42Sp50/eEF1a在正常和gsdf缺失型青鳉性腺中的表达差异

Identification of 42Sp50 and eEF1a expression in normal and gsdf-deficient ovary in medaka(Oryzias latipes)

青鳉(Oryzias latipes)性腺体细胞衍生因子gsdf敲除可导致XY雌性化,但Gsdf的生物学作用和靶基因仍不清楚。用Label Free蛋白组学结合MS质谱分析,定量比对正常的XX卵巢、XY精巢及gsdf缺失XY卵巢中的蛋白质表达谱,发现真核翻译延伸因子1(eEF1)的卵巢异构型42Sp50的表达量,在gsdf缺失卵巢中显著升高。通过实时定量PCR扩增,定量分析了42Sp50在正常XX gsdf^(+/+)卵巢、XX和XY型gsdf^(-/-)卵巢中的表达。用抗人EEF1A抗体的免疫荧光技术,鉴定了青鳉eEF1a和/或42Sp50在生殖细胞性分化及性成熟期,gsdf缺失卵巢同正常卵巢对照组的细胞内表达变化,验证了蛋白组学发现的在gsdf^(-/-)卵巢中有大量卵细胞高表达42Sp50。青鳉eEF1a和42Sp50氨基酸序列高度同源,但在氨基酰基结合囊和核糖体结合部位有显著差异,为此推测Gsdf信号可能直接或间接地影响XY生殖细胞表达42Sp50和雌性分化。同时建立了双色细胞荧光免疫技术用于鉴别青鳉XX型和gsdf缺失的XY型卵细胞,为研究脊椎动物配子发生的分子机制提供了新线索。

XY female can be created by genetic manipulation of the gonadal soma derived factor(gsdf),through transgenic over-expression or targeted disruption of gsdf in medaka(Oryzias latipes),however,the bio-function and target genes of gsdf are still unclear.We used Label Free proteomics combined with Mass Spectrometry(MS)to quantitatively compare the protein expression profiles of normal XX ovaries,XY testis,and gsdf-deficient XY ovaries.Result showed that the protein level of ovarian isoform(42Sp50)of eukaryotic translation elongation factor 1a(eEF1a)was significantly enhanced in gsdf depletion ovaries,in contrast to the normal male or female.The transcripts of 42Sp50 were also significantly enriched in XY gsdf deficiency ovaries by real-time PCR quantitative analysis,in contrast to less expression in normal XX ovaries or rare in XY testis.The amino acid sequence of medaka eEF1a was highly homologous to human EEF1A,but significantly different from medaka 42sp50 in the region of amino-acyl binding pocket.The expression of eEF1a including 42Sp50 was examined in gonads during sexual differentiation,and mature gonads of normal female,male and gsdf depletion ovary by immunofluorescence using anti-human EEF1A antibody.Evidence of normal XX distinct from gsdf-deficient XY oocytes by dual-color immunofluorescent analysis provides a new clue for the mechanism of vertebrate gametogenesis.