Role Of Selenoprotein U In Sertoli Cells In Selenium Deficiency Chicken Testis

Selenium?Se?is required for the maintenance of structural integrity of sperms,development of seminiferous tubules and supply of Se to the testis.Se deficiency is associated with the impaired sperm motility and seminiferous tubule degeneration.Se induces its effects through selenoproteins.Most the selenoproteins with known functions contain anti-oxidative and redox regulating properties.Therefore,Se deficiency induces oxidative stress leading to cell injury and/or death.Selenoprotein U?SelU?is a newly identified protein,and expresses widely in human and chicken.Similar to other thiol-dependant redox family of selenoproteins,SelU sequence also possesses a thioredoxin like-fold;however the function is still unknown.Therefore,we designed this study to determine the:?1?Role of SelU in autophagy and apoptosis in chicken Sertoli cells?SCs??in-vitro?;?2?Effect of Se deficiency on the expression of selenoproteins in chicken testis?in-vivo?;?3?Effect of Se deficiency on the oxidative status and histopathology in chicken testis?in-vivo?;?4?Effect of Se deficiency and SelU silencing on the expression of regulatory factors and Junction associated genes in chicken SCs?in-vitro&in-vivo?.Accordingly,for in-vitro experiment,we developed a SCs culture model to investigate the role of SelU in autophagy,apoptosis,regulatory factors and junction associated genes in SCs.Primary SCs were isolated using 6 weeks-old immature laying breed chickens,and cultured on HEPES-buffered F12/DMEM.SCs were identified microscopically by the presence of characteristic morphological features.Later,cells were transfected with specific SelU targeting siRNA to establish SelU-knockdown?SelU-KD?and normal?N?SCs models.Harvested cells were subjected to electron microscopy,qPCR,and western blot.For in-vivo experiment,180 day-old layers chicks were equally divided into two groups'viz.,Control?C group-0.333 mg/kg?and Low?L group-0.033 mg of Se/kg?groups.Testis were collected at day 60,day 90 and day120.Tissues were subjected to H&E staining,enzymatic assays and qPCR.In-vitro results showed that compare to N cells,SelU decreased by 76%and 63%at mRNA and protein level,respectively,in SelU-KD cells as indicative of successful establishment of SelU-KD model.qPCR results showed that the mRNA of Beclin-1,LC3-1,LC3-2,Bcl-2,mcl-1,f-actin,?-tubulin,TSC1 and TSC2 genes increased?P<0.05?in SelU-KD cells as compare to N cells while decreasing?P<0.05?mTOR,caspase3,Bax,PI3K,Akt1 and Akt2.Western blotting revealed an increase?P<0.05?in the proteins of Beclin-1,LC3-1,LC3-2 and Bcl-2in SelU-KD while decreasing the protein levels of mTOR,caspase3,Bax,p-PI3K,PI3K,p-Akt and Akt.Concurrently,extensive accumulation of cytoplasmic autophagosoms,decreased in lipid droplets and presence of lysosomal degradation were observed in SelU-KD cells.The obtained data illustrated that SelU silencing disrupts PI3K-Akt signaling leading to inhibition of mTOR signaling which stimulate Beclin-1 mediated autophagy.Furthermore,SelU depletion upsurge Bcl2 mediated suppression of casp-3 as indicative of inhibition of apoptosis in SelU-KD SCs.Results further indicated that TSC aid to the promotion of autophagy by suppressing mTOR.We concluded that SelU deficient SCs underwent autophagy via inhibition of PI3-AkT-mTOR pathway while suppressing apoptosis in chicken.We further concluded that SelU regulates the survival and functioning of SCs via PI3K-Akt-mTOR pathway.In-vivo results reveal that in normal physiological conditions,Gpx,Txnrd,Slew and Selp expressed relative highly in testis.At day 60,Se deficiency reduced?P<0.05?16selenoproteins including most the antioxidative and redox regulating selenoproteins.Compare to C1,maximum and minimum suppressions were observed in Slew and Selo,respectively.Contrastingly,the mRNA levels of Gpx4,Txnrd3,Selp15,Sepx1,Selp and SPS2 increased in L1 group.At day 90,20 selenoproteins decreased?P<0.05?in Se deficient testis.Gpx3,Slew and Sels reduced relative highly followed by Gpx1,Gpx2,Dio1,Txnrd2,Selm,Selt,Selh and Selu.Similar to L1,the levels of Selp remain high in L2 at day 90.At day 120,Se deficiency significantly decreased?P<0.05?the mRNA expression of all 24 selenoproteins.Maximum decrease was recorded in Gpx3 followed by Slew,Selh,Selm and Selu.Se deficiency gradually suppressed all selenoproteins in a time dependant manner.Antioxidative and Rdx family of selenoproteins was more affected by Se deficiency than other selenoproteins leading to oxidative stress.Further,enzymatic assay results showed that Se deficiency significantly decreased?P<0.05?the activities of SOD and Gpx in a time dependant manner.Maximum decrease was recorded at day 120.Consequently,MDA and H₂O₂ contents significantly increased?P<0.05?in Se deficiency.The results suggested that gradual loss of antioxidative and ROS scavenging capabilities of Gpx and SOD eventually result in tissue accumulation of H₂O₂ and MDA,as indicative of the establishment of oxidative stress.Se deficiency-induced oxidative stress implicated sever histo-architectural changes in testes leading to BTB disruption and hypospermatogenesis.In-addition,combined in-vivo and in-vitro results indicated that IGF-1,IGF-?,GDNF,EGF and SCF express in normal chicken SCs.Se deficiency decreased the expression of IGF-1.However,in-vitro study showed an increase in IGF-1 levels.Additionally;IGF-?increased in both SelU knockdown and Se deficient conditions.Se deficiency and SelU silencing substantially inhibited the expression of GDNF,EGF and SCF in a time dependant manner.Results suggest that Se deficiency and SelU knockdown altered the expression of IGF-1,IGF-?,GDNF,EGF and SCF in SCs via SelU-PI3K-Akt inhibition.Moreover,we showed that occludin,claudin3 and AKAP9 express in SCs in normal conditions.In-vitro and in-vivo study indicate significant decrease in occludin,claudin3,AKAP9,vinculin and?-catenin in SCs.However,?-catenin increased in SelU deficient SCs.Simultaneously,Se deficiency disrupted both ERK and PI3K-Akt pathway signaling in SCs.Both in-vitro and in-vivo findings indicated that the suppression of TJs,AJs and PI3K-Akt/ERK signaling cascades changed TJ and AJ dynamics.Our findings suggested that Se via SelU preserve positive influence on junction associated genes and therefore sufficient levels of Se is essential for the maintenance of BTB integrity.In summary,we concluded that Se regulates the expression of selenoproteins in testes.Se deficiency suppressed the global expression of selenoproteins in chicken testis including SelU.SelU is involved in the survival and functioning of SCs via various mechanisms.In normal testis,SelU suppressed autophagy and regulate the expression of regulatory factors,and thereby,played role in spermatogenesis.SelU strengthens the linkage between the adjoining SCs and maintains BTB integrity by regulating junction associated molecules.Se via SelU preserve positive influence on SCs-regulated spermatogenesis and therefore sufficient level of Se is essential for the maintenance of normal SCs physiology in chicken testis.