Available online 13 February 2024, 104214

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MHV-3 is detected in Leydig cells during acute infection

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MHV-3 infection elicits an inflammatory response within the testicular environment

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MHV-3 infection detrimentally impacts testicular steroidogenesis

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MHV-3 infection alters germ cell differentiation and Sertoli cell barrier

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MHV-3 infection is associated with raised levels of sperm fragmentation

ABSTRACT

Although several testicular alterations promoted by coronavirus infection have been demonstrated, the extent, causes, and players of testicular pathogenesis are not totally understood. The present study aimed to investigate the short-term effects on male fertility of intranasally administered murine hepatitis virus strain 3 (MHV-3), a member of the genus Betacoronavirus, which causes a severe systemic acute infection. This mouse model might be used as a in vivo prototype for investigating the impact of betacoronavirus on the endocrine and exocrine testicular functions with the advantage to be performed in a biosafety level 2 condition. Herein, we performed virological, histopathological, and molecular studies regarding the testicular spermatogenesis and the spermatic quality analyses in an MHV-3-infected C57BL/6 mice. The main outcomes showed that MHV-3 infects mouse testis and induces a testicular inflammatory state, impairing the steroidogenic pathway. The infection led to several alterations in the testicular parenchyma, such as: seminiferous epithelium sloughing, retention of residual bodies, germ cell apoptosis, alterations in intercellular junction proteins, and worse spermatogenic parameters. Moreover, the levels of plasmatic testosterone as well as the quality of sperm production reduced. Therefore, the present data suggest that the viral/inflammatory impairment of the steroidogenic pathway and the consequent imbalance of androgen levels is critical in testicular pathology, disturbing the SC barrier function and the germ cell differentiation. Our study is important for comprehending the effects of beta coronavirus infections on testis function in order to develop treatments that could prevent virus-mediated male infertility.

Section snippetsINTRODUCTION

The testis is specialized to perform two primary functions: sperm production and steroidogenesis (Sharpe et al., 1994, Mäkelä et al., 2019). The spermatogenesis occurs in the seminiferous tubules (ST), where spermatogonial stem cells proliferate, undergo meiosis, and differentiate into spermatozoa through cyclic events (Houda et al., 2021). For that, the testis presents an immune-privileged environment that is required to protect the developing auto-immunogenic germ cells, which can be

Virus strain

MHV-3 strain was kindly provided and sequenced (GenBank accession no. MW620427.1) by Dr. Clarice Arns and Dr. Ricardo Durães-Carvalho from the Universidade Estadual de Campinas (UNICAMP, Brazil) (Garcia et al., 2021).

L929 cells

L929 (ATCC® CCL-1) cells were cultured under a controlled atmosphere (37 °C, 5% CO2) in high glucose DMEM (L929) supplemented with 7% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin.

Plaque assay (cytopathic evaluation)

100 µL of serially diluted virus suspension, and lung and testis homogenates

MHV-3 replicates in the respiratory system and infects testis

MHV-3 intranasal infection triggered lung damage, with a progressive inflammatory cell infiltration over time (Fig. 1A-D). Viral titer was assessed in the mouse lung and testis at 1, 3, and 5 dpi. In the lung, viral loads significantly increased from 1 dpi to 5 dpi (Fig. 1E). Viruses were detected from 3 dpi onwards in the testis, with viral loads being more stably detected at 5 dpi (Fig. 1E). As the virus was not detected at 1dpi, we only evaluated testes on 3 and 5 dpi. Through

DISCUSSION

The murine beta coronavirus MHV-3 has been postulated as a model to recapitulate many aspects of severe respiratory disease, reproducing systemic alterations as seen in patients with COVID-19 (Andrade et al., 2021). Several alterations promoted by SARS-CoV-2 infection have been evaluated in human testes of deceased patients (Costa et al., 2023). However, important features of testicular pathogenesis in beta coronavirus diseases remain unknown in view of the intrinsic difficulty in having a

CONCLUSION

The present data suggest that the impairment of the steroidogenic pathway is critical in testicular pathology. In general, the imbalance of androgen levels had effects on the SC barrier function and germ cell differentiation, leading to alterations in the intercellular junction proteins, spermatogenic parameters, and quality of sperm. According to our findings, LC is a key element for the testicular pathological pathway after acute viral infection and emerges as a potential target for the

FUNDING INFORMATION

This work was supported by grants from the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES; Finance Code 001. Projeto: CAPES – Programa: 9951 – Programa Estratégico Emergencial de Prevenção e Combate a Surtos, Endemias, Epidemias e Pandemias AUX 0641/2020 – Processo 88881.507175 /2020-01), and from the National Institute of Science and Technology in Dengue and Host-Microorganism Interaction (INCT em Dengue), sponsored by the National Council for Scientific and

Uncited references

(Bupp and Jorgensen, 2018, Cheng and Mruk, 2012, Dejucq-Rainsford, 2022, Elmore, 2007, Waris et al., 2005)

Declaration of Competing Interest

"The authors have no conflict of interest to disclose". None of the authors have any commercial or other associations that might pose a conflict of interest in connection with the submitted article.

ACKNOWLEDGMENTS

Technical help from Mara Lívia Santos is highly appreciated. The support from the Image Acquisition and Processing Center (CAPI- ICB/UFMG) was of great importance.

DISCLOSURES

None of the authors have any commercial or other associations that might pose a conflict of interest in connection with the submitted article. The study was performed after ethical approval. Declarations of interest: none.

AUTHORS’ CONTRIBUTION

This study was conceived by AFAF and GMJC. All authors contributed to the design of the study and interpretation

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