【作者】 赵涵；

【导师】 陈子江； Aleksandar Rajkovic；

【作者基本信息】 山东大学 ， 妇产科生殖医学， 2008， 博士

【摘要】 在哺乳动物中,卵巢是一个独特又重要的器官。卵原细胞的分化,始基卵泡的形成和到初级卵泡的转变,是卵泡早期发育过程中最关键的环节。始基卵泡的库存大小,闭锁程度都直接影响到雌性哺乳动物的生殖年龄。一旦始基卵泡变少耗尽,便会绝经;如果病理性的始基卵泡过早衰减,便会导致卵巢早衰。在卵巢发育的正常生理过程中,一系列卵细胞特异性转录因子起到了重要的调控作用,SOHLHI就是其中关键的一员。SOHLH1是在生殖细胞中特异表达的转录调控因子,在小鼠卵巢始基卵泡及初级卵泡中表达,研究证明SOHLH1在卵泡早期发育阶段发挥重要作用,并被列为非综合征型卵巢早衰的候选基因。Sohlh1基因敲除雌鼠表现为始基卵泡向初级卵泡的发育障碍,卵细胞过早丢失,卵巢萎缩,不能生育。在第一章中,我们以Sohlh1基因敲除小鼠为研究对象,应用基因表达谱芯片技术,发现了若干SOHLH1的上下游基因,并联合各种生物网络资源,建立并完善了生殖细胞发育信号通路的网络结构图谱。为证实SOHLH1在卵泡晚期成熟过程中的作用,第二章中,我们构建了Zp3promoter-Sohth1转基因小鼠模型,突破了Sohlh1仅在始基及初级卵泡中表达的局限性,使Sohlh1成功在各级卵泡中表达,通过分析,发现Sohlh1并非卵细胞后期成熟的抑制因子,相反的,Sohlh1的过表达可以促进排卵。基于有效动物模型的表现,推测SOHLH1基因突变可能是卵巢早衰的致病原因之一。为了验证假设,在第三章中,我们对96名美国高加索人种POF患者进行SOHLH1基因突变筛查;另外,我们还筛查了SOHLH1的间接调控基因——FIGLA和GDF9,在中国汉族POF患者中的突变情况。通过对以上这三个生殖细胞特异性基因的扩增测序,我们发现了一系列有致病意义的杂合改变,结合功能实验,进一步说明调控生殖细胞发育的信号通路异常会导致人类卵巢早衰的发病。第一章探索Sohlh1下游转录调控因子目的:Sohlh1是生殖细胞特异性转录调控因子,具有helix-loop-helix（HLH）结构域,Sohlh1基因敲除雌鼠表现为卵细胞过早丢失,卵巢萎缩,成年Sohlh1基因敲除小鼠卵巢仅为正常小鼠卵巢的1/10大,最终以不孕为特点。HLH结构与DNA启动子区域E-Box作用元件相结合调控其它基因的转录表达,Sohlh1作为HLH家族一员,将其敲除,会导致一系列下游基因失调,从而引起卵巢早衰的表现。本实验目的在于探讨Sohlh1敲除小鼠卵巢的过早衰退的途径及筛查Sohlh1的下游基因。方法:通过对减数分裂影响因子及进入减数分裂期卵细胞的染色体形态学分析,并应用基因芯片技术,联合比较Sohlh1缺陷新生小鼠卵巢与另外两个生殖细胞特异因子-Lhx8和Nobox缺陷的新生小鼠卵巢芯片数据,分析Sohlh1基因敲除小鼠卵巢异常表型的原因、Sohlh1与Lhx8和Nobox的关联作用,筛选下游靶基因。结果:Sohlh1基因敲除小鼠卵巢中影响减数分裂因子Dmc1,Spo11,Msh5和Rec8表达未见明显异常,通过对E15.5胚胎Sohlh1缺陷小鼠卵细胞行Scp3免疫荧光分析,与正常对照比亦无明显区别。进一步行基因芯片检测,分析结果显示,Sohlh1敲除新生小鼠卵巢组织中,494个基因或ESTs表达降低,165个基因表达升高2倍以上。联合Lhx8和Nobox缺陷的基因芯片分析发现有73个基因被Sohlh1,Lhx8和Nobox共同调节,而这其中又有33（45.2%）个为生殖细胞或卵细胞特异基因,且存在5倍以上改变;进一步对33个基因进行表达模式分析,发现Sohlh1,Lhx8和/或Nobox的共同靶基因一般于E18（胚胎18天）以后甚至多数于出生后开始表达,例如Gdf9,Nalp4f,Oas1家族基因等。结论:Sohlh1基因敲除小鼠的卵巢早衰表现不是由于减数分裂异常所造成;通过基因芯片的应用,我们找到了Sohlh1及其下游通路上的Lhx8、Nobox基因的共同调节的若干靶基因,对生殖细胞早期发育的调控通路作了进一步的完善。第二章Sohlh1转基因小鼠目的:Sohlh1作为生殖细胞特异性转录因子,在卵泡发育早期过程中起到了重要作用,其表达具有很强的特异性-仅在小鼠卵巢的卵原细胞、始基卵泡和初级卵泡中表达,而次级卵泡以后则无法检测到。我们猜想Sohlh1在高级卵泡中的缺失表达是卵泡后期发育的关键,如果过量表达可能会影响到卵细胞的成熟、排卵及受精方面的异常,于是在本章节的我们的研究目的是构建在次级卵泡中表达Sohlh1的转基因小鼠,并观察评价该转基因鼠的生殖生育能力。方法:Zp3是另一个对卵泡发育和精卵结合起重要作用的卵细胞特异表达因子,在小鼠卵巢的各级卵泡中均有表达。据此,我们应用小鼠Zp3启动子区域,将小鼠Sohlh1开放读码区即转录区衔接于mZp3启动子之后,采用转基因技术获得Sohlh1转基因小鼠。然后对此转基因鼠进行表型分析。结果:应用转基因技术,我们获得了5只Sohlh1转基因小鼠,包括2只雌鼠3只雄鼠;将转基因鼠与野生型鼠交配后对子代进行各项实验检测验证,确定我们成功建立了mZp3-Sohlh1转基因小鼠模型;Sohlh1转基因鼠外观体重均于同胞野生型对照小鼠无明显差异,雌鼠卵巢形态大致正常,应用RT-PCR,抗Sohlh1抗体染色进一步证实Sohlh1成功转入卵细胞中并在次级卵泡中表达;有趣的发现是,携带转基因Sohlh1的卵细胞优先于正常卵细胞排卵,尤其雌性子代中携转基因者明显增高。结论:我们成功构建了Sohlh1于各级卵泡中持续表达的转基因小鼠模型,Sohlh1并非卵细胞后期成熟的抑制因子,相反的,Sohlh1的过表达可以促进排卵,或可用于动物繁殖中某些基因型的优化。第三章生殖细胞特异性基因SOHLH1、FIGLA和GDF9在卵巢早衰发病机制中的作用研究第一节美国高加索卵巢早衰患者SOHLH1基因突变筛查目的:Sohlh1是生殖细胞特异性转录调控因子,具有helix-loop-helix（HLH）结构域,Sohlh1基因敲除雌鼠表现为始基卵泡向初级卵泡的发育障碍,卵细胞过早丢失,卵巢萎缩,不能生育。Sohlh1基因敲除小鼠的表现型极似人类卵巢早衰的疾病表现,于是推想人类SOHLH1基因突变可能与卵巢早衰发病有关。方法:选择96例美国高加索人种POF患者及同类人种对照96人,应用PCR扩增直接测序的方法,对SOHLH1基因的7个外显子包括5’端和3’端非翻译区（UTR）及相应侧翼序列进行突变筛查。