Rictor/mTORC2 Regulates Cytoskeleton Organization Of Testicular Sertoli Cell And Spermatogenesis

BackgroundMammalian spermatogenesis is a complicated process including morphological changes and cell differentiation.The process of primary spermatocyte becoming to mature spermatozoa after a series of meiosis and morphological changes is called spermatogenesis.The mature spermatozoa further developed and maturated in the epididymis and capacitation,becoming the fertilization ability of germ cells.Testicular sertoli cells are the only body cells contacted with spermatogenesis cells in seminiferous tubules;it not only played roles in sertoli the structure,but also in nutrition and regulation.Mammalian BTB is a special structure connected by adjacent sertoli cells,in part by the leydig small vascular endothelial cell tight junction construction,and other testis specific adhesion,dermatomes and gap junctions.When the BTB dysfunction occurred,development and male reproductive ability were interfered with in sperm cells.At present,It has not been completely clarified that the molecular mechanisms of regulating cytoskeleton organization of testicular sertoli cell.The mammalian target of rapamycin(mTOR)is a highly conserved serine/threonine protein kinase.In vivo,mTOR exists in two distinct protein complexes,mTORCl(mTOR complex 1)and mTORC2(mTOR complex 2).mTORCl and mTORC2 regulate a variety of physiological functions by receiving different upstream signals.mTORCl contains mTOR,Raptor,Deptor,PRAS40 and mLST8.mTORCl is sensitive to short-term treatment with rapamycin.mTORC2,like mTORCl,also contains mTOR and mLST8,and it also contains another three unique members:Rictor,mSinl and PRR5/Protor.Although both of them contain the same members,mTORC2 is insensitive to short-term treatment with rapamycin.Less is known about mTORC2 function compared to mTORCl.Current opinions are that it can phosphorylate Akt at serine 473(S473)in the regulation of cell survival.In addition,Rictor is an important upstream factor in the regulation of actin.It may control the total cytoskeleton and dynamic balance through controlling of actin by PKC and small GTP enzyme Rac or Rho.Rapamycin is a specific inhibitor of mTORCl,it is used as an immunosuppressive agent in organ transplant patients,in order to suppress the immune rejection.The quantity of spern in male patients significantly reduced subsequently long-term use of rapamycin,resulting in male fertility being noticeably decreased.This suggests that mTOR plays an important role in male reproductive ability.However,it is unclear how mTOR affects testicular sertoli cells or stromal cell.The above findings suggest that mTORCl plays an important role in spermatogenesis,however,mTORC2 and mTORCl receive different upstream signals to regulate a variety of physiological functions,consequently,the role of sertoli cells mTORC2 in tihe BTB formation and spermatogenesis is still unknown.Recent studies have shown the Rictor,the key protein in mTORC2,participated in the formation of the BTB.Rictor has the same location as the Occludin,ZO-1 and actin.Down regulation of Rictor by using siRNA,microfilament network of primary support cells had been interfered with.However,the study did not report that the abnormal expression of Rictor protein in vivo affects function of testis and male fertility,and sufficient animal experiments will be needed to further elucidate the regulation function and mechanism of Rictor/mTORC2 on the BTB and spermatogenesis.In the study,we built sertoli cell-specific deletion of Rictor mice by using Cre-loxP recombinase system,and in combination with the cadmium chloride-induced BTB injury mice model,we aim to investigate the roles of mTORC2 in the the formation of cell differentiation and polarity of testicular sertoli cell,BTB,tescular development,spermatogenesis and sperm maturation,and elucidate the mechanisms of mTORC2 in regulating BTB and spermatogenesis,providing new knowledge for the regulation of spermatogenesis and experimental support and possible target for the prevention and treatment of spermatogenesis defect.Materials and methods1.We constructed the mice with testicular sertoli cell-specific deletion of Rictor by the application