Biosynthetic mechanisms enabling… (F32GM167148) – Project Grant to The Regents Of The University Of…

Abstract: Oocytes, the specialized cells that give rise to female gametes, are one of the largest cell types in animals, yet the mechanisms that enable their enormous growth in vertebrate species are poorly understood. Somatic cells maintain a constant ratio of DNA per volume of cytoplasm, which is necessary to enable growth, prevent RNA and protein dilution, and efficiently repair DNA damage. Remarkably, oocytes grow hundreds of times larger than somatic cells without increasing DNA content, and cytoplasmic contributions from other cells are insufficient to account for this growth. Achieving large size is critical for oocyte maturation and to provide a stockpile of nutrients for the embryo. IT is not known how oocytes overcome the limitation of DNA content on biosynthesis or whether they employ specialized strategies to avoid macromolecule dilution. To understand how oocytes achieve their massive growth, in aim 1, i will measure transcript-level RNA synthesis rates in growing xenopus frog oocytes of different sizes, revealing the timing of maximum transcription during oogenesis. Excessive cell growth of proliferative cells causes a stereotyped proteome remodeling that underlies their defects in biosynthesis and DNA damage repair. Therefore, in aim 2, i will determine whether oocytes also experience this proteomic shift. Through proteomic analysis i will distinguish whether oocyte- specific molecular changes prevent these deleterious, large-cell phenotypes or whether oocytes avoid this proteome remodeling altogether, and i will identify candidate proteins that enable immense oocyte growth while maintaining genome stability. In ΑIM 3, i will develop an oocyte extract system to test how DNA dilution affects oocyte transcription by manipulating DNA/cytoplasm ratio in vitro. Through immunodepletion experiments, oocyte extracts will enable assessment of candidate factors for their roles in oocyte biosynthesis and DNA damage repair. This project will fill a major GAP in our understanding of oogenesis and will inform female reproductive health. The techniques developed in this proposal will become the foundation of my independent research group focused on oogenesis and biological scaling. The outstanding training environment for developmental cell biology in the laboratory of DR. Rebecca heald at the university of california, berkeley will enable me to learn multiple new experimental techniques and to develop the mentoring, writing, and critical thinking skills essential for my career as an independent researcher. Mentorship of undergraduate students in our lab will provide me hands on experience supervising other investigators and teaching fundamental biological concepts. The critical thinking and creativity involved in the production of original research papers and peer reviews will strengthen my writing. Finally, through seminar and conference attendance and dedicated reading time, i will keep up to date on cutting edge research in cell and developmental biology. This comprehensive postdoctoral training will enable my success in a full-time faculty position.

Biosynthetic mechanisms enabling extreme growth in vertebrate oocytes

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