【作者】 彭辉；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2012， 博士

【摘要】 Nlrp基因家族在小鼠和灵长类的先天免疫系统和生殖系统起着很关键的作用。到目前为止，在小鼠上鉴定出20个Nlrp基因家族的成员。近来利用系统进化分析法在小鼠上鉴别了Nlrp基因家族中与免疫有关的分支，包括7个Nlrp基因，它们分别是Nlrp1a、1b、1c、3、6、10和12。起初对这个基因家族的研究主要集中在凋亡和炎症信号通路上，近年来越来越多的研究表明一些Nlrp基因在小鼠生殖系统中发挥着至关重要的作用。例如， Nlrp5和Nlrp14已经被研究且结果表明母源性敲除/沉默这两个基因都将导致小鼠早期胚胎发育阻滞。因此，我们选择并研究除了Nlrp5和Nlrp14以外的其它Nlrp基因在小鼠上的表达，并深入研究了Nlrp2和Nlrp4g在小鼠早期胚胎发育过程中的功能。1、Nlrp基因家族中免疫相关基因在小鼠上的表达。结果表明： Nlrp1a、Nlrp1b、Nlrp1c、Nlrp3、Nlrp6和Nlrp12在小鼠附植前胚胎中都不表达，而Nlrp10在桑椹胚和囊胚中能检测到微弱的表达。这7个Nlrp基因家族中免疫相关基因都在多种组织中表达，这些基因有一个共同的特点就是都在脾脏和肝脏中表达。Nlrp基因家族中免疫相关基因在小鼠细胞中的表达也各不相同，但有一个共同的特点就是都不在卵母细胞中表达。2、Nlrp4a-Nlrp4f在小鼠发育过程中的表达分析。结果表明：在附植前的发育过程中Nlrp4a、Nlrp4b、Nlrp4d和Nlrp4f有类似的表达谱，它们在GV期卵母细胞和成熟卵母细胞中大量表达，随着合子基因组的激活，这些基因的表达急剧下调并消失。Nlrp4c和Nlrp4e的转录物也是在GV期卵母细胞和成熟卵母细胞中大量表达，受精之后表达下降，但在桑椹胚和囊胚中又重新检测到它们的表达。在组织中的研究表明Nlrp4b和Nlrp4c只在卵巢中表达，而Nlrp4a、Nlrp4d和Nlrp4f不仅在小鼠卵巢中表达，还在睾丸组织中表达，而Nlrp4e的转录物则在多种组织中都能检测到。这些基因都在小鼠卵巢中表达且都随着小鼠年龄的增加而表达下调。在小鼠不同细胞中的表达表明Nlrp4d的转录物不仅在卵母细胞中检测到，在卵丘细胞和精子中也能检测到，而其它的Nlrp4基因都有相同的表达谱，它们都只在卵母细胞中表达。3、Nlrp9a-Nlrp9c在小鼠发育过程中的时空表达。结果表明：小鼠早期胚胎中Nlrp9a、Nlrp9b和Nlrp9c的转录物都是母源性的，合子基因组激活以后完全降解。Nlrp9a在小鼠出生后的第5d开始表达，Nlrp9b和Nlrp9c在E7.5能检测到微弱的表达。Nlrp9a只在卵巢组织中能检测到，Nlrp9b除了在卵巢中表达，还在小肠中能检测到，Nlrp9c在多种组织中表达。Nlrp9a-Nlrp9c在小鼠卵巢中的表达随着小鼠年龄的增加而下降。Nlrp9a和Nlrp9c只在小鼠卵母细胞中表达， Nlrp9b的转录物除了在卵母细胞中能够检测到，在D3ES和F9ES中也能检测到微弱的表达。4、小鼠早期胚胎的发育需要Nlrp2基因的参与。结果表明：在卵泡发生的过程中，Nlrp2mRNA和蛋白都在卵母细胞和颗粒细胞中表达。排卵和受精以后，Nlrp2mRNA伴随着合子基因组的激活而显著下调，在2-细胞期以后的胚胎中检测不到，但NLRP2蛋白的表达却持续到囊胚期。共聚焦显微镜的结果表明NLRP2主要在胞质中定位，免疫电镜分析进一步揭示出NLRP2不仅在胞质中定位，还在细胞核以及核孔附近存在。通过RNA干扰方法在GV期卵母细胞中特异性的沉默Nlrp2，结果表明这些Nlrp2沉默的GV期卵母细胞能够通过MI中期并排出第一极体，但这些成熟的卵母细胞经过孤雌激活后，孤雌胚胎主要阻滞在2-细胞期。进一步的研究显示在合子中母源性的沉默Nlrp2也导致早期胚胎发育阻滞。此外，在合子中过表达Nlrp2，合子能够发育成形态正常的囊胚，但在这些囊胚中增加了细胞凋亡。5、母源性沉默Nlrp4g阻滞早期胚胎发育。结果表明：Nlrp4g转录物和蛋白在小鼠卵巢中特异性表达，这种表达限制在不同发育阶段的卵泡卵母细胞中，且Nlrp4g转录物和蛋白的表达随着小鼠年龄的增加而显著下降。无论是在正常受精的胚胎还是在孤雌胚胎中，Nlrp4g转录物都伴随着合子基因组的激活而显著下调，但NLRP4G蛋白的表达却持续到囊胚期。共聚焦显微镜分析表明NLRP4G定位在不同时期胚胎的胞质中，而免疫电镜进一步证实这个蛋白存在于胞质溶胶，并不在胞质细胞器中定位。通过RNA干扰方法在GV期卵母细胞中特异性沉默Nlrp4g并不影响卵母细胞成熟，但是在合子中母源性的沉默Nlrp4g则导致早期胚胎主要阻滞在2-细胞期和8-细胞期之间。这些结果证明了Nlrp4g是一个卵母细胞特异性基因，在早期胚胎发育过程中需要Nlrp4g的参与。更多还原

【Abstract】 The Nlrp gene family plays an essential role in the innate immune and reproductivesystems in the mouse and primates. To date,20Nlrp family members have been identified inthe mouse. Recently, the phylogenetic analyses identified a well supportedimmunization-related clade including seven Nlrp genes: Nlrp1a、1b、1c、3、6、10and12.Initially, studies on the function of this family were mainly in apoptotic and inflammatorysignaling pathways. However, a rapidly growing number of recent researches showed thatsome Nlrp genes play key roles in reproductive systems. For instance, Nlrp5(Mater) andNlrp14have been investigated. It is show that these genes are required for early embryonicdevelopment in the mouse. Thus, we selected and investigated the expression of Nlrp genefamily and further researched the function of Nlrp2and Nlrp4g in early embryonicdevelopment in the mouse.1. Expression analyses of immunization-related Nlrp genes in the mouse. The resultsindicated that no expression of Nlrp1a、Nlrp1b、Nlrp1c、Nlrp3、Nlrp6and Nlrp12in earlyembryonic development, while faint expression of Nlrp10was detected in the morula andblastocyst stages. The immunization-related Nlrp genes were expressed in multi-tissues, whilethey were all expressed in the spleen and liver. These genes have the different expressionpattern in the different mouse cells, with a common feature that no expression of these genesin the oocytes.2. Expression analyses of Nlrp4a-Nlrp4f during mouse development. The results showedthat these genes have the similar expression patterns during preimplantation development.They were enriched in the GV stage and MII oocytes, and degraded after fertilization, butNlrp4c