Comparative Analysis of Aneuploidy and Cellular Fragmentation Dynamics in Mammalian Embryos

项目摘要 在减数分裂和/或有丝分裂过程中出现的全染色体丢失和获得（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因素，以及它们的 在整个北真兽亚目的哺乳动物中，流行率变化很大。尽管人类，非人类灵长类， 和牛胚胎都具有相对高的非整倍体发生率，小鼠胚胎很少表现出非整倍体， 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 成熟的减数分裂和有丝分裂染色体分离错误同样普遍，但具体的 在哺乳动物物种中，有丝分裂非整倍性对胚胎损失的贡献仍然不清楚。蜂窝 碎裂（CF），细胞质体在胞质分裂期间夹断胚胎的动态过程， 通常与非整倍体有关，并且已经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的胚胎显示出CF的高发病率， 猪和牛胚胎分别显示出中等和低频率，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可以包裹 DNA可能来源于在微核中错误分离的染色体的包囊， 减数分裂或有丝分裂。然而，它仍然是未知的，如果染色体隔离通过CF是一个进化共享的， 过程中纠正胚胎非整倍性，或者如果有物种差异CF动力学。的总目标 这项建议是利用哺乳动物间非整倍体和CF频率的自然多样性， 使用高分辨率测序研究微核、非整倍体和CF的分子机制 接近。对于目标1，我们将进行活细胞成像、单细胞/CF DNA测序和 拷贝数变异（CNV）分析，以确定非整倍体和染色体的精确频率 在灵长类动物、马、猪和牛胚胎中通过CF包封。目标2将侧重于识别差异 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，我们提出操纵先前发现的和/或 新发现的小鼠和牛胚胎中差异表达的CF相关基因。我们将评估 用真实的- 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 也可以通过将有或没有基因操作的鼠胚胎转移到假怀孕小鼠体内进行评价。 雌性受体小鼠。总的来说，这项研究将极大地促进我们对分子生物学的理解。 在早期哺乳动物胚胎发生过程中染色体错误分离的机制， 由此可以应用于提高农业上重要物种和人类的繁殖效率， 体外受精（IVF）通过减少胚胎丢失的发生率获得成功。

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.