Expression Analysis Of Nlrp Gene Family And Function Of Nlrp2and Nlrp4g In Early Embryonic Development In The Mouse

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.