SARS-CoV-2 Triggers an NF-kB-Driven Proliferative Response in Epididymal Clear Cells of K18-hACE2 Mice

Abstract: We examined SARS-CoV-2 infection in the epididymis of K18-hACE2 mice, focusing on the cauda region. Thirty mice were divided into control (CG), 2-day infection (2D), and 5-day infection (5D) groups. The animals were intranasally infected with SARS-CoV-2. The 5D group showed a significant increase in IL-6, TNF-a, and IL-1b, high mRNA levels of Ifna, Ifnb, and Ifng, and infiltration of CD8+ T cells. SARS-CoV-2 directly infects smooth muscle cells, leading to atrophy of the smooth muscle layer. The infection impaired the basement membrane and downregulated blood-epididymis barrier-related genes Ocln and Jam-a. Clear cells exhibited increased apical V-ATPase and immunoexpression of hACE2, spike, and nucleocapsid. Interestingly, the infected groups showed a greater number of juxtaposed clear cells with PCNA- and Ki67-positive nuclei. The findings also revealed a newly identified NF-kB-driven inflammatory-proliferative response in clear cells. By combining in vivo and in silico analysis of a parallel LPS-stimulation RNA-seq dataset, we uncovered significant co-enrichment of the TNF-aNF-kB pathway alongside cell cycle pathways (G2/M, E2F). The protein-protein interaction network identified key nodes linking the inflammatory NF-kB pathway to the cell cycle machinery. This in silico model was confirmed in vivo, demonstrating co-localization of phosphorylated NF-kB and PCNA in the CC rows. Alterations in spermatozoa, including decreased PNA labeling, ultrastructural changes, and reduced Crisp1 mRNA, were also observed. Therefore, SARS-CoV-2 infection causes structural and functional changes in the epididymal cauda, leading to smooth muscle cell death and basement membrane disruption. Overall, the results suggest that SARS-CoV-2 infection induces dysfunction in the cauda, a microenvironment essential for sperm maturation and storage. Lay Summary: SARS-CoV-2, the virus that causes COVID-19, can reach many organs beyond the lungs, including the male reproductive tract. We studied how this virus affects epididymis, a sperm- carrying tube where sperm are mature and stored, in a genetically modified mouse model that expresses the human ACE2 receptor, which allows SARS-CoV-2 infection. We demonstrated that infection triggers a strong local inflammatory response and impairs the muscular wall and spermatozoa. The virus also infects specialized clear cells that control the epididymal fluid pH where sperm are stored. Following infection, clear cell groups showed proliferation and possible acidification of the sperm storage microenvironment. All these combined changes suggest that the normal environment required for sperm maturation is disrupted. Our findings raise concern that severe COVID-19 may have lasting effects on male fertility and highlight the epididymis as an important but overlooked target organ of this infection.