【作者】 田雨；

【导师】 周立全；

【作者基本信息】 华中科技大学 ， 妇产科学， 2023， 硕士

【摘要】 目的:小鼠生殖细胞发育和植入前胚胎发育过程存在复杂而精密的调控,rRNA合成通路也涉及其中。PPAN(Peter Pan Homolog)在保障核糖体RNA(Ribosomal RNA,rRNA)的合成过程中发挥关键作用,并且在小鼠雌雄生殖系统和早期胚胎发育阶段均存在高水平转录,但其在哺乳动物雌雄生殖细胞发育和植入前胚胎发育阶段的功能均尚未被揭示。本研究旨在明确PPAN缺失对小鼠生殖细胞发育和植入前胚胎发育过程的影响,探讨PPAN参与维持生殖细胞发育和植入前胚胎过程的功能机制。方法:本研究通过Cre-loxP系统构建雄性和雌性生殖细胞条件性PPAN基因敲除小鼠模型(Stra8-cKO和Zp3-cKO),在小鼠雌雄生殖细胞中特异性敲除PPAN基因,研究PPAN在小鼠生殖细胞发育和植入前胚胎发育过程的功能。具体过程通过组织形态学分析、生育力检测、组织切片染色等确定生殖细胞发育和早期胚胎发育过程是否受到影响,通过免疫荧光染色、TUNEL、qRT-PCR检测生殖细胞在增殖、分化、发育过程的变化,通过免疫荧光染色、RNA-Seq和ChIP-Seq分析PPAN缺失导致生殖细胞和植入前胚胎发育异常的分子机制。结果:本研究确认PPAN在小鼠雌雄配子和早期胚胎中均存在较高水平表达,Stra8-cKO小鼠的睾丸和Zp3-cKO小鼠的卵巢中,PPAN表达被显著抑制,同时,5.8 S rRNA、18S rRNA和28S rRNA等成熟rRNA的表达水平被显著下调。Stra8-cKO小鼠表现为雄性小鼠生育能力下降,具体表现为睾丸缩小,生精细胞增殖和凋亡活动异常,精母细胞减数分裂轻度阻滞,精子生成减少、精子质量严重下降和表观遗传修饰发生改变。Zp3-cKO小鼠卵母细胞发育成熟未出现明显异常,但早期胚胎中PPAN的耗竭导致胚胎发育阻滞在桑葚胚阶段。同时,RNA-Seq结果提示在小鼠胚胎敲除PPAN 和小鼠胚胎干细胞(Mouse Embryonic Stem Cells,mESCs)中敲降 PPAN,对转录组影响较小,ChIP-Seq也提示PPAN在mESCs中没有DNA结合活性,因此,PPAN的缺失应当主要通过影响rRNA合成以及在转录后水平影响雄性生殖细胞发育和植入前胚胎发育过程。结论:本研究首次明确了 PPAN在小鼠生殖细胞发育和植入前胚胎发育过程中所发挥的生理功能。PPAN敲除会损伤小鼠雄性生殖细胞发育过程,引发表观遗传修饰发生改变,PPAN敲除未对小鼠雌性生殖细胞发育产生明显影响,但PPAN纯合敲除导致小鼠早期胚胎在桑葚胚阶段发育停滞。在小鼠mESCs中,PPAN敲降对转录组影响较少,也不具备DNA结合活性。因此,PPAN可能通过保障rRNA合成通路在转录后水平和表观遗传水平,参与调控雄性生殖细胞发育和早期胚胎发育。第一部分:PPAN基因的保守性分析和表达谱研究目的:分析PPAN基因在不同物种之间的保守性和同源性,并研究其在小鼠各器官、各类生殖细胞以及早期胚胎发育各阶段的表达水平差异。方法:利用公共数据库并结合生物信息学相关方法,研究PPAN基因在人、猕猴、小鼠等物种之间的保守性和同源性;利用公共转录组数据和qRT-PCR实验技术,在转录水平,明确PPAN基因在小鼠各器官、各类生殖细胞以及早期胚胎发育各阶段的表达水平差异。结果:我们分析了 PPAN蛋白的氨基酸序列在果蝇、非洲爪蟾、智人、猕猴、猪、小鼠和大鼠等多个物种之间的保守性。我们确认PPAN的氨基酸序列在上述物种间具有较好的保守性。同时我们通过qRT-PCR确认了 PPAN在卵母细胞、精原干细胞中有高转录水平,而在除去2细胞阶段的早期胚胎其他阶段也均具有较高的转录水平。结论:PPAN蛋白质氨基酸序列在多个物种中具有较高的保守性;同时PPAN基因在卵母细胞、植入前胚胎的多数阶段以及精原干细胞中有较高水平转录。第二部分:PPAN在小鼠雄性生殖细胞发育中的功能研究目的:解析PPAN在小鼠雌性生殖细胞发育过程中的生理功能。方法:利用Stra8-Cre小鼠构建雄性生殖细胞PPAN条件性敲除小鼠模型。利用生育力测试、组织形态学分析、免疫荧光染色等方法解析PPAN在雄性生殖细胞发育过程的生理功能。结果:我们利用Stra8-Cre工具小鼠成功构建了雌性生殖细胞特异性敲除PPAN的小鼠模型,Stra8-cKO。Stra8-cKO小鼠产仔数减少,精子发生受损和精子质量严重下降。结论::PPAN在小鼠雄性生殖细胞发育过程中发挥关键作用。第三部分:PPAN在小鼠雌性生殖细胞和植入前胚胎发育中的功能研究目的:解析PPAN在小鼠雌性生殖细胞和植入前胚胎发育过程中的生理功能。方法:利用Zp3-Cre小鼠构建雌性生殖细胞PPAN条件性敲除小鼠模型。利用生育力测试、组织形态学分析、RNA-Seq等方法解析PPAN在雌性生殖细胞和植入前胚胎发育中的生理功能。结果:利用Zp3-Cre工具小鼠成功构建了雌性生殖细胞特异性敲除PPAN的小鼠模型,Zp3-cKO,并获取了 PPAN敲除的合子胚胎。Zp3-cKO小鼠生育力正常,但合子阶段敲除PPAN导致植入前胚胎在桑葚胚阶段发育停滞。结论:PPAN在小鼠雌性生殖细胞发育过程中不发挥关键作用,母源性PPAN对于小鼠胚胎植入前正常发育不必要,合子阶段的PPAN表达对于小鼠胚胎植入前正常发育是必须的。更多还原

【Abstract】 Objective:There exists a complex and precise regulation in the development of mouse reproductive cells and pre-implantation embryo development.PPAN(Peter Pan Homolog)plays a critical role in ensuring the synthesis of ribosomal RNA(rRNA),and is highly transcribed in both male and female reproductive systems as well as early embryonic development in mice.However,its function in mammalian reproductive cell development and pre-implantation embryo development remains unknown.This study aims to clarify the impact of PPAN deficiency on mouse reproductive cell and pre-implantation embryo development,and explore the functional mechanism