SUMMARY Fertilization of aneuploid gametes often leads to pregnancy loss or disorders such as Down’s syndrome. Aneuploidy occurs in 10-30% of human gametes and typically arises from chromosome segregation errors during meiosis. Meiotic events such as homologous chromosome pairing, double strand breaks (DSBs), recombination and the formation of crossovers, ensure proper chromosome segregation. Nevertheless, the mechanisms that regulate these events are incompletely understood. Consequently, there are currently few to no strategies to predict or prevent aneuploidy during gametogenesis. To overcome some limitations in the field, we established a fluorescence reporter-operator system (FROS) based on the lac operator-lac repressor (lacO-lacR) paradigm, which enables protein targeting to distinct genomic regions. Our long-term goal is to elucidate the mechanisms that regulate chromosome segregation during meiosis and thus ensure the formation of gametes with a normal karyotype. Aim 1 use FROS to dissect rapid chromosome motions in mouse spermatocytes at prophase I. We will also combine FROS with long-term 3D measurements of chromosome motions in seminiferous tubules to directly test whether homologous chromosome pairing arises from increasingly productive interactions (reeling in), versus reiterative rounds of transient interactions (catch and release). Additionally, we will analyze mutant spermatocytes by FROS to determine how key players, including components of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the synaptonemal complex, affect homologous and non-homologous interactions at specific loci. Dynein and microtubules interact with the LINC complex and are known to contribute to RPMs. Aim 2 will identify additional motors and cytoskeleton components that support these movements, building on our preliminary data uncovering kinesins as candidate molecular motors involved in generating RPMs. We will extend these findings with unbiased proteomic approaches, functional assays and FROS to decipher the dynamic forces that govern homolog pairing. Aim 3 will investigate the mechanism underlying the association between ANKRD31 and pro-DSB factors at recombination hotspots. Using FROS, we will target ANKRD31-GFP-lacI to the lacO region in spermatocytes, which we predict will recruit pro-DSB factors and recombination proteins. We will also analyze the effect of ANKRD31-GFP-lacI on downstream recombination intermediates at lacO sites. The proposed research will for the first time discern how chromosome context affects the mechanisms underlying chromosome segregation and thus advance new knowledge of fundamental meiotic processes and the causes of aneuploidy.