In a new study conducted at the University of Hawaiʻi at Mānoa, researchers from the John A. Burns School of Medicine (JABSOM) have reported on generation of new mouse models, in which a key male fertility gene, ZFY, was “tagged” to enable molecular investigations of its function.
The study was led by Professor Monika Ward from the Department of Anatomy, Biochemistry & Physiology and the Yanagimachi Institute for Biogenesis Research (YIBR). The team has been investigating a zinc finger Y-encoded gene called Zfy. This gene, encoded on Y chromosome in both mice and humans, is considered a male fertility factor. In mice, Zfy is present as two copies, Zfy1 and Zfy2.
The new paper is a continuation of the team’s quest to explore the role of Zfy. Three years ago, the researchers used CRISPR-Cas9 to produce knockout mice specifically lacking Zfy1, Zfy2, and both Zfy1 and Zfy2 genes, the latter called Zfy DKO (Zfy double knockout). They observed that Zfy DKO males were completely infertile and had severely abnormal sperm.The team then applied assisted reproduction technologies (ART) - intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) - to produce more infertile Zfy DKO males. Earlier this year the team reported on the molecular consequences of Zfy loss, demonstrating that without Zfy hundreds of genes become deregulated, expressed either too strongly or too weak. Among these genes are those responsible for sperm production, DNA packaging and organization, and cell death loss.
In a new study accepted for publication in BMC Genomics the team used CRISPR-Cas9 to add “molecular tags” to Zfy1 and Zfy2 genes. Such tags are short, recognizable peptide sequences that can be added to proteins by genome editing to easily detect, track, and purify these proteins using specific anti-tag antibodies.
Using the newly developed mice, XYZfy1-HA and XYZfy2-HA, the team was able to characterize, for the first time, the pattern of expression of ZFY1 and ZFY2 proteins in male germ cells.
“We now know, which male germ cells express which ZFY proteins, and how strongly.” said Dr. Ward. The team also demonstrated that tagged ZFY proteins can be effectively “pulled down” and purified from a mixture of testicular proteins.
“We have been after the ZFY genes for a long time! We knew how important ZFYs are for male fertility for quite a while but were unable to learn how exactly they work. Only recently we started catching glimpses into how these genes work. The new mice represent a much-needed tool to continue the investigations to determine how ZFY controls spermatogenesis. Being able to specifically detect and pull down ZFY1 and ZFY2 will allow us to identify ZFY protein partners.” said Dr. Ward.
“ZFY is thought to be a transcription factor – a protein that binds to specific DNA sequences to control whether other genes are on or off. The new mice will also allow us to identify, which genes are regulated by ZFY1 and ZFY2 in this switch on-off manner.”, she continued.
The creation of mouse models with tagged ZFY represents a significant step forward towards advancing understanding of how male fertility is regulated. The fundamental knowledge obtained through basic research using mice as a model addresses a specific health need - management of male fertility/treatment of male infertility - and has translational implications.
The study details can be found in an article published in BMC Genomics, a peer-reviewed journal focusing on all aspects of genetics, genomics and proteomic
