Role Of AMPK Signaling Pathway In The Expression Of Heat-induced Tight Junction Proteins In Porcine Sertoli Cells

Heat stress refers to a series of physiological responses to high environmental temperature over thermal comfort zones.In summer,heat stress caused by continuous high temperature results in huge loss of the animal husbandry.Due to lack of sweat glands,it is difficult for porcine to evaporate the heat through the skin with abundance of fat under skin.Based on these reasons,hyperthermia causes porcine spermatogenesis failure,resulting in oligospermia,azoospermia,and increases rate of sperm deformity.Furthermore,due to its poor reproductive performance,pig frozen semen has not been popularized in large area,which makes the influence of heat stress on porcine spermatogenic ability appear more obvious.Thus,a comprehensive study on the effects of heat stress on spermatogenesis of porcine may provide theoretical basis and practical significance to avoid the heat stress-induced spermatogenesis dysfunction.Tight junction,the important compo nent of blood testis barrier,plays a vital role in spermatogenesis.The tight junction at the interface of two adjacent Sertoli cells,which is highly dynamic and undergoes remodeling to allow the passage of preleptotene spermatocytes from the basal compa rtment to the adluminal compartment,where they undergo meiosis and develop into elongating/elongated spermatids.Besides,its barrier function provides a stable microenvironment for spermatogenesis.Heat stress can induce the down-regulation and translocation of tight junction proteins and further dysfunction of tight junction.Most of reports about the effects of heat stress on the tight junction between Sertoli cells are in vivo experiments.Although a few in vitro experiments focused on the effects of heat stress on the tight junction,the subjects were matured Sertoli cells.It is unk nown whether tight junction proteins express in immature Sertoli cells and whether heat stress affects the expression of tight junction proteins in porcine Sertoli cells.Because the tight junction between Sertoli cells are gradually formed,the impacts of heat stress on the tight junction proteins in immature Sertoli cells may affect the assembly of blood testis barrier.In the present study,we explored the effects of heat stress on the tight junction proteins in immature porcine Sertoli cells,and investigated the mechanisms.AMPK regulates energy metabolism in eukaryotic cells and participates in the regulation of tight junction in epithelial cell: activated AMPK promotes the assembly of tight junction and silence of AMPK leads to dysfunction of tight junction.It is unknown whether AMPK regulate the expression of tight junction proteins in Sertoli cells.Heat stress often leads to the increased level of intracellular oxidative stress,which causes the cellular oxidative damage.Previous studies showed that ROS can regulate the activity of AMPK through affecting the expression of CaMKK?.Based on the above background,we hypothesized that whether heat stress-induced oxidative stress affect the expression of tight junction proteins via regulating the activity of AMPK.The results are as follows.1.Establishment of in vitro heat treatment model of porcine Sertoli cells and effects of heat treatment on the expression of tight junction proteinsCompared the effect of two ways of heat treatment(43? water bath for 30 min and 43? gas bath for 30 min)on the viability of Sertoli cells,results showed that the transient heat treatment with gas bath significantly decreased cell viability after heat treatment(reduced by 34.46% compared with control,P < 0.01).The cell viability gradually recovered to normal levels at 48 h after heat treatment.Transient heat treatment with water bath decreased cell viability by 57.94%(P < 0.01);however,the viability further decreased and did not recover at 48 h after heat exposure(29.49% of control,P < 0.01).Therefore,we used the gas bath method for heat treatment for subsequent studies.The mitochondrial membrane potential(MMP)was measured as an indicator of the early apoptosis rate of Sertoli cells.The early apoptosis rate of Sertoli cells increased by 3.68× the control after heat treatment(P < 0.01).However,the early apoptosis rate gradually decreased with recovery time,and recovered to the normal level at 48 h after heat treatment.Although tight junction