结果:我们发现了16个包括已知的SNP在内的杂合子改变,其中c.-15 G＞A位于Sohlh1基因的启动子区域,在1例患者及3例对照者中均有发现。仅在POF患者中发现的是1例位于第4外显子上、HLH结构域下游的c.423A＞G导致K120R氨基酸改变;1例第5外显子上的c.598C＞T无意突变,及1例3’UTR区c.1303T＞G和2例c.1623A＞G,而这些均未在对照组出现。另外,我们于第6个外显子上和3’UTR区新发现了若干改变,但大多都在对照组中也有出现,可能是较为罕见的核苷酸多态。结论:c.423A＞G所致的K120R,在灵长目动物中属于保守序列,可能会与POF的发病有一定关系;另外,3’UTR是microRNA的结合部位,SOHLH1基因3’UTR所出现的大量SNPs改变,很可能会影响到一系列microRNA的靶定结合,从而致SOHLH1的表达失衡,这或可是导致卵细胞过早丢失的另一原因,有待于进一步的研究。第二节中国汉族卵巢早衰患者FIGLA基因突变筛查目的:卵巢早衰（POF）是引起高促性腺激素性不孕的一常见疾病,在女性人群中的发病率为1-2%。卵巢早衰的病因具有多样性,部分原因仍考虑为单基因突变引起。而FIGLA,是目前所知的最早调控卵细胞发育成熟的转录因子,Figla基因缺陷雌性小鼠卵巢中无法形成始基卵泡,亦无卵细胞透明带形成,故推测FIGLA基因突变可能会关系到POF。方法:选择就诊于我们生殖中心的100例汉族POF妇女,及304例对照进行血样DNA提取,PCR特异扩增包含有FIGLA的5个外显子的区域,进行直接测序（ABI PrismSequencer 3130XL Applied Biosystems）;后应用酵母双杂交实验证实其中一缺失突变影响FILGA与TCF3的结合。结果:经过突变筛查,我们新发现了3个杂合改变:c.11C＞A（p.A4E）在2例POF患者及1例对照中出现,c.15-36del（p.G6fsX66）和c.419-421delACA（p.140delN）仅从POF患者中得到,而未出现在304例对照组中。c.15-36del缺失了22bp核苷酸,导致了移码突变,使FIGLA仅在翻译了与原蛋白相同的6个氨基酸以后错译至66个氨基酸后终止,从而有效的导致了所谓的半剂量效应,即FIGLA效量减半。另一缺失突变c.419-421delACA（p.140delN）在酵母双杂交实验中被证实破坏了FIGLA与TCF3基因的结合,FILGA与TCF3结合后方可启动下游基因ZP2（另一卵细胞发育关键因子）的表达,所以认为此突变对卵细胞发育具有很大程度的显性负效应。结论:在中国汉族的POF人群中,有部分的发病与基因FIGLA突变有关。第三节中国汉族卵巢早衰患者GDF9基因突变筛查目的:卵巢早衰（Premature Ovarian Failure,POF）是指青春期后、40岁以前发生的非生理性的月经停止,伴有促卵泡素（FSH）、黄体生成素（LH）水平的升高、雌激素（E₂）水平的下降以及临床上表现的潮热、不育和生殖器官的萎缩等综合症。而生长分化因子9（GDF9）是由卵细胞分泌的,对卵泡发育起重要作用的生殖细胞生长因子。本研究旨在探求基因GDF9突变是否与国人POF有关。方法:收集2003年5月～2006年5月在山东大学山东省立医院生殖医学中心就诊的卵巢早衰患者100例,及有正常生育史和月经史对照96人的外周血。其中卵巢早衰患者的采集符合如下标准:年龄在40岁以下,超过3个月经周期无月经来潮;至少两次不同日血清FSH＞40 IU/L,阴道B超监测双侧卵巢未见储备卵泡,并除外染色体异常及免疫性疾病。提取外周血DNA,于GDF9编码区设计3对特异性引物行PCR扩增,先用变性高效液相色谱法（Denaturing High-Performance Liquid Chromatography,DHPLC）检测是否存在杂合双链。如果检测出杂合双链,再用PCR产物直接测序寻找突变位点。结果:在100例POF患者中,我们新发现了GDF9编码区3个位点的非同义突变:c.436C＞T（p.Arg146Cys）,c.712A＞G（p.Thr238Ala）和c.1283G＞C（p.Ser428Thr）,其中c.436C＞T和c.1283G＞C同时也在对照组中出现。c.712A＞G仅存在于POF患者中,该患者为一30岁的继发性闭经女性,无家族性疾病史,FSH 84.6IU/L,LH 39.2IU/L,经阴B超显示子宫缩小（41×33mm）,双侧卵巢较实（大小分别为17×12mm和18×14mm）,无明显卵泡。另外,我们还发现了3个同义突变位点,其中两个也出现在对照组中。结论:我们在100例POF患者中发现了6个位点的突变,其中4个为新发突变,2个为已知突变。新发现突变中两个也同样出现在对照组中,未出现在对照组中的一个（c.588A＞C）为同义突变,另一个c.712A＞G致丙氨酸错译为苏氨酸（p.Thr238Ala）,该位点属于GDF9蛋白编码功能保守区,推测此突变可能会影响GDF9蛋白功能。更多还原

【Abstract】 ChapterⅠ.Screening Downstream Targets of Sohlh1OBJECTIVE:Sohlh1（spermatogenesis and oogenesis specific basic helix-loop-helix transcription factor 1）,an oocyte-specific helix-loop-helix（HLH）gene,plays a critical role during early folliculogenesis.Females lacking of Sohlh1 in mice appear to have normal embryonic gonadogenesis,but form imperfect primordial follicles that not progress to primary follicles.The objective of this study is to investigate if meiosis perturbations account for the rapid oocytes loss and also to identify the downstream targets of Sohlh1.METHODS:RT-PCR and immunofulorescence were carried out to