【作者】 秦莹莹；

【导师】 陈子江； Rajkovic Aleksander；

【作者基本信息】 山东大学 ， 妇产科生殖医学， 2007， 博士

【摘要】 第一章转录因子NOBOX敲除小鼠新生卵巢芯片结果分析目的：Nobox是生殖细胞特异性转录调控因子，具有同源盒结构域，Nobox基因敲除雌鼠表现为初级卵泡发育障碍，出生后14天卵巢被纤维组织填充，导致不孕。Nobox作为同源盒蛋白家族成员，通过DNA-同源盒结构域相互作用调控下游基因的转录表达，在卵子早期发生过程发挥重要作用。研究目的是确定Nobox调控的下游基因，探讨Nobox对其他卵巢优势表达基因在分子水平的调节。方法：通过基因芯片和实时定量PCR技术，分析Nobox基因敲除新生小鼠卵巢中基因的异常表达。结果：基因芯片结果显示，Nobox敲除新生小鼠卵巢组织中，29个卵子优势表达基因表达水平较野生型降调≥5倍；5个卵子优势表达基因表达量升高5倍以上。以Oct4、Rspo2、Nalp4c、Jag1、Sall4、Stra8、Dmrt1、Tekt2、1700019D03Rik和Lin28作为候选基因，利用实时定量PCR技术验证基因芯片结果，候选基因在两种分析系统中表达变化趋势一致。结论：敲除Nobox基因会不同程度的改变卵巢特异性基因的表达，Nobox是调节卵子发生的主导转录调控因子之一。通过对Nobox调控基因的研究将有助于我们充分认识生殖系统的基因表达调控及卵子发生异常疾病的发病机制。第二章NOBOX基因突变与卵巢早衰的相关性研究目的：同源盒基因NOBOX在卵子中特异性表达，在卵泡早期发育中发挥重要作用，是非综合型卵巢早衰的候选基因之一。本研究目的是探讨NOBOX基因突变与卵巢早衰发病机制的相关性。方法：以就诊于Baylor College of Medicine的96名高加索卵巢早衰患者为研究对象，同时采集278例健康、卵巢功能正常的生育期女性外周血做为正常对照。利用直接测序法筛查卵巢早衰患者NOBOX基因突变情况，并利用凝胶迁移滞后实验探讨错义突变对DNA-蛋白质结合功能的影响。结果：直接测序结果发现7个已知的单核苷酸多态和4个新发突变。新发突变包括2个单核苷酸多态、1同义突变和1个错义突变；其中p.Arg355His和p.Arg360Gln均发生在高度保守的同源盒结构域内。凝胶迁移滞后实验证实p.Arg355His突变蛋白结合DNA的能力明显降低，同时对野生型蛋白功能具有显性负效应。结论：NOBOX基因突变是一部分卵巢早衰患者的致病原因。第三章NANOS3、LHX8基因突变在卵巢早衰发病机制中的作用研究第一部分NANOS3基因突变与卵巢早衰的相关性研究目的：作为最早发现于果蝇的母源性基因，Nanos3具有编码RNA结合蛋白的功能，在生殖细胞向性腺迁移分化的过程中起重要作用。Nanos3基因敲除小鼠表现为卵巢萎缩、各级卵泡缺乏及不孕。人NANOS3基因在生殖细胞中特异性表达，并与小鼠Nanos3具有高度同源性，提示NANOS3可能是卵巢早衰(premature ovarian failure，POF)的候选基因。本研究通过对168例POF患者进行NANOS3基因突变筛查，以探讨NANOS3与POF病因的相关性。方法：应用变性高效液相色谱分析(denaturing high-performance liquidchromatography，DHPLC)(WAVE System 3500 Transgenomic Ltd，Omaha，NE)对80例中国汉族及88例美国高加索人种POF患者NANOS3基因的两个外显子进行突变筛查，对DHPLC图形异常的PCR扩增产物则直接行DNA测序分析(ABI PRISM 310 AppliedBiosystems，Foster City，CA)。结果：结果显示66例POF患者DHPLC图形出现双峰或对称性改变(汉族45例、高加索人种21例)，提示存在DNA序列变异。对PCR扩增产物进行测序分析发现第一外显子356位点发生碱基替换(c．356A＞G)，检索NCBI SNPs Database证实为已知同义单核苷酸多态(rs2016163)。其中杂合子、纯合子在汉族及高加索人种POF组发生频率分别为42.5％、13.7％和14.8％、9.1％。NANOS3其他编码序列区未发现致病突变。结论：本研究没有在中国汉族和美国高加索人种卵巢早衰患者中发现NANOS3致病突变，因此推测在中国汉族和美国高加索人群中NANOS3基因突变与卵巢早衰没有明显的关系。第二部分95例高加索卵巢早衰妇女LHX8突变分析研究目的：LHX8(LIM homeobox 8)是一种在生殖细胞中特异性表达的转录调控因子，具有富含半胱氨酸-组氨酸的LIM结构域，在哺乳动物卵子发生过程中发挥重要作用。本研究通过筛查LHX8基因在95例高加索卵巢早衰患者中的突变情况，以探讨LHX8在人类卵巢早衰发病机制中作用。方法：以95名就诊于Baylor college of medicine的美国高加索人种卵巢早衰患者为研究对象，同时收集94名卵巢功能正常的育龄期高加索女性外周血做为对照组，对LHX8编码序列进行直接测序分析。结果：在内含子3和3’非翻译区(3’untranslated region，3’UTR)分别发现新发单核苷酸多态：c.769+10G＞T和c.1787A＞G。两种多态在POF患者和正常对照人群中的携带频率差别没有统计学意义(p＞0.05)。结论：本研究结果提示LHX8基因突变不是美国高加索卵巢早衰妇女的常见病因。更多还原

