The Role And Mechanism Of TOCP In The Damage Of Mature Sperm In Mice

Objective:Tri-ortho-cresyl phosphate(TOCP),a common plasticizer,is widely used in our daily life and has raised increasing public concerning about its reproductive toxicity.Previous studies have shown that TOCP influences sperm production as well as spermatogenesis.However,whether it has effects on the mouse sperm functions has not been reported in literature.In this study,we observed the effects of TOCP on mouse sperm functions by using chronic TOCP exposed mouse model and the method of invitro incubation.Furthermore,we explored the effects of sperm-specific ion channels including CatSper and Slo3 on mouse sperm functions.New insight and method could be accumulated to the molecular basis of male reproductive toxicity induced by plasticizer such as TOCP and the clinical diagnose and treatment of relevant reproductive diseases.Methods:1.Establishing chronic TOCP exposed mouse model by means of intragastric administration in adult male mice with 0,100,200 and 400 mg/kg/d TOCP solution.2.Observing the reproductive toxicity of TOCP in testicular tissues by investigating testis index and HE staining in poisoned mice.3.For the followed experiments in vitro,sperm were incubated with final concentration of 0,0.25,0.5,200 1mmol/L TOCP for 60 min,respectively.4.Examining the viability and motility of sperm in chronic TOCP exposed mouse model and in vitro incubation model by computer-assisted sperm analysis(CASA)system.5.Evaluating spontaneous or P4 induced acrosome reaction(AR)occurrence of mouse sperm in chronic TOCP exposed mouse model and in vitro incubation model by chlortetracycline(CTC)staining.6.Detecting the protein expression changes of sperm-specific calcium channel(CatSper1-4)and potassium channel(Slo3)in chronic TOCP exposed mouse by using Western Blot7.Using sperm patch-clamp technique,currents of CatSper and Slo3 channel(ICat Sper and IKSper)were recorded in chronic TOCP exposed mouse model and in vitro incubation model,respectively Result:1.Histopathological examination of testes showed that structure of testicular seminiferous tubules without obvious destructions in the 100mg/kg/d TOCP treated group,while became significantly disorganized in the 200mg/kg/d and 400mg/kg/d TOCP treated groups,in which the spermatogenic cells shed and the testicular index decreased significantly(p<0.01;p<0.001).2.CASA examination showed that TOCP inhibited sperm viability and motility significantly with dose dependent in chronic feeding group as well as acute incubation group(p<0.05).3.CTC staining results showed that 400mg/kg/d TOCP exhibited significant inhibition on the rate of spontaneous and P4 induced AR in chronic feeding group(p<0.05).0.5mmol/L TOCP exhibited significant inhibition on the rate of P4 induced AR in acute incubation group(p<0.05),leaving the spontaneous acrosome reaction unaffected.1 mmol/L TOCP exhibited significant inhibition on the rate of both spontaneous and P4 induced AR in acute incubation group(p<0.05).4.Western Blot results showed that CatSper1,CatSper2,CatSper3,and Slo3 proteins in the mouse testes of chronic feeding group reduced significantly(p<0.001)with increase of TOCP dosage,while CatSper4 protein exhibited significant decrease only in 400mg/kg/d TOCP treated group.5.Whole-cell recording results showed that TOCP had significant inhibition effect on ICatSper of sperm in the chronic feeding group(p<0.001),and without current change in 400mg/kg/d treated group(p<0.01).While TOCP had significant inhibition effect on ICatSperr of sperm in the acute 1mmol/L TOCP incubation group(p< 0.05),with no effect on IKSper.Conclusion:1.TOCP,a plasticizer,impairs the physiological functions of mouse sperm.2.The inhibition of sperm-specific ion channels including CatSper and Slo3 is involved in the mechanism of functional damage induced by TOCP in mouse sperm.