyces/metabolismo , Animais , Cães , Vírus da Influenza A/efeitos dos fármacos , Células Madin Darby de Rim Canino , SARS-CoV-2/efeitos dos fármacosRESUMO
Severe emerging and re-emerging viral infections such as Lassa fever, Avian influenza (AI), and COVID-19 caused by SARS-CoV-2 urgently call for new strategies for the development of broad-spectrum antivirals targeting conserved components in the virus life cycle. Viral lipids are essential components, and viral-cell membrane fusion is the required entry step for most unrelated enveloped viruses. In this paper, we identified a porphyrin derivative of protoporphyrin IX (PPIX) that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses including Lassa virus (LASV), Machupo virus (MACV), and SARS-CoV-2 as well as various subtypes of influenza A viral strains with IC50 values ranging from 0.91 ± 0.25 µM to 1.88 ± 0.34 µM. A mechanistic study using influenza A/Puerto Rico/8/34 (H1N1) as a testing strain showed that PPIX inhibits the infection in the early stage of virus entry through biophysically interacting with the hydrophobic lipids of enveloped virions, thereby inhibiting the entry of enveloped viruses into host cells. In addition, the preliminary antiviral activities of PPIX were further assessed by testing mice infected with the influenza A/Puerto Rico/8/34 (H1N1) virus. The results showed that compared with the control group without drug treatment, the survival rate and mean survival time of the mice treated with PPIX were apparently prolonged. These data encourage us to conduct further investigations using PPIX as a lead compound for the rational design of lipid-targeting antivirals for the treatment of infection with enveloped viruses.
Assuntos
Antivirais/uso terapêutico , Infecções por Orthomyxoviridae/tratamento farmacológico , Protoporfirinas/uso terapêutico , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/síntese química , Antivirais/metabolismo , Antivirais/farmacologia , Arenavirus do Novo Mundo/efeitos dos fármacos , Chlorocebus aethiops , Cães , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus Lassa/efeitos dos fármacos , Células Madin Darby de Rim Canino , Masculino , Lipídeos de Membrana/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Protoporfirinas/síntese química , Protoporfirinas/metabolismo , Protoporfirinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Células Vero , Envelope Viral/efeitos dos fármacosRESUMO
CRISPR/Cas9-mediated genome editing technology is a simple and highly efficient and specific genome modification approach with wide applications in the animal industry. CRISPR/Cas9-mediated genome editing combined with somatic cell nuclear transfer rapidly constructs gene-edited somatic cell-cloned pigs for the genetic improvement of traits or simulation of human diseases. Chinese Bama pigs are an excellent indigenous minipig breed from Bama County of China. Research on genome editing of Chinese Bama pigs is of great significance in protecting its genetic resource, improving genetic traits and in creating disease models. This study aimed to address the disadvantages of slow growth and low percentage of lean meat in Chinese Bama pigs and to knock out the myostatin gene (MSTN) by genome editing to promote growth and increase lean meat production. We first used CRISPR/Cas9-mediated genome editing to conduct biallelic knockout of the MSTN, followed by somatic cell nuclear transfer to successfully generate MSTN biallelic knockout Chinese Bama pigs, which was confirmed to have significantly faster growth rate and showed myofibre hyperplasia when they reached sexual maturity. This study lays the foundation for the rapid improvement of production traits of Chinese Bama pigs and the generation of gene-edited disease models in this breed.
Assuntos
Sistemas CRISPR-Cas , Miostatina/genética , Porco Miniatura/genética , Animais , Feminino , Técnicas de Inativação de Genes/veterinária , Masculino , Fibras Musculares Esqueléticas/fisiologia , Técnicas de Transferência Nuclear/veterinária , Carne de Porco , Suínos , Porco Miniatura/crescimento & desenvolvimentoRESUMO
Marine environments are known to be a new source of structurally diverse bioactive molecules. In this paper, we identified a porphyrin derivative of Pyropheophorbide a (PPa) from the mussel Musculus senhousei (M. senhousei) that showed broad anti-influenza A virus activity in vitro against a panel of influenza A viral strains. The analysis of the mechanism of action indicated that PPa functions in the early stage of virus infection by interacting with the lipid bilayer of the virion, resulting in an alteration of membrane-associated functions, thereby blocking the entry of enveloped viruses into host cells. In addition, the anti-influenza A virus activity of PPa was further assessed in mice infected with the influenza A virus. The survival rate and mean survival time of mice were apparently prolonged compared with the control group which was not treated with the drug. Therefore, PPa and its derivatives may represent lead compounds for controlling influenza A virus infection.
Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Bivalves/química , Clorofila/análogos & derivados , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírion/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/isolamento & purificação , Betacoronavirus/crescimento & desenvolvimento , Betacoronavirus/metabolismo , Clorofila/química , Clorofila/isolamento & purificação , Clorofila/farmacologia , Cães , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Bicamadas Lipídicas/antagonistas & inibidores , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Células Madin Darby de Rim Canino , Masculino , Camundongos , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Vírus Sinciciais Respiratórios/metabolismo , SARS-CoV-2 , Alimentos Marinhos , Análise de Sobrevida , Vírion/crescimento & desenvolvimento , Vírion/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
Actinobacteria are historically and continued to be an important source for drug discovery. The annual epidemics and periodic pandemics of humans induced by influenza A virus (IAV) prompted us to develop new effective antiviral drugs with different modes of action. An actinobacterium of Streptomyces sp. SMU 03 was identified from the feces of Elephas maximus in Yunnan Province, China. By employing an H5N1 pseudo-typed virus drug screening system, the anti-IAV effect of the dichloromethane extracts (DCME) of this bacterium was investigated. DCME showed broad and potent activities against several influenza viruses, including the H1N1 and H3N2 subtypes and influenza B virus, with IC50 values ranging from 0.37 ± 0.22 to 14.44 ± 0.79 µg/mL. A detailed modes-of-action study indicated that DCME might interact with the HA2 subunit of hemagglutinin (HA) of IAV by interrupting the fusion process between the viral and host cells' membranes thereby inhibiting the entry of the virus into host cells. Furthermore, the in vivo anti-IAV activity test of DCME showed that compared with the no-drug treated group, the survival rates, appearances, weights, lung indices and histopathological changes were all significantly alleviated. Based on these results, the chemical constituent study of DCME was then investigated, from which a number of antiviral compounds with various structural skeletons have been isolated and identified. Overall, these data indicated that the DCME from Streptomyces sp. SMU 03 might represent a good source for antiviral compounds that can be developed as potential antivirus remedies.
Assuntos
Antivirais/farmacologia , Elefantes/microbiologia , Streptomyces/química , Animais , Antivirais/isolamento & purificação , China , Cães , Fezes/microbiologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Estrutura Molecular , Streptomyces/isolamento & purificaçãoRESUMO
Oocyte in vitro maturation (IVM) plays a pivotal role in in vitro embryo production. However, the efficiency of IVM is still low and needs to be further improved. In the present study, we evaluated the beneficial effects of mogroside V, an extract derived from Siraitia grosvenorii, on oocyte IVM. Porcine cumulus-oocyte complexes were cultured in IVM medium supplemented or not supplemented with mogroside V for 40â¯h. We found that mogroside V supplementation increased the percentage of oocyte first polar body extrusion and improved subsequent blastocyst formation after parthenogenetic activation. Furthermore, mogroside V reduced the levels of reactive oxygen species (ROS) and increased the mRNA expression of oxidative stress-related genes (SOD, CAT and SIRT1). Moreover, mogroside V supplementation enhanced the mitochondrial content, mtDNA copy number, mitochondrial membrane potential (ΔΨm), ATP generation, and the relative mRNA expression of mitochondria-related genes (PGC-1α and TFAM). In summary, our findings demonstrate that mogroside V supplementation reduces intracellular ROS levels and enhances mitochondrial function to promote porcine oocyte IVM.
Assuntos
Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/efeitos dos fármacos , Suínos/fisiologia , Triterpenos/farmacologia , Animais , DNA Mitocondrial , Fertilização in vitro/veterinária , Mitocôndrias/fisiologia , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Postovulatory ageing compromises oocyte quality and subsequent development in various manners. We aimed to assay the protective effects of mogroside V on porcine oocyte quality during in vitro ageing and explore the related causes. We observed that mogroside V can effectively maintain normal oocyte morphology and early embryo development competence after prolonged culture for 24 h. Moreover, mogroside V can markedly reduce reactive oxygen species (ROS) levels, alleviate spindle formation and chromosome alignment abnormalities, improve mitochondrial contents, adenosine triphosphate (ATP) levels and the membrane potential (ΔΨm), and reduce early apoptosis in aged oocytes. We examined the molecular changes and found that SIRT1 expression was decreased in in vitro aged oocytes but was maintained by exposure to mogroside V. However, when SIRT1 was successfully inhibited by the specific inhibitor EX-527, mogroside V could not reduce ROS levels or alleviate abnormal spindle organization and chromosome misalignment. In summary, our results demonstrated that mogroside V can alleviate the deterioration of oocyte quality during in vitro ageing, possibly by reducing oxidative stress through SIRT1 upregulation.