【Abstract】 Chapter I. Microarray analysis of newborn mouse ovaries lacking NoboxOBJECTIVE: Nobox （newborn ovary homeobox gene）, an oocyte-specific homeobox gene, plays a critical role during early folliculogenesis and represents a candidate gene for non-syndromic ovarian failure. The objective of this study is to investigate the downstream targets of Nobox.METHODS: We compared the gene expression difference of newborn mouse ovaries between wild type and Nobox knockout using Affymetrix 430 2.0 microarray platform. We also quantified 10 genes that were potentially important during oogenesis using real-time PCR and discussed the possible roles of these genes in oocyte maturation.RESULTS: Genes whose proteins were predicted to be secreted including Jagged 1 （Jag1） and respondin 2 （Rspo2） were down regulated in Nobox knockouts. Nalp4c, a gene that encoded protein involved in apoptotic and inflammatory signaling pathways, was down-regulated in Nobox^-/- ovaries. In addition, pluripotency associated genes, Oct4 and Sall4 were drastically down-regulated while stimulated by retinoic acid gene 8 （Stra8） which was involved in the retinoic acid metabolism, was among few up-regulated genes expressed in oocytes. Testes determining genes, including Dmrt1, Tekt2 and 1700019D03Rik were over-expressed in Nobox deficient ovaries.CONCLUSION: Our findings indicate that Nobox is likely regulator of oocyte-specific gene expression, and suggest that transcription factors preferentially expressed in oocytes may repress male determining gene expression. Chapter II. NOBOX Homeobox Mutations Cause Premature Ovarian FailureOBJECTIVE: NOBOX （Newborn Ovary Homeobox gene）, an oocyte-specific homeobox gene, plays a critical role during early folliculogenesis. Disruption of the murine Nobox gene causes non- syndromic ovarian failure in Nobox^-/- females. We investigate whether NOBOX has mutations causing premature ovarian failure （POF）.METHODS: We sequenced NOBOX gene in 96 Caucasian women with POF, followed by electrophoretic mobility shift assay （EMSA） to verify the effect of novel mutations on protein-DNA.RESULTS: Seven known SNPs and 4 novel variations were discovered. Two of the latter, p. Arg355His and p. Arg360Gln, resides in the highly conserved homeodomain region. Using EMSA, we confirmed that mutation p. Arg355His in mouse decreases the affinity of homeodomain binding to the Nobox DNA binding elements, producing a dominant negative effect that disabled the DNA binding activity of wild type NOBOX.CONCLUSION: Our findings suggest that NOBOX mutations contribute to a subset of women with POF. Chapter III. Mutation analysis of NANOS3 and LHX8 in Womenwith Premature Ovarian FailurePart I. Mutation Analysis of NANOS3 in 80 Chinese and 88 Caucasian Women with Premature Ovarian FailureBACKGROUND AND OBJECTIVE: NANOS3 encodes an RNA binding protein and has a conserved function in germ cell development. Our objective was to investigate whether mutations in NANOS3 are present in Chinese and Caucasian women with premature ovarian failure （POF）.METHODS: Mutation Analysis of NANOS3 in 80 Chinese and 88 Caucasian women with POF were performed using denaturing high-performance liquid chromatography （DHPLC）, followed by sequencing and restriction fragment length polymorphism （RFLP）.RESULTS: A known synonymous single nucleotide polymorphism （SNP） （rs 2016163） in exon 1 was identified through sequencing 80 Chinese and 88 Caucasian women with POF. No additional SNPs or mutations were found in exons encoding for NANOS3.CONCLUSION: Our findings suggest that mutations in NANOS3 exons are rare in both Chinese and Caucasian women with POF. Part II. Perturbations of LHX8 Do not Explain Caucasian Women with Premature Ovarian FailureOBJECTIVE: LHX8 （LIM homeobox 8） encodes a LIM homeodomain transcriptional regulator preferentially expressed in germ cells and is a critical regulator of mammalian oogenesis. We investigated whether nucleotide changes are present in the LHX8 gene of Caucasian women with premature ovarian failure （POF） as compared to control women.METHODS: Mutation of LHX8 was studied in 95 Caucasian women with POF and 94 controls by seqencing.RESULTS: Two novel single nucleotide polymorphisms （SNPs） were discovered in intron 3 （c.769+10G>T） and 3’ untranslated region （c.1787A>G） of the LHX8 gene. These polymorphisms were also found in controls with frequencies that were not statistically different from POF women.CONCLUSION: Mutations in the LHX8 exons are uncommon in Caucasian women with POF.更多还原