of PPAN in maintaining reproductive cell and pre-implantation embryo development.Method:In this study,conditional PPAN-knockout mice models(Stra8-cKO and Zp3-cKO)were constructed using the Cre-loxP system,specifically knocking out the PPAN gene in male and female germ cells of mice.The function of PPAN in germ cell development and pre-implantation embryo development in mice was studied.The process was determined through histological analysis,fertility testing,and tissue section staining to confirm whether germ cell development and early embryo development were affected.Changes in proliferation,differentiation,and development were detected through immunofluorescence staining,TUNEL,and qRT-PCR.The molecular mechanism of abnormal germ cell and preimplantation embryo development caused by PPAN depletion was analyzed using immunofluorescence staining,RNA-Seq,and ChIP-Seq.Result:This study confirmed that PPAN is expressed at a high level in both male and female germ cells and early embryos in mice.PPAN expression was significantly inhibited in the testes of Stra8-cKO mice and in the ovaries of Zp3-cKO mice.At the same time,the expression levels of mature rRNAs such as 5.8S rRNA,18S rRNA,and 28S rRNA were significantly downregulated.The development and maturation of oocytes in Zp3-cKO mice did not show obvious abnormalities,but depletion of PPAN in early embryos led to developmental arrest at the early blastocyst stage.The fertility of male mice in Stra8-cKO was decreased,with smaller testes,abnormal proliferation and apoptosis activity of germ cells,mild meiotic arrest of spermatocytes,decreased production of sperm,severe decrease in sperm quality,and changes in epigenetic modifications.Meanwhile,RNA-Seq results showed that PPAN knockdown had a relatively small impact on the transcriptome in mouse embryos and mouse embryonic stem cells(mESCs),and ChIP-Seq indicated that PPAN had no DNA-binding activity in mESCs,indicating that depletion of PPAN mainly affects rRNA synthesis and development of male germ cells and pre-implantation embryo development at the post-transcriptional level.Conclusion:This study clarifies for the first time the role that PPAN plays in the development of germ cells and pre-implantation embryos in mice.Depletion of PPAN impairs the development of male germ cells in mice and leads to the arrest of preimplantation embryo development,while it does not significantly affect the development of female germ cells.In mESCs,PPAN knockdown has a relatively small impact on the transcriptome and no DNA-binding activity,suggesting that PPAN may participate in regulating the development of male germ cells and early embryo development at the posttranscriptional level.Part Ⅰ:The Conversation and Expression Profile of PPANObjective:To analyze the conservation and homology of the PPAN gene in different species,and to study its expression level differences in various