detect meiosis genes Dmc1,Spo11,Msh5,Rec8 and Scp3 expression in Sohlh1 null mice ovaries or oocytes.Then we compared the gene expression difference of newborn mouse ovaries between wild type and Sohlh1 knockout using Affymetrix 430 2.0 microarray platform.The microarray transcripts profiles from Sohlh1,Lhx8 and Nobox null mice were combined and analyzed to find the common downstream target genes.RESULTS:The relative levels of Dmc1,Msh5,Spo11,or Rec8 transcripts were not significantly different between the wild type and Sohlh1 null ovaries.Also no appreciable differences are noted with staining of Scp3 between wild type and Sohlh1 null oocytes. Further we compared Sohlh1 deficient and wild-type ovaries using Affymetrix 430 2.0 microarray platform.494 genes were down-regulated and 165 gene were up-regulated more than 2-fold in Sohlh1 null ovaries.Combining of Lhx8 and Nobox null ovary arrays,we identified a common set of 73 transcripts whose expression level were affected in all Sohlh1, Lhx8 and Nobox deficient mice;and among them,33（45.2%）were preferentially expressed in oocytes with more than 5 fold change.By exploring functional annotation,gene expression patterns analysis and cis-acting sites screening,we discoved a subset of target genes were regulated in the SOHLH1-LHX8-NOBOX pathway.CONCLUSION:These results indicate that meiotic components currently known to disrupt early oogenesis are not affected by Sohlh1 deficiency and suggest that oocyte differentiation can be governed by factors independent of meiosis.Downstream transcripts like LHX8, NOBOX,GDF9,PADI6,NALP and OAS1 famlily should be account for the failure in maintenance and differentiation of the oocyte during early folliculargenesis in Sohlh1 dificient mice. ChapterⅡ.Sohlh1 Transgenic MouseOBJECTIVE:Mammalian oocytes have been proposed to have important roles in the orchestration of ovarian follicular development and fertility and Sohlh1 is one of the oocyte-specific genes expressed maily in early stage of follicles.To determine whether over-expressed Sohlh1 have functions after the transition from primordial follicles to primary follicles,we generated a Sohlh1 transgenic(Sohlh1^tg)mouse model.MATERIALS AND METHODS:The full coding region of mouse Sohlh1 cDNA was subcloned into the SmaI site of a routine Zp3 promoter-BGH poly-A expression vector.After injection into the male pronucleus of one cell zygotes from F2 of C57 mice,the embryos were transferred to oviducts of foster mothers and correct transgenic mice were indentified by PCR genotyping.Subsequent studies were carried out to investigate the phenotype of Sohlh1 transgenic mice.RESULTS:By RT-PCR and immunohistochemistry probed by anti-Sohlh1 antibodies, transgenic active Sohlh1 is detected through all