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: 10596997
• 负责人: Lucia Carbone
• 金额: $ 52.59万
• 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/10596997
• 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; Cytoplasm; Cytoskeleton; 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; Maternal Age; Measures; Meiosis; Messenger RNA; Methods; Microinjections; Microtubules; Mitosis; Mitotic; Mitotic Chromosome; Molecular; Monitor; Mus; Pathway interactions; Ploidies; Pre-implantation Embryo Development; Pregnancy; Prevalence; Primates; Process; RNA Decay; Resolution; Rhesus; Role; Spontaneous abortion; Testing; 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; micronucleus; mosaic; natural Blastocyst Implantation; nonhuman primate; offspring; overexpression; 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.

项目概要 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（非整倍性）是主要的 自然和辅助生殖中胚胎丢失和自然流产的因素及其影响 整个 Boreoeutheria 树中的哺乳动物的患病率差异很大。虽然是人类，非人灵长类动物， 牛胚胎的非整倍体发生率都比较高，鼠胚胎很少出现非整倍体， 马和猪的胚胎仍在等待高分辨率全基因组方法的研究。现在是 众所周知，减数分裂和有丝分裂染色体分离错误同样普遍，但具体的 有丝分裂非整倍性对哺乳动物物种胚胎丢失的影响尚不清楚。蜂窝网络 碎片（CF），细胞质体在胞质分裂过程中从胚胎中夹断的动态过程， 通常与非整倍性相关，并且已在体外和体内衍生的胚胎中观察到 几种哺乳动物，尽管程度不同。虽然灵长类动物和马胚胎 CF 的发生率很高， 猪和牛胚胎分别显示出中频和低频，而小鼠胚胎则没有 通常显示 CF。我们最近用人类和恒河猴胚胎证明，CF 可以包围 DNA可能起源于错误分离的染色体在微核中的封装 减数分裂或有丝分裂。然而，目前尚不清楚通过 CF 进行的染色体隔离是否是一种进化共享的方法。 纠正胚胎非整倍性的过程，或者 CF 动力学是否存在物种差异。总体目标为 该提案旨在利用哺乳动物非整倍体和 CF 频率的自然多样性并进行研究 使用高分辨率测序研究微成核、非整倍性和 CF 的分子机制 接近。对于目标 1，我们将结合活细胞成像、单细胞/CF DNA 测序和 拷贝数变异 (CNV) 分析可确定非整倍体和染色体的精确频率 CF 在灵长类动物、马、猪和牛胚胎中的封装。目标 2 将侧重于差异化识别 在同一哺乳动物内和跨同一哺乳动物的片段化和非片段化胚胎之间表达基因 使用RNA测序。在目标 3 中，我们建议操纵先前发现的和/或 新发现的小鼠和牛胚胎中差异表达的 CF 相关基因。然后我们将评估 使用真实的体外基因敲除或过度表达对植入前胚胎发育的影响 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 也可以通过将经过或不经过基因操作的小鼠胚胎转移到假妊娠来进行体内评估 雌性受体小鼠。总体而言，拟议的研究将极大地增进我们对分子的理解 早期哺乳动物胚胎发生过程中染色体错误分离的机制 从中可以应用于提高农业上重要物种和人类的繁殖效率 通过减少胚胎丢失的发生率来实现体外受精（IVF）的成功。