| Our previous study has suggested that monochromatic light could affect skeletal muscle growth and development in posthatched broilers.That is,the green light promotes broiler skeletal muscle growth during the early stage(P1 to P21),and blue light enhances growth during the later stage(P21 to P49).Traditional embryologist believes that formation and maturation of skeletal muscle myofiber occur in the embryonic period,and the myofiber number is fixed before hatched.Thus,we guess whether monochromatic light in embryonic period will have a better effect on promoting muscle growth and muscle fiber development in chick embryo.To answer this question,EO fertilized eggs of AA broilers were incubated under dark(D)or blue(480 nm,B),red(660 nm,R),green(560 nm,G)and white(400-760 nm,W)light condition by using LED for 21 days in this study,respectively.The internal mechanism from views of myogenic regulatory factors and muscle growth was discussed.The main results are as follows:1.In ovo exposure to monochromatic lights affect posthatch muscle growth and satellite cell proliferation of chicksIn this study,the broilers in G group had a heavier hatching weight(6.7%-8.6%),body weight(P1-P21,4.3%-25.2%)and a larger muscle index(9.5%-47.6%in pectoral muscle and 12.4%-50.0%in gastrocnemius muscle),and an increase(10.1%-103.9%)in the myofiber cross-sectional area on posthatch day P1 to P10 relative to other illumination treatments(W-,R-,B-and D-groups).Furthermore,we designed both in vivo(PCNA immunohistochemical staining)and in vitro(satellite cell culture)experiments to analyze the effects of in ovo illumination with various light spectra on the proliferation and differentiation of satellite cells in posthatch broilers.Our results showed that the number of PCNA positive cell(36.4%-44.5%in pectoral muscle,35.9%-42.0%in gastrocnemius muscle)and the relative(6.0%-59.3%)and absolute numbers(6.6%-77.0%)and the mitotic activity(12.5%-61.8%)of satellite cells isolated from pectoral and gastrocnemius muscles were larger in the G group relative to the other groups on posthatch days 1-10.Second,blue and white light illumination in incubation period can increased these dates above than dark group in some extent.2.Effect of monochromatic light during Incubation on proliferation and differentiation of satellite cell and its molecular mechanismsMyogenise:We detected Paired-box 7 and MRFs mRNA expression on P1,P4,P7 and P10 in five light groups.These findings showed that green light illumination in incubation period significantly increased mRNA expression of Pax7(12.36%-45.53%),Myf5(5.04%-36.99%),MyoD(6.56%-31.67%)and Myogenin(11.86%-54.26%)than those of other groups,respectively.Growth factors:RT-PCR was used to detecte mRNA expression of the positive growth factor HGF and the negative growth factor MSTN.Westenblot was used to detecte protein expression of c-Met and GDF-8.These findings showed that green light illumination in incubation period significantly increased HGF mRNA expression(16.19%-69.91%)and c-Met protein relative expression(21.49%-96.43%),decreased MSTN mRNA expression(11.36%-36.49%)and GDF-8 protein relative expression(9.47%-40.88%)than those of other groups,respectively.3.IGF-1 mediated monochromatic light-regulated satellite cell proliferation in embryonic periodIGF-1 is a key growth factor for regulating skeletal satellite cell proliferation.In this study,plasma IGF-1 level was increasing from P1 to P21.The plasma IGF-1 level of the G group was significant higher 7.02%-47.15%than other groups.GH is the upstream of IGF-1.Plasma GH was highest in the G group,which was higher 9.39%-59.74%han other light group.The IGF-1R mRNA and protein expression had similar trend with plasma IGF-1 level both in pectoralis and gastrocnemius muscles.Additionally,the plasma IGF-1 level was significantly positively correlated with.Additionally,Pearson analysis showed that the IGF-1 level was significantly positive correlated with the total number(P ＝0.001,r = 0.989)and PCNA positive cell percentage(P = 0.001,r = 0.990)of satellite cells.In vitro experiment in which adding 0-200 ng/mL concentration rhIGF-1 to the cultured satellite cells induced a dose dependent increase in proliferation,and satellite cell of G group was more sensitive to the IGF-1.Pearson analysis showed that the plasma IGF-1 level was significantly positive correlated with Pax7(P=0.004,r = 0,976),Myf5(P = 0.005,r = 0.973),MyoD(P = 0.004,r = 0.977),Myogenin(P = 0.006,r=0.970)and growh factor HGF mRNA expression(P = 0.953,r = 0.012),meanwhile plasma IGF-1 level was significantly negtive correlated with MSTN mRNA expression(P = 0.048;r =-0.882).These dates suggest that IGF-1 was involved in the effect of in ovo monochromatic green light photostimulation on posthatch proliferation and differentiation of satellite cells.4.IGF-1 mediated monochromatic light-regulated satellite cell proliferation through PI3K/Akt pathway in incubation periodThe p-Akt protein expreesion of pectoral muscle in green light group was highest among five light groups,and the ratio of p-Akt/t-Akt was higher 17.10%-60.26%than other groups.Exogenous IGF-1 of 100ng/mL concentrate was enough to promote proliferation of satellite cells and AKT activity.The effect of IGF-1 can be blocked by 10μM LY294002(an inhibitor of PI3K)in vitro.Besides,compared with control(saline),p-Akt level of pectoral muscle was decreased(32.40%in D group and 40.31%in G group),accompanied with the reduction of body weight(17.39%in D group and 28.53%in G group)and pectoral muscle weight(34.54%in D group and 39.98%in G group)when 200μg/200μL LY294002 was injected in broiler pectoral muscle.The result indicated that IGF-1 mediated monochromatic green light during incubation promoted skeletal muscle satellite cell proliferation through PI3K/Akt pathway.5.Melatonin mediates monochromatic light-induced satellite cell proliferation and muscle growth in chick embryoOn P1,P4,P7,P10 and P21,plasma melatonin in green light group was higher 12.46%-58.61%than other groups.Compare green light group with dark group on E15,E18 and E21,green light stimulation promoted pineal gland development,AANAT mRNA expression(12.85%-15.95%)and plasma melatonin level(13.13%-18.20%)than those in dark group on E18 and E21.To study the role of melatonin in the monochromatic green light-induced satellite cell proliferation,the E8 chick embryos were injected with 20μg/20μL PACAP6-38(inhibitors of PACAP)and saline,were exposed to dark condition(D-group)and green light(G-group)in incubation.The results showed plasma melatonin,GH and IGF-1 decreased,following myofiber area,bigger myofiber percentage,PCNA positive cell percentage,absolute and relative number of satellite cell,proliferation of satellite cell,Pax7 mRNA,MRFs(Myf5,MyoD and Myogenin)mRNA,HGF mRNA and IGF-1R mRNA were decreased in different extent.Meanwhile,MSTN mRNA had increased.In PACAP6-38 treatments,the difference between green light group and dark group had gone,compared to saline treatments.In vitro,adding 250 pg/mL concentration exogenous melatonin significantly promoted the proliferation of satellite cells.Conclusion,green light illumination effectively enhanced growth factors expression following enforces the satellite cell myogenic programme in chick embryonic myogenesis,which depend on enhanced melatonin secretion.Conclusion:During the incubation of chick embryo irradiated by monochromatic light(151x),green light stimulation can significantly promote the development of skeletal muscle and satellite cells proliferation.During incubation,monochromatic green light photostimulation can enhance melatonin secretion of pineal gland in chick embryo.Furthermore,melatonin promoted the secretion of GH and IGF-1.IGF-1 binding to its receptor IGF-1R and then activated PI3K/Akt pathway,following promoted Pax7,Myf5,MyoD,Myogenin and HGF mRNA expression,decreased MSTN mRNA.Finally,the proliferation and differentiation skeletal muscle satellite cells were promoted,contributing to skeletal muscle development. |
PDF Full Text Request