Studies On Mechanism Of Notch-FGF4 Signaling Dysregulation In Mouse Germ Cells Results In Cystic Dilation Of The Rete Testis

Cystic dysplasia of the rete testis(CDRT)was first reported in1973,since then there were almost 60 cases.It was a rare disease which resulted into the testis swell in child.The main manifestation of CDRT was irregular cysts dilation of rete testis(RT)in the tes-ticular mediastinum preadolescence that squeezed and replaced the normal testes tissues.Due to the lesions would invade the whole tes-tis and destroy the testicular structure,orchiectomy was the major treatment at present.It widely recognized that CDRT resulted from disorder connections between efferent ductules and RT at about 4 to 5weeks of gestation.However,the detail mechanism of CDRT was still unknown.Notch signaling was evolution conserved regulatory mechanism be-tween cells.At the early stage of embryonic development in mammal testes,Nb(Numb)/Nbl(Numblike)played an important role in the devel-opment of the testicular somatic cells through inhibiting Notch sig-naling.It was reported constitutive activation intracellular domain of Notch1(NICD1)in mice testes lead to the biological phenotype of CDRT.Nevertheless,constitutive activation NICD1 in Sertoli cell or Leydig cells did not cause cystic dilation of RT in mice.Furthermore,there was still on reports about over-activation Notch signaling dis-turbing the development of RT.In addition,cross-talk between the Notch and fibroblast growth factor(FGF)signaling pathways plays important roles in modulating organogenesis,cell differentiation and migration.FGF2,FGF4 and FGF8 protein were produced by germ cells and secreted into the RT fluid to perform functions.However,there was no cystic dilation in Fgf2 and Fgf8 gene knockout mice.A gradient of FGF8 protein could be responsible for guiding migration of the Wolffian duct to the cloaca,and then forming a lumen in chick.Otherwise Fgf4 and Fgf8 gene were functionally redundant during somitogenesis.We speculated FGF4 pro-tein secreted by germ cells could modulate the development of RT.In this study,we established two mice models which made selec-tive deletion of Nb/Nbl and Fgf4 gene in the testicular germ cells to investigate the regulator relationship between Notch signaling andFGF4 in germ cells,the influences of FGF4 protein to the development of RT and elucidate the mechanism how abnormal Notch-FGF4 signaling lead to cystic dilation of RT in preadolescence mice.Method:1.Tex-Cre female mice were bred with floxed Nb/Nbl and floxed Fgf4 male mice to obtain Tex-Cre:Nb^f/f/Nbl^f/f or Tex-Cre:Fgf4^f/f mice with selective deletion of Nb/Nbl and Fgf4 in germ cells,and identi-fied the phenotype of mice.Detected the expression levels of Nb/Nbl,Hey1,Fgf4,Nb/Nbl,activated Notch1 and FGF4 in the germ cells by RT-PCR and Western blot.Observed the histological struc-tures and the distribution of Nb/Nbl and other special proteins in Tex-Cre?Tex-Cre:Nb^f/f/Nbl^f/f and Tex-Cre:Fgf4^f/f testes at 3 months us-ing H&E and immunohistochemistry analysis.Injected solution of Evans blue into the RT of Tex-Cre and Tex-Cre:Nb^f/f/Nbl^f/f mice at 3 months old respectively for tracer dye studies.2.Treated murine spermatogonia cell line GC1-spg(GC1)with DAPT(the inhibitor of Notch receptor activation)or p3XFlag-NICD1(encode NICD1 protein)respectively,then detected the expression levels of Hey1(the target gene of Notch signaling),Fgf4,activated Notch1,HEY1 and FGF4 in GC1 using real-time PCR and Western blot analysis.3.Isolated and purified WT(wild type)mouse testicular germ cells and treated with DAPT.Then detect the expression levels of Hey1,Fgf4,activated Notch1 and FGF4 with real-time PCR and Western blot analysis.4.Detected the expression levels of Fgf4 and FGF4 protein by RT-PCR and Western blot in WT mouse testes at different age.And ob-served the distribution of FGF4 protein in the testes of each group by immunohistochemistry analysis.5.Testicular explant culture of WT mice with recombinant human FGF4 and/or fibroblast growth factor receptor(FGFR)inhibitor treat-ment for 72 hr,then proceeded histological and immunohistochemical analysis to observe the histological structures and the distribution of special proteins in the testes.Results:1.RT-PCR and Western blot showed that there is no expression of Nb/Nbl gene and Nb/Nbl protein in Tex-Cre:Nb^f/f/Nbl^f/f testicular germ cells,while the