| Insulin-like growth factor(IGFs)is a key factor regulating growth and reproduction.Growth hormone(GH)is associated with GH receptor(GHR)in tissues.The regulation mechanism of IGF-3 has been a hotspot in the research of growth and reproduction.In this study,Epinephelus coioides were used as subjects to explore the effects of IGF-3 on the maturation of ovarian follicle cells,the insulin-like growth factor receptors(IGFRs)corresponding to IGF-3,and their complete signaling pathways.And how they interact with transcription factors,how to affect transcription factors and reproductive and sex differentiation related factors to regulate fish growth and reproductive development.This research focuses on research from the above mentioned aspects,and has initially explored the functional impact of IGF-3 on the ovarian maturation and development of Epinephelus coioides,and further studied the relationship between IGF-3 expression regulation and production practices in fish.The main results of this paper are as follows:1.In this study,three IGFs receptors: IGF-1Ra,IGF-1Rb and IGF-2R cDNA sequences are successfully cloned in Epinephelus coioides by gene cloning.The results of sequence analysis indicate that only IGF-1Ra and IGF-1Rb have the tyrosine kinase domain,which is essential for IGF signaling pathway,and IGF-2R does not have this domain.IGF-1Rs belong to the tyrosine kinase receptor class and contain two transmembrane domains.IGF-2R is a multifunctional protein receptor capable of binding the cell surface insulin-like growth factor IGF-2 and mannose-6-phosphate labeled proteins in the trans-Golgi network.Also known as the cation-independent mannose-6-phosphate receptor(CI-MPR).The results of phylogenetic tree and protein structure analysis showed that IGF-1Ra and IGF-1Rb share a common ancestor,while IGF-2R is different from IGF-1Rs.It is evolved from other genes before IGF-1Rs.Ligand receptor binding experiments demonstrated that IGF-1Ra can be activated by IGF-3 protein and initiate downstream signaling pathways.Experimental data show that the IGF-3 receptor is more likely to be IGF-1Ra.2.Four oocytes of differentiating grouper are separated under a microscope,and the follicular cells are incubated at different times according to the time and concentration gradient using recombinant IGF-3 protein.The hCG is used as a control to detect blastocysts.The germinal veside breakdown(GVBD)was investigated to investigate the effect of artificial recombinant protein IGF-3 on promoting follicular maturation at different phases.The effects of artificial recombinant IGF-3 on ovarian maturation of Epinephelus coioides are evaluated by counting.The results show that: artificial recombinant IGF-3 protein can promote the maturation of ovarian follicle cells in Epinephelus coioides.For follicular cells in the early stage of development in the PV stage and EV stage,in vitro incubation of IGF-3 requires a slower process.Under the experimentally designed concentration,GVBD cannot be significantly promoted within 24 h;Ovarian follicular cells during the FG period,and artificially reconstituted IGF-3 protein can significantly promote its GVBD,and there is a time-dose interaction relationship.In the range of incubation concentrations designed in this study,with the increase of incubation dose and incubation time,the effect of artificial recombinant IGF-3 protein on the ovarian follicular cell GVBD of Epinephelus coioides is more obvious,and analysis is likely to be possible.There is a dose and time-complementary effect.This experiment further demonstrat that IGF-3 plays an important role in the maturation of Epinephelus coioides ovary.3.The effect of IGF-3/IGFRs system on the reproductive regulation of Epinephelus coioides is studied in vitro.It is found that IGF-3 can inhibit the expression of LH? mRNA in ovarian cells.The results of the study indicate that the IGF-3/IGFRs system has a negative regulatory effect on the reproductive axis. |
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