Sertoli Cell-specific Disruption Of Rheb Gene Causes Impaired Spermatogenesis In Mice

Background:Rheb is an important upstream regulatory protein of mTOR signaling pathway.Rapamycin,a pharmacological inhibitor of mTORC1 can directly combine mTOR inhibiting its activity and GDP-bound Rheb has a same inhibitory effect with rapamycin.Clinical use of rapamycin as organ transplantation immunosuppressive agent causes male patients infertility,which proves Rheb-mTOR signaling pathway is indispensable in the process of male reproduction.Recently,researchers have reported the function of Rheb in male germ cells in the process of spermatogenesis,but its role in the regulation of testicular sertoli cells still unknow and further laboratory evidence and related mechanism research is needed to expound it.Objective:To investigate the function and underling mechanisms of Rheb protein in sertoli cells in testicular development and spermatogenesis.Methods:1.Use of Amh-cre mice(mice specifically expressing cre enzyme in testis sertoli cells)and RhebL/L mice specifically knockout Rheb gene in sertoli cells and investigate the growth of testes,sperm production process and dynamics and the impact of fertility.2.Intraperitoneal injection of busulfan on the basis of Rheb ablation to construct sperm regeneration model and to research the growth of testes,sperm production process and dynamics and the impact of fertility in pathological state.3.Use of flow cytometry,western blotting and immunohistochemical methods to study percentage changes of spermatogenetic cells at all levels in the testis samples and to find out the key essence of cell types regulated by Rheb in sertoli cells.4.Use of RNA-seq and Real-time PCR to research related signaling pathways and molecular mechanisms during Rheb ablation in testes.Results:1.Identification of Rheb gene knock out in sertoli cells.Rheb specific deletion mice Amh-cre;RhebL/L were constructed through crossing the RhebL/L mice with Amh-cre mice.Rheb mutation was confirmed respectively from the level of DNA,RNA and protein and the results show that specific knockout occurs in testicular tissue in mice reminding us of a successful Rheb deletion model.2.Rheb mutation effects on Testicular development and spermatogenesis.Rheb knockout mice show testicular dysplasia and the testes were decreased significantly with the volume and weight of about 50%to the control group.Epididymis was also smaller by the age of adult.Mice with age of 1 month present a large number of giant multinucleated cells in seminiferous tubules.Detection of sperm count and motility found that Rheb knockout mice show a significant reduction in the number of sperm in caudul epididymis about 50%of the control group,but the sperm motility has no obvious change.Sperm head abnormity was found in the microstructure of Rheb knockout mice testes and sperm abnormity was also significantly increased after sperm counting in the cauda of epididymis using HE staining.But the fertility rate were not significantly affected during a 2 to 9 months detection in adult Rheb mice and the control group.3.Fertility of Rheb knockout mice were declined during pathological condition.Through the injection of busulfan,sperm damage model is constructed.The sperm counts and activity of Rheb knockout mice in pathological state were hit significantly.Sperm counts were down to the base line of fertility causing infertility,and the control group was not affected.4.Related regulating mechanisms of Rheb in sertoli cells during testis development and spermatogenesis.Significant decrease of testes in Rheb mutation mice was due to the reduction of testicular seminiferous tubules size and number.Spermatocytes decreased significantly during the detection of flow and Sycp3 labled spermatocytes by Immunohistochemistry were also decreased.Cell apoptosis and proliferation were tested using TUNEL and Ki67.The results show us that spermatocytes apoptosis were increased but proliferation was not affected.Through RNA-seq and Real-time PCR,the differentially expressed genes in the testis were tested and found that the expression of Tf,a sertoli cell specific secreted gene was markedly increased and the related iron balance within the testis was affected.Also we detected the significant increase of apoptosis related factor IGFBP3.Conclusion:Rheb in sertoli cells is essential for the process of testicular development and spermatogenesis.Rheb ablation leads to abnormal meiosis and obvious spermatocytes apoptosis.This process is at least partly regulated by the Tf regulation pathways.