Interactions of ethane dimethanesulfonate (EDS) with the leydig cell lines R2C, MA-10, and MLTC-1: an in vitro approach to understand the molecular mechanism of EDS action

• Grant number: 22/16531-4
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: March 01, 2023
• End date: June 30, 2023
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Wilma de Grava Kempinas
• Grantee: Jorge Willian Franco de Barros
• Supervisor: Jacques J. Tremblay
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Institution abroad: Université Laval, Canada
• Associated to the scholarship: 21/09882-2 - Population control of synanthropic animals: morphological, functional and biochemical aspects of the contraception promoted by new routes of administration of ethane dimethanesulfonate to male Wistar rats, BP.DR

Abstract

Ethane dimethanesulfonate (EDS) is a molecule with known selective cytotoxicity on adult Leydig cells after gavage, intraperitoneal, and intratesticular injections, which leads to male androgen deprivation and infertility. After EDS exposure in adult male rats, the adult Leydig cells repopulate the testis from the stem Leydig cell pool and start to produce androgen hormones again. In vitro studies with immortalized Leydig cell lines, derived from rats and mice, showed similar effects of cell death promoted by EDS, and indicate that steroidogenesis is first compromised before the apoptosis process. Thus, this study aims to investigate, through in vitro methods and molecular analyses, the mechanisms exerted by EDS on different Leydig cell lines (R2C cells, rat-derived; MA-10 and MLTC-1, mouse-derived), and the implications of this interaction on target genes related to oxidative stress and endocrine function (steroid hormones and INSL3). For this purpose, different Leydig cell lines will be incubated for 3 hours (early effects) or 24 hours (late effects) with either vehicle (0.5% DMSO) or two doses of EDS (for mouse cells less sensitive to EDS, 10 nM and 20 nM; for rat cells more sensitive to EDS, 2 and 10 nM). The various Leydig cell lines will be transiently transfected with the promoter reporter plasmids (Gsta3, Star, Cyp11a1, or Insl3 promoter constructs) then, they will be treated with EDS or DMSO (vehicle) with or without 8Br-cAMP, which stimulates steroidogenesis. At the end of the treatment period, cells will be lysed, lysates collected, and luciferase measurements will be performed. DNA binding proteins in these Leydig cell lines will also be evaluated by DNA precipitation approach coupled with mass spectrometry, to identify the transcription factor(s) targeted by EDS. This study will allow a better understanding of the mechanisms of EDS action in Leydig cells, employing modern methods in reproductive biology and toxicology, contributing to the basic sciences, as well as improving the knowledge of this molecule as an approach to fertility control to be applied in synanthropic, farmed, zoo, and pet animals. (AU)