The Function Study Of Exosome And Y-box Protein 2in Spermatogenesis

Objective: In the early stages of spermatogenesis,spermatogonial stem cells(SSCs)self-renew and continually produce spermatogonia,which subsequently become spermatocyte and finally differentiate into mature spermatozoa.The molecule mechanism and regulatory factors involved in the self-renewal of spermatogonial stem cells remains poorly understood.In the late stages of spermatogenesis,spermatocytes complete meiotic division and differentiate into haploid spermatids.Transcription of spermatids ceases during the development of haploid spermatogenic cells.mRNAs essential for haploid spermatid differentiation are synthesized and are stored by RNA binding proteins until these mRNAs are needed and then translation occurs.However,the molecular mechanism that allows these mRNAs to become translationally competent remains unknown.Method: Exosomes were tracked in the testes from different species and mice at different days post-partum.Exosomes were separated by two-step enzyme digestion and differential ultracentrifugation.Western blot analysis and immunoelectron microscopy were used to detected the source of these exosomes.SSCs were exposed to exosomes to explore whether exosomes effect the proliferation and differentiation of SSCs.GST pull-down assay was performed to identify the testis proteins interacting with YBX2.Western blotting and immunofluorescence was used to verify the expression of PAIP1 in mouse testes.Co-immunoprecipitation assays were used to identify the YBX2 binding sites in PAIP1.Testis mRNAs bound by the YBX2–PAIP1 complex were profiled by sequential RNA immunoprecipitation.Luciferase activities were assayed to further determine the translational role of the interaction between PAIP1 and YBX2.Results: Exosomes are present in spermatogonia of mouse,rat,and human testes.Exosomes are tracked in the spermatogonia from mice at different days post-partum.Western blot analysis of exosomes reveals that they were all detected together with spermatogonial membrane proteins.Further,immunoelectron microscopy shows that GFR?1 proteins are detected on testicular exosomes in the testis sections from mice.After exposed to testicular exosomes,the proliferation of SSC were suppressed.RNA sequencing indicates testicular exosomes alter the expression patterns of genes linked to SSC self-renewal.YBX2 interacts with PAIP1 in vitro and in vivo.In murine testes,PAIP1 is highly expressed and starts being expressed in testis from pachytene spermatocyte to elongating spermatids.YBX2 alone significantly represses translation activity of Luciferase.Co-expression with PAIP1 and YBX2 can re-initiate the translation of Luciferase mRNAs.Conclusion: Testicular exosomes are secreted by spermatogonia and repressed SSCs proliferation by influenced the expression patterns of genes involved in SSC self-renewal.The translational enhancer PAIP1 is highly expressed in murine testes and located in pachytene stage of spermatocyte and elongating stage of spermatids.In vitro studies indicates that PAIP1 stimulates translation of mRNAs stored by YBX2 via its interaction with YBX2.