Evaluation of immunological profile, spermatogenesis and steroidogenesis in SARS-CoV-2-infected K18-hACE2 mice

• Grant number: 21/09328-5
• Support Opportunities: Scholarships in Brazil - Doctorate
• Effective date (Start): August 01, 2022
• Effective date (End): February 28, 2026
• Field of knowledge: Biological Sciences - Morphology - Histology
• Principal Investigator: Estela Sasso Cerri
• Grantee: Salmo Azambuja de Oliveira
• Host Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

The ACE2 and TMPRSS2 proteins allow the entry of the SARS-CoV-2 Virus into the host cell, causing COVID-19, a severe acute respiratory syndrome. In addition to lung cells, these proteins are present at considerable levels in testicular cells. Some studies have demonstrated the presence of the virus in the semen as well as low levels of testosterone and testicular alterations in patients with COVID-19. The testis is a complex organ responsible for steroidogenesis and spermatogenesis for the maintenance of male fertility. Increased cytokines induced by viral infections can affect this organ and impair spermatogenesis, steroidogenesis and sperm integrity. The recent creation of a new K18-hACE2 transgenic mouse has contributed to the study of COVID-19, as these animals express human ACE2 (hACE2). The spike protein of the virus binds to hACE2 in the cells of these animals, infecting them and causing the disease. The aim of this study is to verify which cells express hACE2 and if they are infected by the virus in the testes of K18-hACE2 mice infected by SARS-CoV-2. The presence of inflammatory infiltrate and pro-inflammatory cytokines will also be evaluated, as well as the impact of the infection on the structural and functional integrity of this organ. Twelve adult male rats will be distributed into two groups (n=6): control group (CG) and five-day group (5DG). The G5D animals will be inoculated with the virus intranasally. The CG animals will be inoculated with DMEM culture medium. Testes will be processed and embedded in paraffin and historesin for analysis of tubular and epithelial areas, and quantification of Sertoli cells, spermatocytes and impaired seminiferous tubules. Samples too will also be embedded in Araldite for ultrastructural analysis by Transmission Electron Microscope (TEM), and frozen (-80°C) for molecular analysis (Western blot and qPCR). Immunofluorescence or immunohistochemistry reactions will be performed for detection of: ACE2, TMPRSS2, viral protein spike, IL-1±, IL-1², IL-2, IL-6, INF³, NF-kB, iNOS, StAR, TNF-±, connexin-43, Ki-67, CD8, CD4, CD68 and CD163. TUNEL method will be performed for detection of cell death. The protein levels of TMPRSS2, ACE2, iNOS and NF-kB will be detected by Western Blot while testosterone, IL-1², IL-6 and TNF-± levels will be measured by ELISA. Data will be statistically analyzed by Student-T Test (pd0.05). The evaluation of ultrastructural changes, as well as confirmation of the presence of the virus in tissues/cells will be performed by means of TEM and real time RT-PCR (AU)