Investigations Of The Effects Of Selected Y-chromosome Encoded Gene Deletion And Viral Infection On Testicular Function In A Mouse Model

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Murine and human testes are affected by both Y-chromosome encoded gene loss and the direct or indirect effects of some viral infections. Primary focus into the impact of Y-chromosome gene loss and viral infection on male fertility has been primarily focused on the Y-chromosome encoded testis determining factor, Sry, and viruses that transmit vertically, like ZIKA virus (ZIKV). Zinc finger Y encoded genes (Zfy), termed Zfy1 and Zfy2 in mouse and ZFY in humans, were once suspected to be the testes determining factor (TDF). This was proven incorrect when the true TDF was identified as another Y-chromosome gene, SRY (sex-determining region Y encoded). Subsequent research using mouse models with Zfy deficiencies and Zfy transgenic additions have indicated significant contributions from these genes to other aspects of male fertility in the mouse. However, complete knockout (KO) of Zfy genes has not been conducted. Existing data from testicular biopsies obtained from men who succumbed to SARS-CoV2 (COVID-19) infection, many with underlying conditions, revealed extensive testicular damage in the absence of active viral infection of the testes, despite the presence of the viral cellular receptor, angiotensin converting enzyme 2 (ACE2). However, the effect of SARS-CoV-2 infection on testicular function has not been researched in a well-controlled mouse model. The projects herein are based on the hypothesis that complete Zfy gene loss and SARSCoV-2 viral infection have profound effects on testicular function in the mouse. To assess effect of Zfy gene loss, select spermiogenic phenotypes were assessed in mice with complete Zfy1 (Zfy1 KO), Zfy2 (Zfy2 KO), and both Zfy homologs (Zfy DKO) knockout developed with CRISPR/Cas9 technology. Assessment of the testicular effect of COVID-19 disease was performed using ii transgenic K18-hACE mice expressing human ACE in epithelia, infected with SARS-CoV-2, and assessed 3-, 5-, 8-, and 14-days post infection (DPI). The analyses of sperm and testes Zfy KO mice using silver-stained sperm spreads and light microscopy and transmission electron microscopy (TEM) revealed defects in sperm morphogenesis in Zfy2 KO and Zfy DKO males, but not Zfy1 KO males. The analyses of testicular PAS-stained testicular sections from Zfy DKO males revealed seminiferous tubule abnormalities. Histopathological assessment of testes from K18-hACE mice at 5 different DPI revealed significant increase in seminiferous tubule abnormalities and increase in germ cell apoptosis males with DPI of at least 5. This data shows that the testes are highly susceptible to loss of Zfy1 and Zfy2, and to SARSCoV-2 infection, illustrating the fragility of male fertility. The novel mouse models utilized to examine the effect of Zfy deletion and COVID-19 disease allow for the assessment of attributed solely to intended manipulations (gene knockout or viral infection). This provides insight for further investigations to elucidate the mechanisms of Zfy gene contribution and COVID-19’s lasting impact on mice and by extension possibly mankind.