| Cell autophagy and endocytosis are necessary physiological processes for normal life activities of cells,and their activities are affected by many factors.As an important regulator in mammals,growth factors can participate in follicular development,oocyte maturation,and early embryo development through various modes of action.However,its related researches on regulation of autophagy and endocytosis programs in animal reproduction are still very rare.The yak(Bos grunniens)is an important economic animal breed in the plateau.Its seasonal estrus and low fertility have been one of the main reasons for the difficult economic development in pastoral areas.Cumulus cells(CCs),growing tightly around the oocyte,have their own autophagy and endocytosis activities that play an important role in follicular development and oocyte maturation.In this study,yak cumulus cells were selected as the research object to explore effects of epidermal growth factor(EGF)and insulin-like growth factor-1(IGF-1)on its autophagy and endocytosis.0(control group),50,100,150,200 ng/mL EGF and appropriate concentration(10 ?mol/L)epidermal growth factor receptor(EGFR)inhibitor Gefitinib and 0(Control group),50,100,150,200 ng/mL IGF-1 were respectively applied to cultured yak cumulus cells.Extracted total RNA and protein.The relative expressions of Autophagy related gene 5(Atg5),Bcl-2 homologous domain protein(Beclin1),Microtubule-associated protein light chain 3(LC3),Caveolin1(Cav1)and Caveolin2(Cav2)at various EGF and IGF-1 concentrations were determined using qRT-PCR and Western-blot techniques.And immunofluorescence technology were used to compare the expression localizations of 5 proteins in cells analysis.The results were as follows:1)The yak cumulus cells were successfully cultured in vitro.It was identified that the autophagy-related proteins Atg5,Beclin1,LC3,and endocytosis-related proteins Cav1 and Cav2 were expressed in yak cumulus cells and mainly distributed in the cytoplasm.2)The expression of autophagy gene Atg5 decreased in yak cumulus cells treated with different concentrations of EGF compared with the control group(0 ng/mL),and the expressions of Atg5 protein and Atg5-Atg12 complex were negatively correlated with the concentration of EGF,the expression level of each treatment group was significantly lowerthan that of the control group(0 ng/mL)(P <0.05).The expressions of autophagy genes Beclin1,LC3 and proteins Beclin1,LC3?,LC3?,LC3?/LC3? first decreased and then increased,and all reached the lowest level at 100 ng/mL(P <0.05).The expression of LC3 I protein in the 150 and 200 ng/mL treatment groups was significantly higher than that in control group(0 ng/mL)(P <0.05).The relative expressions of Cav1,Cav2 mRNA and protein were same positively correlated with EGF concentrations and were highest at 200ng/mL(P <0.05).3)After inhibiting EGFR,the relative expression of LC3 mRNA and protein were significantly increased(P <0.05),and the expression of other autophagy genes and proteins were not significantly changed(P >0.05).The relative expressions of Cav1,Cav2 mRNA and protein were significantly increased(P <0.05).4)The relative expressions of autophagy-related genes Atg5,Beclin1,LC3 and proteins Atg5,Atg5-Atg12,Beclin1,LC3?,LC3?,LC3?/LC3? all decreased first then increased after treated with different concentrations of IGF-1 and reached the lowest in the 100 ng/mL treatment group,which is significantly different from other treatment groups(P <0.05).The relative expression levels of Cav1 and Cav2 mRNA and protein are both concentration-dependent with IGF-1,and the expression levels decreased first and then increased with the elevation of IGF-1 concentration.In summary,the addition of EGF and IGF-1 in cultured yak cumulus cells can affect the expression of autophagy and endocytosis-related genes and proteins,and the effects are related to the concentrations of EGF and IGF-1.The two kinds of growth factors which at a certain concentration can inhibit autophagy of yak cumulus cells,and 100 ng/mL is the optimal concentration.Between the expressions of endocytosis-associated caveolin proteins Cav1,Cav2 and concentrations of EGF and IGF-1 have a positive correlation.Two kinds of growth factors significantly increased the expression of Cav1 and Cav2 at a concentration of150 ~ 200 ng/mL(P <0.05).EGF and IGF-1 in this concentration range have a good promotion effect on Cav1 and Cav2 expression.EGF and IGF-1 participate in the biological function regulation of yak cumulus cells autophagy and endocytosis.The above research results provide a theoretical basis for further study of the role of growth factors in theautophagy and endocytosis of animal reproduction-related cells,and their mechanism of oocyte maturation and embryo development. |
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