of Cre-loxP system.In order to know the genotype of mice by using PCR and agarose gel electrophoresis.In order to identify the knockdown effect by Western blot and immunohistochemistry.2.Analysis of effect of Rictor/mTORC2 on mice testis and epididymis tissue morphology and structure by HE staining,immunohistochemistry and immunofluorescence.3.Detection of seminiferous tubules apoptosis by TUNEL in Rictor/mTORC2 of mice.4.Detection of the effect of Rictor/mTORC2 on the changes of serum hormone in mice by blood biochemical analysis.5.Detection of the effect of Rictor/mTORC2 on the cycle of seminiferous tubules cells by the flow cytometer.6.Detection of the effect and mechanism of Rictor/mTORC2 on the testicular sertoli cell cytoskeleton and the BTB by immunofluorescence technique,Western blot technique,Pulldown and immunoprecipitation.7.Establishment of the BTB damage model by the treating normal mice with cadmium chloride.Results1.Rictor is expressed in Testicular sertoli cells during testicular development and down-regulated in a cadmium chloride(CdC12)-induced BTB damage model.To explore the role of Rictor/mTORC2 in spermatogenesis,we firstly characterize the expression of Rictor in seminiferous epithelium of mice of 2 weeks,4 weeks and 8 weeks.Immunohistochemical analysis showed that Rictor was firstly expressed at the center of seminiferous tubule at the age of 2 weeks.As testicular development proceeded,Rictor was predominantly expressed near the basement membrane of the seminiferous tubules,consistent with the localization of Testicular sertoli cells and BTB.This expression of Rictor implicates the important of its role in testicular development and function.CdCl2 leads spermatogenic defects via demaging BTB.To further examine the correlation of Rictor with BTB function and spermatogenesis,we established a CdC12-induced BTB disruption mouse model.BTB construction was damaged following 3 d of treatment with CdCl2,with an excess of vacuoles near the basement membrane of seminiferous tubules.No visible spermatozoa were observed in the epididymis of CdC12-treated mice.In addition,a decrease in Occludin and N-cadherin protein levels was observed in CdC12-treated testes.These data indicated the successful generation of the BTB disruption model.Immmunohistological staining showed that Rictor was significantly reduced after CdC12 treatment,demonstrating the important roles of Rictor in Testicular sertoli cells in maintaining BTB integrity.Moreover,we observed significant up-regulation of FBXW7 protein level in CdC12-treated testes.These results support the importance of Rictor in maintaining BTB integrity and spermatogenesis.2.Generation of mutant mice with Testicular sertoli cell-specific deletion of Rictor.Loss of Rictor disorded development of testis and epididymis.To further investigate the roles of Rictor/mTORC2 in Testicular sertoli cells with regard to BTB integrity and spermatogenesis,we generated mutant mice with Testicular sertoli cell-specific ablation of Rictor,by crossing Rictor loxP/loxP mice with transgenic mice with anti-Mullerian hormone(Amh)promoter-mediated Cre.Immunohistochemical analysis showed that Rictor expression was completely absent in the seminiferous epithelium of Rictor-cK0 mice,suggesting complete Cre-mediated recombination of the floxed Rictor gene.Immunoblotting of Rictor also showed a significant decrease in its expression in mutant mice compared with the controlled specimen.Taken together,these results demonstrate successful deprivation of the Rictor gene in Testicular sertoli cells in cKO mice.Next,we focused on seminiferous tubule development in cKO mice.Although there were no obvious differences in physical appearance and body weight between cKO mice and control littermates at any age,the testicular weight of cKO mice significantly decreased to 30%of that in the controlled mice(27.613.4 mg vs 89.0±0.8 mg,p<0.01).The epididymis in cKO mice was also obviously smaller than that in control mice(14.0±1.6 mg vs 24.7±1.1 mg,p<0.01).These findings indicate defective testicular development in cKO mice.To verify whether loss of Rictor in Testicular sertoli cells impedes seminiferous tubule development,we compared the seminiferous tubules between