In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ injury including testicular inflammation, reduced testosterone, and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells, however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury could be initiated by direct virus infection or exposure to systemic inflammatory mediators or viral antigens. We characterized SARS-CoV-2 infection in different human testicular 2D and 3D culture systems including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not productively infect any testicular cell type. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma decreased cell viability and resulted in the death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 Envelope protein caused inflammatory response and cytopathic effects dependent on TLR2, while Spike 1 or Nucleocapsid proteins did not. A similar trend was observed in the K18-hACE2 transgenic mice which demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis that correlated with peak lung inflammation. Virus antigens including Spike 1 and Envelope proteins were also detected in the serum during the acute stage of the disease. Collectively, these data strongly suggest that testicular injury associated with SARS-CoV-2 infection is likely an indirect effect of exposure to systemic inflammation and/or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ failure including testicular injury and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury can likely result either from direct virus infection of resident cells or by exposure to systemic inflammatory mediators or virus antigens. We here characterized SARS-CoV-2 infection in different human testicular 2D and 3D models including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not establish a productive infection in any testicular cell types. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma depicted a significant decrease in cell viability and death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 envelope protein, but not Spike or nucleocapsid proteins led to cytopathic effects on testicular cells that was dependent on the TLR2 receptor. A similar trend was observed in the K18h-ACE2 mouse model which revealed gross pathology in the absence of virus replication in the testis. Collectively, data strongly indicates that the testicular injury is not due to direct infection of SARS-CoV-2 but more likely an indirect effect of exposure to systemic inflammation or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

Loss of mouse Y chromosome gene Zfy1 and Zfy2 leads to spermatogenesis impairment, sperm defects, and infertility.

Using mice with Y chromosome deficiencies and supplementing Zfy transgenes, we, and others, have previously shown that the loss of Y chromosome Zfy1 and Zfy2 genes is associated with infertility and spermiogenic defects and that the addition of Zfy transgenes rescues these defects. In these past studies, the absence of Zfy was linked to the loss of other Y chromosome genes, which might have contributed to spermiogenic phenotypes. Here, we used CRISPR/Cas9 to specifically remove open reading frame of Zfy1, Zfy2, or both Zfy1 and Zfy2, and generated Zfy knockout (KO) and double knockout (DKO) mice. Zfy1 KO and Zfy2 KO mice were both fertile, but the latter had decreased litters size and sperm number, and sperm headshape abnormalities. Zfy DKO males were infertile and displayed severe spermatogenesis defects. Postmeiotic arrest largely prevented production of sperm and the few sperm that were produced all displayed gross headshape abnormalities and structural defects within head and tail. Infertility of Zfy DKO mice could be overcome by injection of spermatids or sperm directly to oocytes, and the resulting male offspring had the same spermiogenic phenotype as their fathers. The study is the first describing detailed phenotypic characterization of mice with the complete Zfy gene loss. It provides evidence supporting that the presence of at least one Zfy homolog is essential for male fertility and development of normal sperm functional in unassisted fertilization. The data also show that while the loss of Zfy1 is benign, the loss of Zfy2 is mildly detrimental for spermatogenesis.