stages of ovarian follicles.Sohlh1^tgfemales can fertilze and Sohlh1^tgoocytes were matured and ovulated early than wild type,or, alternatively,the pups from Sohlh1^tgfemales are preferrecially carring Sohlh1^tg,especially in female pups.CONCLUSION:We successfully constructed mZp3-Sohlh1 transgenic mouse strain.The results from the current study indicate that Sohlh1 overexpression during the late follicle stages can promote ovulation. ChapterⅢ.Germ-cell Specific Genes-SOHLH1,FIGLA and GDF9 Mutations in Women with Premature Ovarian FailureSectionⅠ.Mutation analysis of SOHLH1 in Caucasian Women with POFOBJECTIVE:Sohlh1 deficiency in female mice leads to unfertility because of postnatal oocyte loss and failure in transition from primordial to primary follcles.The phenotype mimics human premature ovarian failure（POF）well.The aim of this study is to investigate whether SOHLH1 mutation is associated with human POF.MATERIALS AND METHODS:We have directly sequenced SOHLH1 in a cohort of 96 Caucasian POF patients presenting with secondary amenorrhea and high FSH levels and in a control population including 96 women with regular menstrual cycles who had at least one child.RESULTS:We have identified 16 novel heterozygous variants.One alteration c.423A＞G（K120R）in the downstream of HLH domain is conserved among vertebrate species and also not presented in control samples.On the other hand,several variants were detected in the 3’UTR region,including some only appeared in POF samples and some rare SNPs in both POF and controls.CONCLUSION:We propose c.423A＞G（K120R）mutation may be involved in POF; nonetheless,a bunch of SNPs identified in SOHLH1 3’UTR region may impact the binding of mircoRNAs which have potential roles in regulating m RNA translation and protein inhibition. SectionⅡ.Mutation analysis of FIGLA in Chinese Women with POFOBJECTIVE:Premature Ovarian Failure（POF）is a common cause of hypergonadotropic ovarian failure and infertility,affecting 1-2%of women.POF is a genetically heterogenous disease,with few known causative genes.We utilized a candidate gene approach to study if FIGLA,a transcriptional regulator preferentially expressed in the ovary,is mutated in Chinese women with POF.MATERIALS AND METHODS:100 Chinese POF women,as well as 304 control age-matched women,were recruited for this study.The coding regions of FIGLA gene were amplified using polymerase chain reaction（PCR）with 4 pairs of specific primers. Sequencing was performed after PCR amplification on ABI Prism Sequencer 3130XL （Applied Biosystems）.To further test whether the missense or deleted（c.11C＞A and c.419-421delAAC）alleles affect FIGLA’s ability to dimerize with itself or TCF3,we employed a yeast two-hybrid strategy to study protein-protein interactions.RESULTS:Three