| In an organism,cells communicate with each other through the way of extracellular vesicles.Exosome is a kind of extracellular vesicles with double membrane structure in 50-150 nm,which can be detected in various body fluids.The exosomes can regulate the biological activity of different cells by loading protein,polypeptide,mRNA,long noncoding RNA,miRNA,cyclic RNA and tsRNA.At present,many studies have shown that the exosome plays an important role in tumor formation,migration and metabolism.In the process of spermatogenesis,spermatogonial stem cells,in addition to forma a differentiated fate of the spermatogonia,but also produce the same one with their own.The factors that secret from Sertoli cells play an important role in the maintenance and renewal of spermatogonial stem cells in the "niche" microenvironment of testicular seminiferous tubules.There are a large number of extracellular vesicles in epididymal fluid and seminal plasma,where the vesicles promote the deformation and maturation of spermatozoa by interaction with sperm.Therefore,extracellular vesicles play an important role in spermatogenesis.However,little is known about the regulation of spermatogonial stem cells in extracellular vesicles from Sertoli cells.In this study,we have identified the extracellular vesicles from Sertoli cells,and explored the effects of vesicles on the spermatogonial stem cells:(1)The testis of mice at different developmental stages were collected.The CD63 protein,a marker of exosomes,was detected by immunohistochemical method in the seminiferous tubules.We found that in mice at different developmental stages,CD63 was mainly located in the cytoplasm of Sertoli cells,and granular distribution,at the same time,the expression of CD63 in mice before 7 days postpartum was very high,which indicates that positive capsule containing CD63 play an important role in the process of spermatogenesis.(2)Through gradient centrifugation and ultracentrifugation,we isolated and purified the extracellular vesicles released from primary Sertoli cells for the first time.In the electron microscope,vesicles with double membrane structure can be observed.Nano particle tracker measured the size of the vesicle in 76 nm.The specific protein CD63 was in the vesicles by Western blot analysis.Therefore,this kind of vesicles were identified as exosomes from the morphological characteristics,particle size and marker proteins.(3)The DiI was used to label the exosomes(named as Dil-exo),and it was found that the DiI-exo could be attached to the surface of the primary spermatogonial stem cells and C18-4 cells.(4)We cultured cells for 16 h in serum-free medium with 100 ng/ ?L exosomes,and the cells without exosomes as control group.It was found that the C18-4 cells and the primary spermatogonial stem cells were in good state,the cells were plump and the surface was smooth after the addition of the exosomes,while the control group had granules on the surface of the cells,besides,the cells shrank and became worse.Therefore,this study was the first time to isolate and purify the exosomes from Sertoli cells.With phenotypic analysis,we detected exosome attached to the surface of spermatogonial stem cells and maintain the survival of spermatogonial stem cells.Through this study,we can further reveal the mechanism of Sertoli cells to regulate spermatogonial stem cells,and open up a new perspective for understanding the function of spermatogonial stem cells. |
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