Histopathological evaluation of testis, epididymis and submandibular gland of SARS-CoV-2-infected K18-hACE2 mice

Abstract

ACE2 and TMPRSS2 proteins allow the SARS-CoV-2 virus entry into the host cells, inducing Covid-19- associated respiratory distress syndrome. Besides lung cells, these proteins are also found in salivary glands, testes and epididymis. Saliva is a common medium for virus transmission, and the salivary gland is a site of viral infection, replication and transmission. Studies have demonstrated the presence of SARS-CoV-2 in semen as well as low levels of testosterone and testicular and epididymal histopathological changes in Covid-19 patients. Testes and epididymis are complexes organs with specific and exclusive functions that maintain and guarantee spermatogenesis, sperm maturation and storage, essential processes for male fertility. The increase of cytokines caused by viral infections may impair these organs, disturbing spermatogenesis, steroidogenesis and sperm integrity. The creation of a recent K18-hACE2 transgenic mouse has contributed to the study of Covid-19. These animals express the human ACE2 (hACE2), which allow the binding of viral spike protein to the hACE2 in the cells of these animals, infecting them and inducing the respiratory syndrome. In this study, we purpose to evaluate, in submandibular glands, testes and epididymis of SARS-CoV-2-infected K18-hACE2 mice, which cells express hACE2 and if they are infected by the virus. The presence of inflammatory reaction and pro-inflammatory cytokines in these organs as well as the impact of infection on structural and functional integrity will also be evaluated in these organs. Twelve K18-hACE2 adult mice will be distributed into two groups (n=6): control group (CG) and 5-day group (5DG). The animals from 5DG will be inoculated intranasally with SARS-CoV-2, and the animals from CG will be inoculated with DMEM. After 5 days, the organs will be collected, immersed in fixative solution and embedded in paraffin for morphological and morphometric analyses. Testicular and epididymal fragments will also be embedded in Araldite for ultrastructural analyses under transmission electron microscopy (TEM). Samples will also be frozen (-80ºC) for molecular analyses. In the histological sections, immunohistochemistry and/or immunofluorescence will be performed for detection of ACE2, TMPRSS2 and viral spike protein in all organs. The immunoexpression of cytokines (IL-1±, IL-1², IL-2, IL-6, IFN-³, TNF-±) and the incidence of cell death by TUNEL method will also be evaluated. In testes, the testicular levels of TMPRSS2, ACE2, IFN-³, MIF, iNOS e NF-kB will be evaluated by Western Blot whereas the levels of testosterone, IL-1², IL-6 e TNF-± will be evaluated by ELISA. The tubular and epithelial areas will be measured and the number of Sertoli cells, spermatocytes and damaged seminiferous tubules will be quantified. Immunofluorescence and/or immunohistochemical reactions will be performed for detection of: Ki-67 (mitosis/meiosis), connexin-43 (hematotesticular barrier protein), StAR (steroidogenic protein), NF-kB, iNOS, T lymphocytes (CD8, CD4) and macrophages (CD68, CD163). In epididymal sections, besides cytokines, the number of CD11c-positive dendritic cells and F4/80-positive macrophages, as well as the immunoexpression of V-ATPase (proton pump) in clear cells will also be analyzed by immunofluorescence. In the salivary gland sections, the volume density of acini, ducts and glandular stroma as well as the immunoexpression of EGF and V-ATPase in the glandular ducts will be evaluated. Data of quantitative analyses of reactions will be subjected to the statistical analysis by the Student-t test (pd0,05). The ultrastructural changes of testes and epididymis as well as the possible localization of the virus in these organs will be evaluated by TEM and RT-qPCR. (AU)