Role Of Fzr1 In Embryogenesis

The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that targets many cell cycle associated proteins for degradation by the 26S proteasome. APC activity is in part controlled by binding to one of its co-activators, and here the focus of the study was on the co-activator Fizzy-Related 1 (Fzr1). When this thesis was started, antisense knockdown studies had shown that Fzr1 is involved in the maintenance of Germinal Vesicle (GV) stage arrest; the fidelity of chromosome segregation during the first meiotic division; and measurable APC^Fzr1 activity had been observed following fertilization. Although these antisense experiments had used controls, it was still appropriate to question whether any of these observations were due to off-target effects. Moreover, it was important to ask how relevant Fzr1 was to the process of meiosis in vivo, given thus far only in vitro studies had been performed. During the course of my studies, a transgenic knockout of Fzr1 was independently created, and using embryos from these mice, it was found that this APC activator has a crucial role in endoreduplication, and was needed for trophoblast giant cell formation and placentation. However, due to the presence of maternal Fzr1 contribution, its role in early preimplantation embryogenesis or meiosis could not be addressed. Here in this thesis, I created an oocyte-specific Fzr1 knockout from breeding mice that had loxP elements inserted into the Fzr1 gene with those carrying the Zp3-Cre recombinase promoter. Breeding results in progeny that have an oocyte-specific deletion in their Fzr1 gene. I was able to determine that knockout females produce viable metaphase II eggs capable of generating live pups when fertilized. Therefore, even though measurable activity of APC^Fzr1 has been detected at various stages of meiosis, this APC activator was not an essential meiotic gene. I investigated an optimal culture media suitable to sustain preimplantation development of B6CBF1 hybrid and C57Bl6 fertilized embryos and parthenotes to the blastocyst stage. Using parthenotes from knockout females, I was able to show that Fzr1 is involved in the process of pronuclear fusion at syngamy in zygotes. Its loss also resulted in delayed and asynchronous cleavage divisions of blastomeres; which led either to arrest of embryos at the 2-cell stage or premature initiation of compaction in 4-cell stage embryos that then failed to develop to form blastocysts. Additionally, I developed a miRNA construct that achieved Fzr1^-/- knockdown in embryos and also showed a 2-cell arrest phenotype. Examination of 1- and 2-cell Fzr1^-/- parthenotes also showed an increased incidence of chromosomal instability, demonstrated by the formation of micronuclei and fragmented DNA. In summary, work of this thesis found that Fzr1 is not an essential meiotic gene, as knockdown studies may have suggested, but instead was remarkably essential for early embryonic development.