Roles And Mechanisms Of Raptor In The Regulation Of Sertoli Cell Cytoskeletal Organization And Polarity

Background and aimsIn mammals,SCs are highly polarized cells that extend from the basement membrane to the lumen of the seminiferous tubules,and maintenance of this polarity is essential for testis development and spermatogenesis[1].Cytoskeletal proteins are known to determine cell shape and maintain cell junctions,which in turn help to maintain normal epithelial function and morphology.Cytoskeletal networks are therefore believed to provide the foundation for the integrity of the BTB and SC polarity.Mechanistic target of rapamycin(mTOR)is a highly conserved serine/threonine kinase that nucleates at least two distinct multiprotein complexes,mTOR complex 1(mTORC1)and mTORC2[2].mTORC1 includes mTOR,regulatory associated protein of mTOR(Raptor),and others factors and is a sensitive target of rapamycin.Raptor acts as a scaffold for recruiting mTOR substrates.mTORC1 integrates both intracellular and extracellular signals,including growth factors,nutrients,energy levels,and cellular stress[3].In response to these stimuli,mTORC1 is activated by two families of RAS-related small GTPases,Rheb and Rags[4].Rheb is suppressed by tuberous sclerosis complex 1/2(TSC1/2),a functional complex with GTPase-activating protein(GAP)activity for Rheb[4,5].These components comprise the canonical TSC-mTORC1 pathway.In contrast,mTORC2 is insensitive to rapamycin and contains the rapamycin-insensitive subunit,cictor[6].Rapamycin is a specific inhibitor of mTORC1.It is used as immunosuppressive agent in organ transplant patients to suppress the immune rejection.The quantity of sperm in male patients was significantly reduced after long-term use of rapamycin,and male fertility was significantly decreased.A recent in vitro study of primary cultured SCs showed that ribosomal protein S6,a downstream molecule of mTORC1 signaling,was a major regulator of actin organization in SCs and BTB dynamics[7].They subsequently indicated a mechanism involving Akt-mediated regulation of matrix metalloproteinase-9,which affected BTB dynamics,or Arp3-mediated actin microfilament organization[8,9].An in vivo study using a conditional knockout(KO)strategy also demonstrated essential roles of TSC1/TSC2 in the maintenance of SC polarity and spermatogenesis,suggesting the importance of mTORC1 signaling in SC functions and spermatogenesis[10].Furthermore,another study showed that TSC1/TSC2 could function independently of canonical mTORC1 signaling[11].A recent in vivo study demonstrated that Rictor regulated the cytoskeletal organization of SCs in both mTORC2-dependent and-independent ways[12],consistent with the results of other studies using different experimental systems[13,14].These reports cumulatively suggest that the roles of key components of mTOR signaling in SC functions and spermatogenesis require further clarification.The current study therefore aimed to clarify the detailed functions of mTORCl signaling in SC and testis development by deletion of the Raptor(key component of mTORC1)and Rheb(upstream activator for mTORC1)genes,respectively,using the Cre-LoxP system.Materials and methods1.We constructed the mice with testicular sertoli cell-specific deletion of Raptor and Rheb,respectively,by the application of Cre-loxp system.2.Analysis of effect of Raptor and Rheb,respectively,on mice testis and epididymis tissue morphology and structure by HE staining,immunohistochemistry and immunofluorescence.3 Detection of the effect of Raptor and Rheb,respectively,on the testicular sertoli cell cytoskeleton and the BTB by transmission electron microscopy,immunofluorescence technique,Western blot technique and Pull down and immunoprecipitation.4.Performed whole-transcriptome sequencing and screening assays of whole testes to identify differentially expressed genes and the mechanisms.Result1.We examined the effects of Raptor deletion on testis development from the neonatal period to adulthood.Testis mass was similar in SCRaptorKO and control mice at 1 dpp,but started to decline after 5 dpp.Testis mass in the SCRaptorKO mice had decreased to<10%in adult mice,and epididymis mass to 25%,compared with the controls.Furthermore,no spermatozoa were observed in the epididymides in the SCRaptorKO mice,leading to male infertility.Consistently,histological examination showed that the seminiferous tubules in SCRaptorKO mice were morphologically indistinguishable from those in control mice at 1 dpp;however,many tubules had started to degenerate by 5 dpp,showing age-dependent changes,and most tubules displayed degeneration and loss of tubular structures by 15 dpp,accompanied by massive GC loss.By 2 months old,increasingly severe atrophy of the seminiferous tubules was observed,evidenced by complete loss of the tubular structures and GCs.Collapse of the tubular architecture in SCRaptorKO mice was confirmed with immunofluorescent stained.2.We investigated the location of SCs in SCRaptorKO mice by immunofluorescent staining for the SC nuclear marker,Wilm's tumor 1(WT1)[15,16].Notably,many of the remaining SCs in SCRaptorKO mice were scattered over the seminiferous