GADD45G在雄性性别决定中的功能

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,GADD45G Functions in Male Sex Determination by Promoting p38 Signaling and Sry Expression,GADD45G,通过促进,p38,信号和,Sry,表达在雄性性别决定中起作用,王绪海,基础兽医,实验方法,四,.,讨论,三,.,结果,二,.,摘要,一,.,设想,五,.,一、摘要,Gadd45g display complete,male-to-female sex reversal.,p38 MAPK signaling is impaired in Gadd45g mutants.,GATA4,which is required for Sry expression.,二、材料和方法,1.Animals,Staging,and Dissection,2.Histology and In Situ Hybridization.,3.Plasmids,，,pCS2+vector,4.,Fluorescence-Activated Cell Sorting,5.,RNA Extraction,Quantitative RT-PCR,6.Western Blot Analysis,7.,Chromatin,Immunoprecipitation,三、结果,1.Gadd45g,突变体鼠显示了雄性到雌性的性逆转,Gadd45g,纯合子变异鼠,显示了强的雌性性别偏移,获得了,3.5%,的雄性,雄性到雌性的性逆转,1.Gadd45g,突变体鼠显示了雄性到雌性的性逆转,XY mice indistinguishable,from those of wild-type females,Gadd45g-/-;XY gonads at,this stage lacked testis,cords and were smaller,as is characteristic of,female embryonic gonads,1.Gadd45g,突变体鼠显示了雄性到雌性的性逆转,XY mice resulted from,defective fetal testis development,using the number of tail somites(ts),for accurate staging,Sexual fate is determined in the bipotential,gonad at 915 ts and gonadal somatic cells,differentiate from 16 ts onward,1.Gadd45g,突变体鼠显示了雄性到雌性的性逆转,not affect the specication of,the bipotential gonadal Anlage,bipotential markers,was mostly,unchanged in,Gadd45g-/-,1.Gadd45g,突变体鼠显示了雄性到雌性的性逆转,Sox9,a key regulator of Sertoli,cell differentiation,，,Was,not expressed in,Gadd45g-/-,Dhh and Amh,which are,expressed in Sertoli cells,and found that both markers were,absent in,Gadd45g-/-,The early male gonad specication is permanently interrupted in Gadd45g-/-.,1.Gadd45g,突变体鼠显示了雄性到雌性的性逆转,Gadd45g-/-,XY gonads expressed female marker genes,Wnt4 and Rspo1 are rst expressed,in the bipotential gonad and are,subsequently required for female development,1.Gadd45g,突变体鼠显示了雄性到雌性的性逆转,Foxl2 and Fst are induced at E11.5 and are specically in,females required for granulosa cell and follicle development.,Both were expressed in,Gadd45g-/-.,XY gonads,2.Gadd45g,对于正常,Sry,表达是必不可少的,The results raised the question of,where and when Gadd45g is expressed,in the early male embryonic gonad,2.Gadd45g,对于正常,Sry,表达是必不可少的,prominent Gadd45g expression by in,situ hybridization(ISH)in the whole gonad at 11/12 ts,when Sry is expressed only in the center of the gonad,2.Gadd45g,对于正常,Sry,表达是必不可少的,Gadd45g,，,at 9/10 ts,，,by qPCR,Sry was not present.,Gadd45g,，,strongly upregulated 13/14 ts.plateaued until 17/18.,2.Gadd45g,对于正常,Sry,表达是必不可少的,Sry expression in the male embryonic,gonad reproduced the described kinetics.,peaking at 17/18 ts and followed by rapid downregulation,2.Gadd45g,对于正常,Sry,表达是必不可少的,Sox9 was upregulated at 15/16 ts and,stayed expressed in the genital ridge,as reported,The expression of Gadd45g before Sry suggests that,GADD45G might play a role in regulating Sry.,the normal onset of Sry expression,was delayed by approximately 2 ts in,heterozygotes and 4 ts in homozygous mutants,2.Gadd45g,对于正常,Sry,表达是必不可少的,Sry expression in,Gadd45g-/-,;XY mice was,reduced to 25%of wild-type level during the critical,time window between 11 ts and 15 ts.,qPCR analysis,Sox9 is never expressed,above basal levels in,Gadd45g-/-,;XY mice,Sox9 was strongly induced at 15 ts in XY wild-type embryos,whereas this induction was delayed in heterozygotes,2.Gadd45g,对于正常,Sry,表达是必不可少的,sex reversal in Gadd45g mutant mice,caused the delayed and reduced Sry expression,2.Gadd45g,对于正常,Sry,表达是必不可少的,The specic expression of Gadd45g in the early embryonic,gonad and its requirement for normal Sry expression suggested,that Gadd45g may be coexpressed with Sry and hence cell-,autonomously regulate Sry expression.,raised GADD45G antibodies,；,could not detect the protein by immunouorescence,3.Gadd45g,与,Sry,和,Sox9,共表达,mRNA colocalization,;,double-uorescence ISH;coexpression with Sry;,72%of Gadd45g+,cells also coexpressed Sry,Gadd45g+cells,were observed throughout the,genital ridge at 13/14 ts,the germ cell marker Oct4,，,never coexpressed with,Gadd45g in the same cells,Sox9 was coexpressed in 25%,of Gadd45g+cells at 15/16 ts,The results indicate that Gadd45g,is coexpressed with Sry and Sox9,in somatic cells,reporter mice that express EGFP under the control of a SF1,promoter,marking SF1+somatic cells in the male gonadal Anlage,puried SF1+cells by uorescence-activated cell sorting(FACS),from dissociated genital ridges of 8 ts and 18 ts embryos,detected a GFP negative,a weakly GFP-expressing(GFP+),and a highly GFP-expressing population(GFP+),3.Gadd45g,与,Sry,和,Sox9,共表达,endogenous SF1,expression was high in the GFP+population,Low in the GFP+population at 8 ts,and absent in the GFP negative population,The GFP negative cells most likely,represent mesonephric and primordial germ cells,3.Gadd45g,与,Sry,和,Sox9,共表达,3.Gadd45g,与,Sry,和,Sox9,共表达,18 ts,，,Gadd45g,Sry,and Sox9 expression;,in the GFP+population,contained pre-Sertoli cells;,undetectable in GFP+and GFP-cells,Oct4 was expressed only in the GFP-,and GFP+,but not the GFP+,Gadd45g-expressing cells coexpress Sry,and Sox9 and hence repr