The goal of studies described in this amended R01 application is to explore the role of the DNA licensing factor, ORC4, in sequestering chromosomes into polar bodies via cage formation during female meiosis. These studies were considered highly innovative and with fundamental significance for understanding the mechanism of female meiotic division and oocyte maturation, as well as other possible forms of asymmetric cell division. Other strengths of the application include the outstanding qualifications and record of productivity of the Principal Investigator, the recruitment of a strong collaborative team with complementary expertise, the highly conducive research environment at the University of Hawaii, the convincing premise in the form of previous publications and preliminary data confirming the underlying functionalities of ORC4, the availability of many key reagents and resources required to pursue the project goals, adequate attention to elements of scientific rigor and a strong response to the previous critiques. In addition to these abundant strengths, some expressed concerns with an underdeveloped approach, including lack of guidance as to the mechanistic implications for experiments involving ORC4 depletion, beyond previous observations, concerns that overexpression of the EGFP-ORC-4 fusion protein may induce cell stress or lethality, lack of feasibility of purification of ORC4 interacting partners and the absence of sufficient insight on the role of ORC4 in other forms of asymmetric cell division. While these and other minor weaknesses slightly reduced the perceived merit of the application, the panel ultimately concluded that the very high novelty and fundamental mechanistic insights to be gained will exert a major impact on the fields of oocyte development and developmental biology. rtility.
We are studying how half of the chromosomes are evicted from the developing oocyte to prepare first for fertilization then for development immediately after fertilization. This work has direct significance for investigating the biology of (1) oocyte development, (2) asymmetric cell division in mammalian development, (3) the function of the DNA replication licensing ORC proteins, and (4) vesicle trafficking. This work also has implications for human infertility and assisted reproductive technologies in which oocyte maturation is manipulated in vitro.
