The Second Extracellular Loop 22AA Of Occludin Protein Reversibly Regulates Tight Junctions By Down-regulating Occludin And Inducing Apoptosis

In the testis,there is a blood testis barrier(BTB)between the seminiferous tubules and blood vessels.Sertoli cell tight junction(TJ)is an important part of BTB.In recent years,studies have found that abnormal support cell tight junctions(TJ)leading to the destruction of BTB is an important cause of male infertility.Occludin protein is an important structural protein that supports cell tight junctions(TJ)in the blood-testis barrier(BTB).Studies have found that the 22-amino acid peptide(22AA)of the second extracellular loop of Occludin can reversibly regulate tight junctions through Occludin protein.However,its regulatory mechanism is still unclear.In this study,the following questions were studied in depth by constructing a mouse model,combining Western Blot,Real time PCR,immunofluorescence double labeling,cell smear and other biomolecular technologies.Does 22 AA affect the expression and localization of Occludin and the expression of Occludin-related miR(miR-122-5p)?How to dynamically affect the number of supporting cells and sperm cells?Does 22 AA affect the number of offspring?1.Construct a mouse model by injecting 22 AA blocking peptide(the structure is the same as the second extracellular loop of Occludin)into the testis tissue of adult male KM mice(6-8 weeks old).The mice were random Ly divided into six groups,six in each group.2.Through Western Blot and immunofluorescence to detect the expression and localization of Occludin protein after TJ collapse and reconstitution,it was found that the Occludin protein of the control group was mainly distributed in the basement membrane of seminiferous tubules and supporting cell TJ.In the first 27 days after injection of the blocking peptide(22AA),the expression of Occludin protein gradually decreased,the distribution decreased,and the morphology was gradually incomplete.After 37 days,Occludin began to recover a small amount of expression.The morphological structure of Occludin on day 47 was basically similar to that of the control group.QPCR detection of Occludin-related miR(miR-122-5p)expression revealed that miR-122-5p was negatively correlated with Occludin protein expression.22 AA can reversibly down-regulate the expression of Occludin protein and up-regulate the expression of miR-122-5p.3.The morphological results and apoptosis of Sertoli cells and sperm cells were detected by immunofluorescence double labeling.It was found that there were a large number of Sertoli cells and sperm cells in the seminiferous tubules of the control group,with clear boundaries and a small number of apoptotic cells Bax.In the first 27 days after the injection of the blocking peptide(22AA),the Sertoli cells and sperm cells gradually became incomplete.On the 27 th day,the Sertoli cells and sperm cells were almost incomplete,the expression gradually decreased,and the expression of the apoptotic protein Bax gradually increased.After 37 days,the expression began to resume.Sertoli cells and sperm cells began to distribute in a small amount in the basal layer of the seminiferous tubules,and the apoptotic protein Bax decreased,indicating that TJ began to recover and rebuild.22 AA can reversibly promote the apoptosis of Sertoli cells and sperm cells and destroy the morphology of Sertoli cells.4.The number of sperm after TJ collapse and reconstruction was detected by cell smears,and the fertility analysis of mice was performed.It was found that in the first27 days after injection of the blocking peptide(22AA),the number of sperm was greatly reduced,and the number of litters in the mice also gradually decreased.,Began to recover after 37 days,the number of sperm gradually increased,and the number of litters also gradually increased.There are significant differences in the number of sperm and litter size in each group of mice.22 AA can reversibly change the number of sperm and affect the fertility of male mice...