IDENTIFYING THE MOLECULAR FUNCTION OF MOUSE ZFY1 AND ZFY2 IN MALE REPRODUCTION
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2024
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Abstract
The mouse zinc finger genes Zfy1 and Zfy2 are essential for normal spermatogenesis. We recently produced Zfy1/2 double-knock-out (Zfy DKO) mice and observed severe defects in male fertility. The mechanism by which Zfy contributes to reproduction remains unknown but based on its predicted peptide sequence and the phenotype of Zfy DKO mice, we hypothesize that ZFY is a transcription factor that regulates genes essential for reproduction. To determine which genes are regulated by ZFY, we (1) performed transcriptome analysis on Zfy KO germ cells and (2) created tagged Zfy knock-in mice to identify ZFY co-interactors. Bulk RNA sequencing analysis was performed on XY, Zfy1 KO, Zfy2 KO, and Zfy DKO primary spermatocytes (SC1), secondary spermatocytes (SC2) and round spermatids (RS) cells. Ontology and gene set enrichment analysis revealed deregulation of apoptosis, chromatin re-organization, and spermatogenesis pathways in Zfy KO germ cells. Follow-up assays revealed that Zfy KO mice produce sperm with poorly reorganized chromatin and motility deficiencies, and an increase in apoptotic spermatocytes was observed in Zfy DKO testis cross sections. Next, CRISPR/Cas9 genome editing was used to create five distinct Zfy knock-in (KI) models (XYZfy2-FLAG, XYZfy2-3xFLAG, XYZfy1-HA, XYZfy2-HA, and XYZfy1-HA,Zfy2-MYC), and to generate knockouts of two other Y chromosome genes, Prssly and Teyorf1 KO mice to establish if these genes are responsible for similar spermatogenesis phenotypes as those of Zfy KO males. Tagged ZFY protein expression was detected in XYZfy1-HA and XYZfy2-HA mouse testis. A pilot immunoprecipitation followed by tandem mass spectrometry (IP-LC/MS) with a single XY and XYZfy2-HA testis confirmed enrichment of ZFY2 and identified H2AX as a potential binding partner. However, follow-up IP-LC/MS and chromatin immunoprecipitation (ChIP) assays have been inconclusive in determining the biochemical function of ZFY. Phenotypical analysis of Prssly and Teyorf1 KO males revealed that they are fertile with no deficiencies in spermatogenesis. In conclusion, this work described in this dissertation has revealed novel functions of Zfy, and the knock-in lines can be used for further study of Zfy function. Upon completion, this work will advance understanding of the function of Zfy in mice, which could in turn inform our knowledge on the homologous human ZFY homologue.
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Biology, genome engineering, male development, male fertility, spermatogenesis, Y chromosome
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205 pages
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