The alteration in myometrial mRNA transcription of the regulatory genes of DNA methylation in mare with endometrosis.

Overview

• This research investigates changes in the expression of genes involved in DNA methylation in the uterine muscle (myometrium) of mares with endometrosis, exploring how these changes might affect the gene for the oxytocin receptor, which regulates uterine contractions.

Background and Purpose

• Myometrial contractile activity is critical for properly cleaning the uterus after breeding or infection; compromised contractions can lead to persistent uterine inflammation (endometritis) and fibrosis (endometrosis) in mares.
• Oxytocin (OXT) stimulates myometrial contractions via binding to its receptor (OXTR), making OXTR expression important for uterine health.
• Epigenetic modifications such as DNA methylation can regulate gene expression; enzymes like DNA methyltransferases (DNMTs) add methyl groups, whereas ten-eleven translocation enzymes (TETs) can remove them, dynamically shaping gene activity.
• Though epigenetic regulation of uterine gene expression has been studied in humans, little is known about these mechanisms in mares, particularly relating to myometrial expression of DNMTs and TETs and their role in diseases like endometrosis.
• The study aims to assess mRNA expression levels of DNMTs (DNMT1, DNMT3A, DNMT3B), TETs (TET1, TET2, TET3), and the OXTR gene, as well as DNA methylation status at specific regions of the OXTR gene, in the myometrium of mares with varying severities of endometrosis during different phases of the estrous cycle.

Methods

• Myometrial tissue samples were collected post-mortem from mares categorized by the severity of endometrial pathology using Kenney and Doig classification (categories I, IIA, IIB, III).
• Samples were taken during two distinct phases of the estrous cycle: mid-luteal phase (n=23) and follicular phase (n=20).
• Quantitative PCR (qPCR) was used to measure mRNA levels of OXTR, DNMT1, DNMT3A, DNMT3B, TET1, TET2, and TET3.
• DNA methylation at CpG islands in exons 1 and 2 of the OXTR gene was analyzed by bisulfite pyrosequencing to determine methylation patterns at nucleotide-level resolution.

Key Findings

• There were no significant overall differences in OXTR mRNA expression or promoter region DNA methylation across the endometrosis categories.
• However, increased DNA methylation was observed at a specific CpG site (position 6 in exon 2) of the OXTR gene.
• The mRNA levels of certain DNMT and TET enzymes—specifically DNMT1, TET2, and TET3—were significantly altered depending on both the phase of the estrous cycle and the severity of endometrosis (statistically significant with P < 0.05).
• These variations indicate that epigenetic regulatory enzymes are differentially expressed in the myometrium in a manner related to disease severity and hormonal cycle phase.

Implications and Conclusions

• The findings suggest that DNA methylation regulatory enzymes (DNMTs and TETs) may play a role in modifying myometrial gene expression in response to endometrosis and physiological hormonal changes during the estrous cycle.
• This epigenetic modulation could affect uterine contractility by influencing the expression of critical genes like OXTR, potentially contributing to the pathology of endometrosis through altered myometrial function.
• The specific increase in methylation at one CpG site of OXTR suggests localized epigenetic changes rather than widespread methylation alterations in the gene.
• Understanding these regulatory mechanisms may provide new insights into the pathogenesis of endometrosis and identify potential molecular targets for therapeutic intervention to improve uterine health and fertility in mares.