Top-Down Proteomics of the Oocyte Secretome and Nuclear Proteome
The purpose of the female reproductive axis is to produce a terminally differentiated haploid egg. At the completion of the complex process of folliculogenesis, an egg is ovulated and will only survive a few hours unless fertilization occurs to produce a totipotent embryo. Successful fertilization and the genesis of a new organism are dependent on the quality of the individual egg and sperm. However, in humans, reproduction is highly inefficient. In fact, it is estimated that approximately 10-25% of clinically recognized pregnancies end in miscarriage, and this is likely (in part) due to poor egg quality. Therefore, determining: a) how folliculogenesis creates a high quality egg and b) what molecular signatures distinguish a high quality egg from a poor quality one is critical for understanding normal fertility, managing fertility, treating infertility, and developing fertility preservation technologies for female survivors of cancer.
In Task 1 of this DBP, we propose to interrogate the intact proteins secreted from developing follicles and mature eggs to create a temporal map of the mammalian folliculogenesis secretome. Previous studies have been limited in scope because an MS-based platform sensitive enough to detect proteins in such small samples did not exist and folliculogenesis could not be recapitulated faithfully in a controlled in vitro environment.
The egg, which is the largest cell in the body, is also one of the most rare, precious, and difficult to obtain. For example, only 30-40 mature eggs can be collected from a single mouse containing <1 μg protein. Thus, samples derived from sources such as follicles and eggs absolutely require the small scale separations proposed in TR&D 1. Additionally the detection of these proteins will require the use of novel, high dynamic range instruments for top-down proteomics presented in TR&D 2, Specific Aim 2. To study the secretome during folliculogenesis, we will employ an in vitro follicle culture system developed through an interdisciplinary collaboration between the Woodruff and Shea laboratories that supports follicular architecture during development (supported by U54 HD076188, Center for Reproductive Health after Disease). In this system, isolated follicles are encapsulated in an alginate-based hydrogel and grown in vitro,,. In alginate hydrogels, mammalian secondary follicles can grow to the antral stages and produce mature eggs that, upon fertilization, can give rise to embryos andultimately live births . Because the embryo derives half of its genome and all of its cytoplasm from the egg,understanding how a good quality egg is developed will fundamentally change assisted reproductive technologies and has implications for stem cell biology.