Spermatogenesis is a carefully orchestrated process in which spermatogonial stem cells differentiate into spermatids and eventually motile sperm. This process requires a series of changes in epigenetic changes, chromatin reorganization, dynamic changes in mRNA 3’UTR length, and temporally regulated patterns of translation. Emerging evidence suggests that precise stage-specific alterations in the epitranscriptome are also required for proper spermatogenesis. For example, spermatocyte-specific depletion of “readers,” “writers,” or “erasers,” of N6-methyladenosine, a modified nucleotide that impacts long noncoding RNA (lncRNA) function and mRNA stability, translation, and splicing, are all associated with stage-specific arrests in spermatogenesis. Additional data suggests that spermatogenesis is also affected by N6, 2’-O- dimethyladenosine (m6Am), a modified adenosine that is found exclusively at the first transcribed nucleotide position of certain mRNAs. Based on these studies, it is clear that epitranscriptomic modifications are required for spermatogenesis. However the mechanisms by which m6A and m6Am regulate spermatogenesis remain unclear. In order to decipher the role of the epitranscriptome in spermatogenesis, the specific aims of this project are: (1) To map m6A in a cell-type specific and quantitative manner during spermatogenesis. We will develop new methods to selectively map m6A in animals, and determine if dynamic changes in m6A control mRNA 3’UTR length. These methods will reveal the dynamics of m6A levels throughout spermatogenesis and if m6A function is involved in orchestrating 3’UTR length dynamics that is characteristic of spermatogenesis. (2) To determine the role of m6A in controlling the epigenome during spermatogenesis. m6A is often enriched in lncRNAs, and it can affect their ability to induce epigenetic gene silencing. We will identify chromatin- associated lncRNAs that contain m6A and determine how m6A affects epigenetic dynamics during spermatogenesis. (3) To determine how YTHDC2 and m6Am regulate spermatogenesis. YTHDC2 is required for meiotic progression by binding and regulating m6A mRNAs. However, YTHDC2 shows relatively weak binding to m6A. We find that YTHDC2 shows high-affinity binding to m6Am. We will map m6Am during spermatogenesis, and determine the function of m6Am by depleting its biosynthetic methyltransferase. We will also determine if the function of YTHDC2 is to regulate the stability or translation of m6Am mRNAs during spermatogenesis. Together, these experiments will reveal the cell-type specific dynamics of the epitranscriptome and will reveal how these epitranscriptomic modifications affect epigenetic states, mRNA stability, mRNA 3’UTR processing and mRNA translation during spermatogenesis.