RESUMO
Superparamagnetic iron oxide nanoparticles (SPIONs) have gained significant attention in biomedical research due to their potential applications. However, little is known about their impact and toxicity on testicular cells. To address this issue, we conducted an in vitro study using primary mouse testicular cells, testis fragments, and sperm to investigate the cytotoxic effects of sodium citrate-coated SPIONs (Cit_SPIONs). Herein, we synthesized and physiochemically characterized the Cit_SPIONs and observed that the sodium citrate diminished the size and improved the stability of nanoparticles in solution during the experimental time. The sodium citrate (measured by thermogravimetry) was biocompatible with testicular cells at the used concentration (3%). Despite these favorable physicochemical properties, the in vitro experiments demonstrated the cytotoxicity of Cit_SPIONs, particularly towards testicular somatic cells and sperm cells. Transmission electron microscopy analysis confirmed that Leydig cells preferentially internalized Cit_SPIONs in the organotypic culture system, which resulted in alterations in their cytoplasmic size. Additionally, we found that Cit_SPIONs exposure had detrimental effects on various parameters of sperm cells, including motility, viability, DNA integrity, mitochondrial activity, lipid peroxidation (LPO), and ROS production. Our findings suggest that testicular somatic cells and sperm cells are highly sensitive and vulnerable to Cit_SPIONs and induced oxidative stress. This study emphasizes the potential toxicity of SPIONs, indicating significant threats to the male reproductive system. Our findings highlight the need for detailed development of iron oxide nanoparticles to enhance reproductive nanosafety.
RESUMO
BACKGROUND: Cellular entry of SARS-CoV-2 has been shown to rely on angiotensin-converting enzyme 2 (ACE2) receptors, whose expression in the testis is among the highest in the body. Additionally, the risk of mortality seems higher among male COVID-19 patients, and though much has been published since the first cases of COVID-19, there remain unanswered questions regarding SARS-CoV-2 impact on testes and potential consequences for reproductive health. We investigated testicular alterations in non-vaccinated deceased COVID-19-patients, the precise location of the virus, its replicative activity, and the immune, vascular, and molecular fluctuations involved in the pathogenesis. RESULTS: We found that SARS-CoV-2 testicular tropism is higher than previously thought and that reliable viral detection in the testis requires sensitive nanosensors or RT-qPCR using a specific methodology. Through an in vitro experiment exposing VERO cells to testicular macerates, we observed viral content in all samples, and the subgenomic RNA's presence reinforced the replicative activity of SARS-CoV-2 in testes of the severe COVID-19 patients. The cellular structures and viral particles, observed by transmission electron microscopy, indicated that macrophages and spermatogonial cells are the main SARS-CoV-2 lodging sites, where new virions form inside the endoplasmic reticulum Golgi intermediate complex. Moreover, we showed infiltrative infected monocytes migrating into the testicular parenchyma. SARS-CoV-2 maintains its replicative and infective abilities long after the patient's infection. Further, we demonstrated high levels of angiotensin II and activated immune cells in the testes of deceased patients. The infected testes show thickening of the tunica propria, germ cell apoptosis, Sertoli cell barrier loss, evident hemorrhage, angiogenesis, Leydig cell inhibition, inflammation, and fibrosis. CONCLUSIONS: Our findings indicate that high angiotensin II levels and activation of mast cells and macrophages may be critical for testicular pathogenesis. Importantly, our findings suggest that patients who become critically ill may exhibit severe alterations and harbor the active virus in the testes.