Assuntos
Senescência Celular , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 1 , Triterpenos/farmacologia , Animais , Carbazóis/farmacologia , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Feminino , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Espécies Reativas de Oxigênio/análise , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/metabolismo , Suínos , Regulação para CimaRESUMO
The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini-pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line-derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha-1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT-PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1-, DBA- and CDH1-positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.
Assuntos
Células-Tronco Germinativas Adultas/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Células-Tronco Germinativas Adultas/citologia , Animais , Linhagem Celular , China , Técnicas de Cocultura , Masculino , Camundongos , Espermatogênese , Suínos , Porco Miniatura , Testículo/citologia , Fatores de Transcrição/metabolismoRESUMO
Dwarfism, also known as growth hormone deficiency (GHD), is a disease caused by genetic mutations that result in either a lack of growth hormone or insufficient secretion of growth hormone, resulting in a person's inability to grow normally. In the past, many studies focusing on GHD have made use of models of other diseases such as metabolic or infectious diseases. A viable GHD specific model system has not been used previously, thus limiting the interpretation of GHD results. The Bama minipig is unique to Guangxi province and has strong adaptability and disease resistance, and an incredibly short stature, which is especially important for the study of GHD. In addition, studies of GHR knockout Bama minipigs and GHR knockout Bama minipig fibroblast cells generated using CRISPR/Cas9 have not been previously reported. Therefore, the Bama minipig was selected as an animal model and as a tool for the study of GHD in this work. In this study, a Cas9 plasmid with sgRNA targeting the first exon of the GHR gene was transfected into Bama minipig kidney fibroblast cells to generate 22 GHR knockout Bama minipig kidney fibroblast cell lines (12 male monoclonal cells and 10 female monoclonal cells). After culture and identification, 11 of the 12 male clone cell lines showed double allele mutations, and the rate of positive alteration of GHR was 91.67%. Diallelic mutation of the target sequence occurred in 10 female clonal cell lines, with an effective positive mutation rate of 100%. Our experimental results not only showed that CRISPR/Cas9 could efficiently be used for gene editing in Bama minipig cells but also identified a highly efficient target site for the generation of a GHR knockout in other porcine models. Thus, the generation of GHR knockout male and female Bama fibroblast cells could lay a foundation for the birth of a future dwarfism model pig. We anticipate that the "mini" Bama minipig will be of improved use for biomedical and agricultural scientific research and for furthering our understanding of the genetic underpinnings of GHD.
Assuntos
Sistemas CRISPR-Cas , Fibroblastos/fisiologia , Receptores da Somatotropina/genética , Porco Miniatura/genética , Animais , Animais Geneticamente Modificados , Linhagem Celular , Feminino , Edição de Genes , Técnicas de Inativação de Genes , Homozigoto , Masculino , Mutação , SuínosRESUMO
NEK5, a member of never in mitosis-gene A-related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA-mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin-dependent kinase 1 in oocytes, resulting in a decrease of maturation-promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1-cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.
Assuntos
Blastocisto/metabolismo , Ciclo Celular , Desenvolvimento Embrionário , Quinases Relacionadas a NIMA/metabolismo , Oócitos/metabolismo , Animais , Blastocisto/citologia , Feminino , Camundongos , Quinases Relacionadas a NIMA/genética , Oócitos/citologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologiaRESUMO
As a natural plant-derived antitoxin, resveratrol possesses several pharmacological activities. This study aimed to evaluate the effects of resveratrol addition on nuclear maturation, oocyte quality during in vitro maturation (IVM) of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer (SCNT). Our experiments showed that the treatment of porcine oocytes with 5 µM resveratrol during IVM resulted in the highest rate of the first polar body extrusion. Treatment of oocytes with resveratrol had no influence on cytoskeletal dynamics, whereas it significantly increased glucose uptake ability compared to the control oocytes. Oocytes matured with 5 µM resveratrol displayed significantly lower intracellular reactive oxygen species (ROS) levels and higher relative mRNA expression levels of the genes encoding such antioxidant enzymes as catalase (CAT) and superoxide dismutase 1 (SOD1). In addition, resveratrol also prevented onset and progression of programmed cell death in porcine oocytes, which was confirmed by significant upregulation of the anti-apoptotic B-cell lymphoma 2 (BCL-2) gene and significant downregulation of the pro-apoptotic BCL2-associated X (BAX) gene. Furthermore, the blastocyst rates and the blastocyst cell numbers in cloned embryos derived from the oocytes that had matured in the presence of 5 µM resveratrol were significantly increased. In conclusion, supplementation of IVM medium with 5 µM resveratrol improves the quality of porcine oocytes by protecting them from oxidative damage and apoptosis, which leads to the production of meiotically matured oocytes exhibiting enhanced developmental potential following SCNT.