novel variants were identified in four POF individuals:c.11C＞A（p.A4E）, c.15-36del（p.G6fsX66）and c.419-421delACA（p.140delN）.The c.15-36delta causes a frameshift mutation with haploinsufficiency.Functional analyses by the yeast two-hybrid assay demonstrated that p.140delN mutation disrupted FIGLA binding to the TCF3 HLH domain.The c.15-36 delta and c.419-421delACA mutations were not present in the 304 control women.CONCLUSION:Our findings show that a subset of Chinese women with sporadic premature ovarian failure harbor mutations in FIGLA.Haploinsufficiency in transcription factors is known to cause many human Mendelian disorders,and c.15-36delta frameshift results essentially in haploinsufficiency by terminating FIGLA open reading frame immediately after the first five amino acids.Moreover,we show that the c.419-421delACA disrupts FIGLA’s interaction with TCF3.High throughput sequencing of genes involved in the FIGLA pathway will be useful to detect other genes that play critical roles in premature ovarian aging. SectionⅢ.Mutation analysis of GDF9 in Chinese Women with POFOBJECTIVE:Growth differentiation factor 9（GDF9）is a germ cell specific growth factor secreted by oocytes and crucial for folliculogenesis.Our goal was to determine if perturbations in the GDF9 gene occur in Chinese women having Premature Ovarian Failure （POF）.MATERIALS AND METHODS:100 POF women and 96 control women,were recruited in the Reproductive Medical Center,Shandong Provincial Hospital,China.Inclusion criteria were defined as twice serum follicle stimulating hormone concentrations greater than 20IU/ml before age of 40 years,and normal karyotype.After genomic DNA was extracted from blood samples,the coding regions of GDF9 were amplified using polymerase chain reaction（PCR）with 3 pair of primers.Heteroduplexes were detected using denaturing high-performance liquid chromatography（DHPLC）.Samples which demonstrated heteroduplex formation on DHPLC were then sequenced directly after PCR amplification on an automated sequencer.RESULTS:In the POF group,we found 3 nonsynonymous SNPs in the GDF9 gene: c.436C＞T（p.Arg146Cys）,c.712A＞G（p.Thr238Ala）and c.1283G＞C（p.Ser428Thr）.Novel mutations c.436C＞T and c.1283G＞C were also discovered in the controls.The only nonsynonymous SNP（c.712A＞G）detected in POF group but not in controls was present in a 30-year-old woman with secondary amenorrhea.In addition,we found 3 synonymous（silent） mutations in the POF group,two also present in controls.CONCLUSION:Although 4 novel SNPs and 2 additional known mutations were found in the POF cases,all except one（c.712A＞G）was either found in controls or was silent.712A＞G mutation was not present in controls.712A＞G mutation in GDF9 may be associated with POF in our study population.更多还原