organs,reproductive cells,and early embryonic development stages in mice.Methods:Using public databases and bioinformatics methods,we studied the conservation and homology of the PPAN gene in humans,macaques,and mice.Using public transcriptome data and qRT-PCR experimental techniques,we determined the expression level differences of the PPAN gene in various organs,reproductive cells,and early embryonic development stages in mice at the transcription level.Results:We analyzed the conservation of the PPAN amino acid sequence in multiple species such as fruit flies,African clawed frogs,humans,macaques,pigs,mice,and rats.We confirmed that the amino acid sequence of PPAN has good conservation among these species.We also confirmed through qRT-PCR that PPAN has high transcription levels in oocytes,spermatogonial stem cells,and most stages of pre-implantation embryos except for the 2-cell stage.Conclusion:The PPAN amino acid sequence has relatively high conservation in multiple species.The PPAN gene has high transcription levels in oocytes,most stages of preimplantation embryos,and spermatogonial stem cells.Part Ⅱ:Function of PPAN in Mouse Male Germ Cells DevelopmentObjective:To elucidate the physiological function of PPAN in mouse male germ cell development.Methods:A conditional knockout mouse model of PPAN in male germ cells was generated using Stra8-Cre mice.The physiological function of PPAN in male germ cell development was analyzed using fertility tests,histological analysis,immunofluorescence staining,and other methods.Results:We successfully generated a male germ cell-specific PPAN conditional knockout mouse model using the Stra8-Cre tool,Stra8-cKO.Stra8-cKO mice had reduced litter size,impaired spermatogenesis,and decreased sperm quality.Conclusions:PPAN plays an important role in mouse male germ cell development.Part Ⅲ:Function of PPAN in Mouse Female Germ Cells Development and Pre-implantation Embryo DevelopmentObjective:To elucidate the physiological function of PPAN in mouse female germ cells and pre-implantation embryo development.Methods:A conditional knockout mouse model of PPAN in female germ cells was generated using Zp3-Cre mice.The physiological function of PPAN in female germ cells and pre-implantation embryo development was analyzed using fertility tests,histological analysis,RNA-Seq,and other methods.Results:We successfully generated a female germ cell-specific PPAN conditional knockout mouse model using the Zp3-Cre tool,Zp3-cKO,and obtained PPAN knockout homozygotes.The fertility of Zp3-cKO mice was normal,but knockout of PPAN during the homozygous stage caused embryonic tissue to grow to the morula stage.Conclusions:PPAN does not play a critical role in the development of mouse female germ cells.The expression of PPAN during the homozygous stage,rather than the maternal PPAN,is necessary for normal pre-implantation embryo development in mice.更多还原