control and cKO mice morphologically.Abnormalities were observed in the seminiferous tubule in cKO mice as early as 4 weeks old.With increased age,more severe defects were observed,such as progressive lesions in seminiferous epithelium,characterized by the absence of seminiferous lumen and spermatids,the presence of an abnormally growing nucleus,and loss of polarity in the whole seminiferous tubule.Some tubules displayed severe vacuolization in seminiferous epithelium,and some complete loss of germ cells.These results suggest that Rictor in Testicular sertoli cells is essential for seminiferous tubule integrity and its deficiency leads to testicular development defects.3.Ablation of Rictor in Testicular sertoli cells impairs spermatogenesis and disorded development of reproductive organs,the phenotype is azoospermia.As we known,the defect of testicular tubular leads spermatogenic defects.As expected,histological examinations showed that in contrast to the epididymis of controlled mice,which were full of spermatozoa,there were no visible spermatozoa in the epididymis of cKO mice as early as 3 months old.There was only undefined round cell debris.After releasing the spermatozoa from the epididymis,we counted 6 the sperm(9.0×106±0.1 vs 0)and tested the fertility(108 vs 0)of control and cKO.We found that cKO mice demonstrated azoospermia and were sterile.Consequently,we explored whether defects in spermatogenesis contributed to the azoospermia seen in cKO mice.The relative distribution of germ cell populations in the testes of control and cKO mice was determined by quantifying the DNA content in propidium iodide-labeled germ cells via cytometric scanning.Three main histogram peaks of DNA content are usually observed,which correspond to haploid(1C,spermatids and spermatazoa),diploid(2C,spermatogonia,preleptotene primary spermatocytes and secondary spermatocytes),and tetraploid cells(4C,spermatogonia,leptotene,zygotene,pachytene and diplotene primary spermatocytes),respectively.The cKO mice exhibited a comparable number of tetraploid cells(p>0.05),an increase in diploid cells(p<0.01)and a decrease in cells with haploid DNA content(p<0.01).We also marked ki-67 and separase.This data indicated that spermatogenic arrest in cKO mice occurs before and on the second meiosis,but the proliferation of germ cells had no difference.Then,TUNEL assays showed the presence of progressive numbers of dying germ cells in the cKO testes.There were many apoptosis cells in cKO when 1 month old.A lot of apoptosis cells filled with tubulars in cKO when 3 months.Apoptosis cells decreased compared with that of 3 months,but the number was still more than the control(p<0.01).The reason may be there were serious defects in testicular tubulars.Hormones play an importmant role in spermatogenesis,such as follicle-stimulating hormone(FSH),luteinzing hormone(LH)and Testosterone(T).We found that T levels in cKO mice were also significantly decreased(p<0.01),but no notable differences were observed for serum FSH and LH levels between the controlled and cKO mice(p>0.05).Testosterone is considered a crucial hormone for the initiation and maintenance of spermatogenesis.When all factors are combined,it was clear that,loss of Rictor in the Testicular sertoli cells causes spermatogenic arrest and excessive germ cell apoptosis,in turn contributing to azoospermia and infertility in cKO mice.4.Elimination of Rictor from Testicular sertoli cells disrupts BTB dynamicsTo explore the possibility of BTB dysfunction in cKO mice,the expression and localization of several key component proteins constituting the BTB were examined by immunofluorescent analysis,including Occludin and ZO-2(TJ proteins),?