levels of Nb/Nbl proteins in the lung and brain were still normal.Immunohistochemical analysis revealed the absent of Nb/Nbl proteins in Tex-Cre:Nb^f/f/Nbl^f/f testicular germ cells.2.Tex-Cre:Nb^f/f/Nbl^f/f testis became significantly enlarged and with large cysts near the cranial pole of the testis at 3 months.In-jection of Even blue into the RT of mutant mice demonstrated incom-plete obstruction in efferent ductules.H&E showed the size and structures of seminiferous tubules and efferent ducts were normal.Immunohistochemical analysis showed there were no significant changes in GCNF-positive spermatogenic cells,GATA4-positive Sertoli cells and Cyp17A1-positive interstitial cells in Tex-Cre:Nb^f/f/Nbl^f/f testis.3.RT-PCR results showed that the expression of the target gene of Notch signaling-Hey1 was significantly elevated,but the expres-sion of Fgf4 gene was reduced in germ cells of Tex-Cre:Nb^f/f/Nbl^f/ftes-tes at 3 months.Western blot showed the level of activated Notch1protein was increased,but the level of FGF4 protein was significant-ly reduced in these cells.4.Real-time PCR results showed the expression of Notch signaling target gene Hey1 was decreased and Fgf4 gene was increased after DAPT treatment.Furthermore,Western blot results showed the levels of ac-tivated Notch1 protein and HEY1 protein were reduced,but the level of FGF4 protein were significantly increased.Meanwhile,they were changed in a dose-dependent manner.5.Real-time PCR results showed the expression of the Notch sig-naling target gene Hey1 was increased,but the expression of Fgf4gene was decreased in GC1 cells after being transfected by p3XFlag-NICD1 which encoded NICD1 protein.Meanwhile,Western blot results showed the level of activated Notch1 protein and HEY1 protein were upregulated,but the level of FGF4 protein was downregulated in these cells.6.RT-PCR and Western blot results showed that Fgf4 gene ex-pressed in various cells in testes,the expression level of Fgf4 gene reached the peak at P10(postnatal day 10)and the level of FGF4 pro-tein began to go up and maintained constant until adulthood in WT mouse.Immunohistochemical analysis evaluated FGF4 protein was dis-tributed in Sertoli cells?Leydig cells and germ cells from neonate to adult.7.Real-time PCR showed the expression of Notch signaling target gene Hey1 was reduced,but the expression of Fgf4 gene was increased in extracted WT mouse testicular germ cells after DAPT treatment.Western blot results showed the level of activated Notch1 protein was reduced,but the level of FGF4 protein was significantly increased.8.RT began to form at about E15.5(embryonic day 15.5)and the lumen was thoroughly opened almost at P1(postnatal day 1)mouse which cultured 72hr.P1 testicular explant culture showed RT was cystic dilation,meanwhile the numbers of DMRT1 positive RT epitheli-ums were increased significantly after FGFR inhibitor LY2874455treatment for 72 hr.Combined recombinant human FGF4 protein mean-while could rescue the dilation of RT,and reduce the numbers of DMRT1 positive epitheliums.Otherwise,there were no significant changes in other special proteins such as GATA4,WT1,SF1,AR,PAX8,ESR1,DAX1 and E-cadherin.9.Selective deletion of Fgf4 gene in Tex-Cre:Fgf4^f/ftesticular germ cells leaded to RT cystic dilation with normal spermatogenesis.Immunohistochemical analysis showed that deletion of Nb/Nbl and Fgf4gene could increase the numbers of DMRT1 positive RT epitheliums,while the distribution of liquid reabsorption related protein such as AQP3?AQP9 and CFTR was normal in the epithelium of efferent ductules.Conclusion:1.Selective deletion of Nb/Nbl gene in testicular germ cells in-duce constitutive activation of Notch signaling pathway.Over-expressed Notch signaling might through downstream target gene Hey1inhibits the expression of Fgf4 gene and reduces the level of FGF4protein in germ cell.2.The reduced level of FGF4 protein in the germ cell does not influence spermatogenesis and the spermatogenic function of testis is normal.3.The reduced level of FGF4 protein inhibits FGF4/FGFR signaling which leads to biological changes of RT epithelium,occur some char-acters of Sertoli cells,destruction of RT structure and cystic dila-tion of RT.4.The cystic dilation of RT which is resulted from the abnormal of FGF4/FGFR signaling could be rescued by supplement of FGF4 protein and the numbers of DMRT1 positive epitheliums could be reduced.