P-002 SARS-CoV-2 tropism spectrum for human testicular cells
Authors: Ribeiro M R , Calado A M , Alves Â , Pereira R , Sousa M , Sá R 
Published: June 2023

Abstract

Study question
In human testicular cells, how are SARS-CoV-2 receptors and their priming proteases for the viral spike (S) protein distributed spatially?

Summary answer
Human testicular tissue expressed both the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) receptors and proteases investigated, key factors for cellular susceptibility to SARS-CoV-2 entry/infection.

What is known already
Male reproductive function is altered on numerous levels by SARS-CoV-2 and coronavirus disease. Angiotensin-converting enzyme 2 (ACE2) is the main SARS-CoV-2 host membrane receptor but requires transmembrane protease serine 2 (TMPRSS2) for S protein priming (activation by proteolysis). Other proteases, like cathepsin L (CTSL), might nevertheless contribute to SARS-CoV-2 entrance. This mostly happens in cells with low or nonexistent ACE2 expression, which calls for alternative receptors like cluster of differentiation 147 (CD147). Although co-expression of receptors and proteases is essential for successful SARS-CoV-2 infection, it is still unknown how viral receptors and accompanying proteases are distributed geographically in testicular cells.

Study design, size, duration
To overcome this restriction, we focused on using immunohistochemistry to map the spatial distribution of the SARS-CoV-2 receptors ACE2 and CD147, as well as their priming proteases for the viral spike (S) protein, TMPRSS2 and CTSL, which are necessary for viral fusion with host cells, in human testicular tissue.

Participants/materials, setting, methods
Laboratory experiments were executed under the Joint Ethics Committee of the Hospital and University (approval number 2020-094 (077-DEFI-078-CE)). Human testicular tissues were obtained from cases presenting Sertoli cell syndrome, maturation arrest, and hypospermatogenesis at the Hospital Pathology department, with five patients per pathology. Formalin-fixed paraffin-embedded tissue sections 4 μm were incubated with anti-antibodies ACE2 (orb638860, 1:100 and 1:150, Byorbit), TMPRSS2 (ab109131, 1:1000, Abcam), CD147 (MA5-29060, 1:2500, Invitrogen) and CTSL (MA5-32602, 1:100, Invitrogen).

Main results and the role of chance
The present study did not intend to study the virus entry proteins in different testicular pathologies. As spermatogenesis is a complex process involving many intricately linked cells, we selected these syndromes specifically to identify individual cells more clearly, Sertoli cells in Sertoli cell-only syndrome cases, spermatocytes in maturation arrest cases and spermatids in hypospermatogenesis cases. The hypospermatogenesis group consisted of cases whose azoospermia was due to duct obstruction, showing conserved spermatogenesis. Interstitial cells, including endothelium, Leydig and myoid peritubular cells, and the seminiferous epithelium (Sertoli cells, spermatogonia, spermatocytes, and spermatids), were discovered to contain ACE2 and TMPRSS2, demonstrating co-expression of both receptor and protease in all testicular cells. All cell types, with the exception of endothelium and peritubular cells, showed the presence of CD147, whereas CTSL was only found in Leydig, peritubular, and Sertoli cells. As a result, only Leydig and Sertoli cells co-expressed CD147 and CTSL. The findings of proteomic databases indicating testicular cells have SARS-CoV-2 receptors and proteases are supported by our discoveries, demonstrating that testicular cells may be directly infected and injured, halting spermatogenesis and acting as viral vectors for the SARS-CoV-2.

Limitations, reasons for caution
Our results support viral tropism for human testicular cells and raise the possibility of testicular manifestations, although this does not necessarily mean that the virus infects testicular cells directly and further studies are needed to discern SARS-CoV-2 infection and transmission targeting alternative receptors and to identify the underlying mechanisms.

Wider implications of the findings
Male reproductive system susceptibility to viral infections has long been demonstrated. To prevent infection by allowing the virus to attach to germ cells, impair sperm production, and increase the risk of meiotic errors and viruses spreading through sperm, our study emphasizes the importance of safe practices in the wider community.

Trial registration number
not applicable