The Role And Mechanism Of FBXO7 In Sperm Release

Background:Spermatogenesis is a highly complicated process of germ cell division and differentiation leading to the production of spermatozoa.Spermatozoa need to be released from seminiferous epithelium,then enter into epididymis for further maturation and to gain mobility.The process of spermatozoa release is called spermiation,disability of spermiation can induce azoospermia.Study on spermiation can provide theoretical basis for infertility treatment and male contraceptives development.Spermiogenesis and spermiation are conserved processes.For example,the process of spermiogenesis can be divided into 16 steps in mice.Spermiation is a multi-step process involving changes in both spermatid and Sertoli cell.Prior to spermiation,the elongated spermatid interacts with the Sertoli cell via a conserved structure known as the apical ectoplasmic specialization(apical ES),which forms at sites of intercellular adhesion between spermatids and Sertoli cells.The apical ES is based on the actin filament bundles of Sertoli cell.It is comprised of hexagonally-packed actin filaments sandwiched between Sertoli cell membrane and the underlying endoplasmic reticulum,where spermatid is anchored to the supporting Sertoli cell.The morphological and ultrastructural events associated with spermiation show dynamic changes,including the remodeling of the spermatid nucleus,removal of Sertoli cell ES junctions and retraction of Sertoli cell cytoplasm.The apical ES and TBCs(Tubuloubulbar complexes)present the dynamic changes in order,and they are the basic structures of spermiation.But many unresolved questions remain,such as how the composition of the adhesion junction changes as spermiation progress and whether there is a 'switch' molecule controlling this process.In eukaryotes,there are two proteolytic routes,the autophagy and ubiquitin-26S proteasome system(UPS).The UPS is thought to identify the target protein for accurate regulation.In recent studies of spermatogenesis,a growing number of findings suggest that ubiquitin may play a key role in the process of spermatogenesis.We focus on a ubiquitin-related protein FBXO7 in the testis as it is a substrate-recruiting subunits of SKP1-Cullin1-FBP(SCF)-type E3 ubiquitin ligases We postulate that it may target specific substrates for UPS-dependent degradation,and participate in the spermiation process.Methods and results:We found that Fbxo7 was expressed in various tissues of mouse by Realtime PCR,and its expression was significantly increased in testis of 3-4 week old mouse.In order to better understand the function of FBXO7,Fbxo7 knockout(KO)mice were generated.Fbxo7-/-mice showed spermiation failure in stage VIII,apoptosis of the retained sperm,azoospermia and completely male infertility,while the relative testes weight and hormones appeared normal.We generated germ cells(Fbxo7F/F Stra8-Cre)and Sertoli cells(Fbxo7F/F AMH-Cre)conditional KO mice,respectively,and found germ cells conditional KO mice showed similar phenotypes to the Fbxo7-/-mice,whereas the Sertoli cells conditional KO were fertile and showed no phenotype compared with Fbxo7-/-mice.Ultrastructure analysis indicated the apical ES junction still exited in the retained sperm,and IF staining also showed the ES junction markers,such as Palladin,Drebrin,ARP3,?-Catenin,EPS8 and Espin were located in the retained sperm.Nevertheless,compared with control groups,we found the protein level of Espin was markedly increased in Fbxo7-/-mice testes via IF and Western blot,while the mRNA level of Espin was not altered.These results indicated that FBXO7 regulate Espin in protein level.Similar results were also been detected in Fbxo 7F/F Stra8-Cre mice.The above data demonstrated that loss of Fbxo7 in germ cells leads to the accumulation of apical ES junction associated protein,Espin,and cause spermiation failure.To elucidate how FBXO7 regulates Espin in testis,we generated anti-FBXO7 antibody.By co-IP assay in vivo,we found FBXO7 could interact with SKP1,suggesting that FBXO7 was involved in E3 ligase SCFFBXO7 complex in testis.In addition,we found that FBXO7 could bind to Espin,and in Fbxo7-/-mice testis,the poly-ubiquitination level of Espin was decreased,thus,leading to the reduction of the degradation of protein.Collectively,these data revealed FBXO7 could ubiquitinate Espin and regulate its degradation process.To explain the aberrant degradation of the apical ES associated protein,Espin,in the absence of FBXO7,we observed the distribution of FBXO7 by IF,and found it mainly located in the elongating/elongated spermatids and partially co-localized with Espin at stage VII.Using high resolution microscopy,we found FBXO7 localized in the apical ES with punctate distribution.Furthermore,the significant increase of Espin was only founded in FbxO 7-/-mice and FbxO7F/F Stra8-Cre mice testes,but not in Fbxo 7F/F AMH-Cre testes.We found that FBXO7 transported from germ cell into Sertoli cell by a specific tunnel,TBC,in the beginning of spermiation.The above data indicated FBXO7 was associated with the degradation of Espin by directly binding to it at stage VII.To test this hypothesis,we used HEK293T cells to investigate the relationship between FBXO7 and Espin.Firstly,we found the expression of FBXO7 and Espin were negatively correlated via co-transfection of the indicated plasmids,and the expression level of Espin was only regulated by proteasome,but not the lysosome pathway by treatment with the indicated inhibitors.Additionally,CHX assay showed FBXO7 could regulate the stability and the poly-ubiquitination level of of Espin.Furthermore,poly-ubiquitination assay in vivo and vitro revealed that FBXO7 could ubiquitinate Espin in K48 ubiquitin chain,and thus mediated the degradation of Espin.Conclusion:In summary,we find that spermiation is regulated by FBXO7.FBXO7 ubiquitinates Espin and mediates the degradation of Espin at the end of spermiogenesis,thus commencing spermiation.At present,FBXO7 is the first identified 'switch' of regulating spermiation.Our findings indicate that the intercellular adhesion junctions between germ cells and Sertoli cells during spermiation are controlled by ubiquitination.Our studies enhance the understanding of spermiation and provide a new target for male contraceptives and treatment of patients with non-obstructive azoospermia(NOA).