Grant: Discovery Grants Program - Individual

During fertilization, sperm deliver a signal that instructs the egg to complete female meiosis and transition to mitotic cell divisions that build the new organism. However, it is unclear which molecular components are involved and how they specify the correct cell divisions. This proposal focuses on a molecular pathway that we discovered, which is required for the meiosis-to-mitosis transition in the nematode C. elegans. Our approach identified oocyte- and sperm-specific components, and we are now poised to exploit our system to further understand this critical pathway. We can perform experiments on C. elegans that would be impossible to do in humans or other mammals. Fertilization, the meiotic divisions, and the first mitotic division all occur within an hour, and all processes can be viewed in utero with high-resolution live-cell microscopy. Specific parameters of each stage can be quantified with precision. Furthermore, the activity of individual genes can be efficiently altered by mutation or RNA interference (RNAi) to assay the effects within a developing organism.Using NSERC-DG funds, we discovered 3 highly-similar genes, memi-1, memi-2, and memi-3, which encode oocyte factors required for meiosis II (MII). MEMI proteins are present during early meiosis, but they are degraded shortly after MII begins, and are undetected in mitotic embryos. Loss of all three genes results in fertilized embryos that abort meiosis I and enter mitosis prematurely (i.e., they skip MII). This is very similar to what happens when immotile sperm cannot fertilize the oocyte. Thus, we hypothesize that MEMI “senses” sperm entry. To our advantage, we have a hypermorphic mutation, memi-1(sb41), which results in “excess” MEMI activity. sb41 interferes with MEMI-1 degradation, resulting in embryos that fail to transition from MII to mitosis properly. Using an RNAi-based suppressor screen of memi-1(sb41), we discovered a sperm PP1 phosphatase, which is a genetic activator of memi and ideal candidate for the elusive sperm signal that triggers MII.In a recent pilot screen for memi-1(sb41) suppressors using EMS-mutagenesis, we recovered the same PP1 phosphatase, as well as many new genes. To further understand the MEMI pathway, we will: 1) identify and characterize the new EMS-induced suppressors of memi-1(sb41). 2) identify post-translational modifications in MEMI proteins that are hypothesized to occur during the meiosis-to-mitosis transition. 3) analyze MEMI physical interactions using proximity ligation and yeast 2-hybrid assays.Although there is limited knowledge of the mechanisms that coordinate the complex cell divisions characteristic of fertilized embryos, our proposed aims have a potential to reveal core components and their molecular roles. Each of the new genes identified will provide ideal training opportunities for independent research projects, at both the graduate and undergraduate level.