Testosterone and estrogen role in testis and epididymis immune response to epididymo-orchitis

• Grant number: 25/01918-9
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: July 01, 2025
• End date: June 30, 2029
• Field of knowledge: Biological Sciences - Morphology - Cytology and Cell Biology
• Principal Investigator: Estela Sasso Cerri
• Grantee: André Acácio Souza da Silva
• Host Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

Epididymo-orchitis is an inflammation of the testicles and epididymis that can compromise male fertility by causing spermatogenic dysfunction, disruption of the blood-testis barrier, and reduced sperm motility. Animal models have shown that bacterial and viral infections trigger exacerbated inflammatory responses, characterized by increased pro-inflammatory cytokines and leukocyte infiltration. The regulation of this inflammatory response by sex hormones remains an underexplored aspect, but studies suggest that estrogen modulates immune activation by promoting a Th2-mediated response, whereas testosterone exerts immunosuppressive effects, dampening inflammatory activity. Understanding how these hormones influence testicular and epididymal immunity may provide new insights into treating infertility caused by inflammatory processes. This study aims to evaluate the role of estrogen and testosterone in modulating the testicular and epididymal immune response during the early phase of epididymo-orchitis induced by intravasal injection of TLR4 (LPS) and TLR3 (Poly I:C) agonists. To achieve this, we will investigate immune cell populations, Leydig cell steroidogenic activity, the integrity of the blood-testis and -epididymis barriers, the activation of hormone-dependent genes, and sperm parameters. A total of 120 male C57BL/6 mice will be used, divided into 12 experimental groups according to hormonal treatment and exposure to inflammatory agents. Estrogen, testosterone, or a combination of both will be administered for seven days using subcutaneous osmotic pumps. Following this period, epididymo-orchitis will be induced via intravasal-epididymal injection of saline, LPS, or Poly I:C, and the animals will be euthanized after 48 hours (the early infection phase). Biochemical analyses of blood serum will be conducted to measure testosterone, estrogen, LH, and FSH levels. Testicular and epididymal tissues will be processed for morphological analysis, immunofluorescence, and flow cytometry to identify structural and functional alterations in these organs, including changes in the blood-testis and -epididymis barriers and immune response. Immunofluorescence and/or Western blot will be used to detect immune cell markers (CD45, CD11c, F4/80, CD8, CD4, CD49b, and Ly6G), steroidogenic activity markers (17¿-HSD), and androgen response markers (AR and ABP). Double-labeling assays will assess interactions between clear cells and mononuclear phagocytes, along with the immunoexpression of inflammatory cytokines in Leydig cells. Confocal microscopy will be employed to quantify interactions between mononuclear phagocytes and clear cells. Additionally, RT-qPCR and Western blot techniques will be used to analyze potential signaling pathways involved in immune activity in response to steroid hormones. Finally, sperm parameters (concentration, motility, and mitochondrial activity) will be assessed and correlated with the expression of genes associated with sperm maturation. This comprehensive analysis will provide valuable insights into the impact of steroid sex hormones on the early inflammatory response in the testis and epididymis.