SUMMARY Meiotic chromosomes undergo a range of motions promoting highly regulated chromosome interactions. These interactions culminate in pairwise associations of maternal and paternal homologous chromosomes, which are later stabilized via the proteinaceous structure called synaptonemal complex (SC) that forms between the homologous chromosomes (synapsis). Chromosome pairing and SC formation are influenced by rapid prophase movements (RPMs). Mutations that affect chromosome motions or SC dynamics can lead to costly phenotypes ranging from problems in reproductive biology and fertility to severe aneuploid-based birth defects. Fundamental questions regarding the processes underlying RPMs, and how RPMs lead to stably homologous chromosome pairing are poorly understood. Equally important, double-strand breaks are essential for meiotic recombination to occur allowing homologous interactions. However, how timing and genome distribution of double strand are controlled is poorly understood. A prominent candidate for this control is the recently identified SPO11 partner, the ANKRD31 protein, whose precise mechanism of action and targets in germ cells are unknown. Molecular studies in mouse have been traditionally limited by the lack of genome engineering tools. This proposal builds on the development of an innovative approach that directs proteins to chromosomal loci in mouse meiocytes in vivo by utilizing a fluorescence reporter-operator system (FROS) using lacO-lacR technology. Our preliminary data shows that this system has the potential to answer questions that have previously been intractable for mouse meiosis relevant to differentiation and maturation of male germ cells. The planned experiments will answer two specific questions: 1) how do chromosome motions promote stable homolog pairing and 2) what is the action mechanism of ANKRD31 in mediating proper timing and location of meiotic double strand breaks? The first Aim will assess the mechanism and regulation of RPMs on homologous chromosome pairing. We will use lacO/lacR-GFP to visualize and quantify chromosome motions using 3D time-lapse movies in mammalian live germ cells. We will test how previously unrecognized chromosome characteristics (e.g. chromosome location within the nucleus and chromosomal level of expression) that modulate RPMs. Additionally, our newly developed long-term seminiferous tubule culture system will allow us to directly test two competing models explaining how homologous chromosomes interact and pair. This aim will lend new insights into the dynamic forces that govern homolog pairing in space and time. Aim 2 will determine the requirements of the ANKRD31 protein in meiotic double strand formation. To this end we plan to generate transgenic mice carrying lacO repeats and ANKRD31- GFP-lacR. Targeting ANKRD31 fusion to specific genomic loci will allow direct evaluation of ANKRD31 effect on local accumulation of SPO11 auxiliary proteins (REC114, MEI4, and IHO1), downstream re...