Regulation of expression of Wfdc (Whey-acidic protein four dissulfite core) genes by inflammatory stimulus in the mouse epididymis: potential roles on immune and reproductive functions

Author(s)

Alexandre Dorth de Andrade

• Document type: Master's Dissertation
• Press: Botucatu. 2019-11-07.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2019-10-04
• Advisor: Erick José Ramo da Silva

Abstract

The epididymis is responsible for transforming immature sperm into functionally competent cells capable of acquiring progressive motility and recognizing and fertilizing the oocyte. In addition, the epididymis is paramount for protecting and storing the male gamete before ejaculation. Changes in epididymal physiology may affect sperm maturation, transport or storage, causing male infertility. Epididymal inflammation, known as epididymitis, is one of the most prevalent diseases of the male urogenital tract, being a relevant factor of male infertility. Its main etiology involves the invasion of bacteria, such as E. coli, through retrograde ascension through the urethra. The epididymis constitutively expresses several elements of the innate immune system, including Toll-like receptors (TLRs), like the TLR4, which is activated by the lipopolysaccharide (LPS) of Gram-negative bacteria, as well as proteins with antimicrobial activity, like the WFDC (Whey-acidic difulfide core)-type protease inhibitors, indicating that this organ is constantly alert to fight bacterial infections. In addition to antimicrobial and protease inhibitory activities, WFDC proteins show anti-inflammatory, immunomodulatory and reproductive functions, being multifunctional proteins in the male reproductive tract. In this study, we tested the hypothesis that Wfdc gene expression in the epididymis is modified by inflammatory stimuli as part of tissue defense mechanisms to microbial aggression. For that, we characterized two mouse models of epididymitis induction induced by ultrapure LPS from E. coli via interstitial of the initial segment and vas deferens, causing inflammatory events in the proximal and distal regions of the epididymis. We processed the initial segment, for the first epididymitis model, and cauda epididymis, for the second epididymitis model, to investigate the effects of LPS on cytokine expression by RT-qPCR and multiplex assays and for histopathological evaluation. Our results show that the epididymis rapidly responds to inflammatory stimuli, but with different patterns and intensity for each organ region, and the initial segment is more sensitive to LPS than the cauda epididymis to increased expression of pro-inflammatory cytokines (IL1B, IL6, TNF e IFNG). In order to investigate the expression profile of the Wfdc genes, we processed the initial segment and the cauda epididymis for the RT-PCR and qPCR assays. Our results show that the expression of Wfdc genes is modified by the inflammatory challenge in the epididymis in a region-specific fashion. We observed a negative regulation of the levels of Wfdc6a_v2, Wfdc7, Wfdc8, Wfdc11, Wfdc15b_v1-2 and Wfdc16 transcripts in the initial segment 6 h after interstitial LPS injection, and Wfdc6a_v1, Wfdc6a_v2, Wfdc6b, Wfdc11, Wfdc15b_v1_v2 and Wfdc16 in the cauda epididymis 24 or 72 h after intravasal LPS injection. On the other hand, we observed a positive regulation of Wfdc2, Wfdc3, Wfdc4, Wfdc5, Wfdc6b, Wfdc10, Wfdc12, Wfdc15a and Wfdc15b_v1-2 transcripts in the initial segment 24 and/or 72 h after interstitial LPS injection, while only Wfdc2 and Wfdc5 transcripts were increased in the cauda epididymis after intravasal LPS injection at the same time-point. Treatment with PDTC (100 mg/kg, i.p.), an inhibitor of NFKB transcription factor, 1 h before inducing epididymitis revealed that up-regulation of some Wfdc transcripts, including Wfdc2, Wfdc6b and Wfdc10 in the initial segment and Wfdc5 in the cauda epididymis were dependent on the activation of this transcription factor. Altogether, our results provide new information on how Wfdc gene expression is modulated in the epididymis during acute inflammation and highlight the possible roles of the Wfdc family in combating inflammation and restoring epididymis homeostasis. Our results suggest that WFDC proteins may be exploited as adjuvant therapeutic targets for the treatment of epididymitis. (AU)

• FAPESP's process: 17/20102-3 - Regulation of expression of Wfdc (Whey-acidic protein four dissulfite core) genes by inflammatory stimulus in the mouse epididymis: potential roles on immune and reproductive functions
• Grantee: Alexandre Dorth de Andrade
• Support Opportunities: Scholarships in Brazil - Master