Comparative Analysis of Aneuploidy and Cellular Fragmentation Dynamics in Mammalian Embryos

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.

Grant Number: 5R01HD105320-05
NIH Institute/Center: NIH
Principal Investigator: Shawn Chavez