In vitro and in vivo investigation of SARS-CoV-2 associated injury and functional impairments in the testis.

Abstract

Traditionally associated with shortness of breath and lung injury, the 2019 severe-acute respiratory syndrome coronavirus 2 pandemic presented several other alarming features in COVID-19 patients, including cytokine storm and injury of other organs such as the kidney and testis. Infected males also experienced symptoms such as orchitis, erectile dysfunction, reduced testosterone levels, and low sperm count. These testicular symptoms are accompanied by leukocyte infiltration, sloughing of the seminiferous tubules, and germ cell death. Cohorts of studies from COVID-19 moderate, severe, and critical patients have revealed additional hormone alterations as well as functional damages to function of the male reproductive tract. These include decreased semen volume, lower sperm count, and decreased testosterone, follicle-stimulating hormone, and luteinizing hormone. Additional sperm qualitative analysis by these studies revealed altered sperm morphology, decreased sperm viability/vitality, and progressive motility as well as increased pro-inflammatory molecules in the semen such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β). Further, lower sperm count, and reduced sperm motility was correlated with increased sperm DNA damage and reactive oxygen species. While most of these parameters recover rapidly upon recovery from the disease, long-term follow up of recovered patients suffering from long-COVID revealed lower testosterone levels and reduced sperm parameters up to 12 months after disease onset. Despite these abnormal findings, SARS-CoV-2 RNA or infectious virions are not detected in the semen of COVID-19 individuals. Therefore, it is still unclear what the mechanism of testicular injury is and what are the specific immune mechanisms responsible for contributing to SARS-CoV-2 testicular short-term and long-term pathogenesis. We here demonstrate that neither human multicellular 3D organoid models nor 2D models consisting of different testicular cell types can support productive SARS-CoV-2 infection. This suggests that the mechanism of testicular injury observed in COVID-19 patients is a bystander effect of infection. Therefore, the objective of this study was to utilize novel in vitro and in vivo tools to define the underlying molecular mechanism and potential consequences of SARS-CoV-2 bystander effects in the testes. For Aim 1, we utilized different 2D and 3D testicular models, optimized in our lab, to characterize the effect of the SARS-CoV-2 infection-derived inflammatory mediators and antigens, on the testicular cells. The K18-hACE2 mouse model has been extensively used to study SARS-CoV-2 pathogenesis and partially recapitulates human disease. Therefore, for Aim 2 we used these hACE2 mice to characterize various aspects of testicular pathogenesis associated with SARS-CoV-2 in vivo. These include histopathology studies during the acute stage and short-term after recovery and the correlation between systemic cytokine storm and different markers of testicular injury and function. For Aim 3 we will use high throughput RNA-seq approach to identify the molecular signatures associated with SARS-CoV-2 infection in the K18-hACE2 mouse testis. The outcome of this study provides significant mechanistic insights into the testicular injury and short-term effects of SARS-CoV-2 on testicular function. It also addresses fundamental gaps in our understanding of how this virus affects male reproductive health. Tools and methodologies developed in this study will also provide a platform for investigating the bystander effects of other viruses such as Ebola virus, Dengue, and West Nile as well as guide therapeutic approaches in the future.