The roles of mouse Y chromosome Zfy genes in male reproduction.

Project Summary The Y chromosome encoded zinc finger gene, Zfy, has once been in the center of attention as a potential candidate for the testis-determining factor. When the fame went to another Y chromosome gene, Sry, Zfy was quickly forgotten, and it has taken more than two decades for it to re-emerge with newly ascribed spermiogenic roles, some of which were crucial for Y chromosome evolution. The mouse Y chromosome has two Zfy copies, Zfy1 and Zfy2, both with potential to act as a transcription factor and with postnatal expression restricted to spermatogenic cells. Current evidence, including some from our lab, supports the role of Zfy genes in male reproduction. Yet, nothing is known about the molecular mechanism whereby ZFY1 and ZFY2 impose their important roles. Our goal is to fill this gap in knowledge and define how ZFY regulate mouse spermatogenesis. We propose, and will test, a hypothesis that that mouse ZFY proteins are essential regulators of spermatogenesis and male fertility. In Specific Aim 1 we will use Zfy KO mice that we recently developed to identify the consequences of Zfy loss on transcript and protein expression in purified spermatogenic cells: primary spermatocytes (sc1), secondary spermatocytes (sc2), round spermatids (rs), and sperm (sp) using RNA-seq and mass spectrometry. In Specific Aim 2, we will use CRISPR/Cas9 to add epitope tags via knock-in to Zfy1 and Zfy2 genes. Using these new mouse models, we will identify ZFY chromatin and protein binding targets in purified spermatogenic cells (sc1, sc2 and rs). In Specific Aim 3, we will integrate the findings from Aim 1 and 2 to define the gene and protein network regulated by ZFY in mouse testis and perform downstream molecular, biochemical, and functional assays on selected genes and pathways regulated by ZFY. In preparation for this project, we performed a pilot RNA-seq analysis and have shown that Zfy loss results in significant changes of germ cell transcriptome. We also developed mice with tagged ZFY proteins and showed that they can be immunoprecipitated. The project will fill a gap in knowledge regarding the molecular function of an essential and conserved Y chromosome- encoded male fertility factor, ZFY, which arose due to inability to reliably detect ZFY proteins and lack of methods to genetically modify Y chromosome. The findings will impact on understanding of how mouse Y chromosome Zfy regulate spermatogenesis, providing insights the role of the human ZFY and the mechanisms underlying human male infertility.