Large-Scale Studies of the Protein Biochemistry Regulating Meiotic Exit and Fertilization

Abstract

Dynamic post-translational modifications are central to coordinating complex biological processes. This is especially evident after fertilization, where the modification or destruction of the egg’s existing proteins is sufficient to guide the embr¬yo through early embryogenesis. In particular, protein phosphorylation and degradation drive the rapid release of the meiotic arrest and numerous other events, such as the blocks to polyspermy. Despite decades of study, the full extent of protein loss and phospho-regulation following fertilization is still unknown. This is largely because comprehensive and quantitative analysis of such biochemical regulation is currently difficult. The following work examines protein and phosphosite dynamics of the fertilization response by mass spectrometry-based proteomics in Xenopus eggs, which have long served as an informative cell cycle and developmental model. To interpret the function of the biochemical changes, it is informative to observe quantitative features like absolute rates and the stoichiometry of modifications. This motivated the development of two analytical approaches: the first to estimate protein concentration from label-free proteomics, the second to calculate the stoichiometry of phosphosites from multiplexed proteomics with confidence intervals. The data suggest that the rapid transition after fertilization is achieved by two contrasting and parallel programs: 1) protein degradation is limited to a few low abundance targets. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. 2) A large amount of protein is released from the egg into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation, which is likely tied to the signal transduction downstream of the calcium-activated kinases. The analytical approaches developed and demonstrated here are broadly applicable to many classes of post-translational modification and for studies of dynamic biochemical regulation of diverse biological systems.