Role And Mechanism Of Fbxw7 In Testis Sertoli Cells In The Regulation Of Spermatogenesis In Mice

Background and aimsSpermatogenesis relies on a continuous and complex process whereby spermatogonial stem cells(SSCs)are transformed into differentiated spermatogonia and spermatocytes,followed by round and elongated spermatids and spermatozoa.Sertoli cells(SCs)are the only somatic cells in the seminiferous epithelium with direct contact with germ cells.SCs decide polarized alignment and the microenvironment in seminiferous tubules,and play crucial roles in spermatogenesis.SCs first differentiate in the fetal gonad,enabling formation of the seminiferous cords,and prevent primordial germ cells from entering meiosis.With puberty,SCs undergo a process of maturation,which allows germ cells to enter into the first spermatogenic cycle and provide critical support,including nutrient supply,hormonal regulation,paracrine interaction,and construction of the blood-testis barrier(BTB).At puberty,SC morphology is constantly changing,with its cytoplasm extending from the basement membrane to the tubule lumen.At this time,adjacent SCs comprise the BTB,which provides an immune privileged environment for germ cells and creates polarity of the seminiferous epithelium.Meanwhile,maturated SCs are the most important nutritional provider for male germ cells,and play critical roles in endocrine regulation during spermatogenesis and spermiogenesis.SCs also secrete various growth factors and chemokines that promote SSC self-renewal and maintenance in the niche.F-box and WD-40 domain protein 7(Fbxw7;also known as Fbw7,Cdc4,Fbxw6,or Fbxo30),is a component of the Skpl-Cdc53/Cullin-F-box-protein complex(SCF/β-TrCP),an E3 ubiquitin ligase that ubiquitinates proteins and triggers proteasomal degradation.Fbxw7 has three subtypes,Fbxw7α,β,and-y,which share the WD40 repeat structure.Different subtypes have distinct localizations:Fbxw7α is localized in the nucleoplasm,Fbxw7β is cytoplasmic,and Fbxw7γ is nucleolar.Fbxw7 is a well-established tumor suppressor,as it is inactivated by mutation in various human cancers.Fbxw7 deficiency induces mitotic defects and chromosomal instability,thus promoting tumorigenesis.In recent years,evidences have emerged demonstrating that Fbxw7 controls the pluripotency of several stem cells/progenitors by regulating the stability of target proteins.A recent in vivo study of SSCs showed that Fbxw7 is expressed in the undifferentiated spermatogonia and in a cell cycle-dependent manner.Fbxw7 overexpression suppressed SSC activity and proliferation,whereas its deficiency enhanced SSC colonization and resulted in accumulation of undifferentiated spermatogonia,and impaired spermatogenesis,suggesting that Fbxw7 plays pivotal roles in the regulation of SSC self-renewal and differentiation,and spermatogenesis.However,no study has unraveled the role of Fbxw7 in SC proliferation,differentiation,or function.Here,by generating conditional mutant mice with SC-specific deletion of Fbxw7,we demonstrate that Fbxw7 is essential for the proliferation and function of SCs.Materials and methods1.We constructed the mice with testicular sertoli cell-specific deletion of Fbxw7,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 identificat the knockdown effect by Western blot and immunohistochemistry and immunofluorescence.2.Changes of morphology of mouse testis and epididymis by Fbxw7 in specific knockout SCs by H&E staining and immunohistochemical analysis3.Immunofluorescence and TUNEL kit were used to detect the effects of Fbxw7 in specific knockout SCs on mouse testicular cell proliferation and apoptosis.4.Using qPCR technology and ELISA kit to detect the effects of specific knockout of Fbxw7 in SCs on testosterone production-related hormone genes and expression in mouse serum.5.Analysis of the expression of mRNA by reverse transcription and qPCR.6.Use sperm counts of all ages and mating of mice to evaluate the effects of Fbxw7 in specific knockout SCs on fertility in mice.Result:After the Fbxw7 was specifically knocked out in Sertoli cells,the mutant mice had no significant difference in body shape or weight at all ages.Testicular volume and weight decrease in a time-dependent manner,with phenotypes starting at four weeks of age,severe atrophy of seminiferous tubules at eight weeks of age,severe vacuolation of seminiferous epithelium,and the complete loss of germ cells with support cells type.Due to excessive loss of germ cells,the size and weight of the epididymis in adult mutant mice were significantly reduced,beginning to show age-dependent decline in reproductive capacity at 2 months of age,and infertility to 7 months of age.Irregular arrangement and abnormal assembly of actin filaments supporting the cytoskeleton system in mutant mice,and the expression and localization of tight junction-associated proteins in the blood-testis barrier became abnormal,showing a discontinuous distribution along the basement membrane,or scattered in the entire seminiferous epithelium,the integrity of the blood-testis barrier is impaired.Functional levels of germ cells(Stra8,Dkkl1,Tnp1),sertoil cells(Amh,Erbb4,Fgf9,Fshr,Gdnf,Shbg Sox9,WT-1),and leydig cells(Hsd3β6,Lhcgr,Hsd17β3,Star,Cyp11α1,Hsd3β1,Cyp17α1)in Fbxw7 mutant mice have generally decreased,but GATA-4 expression has increased abnormallyConclusion:1、Generation of conditional mutant mice with SC-specific deletion of Fbxw72、Loss of Fbxw7 in SCs perturbs testis development and induces testicular atrophy3、Ablation of Fbxw7 in SCs impairs spermatogenesis and causes subfertility in mice4、Fbxw7 deficiency disrupts BTB integrity and cytoskeletal organization in SCs5、Elimination of Fbxw7 in SCs alters the expression of testicular cell markers...