PROJECT SUMMARY/ABSTRACT Generating high quality oocytes is critical for a woman's fertility and health, and for healthy offspring. Nuclear envelope membrane protein 1 (Nemp1) is a transmembrane protein of the inner nuclear envelope that we found is needed for fertility in a wide range of organisms. Loss of Nemp1 in mice leads to near-sterility of females, associated with loss of the oocytes that compose the primordial reserve. The remaining oocytes have poor developmental competence, with defects in chromosome segregation, chromatin compaction and completion of meiosis. GWAS studies show that variants in NEMP1 are associated with early menopause, suggesting the role of Nemp1 in fertility is conserved to humans. Our long term goal is to understand how Nemp1 functions at the nuclear envelope to promote oocyte quality and human fertility. In this grant we will use mouse models to determine why loss of Nemp1 leads to reduced ovarian reserve and poor developmental potential. We will determine when and how the ovarian reserve is lost, and define pathways involved in oocyte loss. We will conduct gain of function and loss-of-function studies, to determine which cells require Nemp1 in the mouse ovary. To identify Nemp1-interacting proteins, we will take advantage of novel mouse lines we have generated, which allow us to conduct affinity-purification coupled mass spectrometry from resting and growing oocytes. Our preliminary studies have revealed that Nemp1 accumulates into extremely large, regular foci at the nuclear envelope, uncovering a novel nuclear envelope structure in growing oocytes. Proteomic analysis will reveal other proteins that localize to these foci (which we term NECs). Super-resolution microscopy and electron microscopy will define the structure of Nemp1 clusters and the adjacent nuclear envelope. FRAP analysis will clarify the stability of the Nemp1 clusters, and associated proteins. Our preliminary data indicate that chromatin compaction is disrupted in Nemp1 mutant oocytes, and that transcription is deregulated. We will use RNAseq to define change in gene expression in Nemp1KO oocytes. We will use ChIP-seq to define changes in histone modifications in mutant oocytes, and determine alterations in 3D chromatin architecture using Hi-C. Our preliminary data indicate that a closely related gene, Nemp2, is upregulated in Nemp1KO oocytes, so we will explore the contribution of Nemp2 to Nemp1 mutant phenotypes. Our preliminary data indicate that the nucleoplasmic tail of Nemp1 can interact with chromatin in cultured cells: we will clarify the regions of Nemp1-chromatin interactions in oocytes, using transgenic DamID approaches. Together these studies will illuminate the function of the nuclear envelope, and the role of Nemp1 in supporting the creation of healthy oocytes.