-catenin and N-cadherin(ES)proteins and actin-related protein complex 2(Arpc2),a component of the Arp2/3 complex that plays a crucial role in inducing changes in the conformation of the actin network from its bundled to debundled state to facilitate TJ and basal ES remodeling.The results showed that all these proteins were uniformly and specifically localized near the basement membranes of seminiferous tubules in control mice,consistent with the localization of Rictor mentioned earlier.In contrast,in cKO mice,the expression and localization of these proteins were altered and scattered over the whole seminiferous epithelium.Interestingly,although their localization was aberrant,their protein levels remained relatively stable in cKO mice,except for an increase in occludin and Arpc2,indicating that loss of Rictor in Testicular sertoli cells alters the localization,but not the expression of BTB proteins.Together,these results suggest that Rictor in Testicular sertoli cells is essential for the integrity and function of the BTB.5.BTB integrity failure after deleting Rictor in the sertoli cell resulted in polarity and skeletal disordersTesticular sertoli cells are highly polarized mesoepithelial cells that extend from the basement membrane to the lumen of seminiferous tubules.Wilm's tumor 1(WT1),a marker for the nucleus of Testicular sertoli cells,was scattered over the seminiferous epithelium in cKO mice,and not specifically localized near the basal membrane as in control mice.These results demonstrated loss of polarity in Testicular sertoli cells and in the whole seminiferous epithelium in cKO mice.Furthermore,the long apical extensions of Testicular sertoli cells are framed by both highly-organized actin and microtubule networks to direct germ cell migration towards the lumen of the seminiferous tubules.The findings of the morphological analysis of seminiferous epithelium and the immunofluorescent assessment of BTB proteins indicated the loss of apical extensions.As postulated,immunofluorescent examination of both tubulin and actin,verified the disruption of organized microtubular and actin arrangement.At present,although we had known Rictor regulates organization of the actin cytoskelet and BTB,the interaction between actin cytoskelet and BTB was not clear.Furthermore,Co-IP experiments using whole testes tissue revealed a notable reduction in the interaction between ?-actin and Occludin,and between ?-actin and?-catenin in cKO mice,indicating a reduced association between the actin cytoskeleton and BTB proteins after Rictor deletion.Previously,it was shown that Rictor regulates organization of the actin cytoskelet on in a rapamycin-insensitive and Raptor-independent pathway,in which PKCa is a mediator of this function.Also,earlier findings in adipocytes showed that Rictor could regulate the actin cytoskeleton in an mTORC2-independent manner via Paxillin,a scaffolding protein that through phosphorylation on Y118 was found to be important in actin regulation.Thus,we determined the expression and/or activity of these key effectors participating in actin organization.In cKO mice,it was found that Y118-phosphorylated-Paxillin level significantly declined,while both S657-phosphorylated-PKC? and total PKC? levels remained unaltered.In addition,the small GTPase,Rac1,was shown to be involved in the modulation of the actin cytoskeleton.By examining a Pulldown essay that specifically recognized the GTP-bound Rac1(the active form of Rac1),it was found that the activity of Racl was obviously decreased in cKO mice,with no reduction in total Racl,indicating that Paxillin and Racl are involved in Rictor/mTORC2-mediated actin homeostasis in Testicular sertoli cells.Taken together,these results demonstrate the central role of Rictor in the regulation of Testicular sertoli cell actin dynamics,and BTB barrier damage in cKO mice was mainly attributed to the disruption of actin organization.Rictor may control Testicular sertoli cell actin organization through regulation of the small GTPase,Racl,and phosphorylation of the actin filament regulatory protein,Paxillin.Conclusion1.Rictor/mTORC2 is necessary for the Testicular sertoli cells in the normal development and maintenance of the BTB,down-regulation of Rictor/mTORC2 lead to the BTB injury.2.Rictor/mTORC2 is required for normal spermatogenesis in testis,Rictor/m TORC2 deletion resulted in spermatogenesis disorder and azoospermia.3.Deletion of Rictor/mTORC2 in the sertoli cell resulted in polarity and skeletal disorders and BTB integrity failure.4.Rictor/mTORC2 may regulate cytoskeleton organization and polarity of sertoli cell through PKC,Paxillin and Racl.5.The sertoli cell-specific deletion of Rictor in mice can be induced for the study of BTB injury in azoospermia.