Meiotic checkpoint pathways and gametocyte quality control



 DESCRIPTION (provided by applicant): Reproductive lifespan in females relies on the size of the resting oocyte pool. Excessive atresia of oocytes occurs in response to defects in meiotic prophase and extraneous DNA damage such as cancer radiotherapy and chemotherapy. For instance, oocyte pools in mouse Spo11 mutants are eliminated within three months because Spo11-/- fails to initiate recombination and pair up homologous chromosomes. In Msh4 mutants, recombination is initiated, but progression of recombination and homolog pairing/synapsis are defective. The oocyte pools of Msh4-/- are depleted within four days of birth. Two distinct branches of the meiotic checkpoint pathway are inferred to respond to defects in recombination or homolog pairing, respectively. Several components of the DNA damage response (DDR) define the meiotic response to unrepaired recombination intermediates. In contrast, the response to defective synapsis requires a meiosis-specific factor, HORMAD1, and provokes transcriptional silencing via a process termed Meiotic Silencing of Unpaired Chromatin (MSUC). I have identified RNF212 as a novel component of the meiotic checkpoint machinery. Strikingly, Rnf212 mutation restores the resting oocyte pools in both Spo11-/- and Msh4-/- backgrounds. Intriguingly, Rnf212 encodes an RING-family E3-ligase that catalyzes a protein modification by the Small Ubiquitin-like Molecule, SUMO. These data imply that RNF212-mediated SUMOylation is a key aspect of the meiotic checkpoint-signaling pathway that leads to oocyte apoptosis. I will investigate the nature and mechanism of RNF212-dependent oocyte quality control in mouse using a combination of genetics, cell biology and molecular approaches. These studies will build the foundation for research in my own laboratory that will pursue a mechanistic understanding of gamete quality control. Aim1 is to determine the efficiency of oocyte rescue in synapsis/recombination mutants and whether RNF212 contributes to physiological post-partum atresia. In the Spo11-/-, Spo11-/-Rnf212-/-, Msh4-/-, and Msh4-/-Rnf212-/- mutant backgrounds, total numbers of oocytes and developing follicles will be quantified in the ovaries of newborns and animals of various ages. Aim 2 is to understand how Rnf212 mutation rescues oocyte pools in synapsis/recombination mutants. A number of models could explain how Rnf212 mutation can bypass oocyte loss in synapsis and recombination mutants. To begin to distinguish between these models, I will perform a detailed characterization of meiotic prophase in fetal ovaries. Aim 3 is to identify targets of RNF212-mediated SUMOylation involved in the meiotic checkpoint. Understanding the role of RNF212 in meiotic checkpoint signaling will require identification of pertinent target proteins. In this aim, I will identify RNF212 targets through a combination of candidate and unbiased approaches using western blot, yeast-two-hybrid and proteomics approaches.