Comparative Analysis of Aneuploidy and Cellular Fragmentation Dynamics in Mammalian Embryos

哺乳动物胚胎非整倍性和细胞破碎动力学的比较分析

• 批准号: 10366610
• 负责人: Lucia Carbone
• 金额: $ 51.79万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10366610
• 关键词: Agriculture; Aneuploid Cells; Aneuploidy; Animals; Appearance; Cattle; Cell division; Cell physiology; Cells; Chromosomal Gain; Chromosomal Loss; Chromosome Segregation; Chromosome abnormality; Chromosomes; Comparative Study; Copy Number Polymorphism; Cytokinesis; DNA; DNA Fragmentation; DNA sequencing; Data; Detection; Development; Embryo; Embryo Loss; Embryonic Development; Encapsulated; Equus caballus; Exhibits; Family suidae; Female; Fertility; Fertilization in Vitro; Frequencies; Gene Expression; Genes; Genomic approach; Goals; Human; Image; Implant; In Vitro; Incidence; Investigation; Link; Macaca mulatta; Mammals; Measures; Meiosis; Messenger RNA; Methods; Microinjections; Microtubules; Mitosis; Mitotic; Mitotic Chromosome; Molecular; Monitor; Mosaicism; Mus; Pathway interactions; Ploidies; Pre-implantation Embryo Development; Pregnancy; Prevalence; Primates; Process; RNA Decay; Resolution; Rhesus; Role; Spontaneous abortion; Testing; Time; Tissue-Specific Gene Expression; Trees; Work; aneuploidy analysis; assisted reproduction; blastocyst; chromosome missegregation; comparative; comparative genomics; differential expression; embryo stage 2; failure Implantation; genetic manipulation; genome analysis; implantation; improved; in vivo; insight; knock-down; live cell imaging; natural Blastocyst Implantation; nonhuman primate; overexpression; pregnant; preimplantation; real-time images; reproductive; reproductive success; species difference; success; transcriptome sequencing; translational applications; whole genome

PROJECT SUMMARY Whole chromosomal losses and gains (aneuploidy) that arise during meiosis and/or mitosis are major contributors to embryo loss and spontaneous miscarriage in natural and assisted reproduction, and their prevalence varies drastically in mammals across the Boreoeutheria tree. Although human, non-human primate, and bovine embryos all have a relatively high incidence of aneuploidy, murine embryos rarely exhibit aneuploidy, and equine and porcine embryos still await investigation with high-resolution, whole-genome methods. It is now well-established that meiotic and mitotic chromosome segregation errors are equally prevalent, but the specific contribution of mitotic aneuploidy to embryo loss amongst mammalian species is still unclear. Cellular fragmentation (CF), the dynamic process by which cytoplasmic bodies pinch off of embryos during cytokinesis, is often associated with aneuploidy and has been observed in both in vitro and in vivo-derived embryos from several mammals, albeit to varying degrees. While primate and equine embryos exhibit a high incidence of CF, porcine and bovine embryos show intermediate and a low frequency, respectively, and mouse embryos do not typically display CF. We recently demonstrated with human and rhesus macaque embryos, that CFs can enclose DNA that likely originated from the encapsulation of mis-segregated chromosomes into micronuclei during meiosis or mitosis. However, it remains unknown if chromosome sequestration via CF is an evolutionary shared process to correct embryo aneuploidy, or if there are species differences in CF dynamics. The overall goal of this proposal is to leverage the natural diversity in the aneuploidy and CF frequency across mammals and study the molecular mechanisms underlying micronucleation, aneuploidy, and CF using high-resolution sequencing approaches. For Aim 1, we will perform a combination of live-cell imaging, single-cell/CF DNA-sequencing, and copy number variation (CNV) analyses to establish the precise frequency of aneuploidy and chromosome encapsulation by CF in primate, equine, porcine, and bovine embryos. Aim 2 will focus on identifying differentially expressed genes between fragmented and non-fragmented embryos within and across the same mammals using RNA-sequencing. In Aim 3, we propose to manipulate the expression of previously discovered and/or newly identified differentially expressed CF-related genes in murine and bovine embryos. We will then assess the impact of gene knockdown or overexpression on preimplantation embryo development in vitro using real- time imaging and single-cell/CF CNV assessment. Implantation potential and subsequent embryogenesis will also be evaluated in vivo by transferring murine embryos with or without gene manipulation to pseudo-pregnant female recipient mice. Overall, the proposed study will greatly advance our understanding of the molecular mechanisms involved in chromosome mis-segregation during early mammalian embryogenesis, the findings from which can be applied to improving reproductive efficiency in agriculturally important species and human in vitro fertilization (IVF) success by reducing the incidence of embryo loss.

项目摘要 在减数分裂和/或有丝分裂期间出现的整个染色体损失和增益（非整倍）是主要的 在自然和辅助繁殖中引起胚胎丧失和自发流产的贡献者，他们 患病率在整个钻孔树上的哺乳动物中差异很大。虽然是人类的非人类灵长类动物，但 和牛胚胎都有相对较高的非整倍性，鼠类胚胎的发生率，很少表现出非整倍性， 以及高分辨率，全基因组方法，马仍在等待研究。现在 确定的减数分裂和有丝分裂染色体隔离错误同样普遍，但特定 有丝分裂性非整倍性对哺乳动物物种中胚胎丧失的贡献尚不清楚。细胞 碎裂（CF），动态过程，在细胞因子过程中细胞质体从胚胎中捏合的动态过程， 通常与非整倍性相关，并且在体外和体内衍生的胚胎中都观察到 几种哺乳动物，尽管程度不同。虽然灵长类动物和马胚胎表现出很高的CF发生率 猪和牛胚胎分别显示中间体和低频率，小鼠胚胎不显示 通常显示CF。我们最近用人和恒河猕猴的胚胎证明，CF可以包围 可能起源于错误隔离的染色体在Micronuclei中的DNA 减数分裂或有丝分裂。但是，尚不清楚是否通过CF染色体隔离是一种进化共享 纠正胚胎非整倍性的过程，或者如果CF动态存在物种差异。总体目标 该建议是利用哺乳动物的非整倍性和CF频率的自然多样性并进行研究 使用高分辨率测序的微核，非核和CF的分子机制 方法。对于AIM 1，我们将执行活细胞成像，单细胞/CF DNA序列和 拷贝数变化（CNV）分析以建立非整倍性和染色体的精确频率 CF在灵长类动物，马，猪和牛胚胎中封装。 AIM 2将专注于差异化 在同一哺乳动物内和跨碎片的胚胎之间表达的基因 使用RNA测序。在AIM 3中，我们建议操纵先前发现的表达和/或 新鉴定的鼠和牛胚胎中差异表达的CF相关基因。然后我们将评估 基因敲低或过表达的影响对植入前的胚胎胚胎发育，并使用房地产 时间成像和单细胞/CF CNV评估。植入潜力和随后的胚胎发生将 也可以在体内通过或不进行基因操纵的鼠类胚胎来评估体内评估 女性接受者小鼠。总体而言，拟议的研究将大大提高我们对分子的理解 早期哺乳动物胚胎发生期间染色体错误分类的机制，发现 从中可以应用于提高农业重要物种的生殖效率，人类的生殖效率 体外受精（IVF）通过降低胚胎损失的发生率而成功。