proteins(C laudin-11,JAM-A,Occludin,and ZO-1)were found to express in immature Sertoli cells,no tight junction was observed between two adjacent Sertoli cells.Both m RN A and protein levels of tight junction proteins were down-regulated by heat treatment,but gradually recovered after removing heat treatment.In addition,C laudin-11 was found to be localized at the plasma membrane,cytoplasm,and the membrane of transitional vesicles in the control cells,whereas it was localized at the membrane of transitional vesicles alone after heat treatment.The expression of C laudin-11 increased at 48 h after heat exposure,and its location was recovered.The above results indicated that heat treatment induced reversible down-expression of tight junction proteins and changed the cellular localization of Claudin-11.2.Effect of AMPK on heat-induced reversible down-expression of tight junction proteinsThe AMPK phosphorylation level significantly decreased after heat exposure(reduced by 60.04% compared with control,P < 0.01).Furthermore,the AMPK activity was gradually increased with recovery time.To detect the mechanism by which heat treatment inhibits AMPK phosphorylation,the factors upstream of AMPK : ATP,LKB1,and CaMKK? were detected.The results showed that heat treatment decreased the ATP concentration in Sertoli cells(48.92% of control,P < 0.01).Furthermore,the CaMKK? expression level was significantly decreased after heat treatment(46.18% of the control,P < 0.01),and showed a similar recovery pattern as AMPK phosphorylation.However,heat treatment did not affect LKB1 phosphorylation(P > 0.05).To further confirm whether CaMKK? is an upstream regulator of the AMPK signaling pathway in heat treatment,STO609 was used to inhibit the activity of CaMKK?.STO609(10 ?mol/L,2 h)was found to further suppress AMPK activity by 62.19%,compared with heat treatment alone(P < 0.01).However,STO609 alone did not significantly affect AMPK phosphorylation.These findings suggested that heat treatment inhibited the activity of AMPK via reducing the expression of CaMKK?.To further explore the role of AMPK in heat-induced reversible down-expression of tight junction proteins,AICAR(activator of AMPK,2 mmol/L for 2 h)was used to treat Sertoli cells.Results showed that pretreatment with AICAR significantly up-regulated both the m RNA and protein levels of tight junction proteins after heat exposure,compared with heat treatment only.Additionally,pretreatment with AICAR resulted in reorganization of C laudin-11,causing it to appear at the plasma membrane and cytoplasm again.AMPK over-expression vector pc DNA-AMPK/ AMPK si RNA were used to over-expression/knock down AMPK,results showed that heat-induced down-regulation of tight junction proteins were remarkably rescued at both the m RNA and protein levels in AMPK over-expressing cells.However,AMPK knockdown caused further decrease of tight junction proteins,and C laudin-11 was almost undetectable after heat treatment.These results indicated that AMPK played a crucial role in heat-induced down-regulation of tight junction proteins.3.Role of oxidative stress in heat-induced reversible down-expression of tight junction proteinsHeat treatment caused increased level of ROS and MDA,and decreased activities of SOD,GSH-Px,and CAT.Furthermore,these changes were reversible and gradually recovered with recovery time.Pretreatment with NAC(1 mmol/L,2 h),Sertoli cells showed a stronger tolerance to heat stress.Heat-induced ROS and MDA level were significantly reduced and the activity of SOD,GSH-Px,and C AT were remarkably increased by NAC.In add ition,NAC lowered the heat-induced early apoptosis rate and increased the viability of Sertoli cells.Furthermore,NAC up-regulated the expression of CaMKK?,and increased the activity of AMPK.These finding showed that heat treatment inhibited CaMKK?-AMPK signaling pathway by inducing the generation of ROS.Furthermore,pretreatment of NAC rescued the down-expression of tight junction proteins induced by heat exposure.In conclusion,heat treatment induced reversible down-regulation and re-localization of tight junction proteins through ROS generation,which decreased the expression of CaMKK? and the activity of AMPK.The conclusion revealed the mechanisms of heat treatment affecting the tight junction in porcine testis and provided theoretical basis for reducing the negative effect of heat stress on the spermatogenesis.