Towards understanding mare endometrosis: ex vivo study on the role of relaxin.

Overview

• This study investigated how relaxin (RLX), a hormone, affects mare endometrium tissue affected by endometrosis, a poorly understood fibrotic uterine condition.
• Using an ex vivo system, the research analyzed changes in inflammatory markers, fibrotic proteins, and cell signaling pathways induced by RLX treatment.

Background and Objective

• Mare endometrosis is a pathological fibrosis of the uterine lining that impairs fertility in horses but its mechanisms are not well understood.
• The hormone relaxin (RLX) is known to influence tissue remodeling and fibrosis in other contexts, but its role in mare endometrosis had not been clearly studied.
• The study aimed to characterize the effects of RLX on equine endometrial tissue exhibiting endometrosis to better understand potential mechanisms and treatment approaches.

Experimental Design

• Endometrial tissues were collected from mares and separated into groups: healthy untreated (H), healthy vehicle-treated (Hveh), endometrotic untreated (E), endometrotic vehicle-treated (Eveh), and endometrotic treated with relaxin at different concentrations (10, 25, 50 nM) (ER).
• Tissues were cultured ex vivo for 72 hours under these treatments.
• Histological examinations were performed to observe tissue morphology and integrity.
• Immunoenzymatic assays measured levels of key signaling molecules and hormones including transforming growth factor β (TGF-β), proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), and progesterone.
• Western blotting analyzed protein abundance and activation states related to cellular processes, signaling pathways, and extracellular matrix remodeling. Target proteins included:
• N-cadherin and cortactin (cell adhesion and cytoskeleton)
• Wnt/β-catenin signaling components and phosphorylation of kinases GSK-3β and AKT (growth and survival signaling)
• Matrix metalloproteinases MMP9 and MMP2 (enzymes involved in matrix remodeling)
• Cyclins D1 and D3 (cell cycle regulators)

Key Findings

• Relaxin at 50 nM reduced TGF-β concentration, a cytokine commonly associated with fibrosis and extracellular matrix deposition.
• Conversely, RLX increased levels of IL-6 and IL-8, which are pro-inflammatory cytokines, indicating a complex role of RLX in inflammatory signaling.
• Progesterone concentration was decreased by RLX treatment, suggesting hormonal modulation by relaxin in endometrial tissue.
• Histological structure of the tissues was not altered by RLX treatment over the 72-hour period, indicating no gross tissue damage.
• Changes in protein abundance and signaling indicated enhanced cell adhesion and cytoskeletal dynamics as seen by increased N-cadherin and cortactin levels.
• Phosphorylation (activation) of β-catenin, GSK-3β, and AKT was increased, highlighting upregulation of Wnt/β-catenin and AKT signaling pathways which regulate cell proliferation, survival, and remodeling.
• Matrix metalloproteinases MMP2 and MMP9 were upregulated, indicating enhanced extracellular matrix degradation and tissue remodeling activity.
• Cyclin D3, a regulator of cell cycle progression, was increased, pointing to potential proliferative responses in endometrial cells treated with RLX.

Conclusions and Implications

• Relaxin exhibits a dual role by modulating both inflammatory and fibrotic pathways within mare endometrium affected by endometrosis.
• The hormone can simultaneously reduce certain fibrosis markers (TGF-β) while increasing some inflammatory cytokines (IL-6 and IL-8), suggesting that RLX’s effect is context-dependent.
• Modulation of signaling pathways related to cell adhesion, survival, and remodeling implies relaxin influences multiple cellular processes that can impact fibrosis progression or resolution.
• The results support a potential therapeutic approach where relaxin, possibly in combination with agonists or antagonists targeting the identified proteins and pathways, could be used to treat endometrial fibrosis in mares.
• This approach holds promise in equine reproductive medicine by improving uterine health and fertility outcomes in mares with endometrosis.