Comparative Analysis of Aneuploidy and Cellular Fragmentation Dynamics in Mammalian Embryos

项目总结 在减数分裂和/或有丝分裂过程中出现的整个染色体的丢失和获得(非整倍体)是主要的 自然和辅助生殖中胚胎丢失和自发流产的贡献者及其 Boreoeutheria树上的哺乳动物的患病率差异很大。尽管人类，非人类的灵长类， 牛胚胎都有较高的非整倍体发生率，小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的全基因组方法的研究。现在是时候了 众所周知，减数分裂和有丝分裂染色体分离错误同样普遍，但特定的 哺乳动物中有丝分裂的非整倍体对胚胎丢失的作用尚不清楚。蜂窝 碎裂(CF)，胞质小体在胞质分裂过程中夹住胚胎的动态过程， 通常与非整倍体有关，在体外和体内来源的胚胎中都观察到了 几种哺乳动物，尽管程度不同。虽然灵长类和马的胚胎表现出很高的CF发生率， 猪胚胎和牛胚胎分别表现出中等频率和低频率，而小鼠胚胎则不显示 通常显示配置文件。我们最近在人类和恒河猴胚胎上证明了CFS可以封闭 可能起源于将错误分离的染色体封装成微核的DNA 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进化共享的 纠正胚胎非整倍体的过程，或在CF动态中是否存在物种差异。的总目标是 这一建议是为了利用哺乳动物中非整倍体和CF频率的自然多样性并进行研究 应用高分辨率测序技术研究微核、非整倍体和CF的分子机制 接近了。对于目标1，我们将进行活细胞成像、单细胞/CFDNA测序和 拷贝数变异(CNV)分析建立非整倍体和染色体的精确频率 CF在灵长类、马、猪和牛胚胎中的包埋。目标2将专注于区别识别 在同一哺乳动物内部和跨哺乳动物的碎片化和非碎片化胚胎之间的表达基因 使用RNA测序。在目标3中，我们建议操纵先前发现的和/或 新发现的在小鼠和牛胚胎中差异表达的CF相关基因。然后我们将评估 基因敲除或过表达对着床前胚胎体外发育的影响 时间成像和单细胞/CFCNV评估。植入潜力和随后的胚胎发育将 也可以通过将带有或不带有基因操作的小鼠胚胎移植到假孕来进行体内评估 雌性受体小鼠。总体而言，拟议的研究将极大地促进我们对分子的理解 哺乳动物早期胚胎发育过程中染色体错误分离的机制 可用于提高农业上重要物种和人类的繁殖效率。 体外受精(IVF)通过降低胚胎损失率而获得成功。

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.