Study Of Function And Regulatory Mechanism Of Pentraxin3 In Mouse Sertoli Cells During Heat Stress Recovery

PTX3 is a multimeric secreted protein belonging to the Pentraxins family which is highly conserved in evolution.Expression of PTX3 is induced by stress stimulation such as endoplasmic reticulum stress,oxidative stress,pathogen infection,and inflammatory cytokines such as IL-1,TNF-?,IL-6.The temperature of spermatogenesis is lower about 2-4°C than core body temperature,while hyperthermia can cause testicular tissue damage,sperm cells disorder in the seminiferous tubules and induce inflammatory cytokine expression,also impair spermatogenesis.The injury caused by transient heat stress can recover after temperature resuming,which indicates that there is a temperature-sensitive mechanism in testis responding to heat stress.Therefore,stress-related protein PTX3 may have a role in regulation of spermatogenesis recovery after heat stress.In mouse sertoli cell culture by detecting the expression pattern of PTX3 and function-related proteins(CEBP-?,p-ERK1/2,apoptosis-related protein,BTB-related protein,secreted proteins such as GDNF,SCF during recovery after heat stress along with RNA interference to knock down PTX3 expression before heat stress,the possible mechanisms of PTX3 functional change during heat stress recovery in sertoli cells were revealed.The main results are as follows:(1)Effects of heat stress on expression and localization of PTX3 in sertoli cells.After heat stress(43°C,1h)treatment,sertoli cells were recovery for different time(0,3,6,12h),results showed that the expression of PTX3 mRNA and protein was significantly increased in 6h after heat stress recovery and decreased subsequently to normal level The localization of PTX3 was also changed from cytoplasm to nucleus(2)Effects of transient hyperthermia on expression on upstream regulators of PTX3.Results showed after heat stress treatment(43°C,1h),the expression of CEBP-? and p-ERK1/2 were significantly increased during heat stress recovery period.Since CEBP-? and p-ERK1/2 are known upstream regulators of PTX3.The possible mechanism of transient heat stress-induced spermatogenesis damage and subsequent self-repair may be related to p-ERK1/2,CEBP-?,PTX3 pathway.(3)The effect of heat stress on apoptosis related gene expression and possible relationship with PTX3.Further study showed that heat stress induced a significant increase of Bcl-2 and CAT mRNA level in sertoli cells,while the increase of Bcl-2 and CAT mRNA level was blocked by knocking down of PTX3 expression,and the cell viability was also significantly decreased by heat stress treatment.These results suggest that increased expression of PTX3 during heat stress recovery period might be related to testis' resistance to hyperthermia and adjustment.(4)The effects of heat stress on BTB-related gene expression in sertoli cells and possible relationship with PTX3.The gene expression of BTB related proteins(N-cadherin,?-catenin and ZO-1)was decreased and then recovered after heat stress.While knocking down of PTX3 expression blocked the decrease of N-cadherin,and ZO-1 mRNA expression while ?-catenin mRNA expression had no significant change during recovery period after heat stress.These suggest that increased expression of PTX3 during heat stress recovery period may also be associated with the recovery and modulation of the blood-testis barrier function of sertoli cells.(5)The effects of heat stress on the gene expression of important functional secreted proteins in sertoli cells and possible relationship with and PTX3.Several important functional proteins secreted by sertoli cells,such as GDNF,SCF and Fas L,and play important roles in the self-renewal,differentiation and apoptosis of spermatogonial stem cells.The mRNA expression of GDNF and Fas L increased at first and then decreased after heat stress.While knocking down PTX3 blocked the expression recovery of these two proteins.This suggests that the expression of PTX3 induced by hyperthermia may also be related to the functional recovery and regulation of the sertoli cells after heat stress.AboveIn conclusion,above results suggest that PTX3 induced in sertoli cells during heat stress recovery may play an important role in the regulation of cell apoptosis and viability,BTB and secretory function,and subsequent functional recovery of sertoli cells.