and Nlrp4e transcripts were detected again at the morula and blastocyst stages. Thetissue distribution of Nlrp4a–Nlrp4f indicated that Nlrp4b and Nlrp4c were only detected inthe ovary; Nlrp4a, Nlrp4d and Nlrp4f were also transcribed in the ovary, as well as in thetestis, while Nlrp4e was expressed in various mouse tissues. Furthermore, expression of thesegenes was downregulated in the ovary with mouse aging. In addition, the expression profilesof Nlrp4a–Nlrp4f in different cells demonstrated that Nlrp4a, Nlrp4b, Nlrp4c, Nlrp4e andNlrp4f were not detected in other cell lines except for oocytes, while Nlrp4d transcripts were detected in oocytes, as well as in cumulus cells and spermatozoa.3. The temporal and spatial expression patterns of Nlrp9a–Nlrp9c during mousedevelopment. In this study, we investigated the temporal and spatial expression patterns ofNlrp9a, Nlrp9b and Nlrp9c and present evidence for the exclusive maternal origin of thesetranscripts, which were present in oocytes and zygotes, immediately downregulated and notdetected after the2-cell stage during preimplantation development. No expression of Nlrp9awas evident during postimplantation embryo development. This transcript was first detected5days postpartum. However, Nlrp9b and Nlrp9c transcripts were first detected at embryonicday7.5. In4-week-old mouse tissues, Nlrp9a expression was detected in ovary, Nlrp9b wasdetected in ovary and intestine, Nlrp9c was expressed in multi-tissues. Furthermore,down-regulation of Nlrp9a–Nlrp9c expression in ovary was connected with mouse aging. Inaddition, Nlrp9a and Nlrp9c transcripts were not detected in other cells except for oocytes.Nevertheless, Nlrp9b expression was detected in oocytes, as well as in D3ES and F9ES.4. Nlrp2is required for early embryonic development in the mouse. Nlrp2mRNAs andtheir proteins are expressed in oocytes and granulosa cells during folliculogenesis. Thetranscripts show a striking decline in early preimplantation embryos before zygotic genomeactivation, but the proteins remain present through to the blastocyst stage. Immunogoldelectron microscopy revealed that the NLRP2protein is located in the cytoplasm, nucleus andclose to nuclear pores in the oocytes, as well as in the surrounding granulosa cells. UsingRNA interference, we knocked down Nlrp2transcription specifically in mouse germinalvesicle oocytes. The knockdown oocytes could progress through the metaphase of meiosis Iand emit the first polar body. However, the development of parthenogenetic embryos derivedfrom Nlrp2knockdown oocytes mainly blocked at the2-cell stage. The maternal depletion ofNlrp2in zygotes led to early embryonic arrest. In addition, overexpression of Nlrp2inzygotes appears to lead to normal development, but increases blastomere apoptosis inblastocysts.5. Maternal depletion of Nlrp4g arrests early embryonic development in the mouse. Nlrp4gtranscripts and proteins are specifically expressed in mouse ovaries, restricted to the oocytesat various follicular stages and decline with oocyte aging. The transcripts show a strikingdecline in both fertilized and parthenogenetic embryos before zygotic genome activation, butthe proteins remain present through to the blastocyst stage. Confocal microscopydemonstrated that this protein was localized in the cytoplasm, Immunogold electronmicroscopy further confirmed that the protein was present in the cytosol rather than in oocytecytoplasmic organelles. Compromising Nlrp4g in GV-stage oocytes via interfering RNA didnot affect oocyte maturation. However, the maternal depletion of Nlrp4g in zygotes resulted in early embryonic arrest. These results provide the first evidence that Nlrp4g is anoocyte-specific gene that is required for early embryogenesis in the mouse.更多还原