epithelium in the tubules with relatively intact tubular structures,compared with being located in an ordered alignment along the basal membrane in the control mice,indicating aberrant location of SCs in SCRaptorKO mice.This was particularly evident at 15 dpp.This finding was further supported by evidence from transmission electron microscopy.These results cumulatively suggest that Raptor deletion disrupted the localization of SCs,and may impair SC polarity from the basement membrane to the seminiferous tubule lumen.3.The cytoskeleton and tight junctions synergistically regulate SC polarity.We explored the possible disruption of SC polarity in SCRaptorKO mice by immunofluorescent analyses of actin and tubulin.Both the actin and microtubule cytoskeleton were disorganized in SCRaptorKO mice from 15 dpp,characterized by irregular arrangement and aberrant assembly of actin filaments and microtubules,respectively.Apical extension and direction seemed to have completely disappeared at 2 months old.We also performed immunofluorescent staining of vimentin as a cytoskeletal marker in SCs.Vimentin showed a flattened and circular distribution along the basal membrane before 5 dpp in control mice,followed by apical extension,and then collection of vimentin around the nuclei of the SCs,pointing towards the lumen of the seminiferous tubules,at 15 dpp,with a characteristic "halo"appearance[17].By 2 months old,vimentin exhibited a mature distribution pattern surrounding the SC nuclei,indicating dynamic cytoskeletal organization inside SCs.However,vimentin failed to form apical extensions at 15 dpp in SCRaptorKO testes,and the directional polarity of vimentin was lost.Vimentin distribution was totally disordered at 2 months old,and its expression in some tubules had completely disappeared.This suggested that Raptor loss disrupted the intrinsic cytoskeletal organization and cell polarity in the SCs,leading to destruction of the cell architecture throughout the seminiferous epithelium.We therefore generated another line of conditional KO mice lacking Rheb expression.Adult SCRhebKO mice exhibited only mild morphological changes in the tubules.Despite a 60%reduction in testis mass and a 70%reduction in the sperm count,SCRhebKO mice maintained integrity in many seminiferous tubules and had many normal sperm in the epididymis and normal fertility,indicating milder phenotypic changes compared with SCRaptorKO mice.Moreover,adult SCRhebKO mice displayed a similar vimentin expression profile to control mice.4.We further investigated the mechanisms underlying the different effects of Raptor and Rheb in the regulation of SC cytoskeletal organization.A pull-down assay specifically recognizing GTP-bound Racl(the active form of Racl)showed that Rac1 activity was markedly reduced in SCRaptorKO mice,but not in SCRhebKO mice,suggesting that Rac1 was involved in Raptor-mediated actin organization in SCs.To determine the existence of potential effectors linking Raptor and Racl,we performed whole-transcriptome sequencing and screening assays of whole testes.The gene encoding cingulin was of particular interest.Immunofluorescent examination showed that cingulin was distributed across the whole seminiferous epithelium in normal testes at 1 dpp,and increased progressively.By 15 dpp,cingulin was accumulated along the testicular basal membrane,with weak expression near the lumen and along the interfaces between the SCs,and this was maintained into adulthood.This expression pattern coincided with the basal and apical ectoplasmic specialization,suggesting that cingulin may be involved in formation of the BTB and the establishment of apical-basal polarity of SCs.Cingulin expression was reduced in SCRaptorKO mice from 10 dpp,and no basal accumulation or polar localization of cingulin was observed at 15 dpp.Cingulin expression had completely disappeared in some tubules by adulthood in SCRaptorKO mice.In contrast,adult SCRhebKO mice continued to show a similar cingulin expression profile to the control mice.Protein levels of cingulin were also significantly reduced in SCRaptorKO but not SCRhebKO mice,indicating that cingulin was involved in Raptor-controlled cellular activity.We therefore examined the expression of Racl GEFs and GAPs associated with junction proteins,such as Asef(also known as ARHGEF4)[18],Richl[19],MgcRacGAP[20],and Vav2[21].Asef gene expression was significantly reduced in SCRaptorKO mice,as shown by qPCR,whereas changes in other GEFs and GAPs were negligible,indicating involvement of a GEF,rather than a GAP,in cingulin-directed modulation of Racl activity.This result was confirmed with western blotting.Conclusion:1.Raptor is essential for Sertoli cells development and spermatogenesis.Ablation of Raptor in Sertoli cells impairs cell architecture of testis,including excessive GC loss and reduced PTMC.Raptor silencing in Sertoli cell causes azoospermia and male infertility.2.Aberrant localization of SCs impairs SC polarity in neonatal SCRaptorKO mice.3.Raptor but not Rheb regulates cytoskeletal dynamics in SCs.4.Raptor silencing reduces Rac1 activity and causes aberrant cingulin expression,impairing cytoskeletal homeostasis.