The Effects Of Heat Stress And Senescence On The Biological Activity Of EGF Were Studied Based On Swine Testicular Cell Model

Epidermal growth factor(EGF)is a heat-resistant single-chain low-molecular polypeptide composed of 53 amino acid residues with a molecular weight of 6.045 Da and an isoelectric point of 4.6.EGF has many important biological functions and roles.However,the biological regulation of EGF on swine testicular cells is not completely clear up to now.In the current research,we mainly studied the following scientific issues;1)The biological activity and characteristics of EGF/EGFR on swine testicular cells(ST);2)The effect of heat stress on the biological activity of EGF/EGFR;3)Effect of senescence on biological activity of EGF/EGFR.Firstly,the cellular behavior of EGF on ST cells was studied in this experiment.The results of indirect immunofluorescence(IFA)showed that ST cells expressed abundant epidermal growth factor receptor(EGFR)which was mainly located on the cell membrane.In addition,there is also a small distribution of EGFR in the cytoplasm.Then the internalization kinetics of EGF and EGFR were studied by confocal laser scanning microscopy(CLSM).The results showed EGF and EGFR could internalize into ST cells in a time and dose dependent manner.Further exploration of the endocytic mechanism of EGF/EGFR found EGF/EGFR can co-localize with clathrin and caveolin,this indicating the internalization of EGF/EGFR may be mediated by clathrin.Western-blot showed EGF/EGFR activated intracellular signal pathway in a time and dose dependent manner.Secondly,the cellular behavior and signal characteristics of EGF under heat stress are not clear in swine testicular cells.We explored the effects of heat stress on EGF-mediated intracellular signaling and cellular behavior in the ST cells model.The results showed heat stress altered the cellular behavior of EGF/EGFR.It was found that under the condition of heat stress,EGF/EGFR did not undergo internalization and mainly localized on the cell membrane.That indicates the heat stress inhibits the internalization of EGF/EGFR.By further studying the effect of heat stress on EGF signaling characteristics,the results showed the ability of EGF to activate EGFR-mediated intracellular signaling is significantly reduced under heat stress conditions.Finally,we explore the effects of senescence on EGF/EGFR cell behavior and signal transduction properties.Western-blot showed leads to a significant downregulation of the activation intensity of EGFR,STAT3,AKT,and STAT5 signaling molecules mediated by EGF/EGFR.The experiments have shown senescence could cause the decrease in the biological activity of EGF/EGFR.In summary,under normal physiological conditions we found EGF and EGFR can be internalized into ST cells in a time-dependent manner.And can activate p-EGFR,p-STAT3,p-AKT and p-STAT5 signaling pathways.On this basis,we further explored the effects of heat stress and senescence on the biological activity of EGF/EGFR.That found both heat stress and aging can lead to a significant down-regulation of intracellular signal transduction mediated by EGF/EGFR.This study laid a theoretical foundation for exploring the physiological regulation of EGF/EGFR on the testis.