Environmental Signaling in Reproduction and Pregnancy

National Institute Of Environmental Health Sciences

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Abstract

The environment impacts the ability of women to give birth to healthy children. The goal of this project is to investigate the molecular mechanisms governing uterine function and health. We have continued to investigate these molecular mechanisms in order to understand the factors governing womens reproductive health. Over the last year we have completed our investigation in: the role of estrogen receptor in the regulation of human endometria gene expression, the role of steroid hormone signaling in human endometrial organoid culture, the role of the progesterone receptor in myometrial function, and have utilized a novel technology to identify cell to cell interactions in situ in the pregnant mouse uterus. These findings shed light on the role of this important regulator of female function in the womens health and the ability to have a successful pregnancy that delivers an infant at the appropriate stage of development. Endometrial health is affected by molecular processes that underlie estrogen responses. We assessed estrogen regulation of endometrial function by determining the sites the estrogen receptor (ESR1) binds in the genome of human endometrial biopsies taken at the proliferative and secretory phase of the menstrual cycle. We then aligned these binding sites with the RNA expression of biopsies taken from these phases of the cycle. We conducted a similar analysis using organoid endometrial cultures which were hormonally stimulated to match the proliferative and secretory phase of the cycle. Genes with ESR1 binding in whole endometrium were enriched for chromatin modification and regulation of cell proliferation. The distribution of ESR1 binding sites in organoids were more similar to the proliferative than the mid-secretory phase ESR1 cistrome when compared to the endometrial biopsies. In the organoids noid estrogen/ESR1 candidate target genes affected formation of cellular protrusions and chromatin modification. Comparison of signaling effected by candidate ESR1 target genes in endometrium vs organoids reveals enrichment of both overlapping and distinct responses. Our analysis of the ESR1 cistromes and transcriptomes from endometrium and organoids provides important resources for understanding how estrogen affects endometrial health and function. This work has been published in the journal Endocrinology. We next investigated the how the chromatin structure of endometrial epithelial cells. The DNA loops and bends to bring regulatory elements together to regulate gene expression. The estrogen receptor (ESR1) and progesterone receptor (PGR regulate genes by binding chromatin at genes and at distal enhancer regions, which interact via dynamic 3-dimensional chromatin structures. Endometrial epithelial cells are the initial site of embryo attachment and invasion, and thus understanding the processes that yield their receptive state is important. Here, we cultured and treated organoids derived from human epithelial cells, isolated from endometrial biopsies, with estrogen and progesterone and evaluated their transcriptional profiles, their PGR cistrome, and their chromatin conformation. above ESR1 regulate gene expression governing proliferation and cellular protrusions. . The impact of Progesterone minimally impacted the organoid transcriptome with the stimulation of target gene expression however attenuation of estrogen-dependent gene responses was observed. PGR bound closely to previously described organoid ESR1 peaks, and most PGR and ESR1 peaks were in inactive compartment regions of chromatin. Overall, this analysis defined the hormonal regulatory units of the organoid genome and addressed the limitation of this in in vitro model when compared to in vivo models to investigate hormone responsiveness. This work was published in the journal Cells. Parturition disorders such as preterm birth and laboring dysfunction impose serious health risks on the mother and the child. The myometrium, the muscle compartment of the uterus, is tasked to hold the increasing weight and size of a growing fetus with minimum contractile activities before parturition. To achieve this, the uterus reinforces its structural integrity by remodeling the myometrium. During the last year we investigated the gene expression program under the control of PGR for this remodeling process in a mouse model. We next determined how modifications of the chromatin regulating gene expression identified genomic elements pertinent to PGR regulation of gene expression. The results of this work demonstrate a novel role of PGR in preparing the myometrium to support pregnancy. The steroid hormone progesterone is essential to suppress premature labor, during the last year we investigated the role of progesterone signaling in myometrial expansion. Ablation of the cognate receptor for progesterone, PGR, in mouse smooth muscles led to subfertility, oviductal embryo retention, and impaired myometrial adaptation to pregnancy. While the overall structure between mutant and control uteri were comparable, mutant uteri manifest showed a decrease in contractility when stimulated with oxytocin, a hormone that regulate myometrial contraction during birth. At mid-pregnancy, the mutant myometrium exhibited discontinuous muscle fibers and disarrayed extracellular matrix at the conceptus site. Analysis of the gene expression of the mutant mid-pregnant uterine wall showed altered profiles in manifests gene expression that resembled that of late-pregnant control tissues. A survey of PGR binding to the myometrial genome and modifications of histone proteins, H3K27ac, on the chromatin as well as chromatin looping defined the regulator elements that may direct gene expression patterns of mid-pregnancy uteri for uterine remodeling. This analysis identified the major muscle and matrix regulators Myocd and Ccn2, along with building block genes of smooth muscle, are PGR direct downstream targets. Cataloging regulatory units associated with progesterone downstream genes reveals distinctive patterns of transcription factor binding motifs in groups of enhancers and identified potential regulatory partners of progesterone receptor outside its occupying sites. Finally, conserved correlations are found between estimated PGR activities and RNA abundance of downstream muscle and matrix genes in human myometrial tissues. This work was published in PNAS Nexus. Normal uterine functions at pregnancy depend on crosstalk among multiple cell types in uterine microenvironments. During the last year we utilized a cutting edge approach to investigate the spatial expression of genes in the Day 7.5 mouse uterus. We then combined this analysis with gene expression at the single cell level to determine the compartmental and cells specific expression of genes at this stage of pregnancy. Functional annotation assays on spatial transcriptomic data revealed uterine microenvironments with distinguished metabolic preferences, immune responses, and various cellular behaviors that are regulated by region-specific endocrine and paracrine signals. This analysis was then used to determine the communication networks between these compartments to determine what growth factors regulate the biology of these cells during pregnancy. This study provided a high-resolution profile with location information at the embryo implantation site to facilitate further investigations on molecular mechanisms for normal pregnancy progression. This work was published in Biology of Reproduction.