The development and maintenance of the germline, the set of highly-specialized cells responsible for passing on genetic material to the following generation, requires the packaging of specific mRNAs into ribonucleoprotein (RNP) granules called germ granules. Throughout the animal kingdom, germ granules play essential roles in germline proliferation, differentiation, and maintenance through post-transcriptional gene regulation. However, the connection between quantitative changes in germ granule mRNA composition and reproductive health remains unknown. The goal of this proposal is to determine how quantitative changes in germ granule mRNA composition influence animal reproduction using Drosophila as a model system. In Drosophila, mRNAs such as nanos (nos) and polar granule component (pgc) are essential for germline development and accumulate within germ granules by forming homotypic clusters. These clusters contain multiple transcripts of a specific mRNA type. Homotypic clusters are generated through a stochastic seeding and self-recruitment process that relies on cis-regulatory sequences found in the 3′UTR called “clustering elements.” Preliminary data demonstrate natural variations in egg production along with striking diversity in germ granule composition among Drosophila species. Specifically, a 50% difference in the number of transcripts in nos and/or pgc homotypic clusters was discovered between species. By developing an algorithm and novel image analysis software, we scored the overall divergence in germ granule mRNA composition between Drosophila species and identified a correlation between increased germ granule divergence and reduced egg production. Interestingly, the nos clustering element identified in eleven Drosophila species had differences in a sequence motif recognized by Rumpelstiltskin, a protein known to influence nos accumulation. Our preliminary results support a hypothesis that clustering element diversity alters the nos content in germ granules, yielding differences in germline development and egg production. To test our hypothesis, Aim 1 seeks to identify variations in germline development among six Drosophila species that exhibit natural differences in egg production by quantifying primordial germ cell development and survival using a novel virtual reality image analysis technique. Aim 2 explores the relationship between germ granule mRNA content and egg production by using single molecule in situ hybridization and 3D image analyses to quantify nos and pgc homotypic clusters in six Drosophila species. Aim 3 seeks to determine whether diversity in the nos clustering element is sufficient to alter nos homotypic clusters, germline development, and egg production by engineering D. melanogaster with clustering elements identified in other species. In humans, mutations in nos orthologs are linked to premature ovarian failure and defects in spermatogenesis. Given the conservation of germ granules and nos, completion of our propo...