Functional Analysis Of IGF-1on Duck Embryonic Muscle Development And Research About Its Molecular Mechanism

The characteristics of birds’ muscle development are similar with other verterbrates. During birds’ embryonic stage, the myofiber number is already fixed for most animals, and the number does not increase in the postnatal period. Hypertrophy of muscle in the postnatal period is due to the lengthening and thickening of muscle fibers. Considering with the avian embryo developing outside the body of the female parent (oviparity) is different from the mammalian embryo, the administration of exogenous nutrients into the eggs, air rooms, albumin or embryo, may enhance egg nutrition and poultry early growth performance and energy metabolism. Many factors could induce avian embryo muscle development, and most of those factors are facilited by upregulation the expression of IGF-1. In order to futher enlucidate the effects of IGF-1on duck embryo muscle development, as well as the molecular mechanism involed in it, we used an in ovo feeding method to inject IGF-1into duck eggs’ albumin, and then analysis the effects of IGF-1on proliferation of myoblast, activation of satellite cell and expression of some metabolism related genes. All these works were going to explain the effects of IGF-1on the above proceedings. The detail contents are as follows:1) Using real time PCR method to analysis the expression of muscle development related genes during muscle development, to primarily understanding the characteristics of duck embryo muscle development and roles of some myogenesis genes involed in it.2) To investigate roles of IGF-1on embryonic muscle development, using immunofluorescence technique to analysis the effects of IGF-1on myoblast proliferation and satellite activation, as well as using real time PCR technipue to analysi the effects of IGF-1on expression of muscle development and protein metabolism related genes.3) Add the PI3K and mTOR inhibitor into the vitrol cultured duck myoblast medium to block the PI3K and mTOR activities, respectively, and then analyze the effects of IGF-1on expression of FST and other maker genes in the signal pathway which IGF-1and FST involed in. These works together were to investigate the roles of IGF-1in myoblast proliferation.4) To obtain the duck recombinant FST protein using prokaryoc expression system, and then analyze the effects of FST on embryo muscle development.The main results and conclusions are as follows:(1) All the MRFs gene family members expressed during the duck secondary muscle development stage, and experienced a regularity pattern. Among of these four members, the expression pattern of MyoD and Myf5, MyoG and MRF4are similar, respectively, and play different roles in muscle differentiation. In addition, the MRFs have a different expression in duck breast muscle and leg muscle tissues, indicating that MRFs may also functioned in formation of different types of myofiber. During the duck secondary muscle development, there is a similar expression between IGF-1and FST, indicating IGF-1may cofunctional together with FST in muscle development.(2) In ovo feeding IGF-1into duck eggs influent the duck embryo body weight, muscle weight, and myofiber parameters including myofiber diameter and myofiber number. Meanwhile, in ovo feeding IGF-1induce myoblast’s proliferation. The expression of MyoG and MRF4in both breast muscle and leg muscle in the IGF-1treatment groups are higher than in the control groups. That suggested that in ovo feeding IGF-1can upregulate expression of MyoG and MRF4, and therefore induces myoblast differentiation, which leads to muscle fiber hypertrophy and ultimately muscle development. Moreover, the roles of in ovo feeding IGF-1on duck breast muscle is different from leg muscle. That maybe because of the leg muscle develops a litter earlier than the breast muscle does, or there are difference of muscle fibers types’ constitution in poultry between breast muscle and leg muscle.(3) In ovo feeding of IGF-1could induce the duck growh around the nenotal stage, especially can inhance the duck muscle fiber hypertrophy and muscle development depending on the effects of IGF-1on activation of satellite cells. IGF-1can upregulate the expression of MyoD and Myf5, and then induce satellite cell activation. The activated satellite cells proliferated first and then fused into myofiber to make muscle development. In addition, the effects of IGF-1on duck breast muscle are different from leg muscle. The effects of IGF-1on satellite activation and proliferation in duck leg muscle is feasibly higher than in breast muscle, indicating IGF-1may paly more roles in the slow type myofibers.(4) After the duck eggs were administrated of IGF-1, the expression EIEF was upregulated, and expression of two maker genes during muscle protein degradation were downregulated. That indicated that in ovo feeding IGF-1can induce muscle development by inducing proteins’ translation processing and inhibiting protein degradation. In ovo feeding IGF-1can also regulate duck muscle protein metabolism, and therefore influent muscle development. The ER protein regulation system may take part in muscle development when influenced by in ovo feeding of IGF-1into duck eggs, however, the exact molecular mechanism need to be further elucidation.(5) In vivo, in ovo feeding of IGF-1can upregulate expression of FST, and expression of FST is induced by IGF-1in a dose-dependent manner. In vitrol, IGF-1can also induce expression of FST. When adding Ly929402into the myoblast’s medium, which is a inhibitor of PI3K, expression of FST were not significant influenced at all, however, the other genes in the IGF-1siginal pathway including IGF-1R, EIEF, mTOR, MAFbx, MurF, MyoG, MRF4, ACVR1, and ACVR2were affected by the inhibitor, experienced a different changement at different levels. If adding RAPA to the myoblast medium at the same time when IGF-1exsited, expression of FST were upregulated and the other genes in the IGF-1signal pathway were changed at different levels. These results together suggested that IGF-1can inhibit the activities of signal TGF β through inducing expression of FST. The PI3K may not take part in the above processings, instead of that, maybe some unknown signal pathways have taken part in the processing, of which IGF-1regulating FST through mTOR. FST is another key factor involed in the complexity cross-talking between signal pathway IGF-1and TGF β, and it may be also the key factor for IGF-1regulating TGF β by inhibiting MSTN.(6) The purified duck recombinant FST protein was obtained in this research, and the protein had some biology avitivities of incuding duck myoblast proliferation. In ovo feeding of duck recombinant FST into duck eggs can not induce embryo muscle development, however, it can upregulate expression of MyoD, Myf5and Pax7and downregulate MSTN, indicating that the roles of duck FST in regulating muscle development is relatively important.