| Oxygen is vital for organism normal physiological activities of most organisms.Animals could acclimatize a short period hypoxia or hyperoxia through adjusting itself metabolism,but their growth,development and reproduction,especially,cardiovascular development will be severely influenced by the change of dissolved oxygen.Fishes,as aquatic animals,are more sensitive to the change of dissolved oxygen concentration.Hypoxia and hyperoxia stresses on fish would result in a series of physiological responses.However,the growth and development of fish still be influenced,although fish generated compensation phenomenon when the stress eliminated.Due to abnormal dissolved oxygen,many problems of growth,development and disease of fish will be produced.To date,the researches about dissolved oxygen stress on fish have focused on adult fish.The study that dissolved oxygen effected early growth and development of fish embryo is few.Especially,the effect of different dissolved oxygen on cardiovascular development and signal pathway in fish embryo.In this study,we used zebrafish(Danio rerio),transgenic zebrafish with heart-labeled and vascular-labeled by green fluorescence protein,as study model.Set three dissolved oxygen conditions that were 2.0±0.5mg/L,6.0±0.5mg/L and 15.5±0.5mg/L,respectively.The morphological structure of embryo,heart and vascular external form,heart rate,the diameter of dorsal aorta(DA)and posterior cardinal vein(PCV),the distance between dorsal longitudinal anastomotic vessels(DLAV)and PCV,the distance between two intersegmental vessels(ISV),parachordal vessels(PAV)development within early 72 hpf were measured and analyzed by fluorescence microscope.Results showed survival rate of zebrafish embryo was 74.18± 4.23% under normoxia,76± 4.09% under hyperoxia,but only 43.98± 1.94% under hypoxia.Hypoxia stress not only lagged embryo growth,but also caused abnormal development on morphology.Such as short embryo length,small brain and eye,yolk malabsorption,delayed yolk extension.Hyperoxia could accelerate embryos growth and development.For instance,hyperoxia could reduce by 2 to 4 hours when embryo developed to post-fertilization 24 hours.Whereas the time of development will be increased 24 to 48 hours under hypoxia.Hyperoxia could increase melanin deposition of embryo,and the diameter eye.The diameter eye were 253.0± 20.7?m in normoxia group and 268.3± 14.7?m in hyperoxia group,respectively.Deformities of zebrafish embryo heart exposed to hypoxia mainly manifested pericardialites and cardiac tube stasimorphy.Although cardiac tube could differentiate into atrium and ventricle,but it couldn`t complete right cyclization process.Long-term hyperoxia had none influence for embryo except reducing heart rate.The diameter of DA were 27.5± 3.6?m under normoxia and 19.1± 3.8?m under hypoxia.The diameter of PCV were 35.7± 5.4?m under normoxia and 23.0± 5.8?m under hypoxia.The distance between DLAV and PCV were 181.7± 5.8?m under normoxia and 143.7± 16.5?m under hypoxia.Therefore,hypoxia could shorten DA and PCV diameter,the distance between DLAV and PCV.Moreover,Hypoxia could induce abnormal growth of ISV.The PAV had specific disappeared under hypoxia.Hyperoxia had none influence for embryo except increasing the diameter of DA(30.8± 5.0?m).To elucidate the mechanism that impact of hypoxia and hyperoxia on cardiovascular development.Used via hybridization in situ and qPCR to test the changes of cardiovascular development related genes expression location and quantity for exploring the mechanism of cardiovascular development under the change of dissolved oxygen.According to DNA sequence information of cardiac myosin light chain 2(Cmlc2),Angiogenic factor with G patch and FHA domains 1(Aggf1),fms-related tyrosine kinase 4(Flt4)genes in NCBI,these cDNA sequences were clone by PCR.To composed DIG-RNA labels for ISH.To test the changes of Cmlc2,Aggf1,Flt4,vascular endothelial growth factor receptor 2(Flk1/VEGFR-2)and Tbx5 gene expression by qPCR.ISH results showed hypoxia stress had induced cardiac cyclization obstacles,although Cmlc2 expressive sites had none influences in different dissolved oxygen.Hypoxia and hyperoxia couldn`t change Aggf1 expression sites and expression quantity.The expression of Flk1 on ISV had specific disappeared under hypoxia,and hyperoxia had none influence on Flk1.Flt4 gene expressive sites on embryo head was incomplete,but it expression quantity clearly increased in trunk.qPCR results showed Cmlc2 were down-regulation under hypoxia and hyperoxia,and had highly significant difference(P<0.01).Hypoxia and hyperoxia were up-regulation Tbx5 genetic expression,and had highly significant difference(P<0.01)and significant difference(P<0.05),respectively.Aggf1 were down-regulation under hypoxia and hyperoxia,and had significant difference(P<0.05).Flk1 were down-regulation under hypoxia and hyperoxia,and had highly significant difference(P<0.01).Hypoxia and hyperoxia were up-regulation Flt4,and had highly significant difference(P<0.01)and significant difference(P<0.05),respectively.qPCR results demonstrated the authenticity of ISH results under hypoxia,and it also detected the changes of these genes expression under hyperoxia.According to these genes functions in cardiovascular development,we speculated hypoxia be likely to impact on cardiac cyclization process via Tbx5 gene.Origin reasons of abnormal cardiac development under hypoxia might be the cardiac cyclization obstacles.Hypoxia could be impact upon vessels development through VGEF/VGEFR pathway and Notch signal pathway.Even though hyperoxia had few impacts on zebrafish embryo development,it had influences on cardiovascular genes expression through VGEF/VGEFR pathway.In conclusion,hypoxia caused embryo development arrest,cardiovascular abnormality,and expression changes of genes involved in cardiovascular development.Hyperoxia promoted slightly embryo development,expression changes of genes involved in cardiovascular development.However,cardiovascular morphological change was not significant.Whether or not it would affect morphological structures and physiological functions of adult zebrafish suffered hypoxia and hyperoxia stresses in early embryo development stage,it remains to be further study. |
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