Project Summary The mouse zinc finger proteins ZFY1 and ZFY2 are essential for male fertility. Although human ZFY’s reproductive role has not yet been determined, the Y-derived Zfy transgenes improve spermatogenesis when added to mouse models with limited Y chromosome contribution. Specifically, Zfy (1) reinstates quality control checkpoints during the pachytene stage and metaphase I of meiosis, (2) promotes the second meiotic division and production of haploid round spermatid, and (3) improves spermiogenesis and assisted reproduction outcome. Nakasuji et al. and the Ward Lab recently produced Zfy1/2 double knock-out (DKO) mice, with both groups observing a complete loss of fertility and severe defects in spermatogenesis. The mechanism by which the homologues contribute to male fertility remains unknown but based on its predicted protein sequence and in vitro assays in human cultured cells ZFY is widely believed to be a transcription factor. Transcriptome and proteome analyses of germ cells from Zfy1/2 DKO mice will help identify which genes are regulated by ZFY1 and ZFY2, and the biochemical function of both proteins can be determined by antibody-based assays such as immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP). The proposed project will investigate the molecular function of ZFY, with the hypothesis that ZFY1 and ZFY2 contribute to spermatogenesis and male fertility by regulating the expression of a cascade of reproduction-related genes. This hypothesis will be tested in two specific aims. In Specific Aim 1, we will perform transcriptome and proteome analyses of primary spermatocytes (ps), secondary spermatocytes (ss), and round spermatid (rs) from Zfy1/2 DKO males to determine which genes are dysregulated in the absence of ZFY. This will involve isolating Zfy1/2 DKO germ cells via fluorescence-activated cell sorting (FACS), extracting RNA and protein from each cell type, and then performing RNA-seq and mass spectrometry (MS). Potential downstream candidates of ZFY1 and ZFY2 will then be identified from the genes dysregulated in DKO mice. In Specific Aim 2, antibody- based assays will be performed to discover the biochemical function of ZFY1 and ZFY2 in the mouse testis. This will require first generating a method to detect ZFY1 and ZFY2 proteins. Thus, in Aim 2.1, zygotes will be targeted with CRISPR/Cas9 technology to create knock-in (KI) mouse models, independently for each homologue, in which targeted ZFY will have a C- terminal HA tag (XYZfy1-HA and XYZfy2-HA). In Aim 2.2, we will confirm that we can specifically recognize ZFY1 and ZFY2 proteins, as well as any potential binding partners, with immunoprecipitation and liquid chromatography followed by tandem mass spectrometry (IP/LC/MS). Finally, in Aim 2.3, purified spermatogenic cells from the KI mice will be used for ChIP-PCR to determine whether ZFY1 and ZFY2 regulate expression of selected downstream candidates identified in Specific Aim 1. Additionally, ChIP-seq wi...