Chromosomal abnormalities, particularly aneuploidies, are prevalent during the earliest cell cycles in pre- implantation human embryos. The high incidence of mitotic errors is puzzling – stable chromosome transmission represents a fundamental process ostensibly honed by natural selection. However, many of the underlying proteins, including centromere proteins that direct chromosome segregation and telomere proteins that preserve chromosome ends, evolve rapidly under positive selection. This paradox of conserved cellular processes supported by unconserved machinery suggests recurrent innovation. A proposed but largely untested resolution to this paradox is that rapid evolution of repetitive DNA drives the evolution of proteins that package this DNA. Under this co-evolution model, constantly changing repetitive DNA compromises viability and/or fertility, spurring adaptation at chromosomal proteins that preserve genome stability. Data from non- mammalian model organisms implicates the very earliest embryonic cycles. Here we consider the distinct challenges posed by sperm-deposited DNA, which enters the egg highly compact and inert and is transformed into competent chromosomes by maternal proteins. We hypothesize that maternally-deposited proteins evolve rapidly to remodel and establish centromeres and telomeres on ever-evolving paternal repetitive DNA. Using mouse as a mammalian model system, we exploit both natural variation in Mus centromeric and telomeric repetitive DNA content and divergent maternal proteins from M. musculus relatives to study the cell biological consequences of ‘mismatched’ paternal repetitive DNA and maternally provisioned proteins. Our hypothesis predicts that maternally-provisioned proteins adapted to repetitive DNA in one species will not function optimally when confronted with divergent paternal centromeres and telomeres of another species. Our specific aims are to (1) establish an in vitro fertilization (IVF) scheme to systematically vary the paternal DNA and (2) replace rapidly-evolving maternal proteins with diverged versions from related species. In each case, we will determine the consequences for centromere and telomere packaging and embryonic genome stability. This innovative, evolution-guided functional approach reveals otherwise invisible genetic and epigenetic determinants of early embryonic viability. Our overall goal is to establish an integrated experimental system that allows us to challenge diverged, maternally provisioned proteins with paternal genomes of varying repeat number and sequence, providing crucial support for a future R01 that investigates how the zygote restores epigenetic symmetry between essential chromosomal loci that diverge genetically between the maternal and paternal genomes. Defining the centromere and telomere factors at the interface of dynamic evolution with cognate repetitive DNA will expose an underappreciated co-evolutionary process in the pre-implantation embryo. Under this model,...