Effects Of PTBP1 On The Integrity Of Blood-testis Barrier And Sertoli Cell Autophagy In Mice

Objectives :Sertoli cells are one of the most important components of the germinal tubules and can be involved in forming the blood-testis barrier.Blood-testis barrier plays an essential role in maintaining the growth and development of germinal cells and spermatogenesis.Polypyrmidine tract-binding protein 1(PTBP1)is a key protein regulating precursor m RNA splicing and selective splicing events,which not only promotes spermatogonia proliferation but also regulates the expression of tight junction-related proteins.PTBP1 can activate PI3K/Akt pathway and inhibit autophagy to promote cell growth,and both the PI3K/Akt pathway and autophagic activity play an important role in sertoli cells to maintain the integrity of blood-testis barrier.Therefore,we further explored whether PTBP1 regulates the integrity of blood-testis barrier by affecting the expression of tight junction-related protein and autophagy,based on the observation that the expression of PTBP1 in sertoli cells decreases significantly with age.Methods :1.The left side testicular tissues from 2 months,6 months and 18 months mice were collected.The histological changes were observed by paraffin section and transmission electron microscope.2.The right side testicular tissues from 2 months,6 months and 18 months mice were collected as well.The expression of PTBP1 was detected by Western blot and real-time q PCR and immunofluorescent staining.3.The tail of epididymis tissues from 2 months,6 months and 18 months mice were collected.The activity of hyaluronidase(HYD)and penetration rate of sperm.4.Primary SCs were isolated and cultured by collagenase IV and trypsin digestion method.To identify cell purity with Sox-9 and WT-1 were detected by immunohistochemistry and immunofluorescence respectively.The expression of PTBP1 in primary SCs was detected by immunofluorescence staining.5.Primary SCs were transfected with recombinant lentivirus Lv-sh-EGFP and Lv-sh PTBP1-EGFP respectively.Three days later,the expression of EGFP was observed under fluorescence microscope.The expression of PTBP1 and TJ related proteins ZO-1,occludin and claudin-5 were detected by Western blot and real-time q PCR.6.Primary SCs transfected with recombinant lentivirus LV-sh-EGFP and LV-sh PTBP1-EGFP respectively were transfected into Trans-well plate.Three days later,the permeability of BTB was detected by the trans epithelial electrical resistant(TEER).7.Primary SCs were transfected with recombinant lentivirus Lv-sh-EGFP and Lv-sh PTBP1-EGFP respectively.Three days later,the expression of PI3K/Akt/m TOR pathway and autophagy related proteins(LC3 ? /LC3 ?,Beclin1 and p62)were detected by Western blot.8.Primary SCs were transfected with recombinant lentivirus Lv-EGFP and Lv-PTBP1-EGFP respectively.Three days later,the expression of EGFP was observed under fluorescence microscope.The expression of PTBP1 was detected by Western blot and real-time q PCR.9.Primary SCs were transfected with recombinant lentivirus Lv-EGFP and Lv-PTBP1-EGFP and LY294002(PI3K pathway inhibitor)respectively.Three days later,the expression of PI3K/Akt/m TOR pathway and autophagy related proteins(LC3?/LC3?,Beclin1 and p62)were detected by Western blot.The expression of TJ related proteins ZO-1,occludin and claudin-5 were detected by Western blot and real-time q PCR.10.The posterior edge of the testicle mediastinum were injected into Lentivirus Lv-EGFP and Lv-PTBP1-EGFP and LY294002 of 6 weeks mice by microinjection.Then supplementary injection was performed on day 7,14 and 28.Four weeks later,the infection structure was observed by frozen section fluorescence microscope.The changes of TJ in testicular SCs were observed by transmission electron microscope.Results :1.The testicular tissue structure was intact,spermatogenic cells were well arranged between 2 months and 6 months mice;The arrangement of spermatogenic epithelium in testis in 18 months mice was disordered,the number of layers further decreased,most spermatogonia showed vacuolar changes,a large number of exfoliated spermatogenic cells could be seen in some lumens,and the connection between SCs and adjacent spermatogenic cells was loose or disappeared;2.The number of PTBP1 positive cells decreased,and the expression of PTBP1 protein and m RNA decreased significantly of 18 months mice.3.The positive rate and activity intensity of sperm HYD and in vitro fertilization rate of 18 months mice decreased significantly.4.Primary SCs was successfully isolated and cultured.Moreover,PTBP1 was expressed in primary SCs.5.Compared with untransfected primary SCs and Lv-sh-EGFP transfected primary SCs,the expression of PTBP1 and TJ related proteins(ZO-1,occludin and claudin-5)at protein level and m RNA level in primary SCs transfected with Lv-sh PTBP1-EGFP decreased significantly.6.The resistance of primary SCs transfected with Lv-sh PTBP1-EGFP decreased,the permeability of BTB in primary SCs transfected with Lv-sh PTBP1-EGFP increased.7.The expression of p-PI3 K and p-Akt and p-m TOR in PI3K/Akt/m TOR pathway decreased.The expression of autophagy related proteins LC3?/LC3? and Beclin1 increased significantly,and the expression of p62 decreased.8.Compared with that of the untransfected primary SCs and Lv-EGFP transfected primary SCs,the expression of PTBP1 at protein level and m RNA level increased significantly in primary SCs transfected with Lv-PTBP1-EGFP.9.Compared with that of the untransfected primary SCs and Lv-EGFP transfected primary SCs,the permeability of BTB decreased,the expression of TJ related proteins ZO-1,occludin and claudin-5 increased at protein level and m RNA level in primary SCs transfected with Lv-PTBP1-EGFP;the expression of p-PI3 K,p-Akt,p-m TOR,LC3?/LC3? and Beclin1 increased,and the expression of p62 decreased in primary SCs transfected with Lv-PTBP1-EGFP.The permeability of BTB increased,the expression of TJ related proteins ZO-1,occludin and claudin-5decreased at protein level and m RNA level in primary SCs transfected with LY294002;the expression of p-PI3 K,p-Akt,p-m TOR,LC3?/LC3? and Beclin1 decreased,and the expression of p62 increased in primary SCs transfected with LY294002.10.Compared with that of the lentivirus testicle without injection,the expression of PTBP1 and TJ related proteins ZO-1,occludin and claudin-5 transfected with Lv-PTBP1-EGFP increased significantly.The expression of PTBP1 and TJ related proteins ZO-1,occludin and claudin-5 transfected with Lv-sh PTBP1-EGFP decreased significantly.Through transmission electron microscope,it was observed that TJ was incomplete or missing after microinjection of Lv-sh PTBP1-EGFP into testicular reticulum.TJ was complete after microinjection of Lv-PTBP1-EGFP into testicular reticulum.Conclusions :In this study,we found that aging could be an important factor leading to the decrease of PTBP1 expression.PTBP1 can affect the expression of TJ-related protein and autophagy by regulating PI3K/Akt/m TOR pathway,leading to changing of the integrity of BTB and the microenvironment with maintaining the normal development of spermatogenic cells.