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 focused on the characterizing the cell populations of human endometrial stroma cells to characterize the in vitro reagents used in our research of the hormones regulation of female reproduction. We then conducted the investigation of the role of regulators or progesterone action in the regulation of uterine function. We have focused on the chromatin architecture gene CTCF, the role of the orphan nuclear receptor COUP-TFII and the transcription factor TRIM28 in the regulation of uterine function. These findings shed light on the role of this important regulator of female function in the women's health and the ability to have a successful pregnancy that delivers an infant at the appropriate stage of development. To define the in vitro reagents used to investigate hormonal regulation of uterine function we systematically analyzed the cell composition and transcriptome of primary human endometrial stromal cells (HESCs) and transformed human endometrial stromal cells (THESCs) using single cell RNA sequencing. We found the individual differences among the primary HESCs and bigger changes between the primary HESCs and THESCs. Cell clustering with or without integration identified cell clusters belonging to mature, proliferative, and active fibroblasts that were conserved across all samples at different stages of the cell cycles with intensive cell communication signals. All primary HESCs and THESCs can be correlated with some subpopulations of fibroblasts in the human endometrium. Our study indicated that the primary HESCs and THESCs displayed conserved cell characters and distinct cell clusters. Mature, proliferative, and active fibroblasts at different stages or cell cycles were detected across all samples and presented with a complex cell communication network. The cultured HESCs and THESCs retained the features of some subpopulations within the human endometrium. This work was published in F&S Sci. In our investigation of the mechanism by which steroid hormones regulate uterine function we first investigated the role of proteins involved in chromatin structure on uterine gene expression and function. Receptors for estrogen and progesterone frequently interact, at distal regions gene promoters to regulate the expression of these genes. One of the protein complexes that bring these regulatory regions near the gene promoter is the Cohesin/CTCF loop extrusion complex. Loss-of-function mutations in CTCF occur in >20% of human endometrial tumors indicates its importance in uterine homeostasis. To better understand how CTCF-mediated enhancer-gene interactions impact endometrial development and function, the Ctcf gene was selectively deleted in female reproductive tissues of mice. Prepubertal uterine tissue in which CTCF was deleted exhibited a marked reduction in the number of uterine glands compared to those without Ctcf deletion. Post-pubertal Ctcf deleted uteri were hypoplastic with significant reduction in both the amount of the endometrial stroma and number of glands. The investigation of gene expression of these mice revealed increased expression of stem cell molecules Lif, EOMES, and Lgr5, and enhanced inflammation pathways following Ctcf deletion. Analysis of the response of the uterus to steroid hormone stimulation showed that CTCF deletion affects a subset of progesterone-responsive genes. This finding indicates (1) Progesterone-mediated signaling remains functional following Ctcf deletion and (2) certain progesterone-regulated genes are sensitive to Ctcf deletion, suggesting they depend on gene-enhancer interactions that require CTCF. The progesterone-responsive genes altered by CTCF ablation included Ihh, Fst, and Errfi1. CTCF-dependent progesterone-responsive uterine genes enhance critical processes including anti-tumorigenesis, which is relevant to the known effectiveness of progesterone in inhibiting progression of early-stage endometrial tumors. Overall, our findings reveal that uterine Ctcf plays a key role in progesterone-dependent expression of uterine genes underlying optimal post-pubertal uterine development. This work was published in FASEB J. We nest attempted to identify proteins that work with the hormone receptros in the regulation of uterine function. Estrogen and progesterone, acting through their cognate receptors the estrogen receptor (ER) and the progesterone receptor (PR) respectively, regulate uterine biology. Using rapid immunoprecipitation and mass spectrometry (RIME) and co-immunoprecipitation, we identified TRIM28 (Tripartite motif containing 28) as a protein which complexes with ER and PR in the regulation of uterine function. Impairment of TRIM28 expression results in the inability of the uterus to support early pregnancy through altered PR and ER action in the uterine epithelium and stroma by suppressing PR and ER chromatin binding. Furthermore, TRIM28 ablation in PR-expressing uterine cells results in the enrichment of a subset of TRIM28 positive and PR negative pericytes and epithelial cells with progenitor potential. In summary, our study reveals the important roles of TRIM28 in regulating endometrial cell composition and function in women, and also implies its critical functions in other hormone regulated systems. This work was published in Nature Comm. In addition to TRIM28 and CTCF we also investigated a orphan nuclear receptor involved in the regulation of endometrial stroma cell function. One of the regulators of endometrial stroma cell function is orphan nuclear receptor NR2F2, which regulates uterine epithelial receptivity and stromal cell differentiation. In order to determine the molecular mechanism regulated by NR2F2, RNAseq analysis was conducted on the uterus of mice with uterine specific ablation of NR2F2 mice at Day 3.5 of pregnancy. This transcriptomic analysis demonstrated Nr2f2 ablation in Pgr-expressing cells leads to a reduction of Hand2 expression, increased levels of Hand2 downstream effectors Fgf1 and Fgf18, and a transcriptome manifesting suppressed progesterone signaling with an altered immune baseline. Identification of the genomic location of NR2F2 binding c demonstrated the majority of NR2F2 occupies genomic regions of major uterine transcription regulators Hand2, Egr1, and Zbtb16. Furthermore, functional analysis of an NR2F2 occupying site that is conserved between human and mouse was capable to enhance endogenous HAND2 mRNA expression w in human endometrial stroma cells. These data establish the NR2F2 dependent regulation of Hand2 in the stroma and identify a cis-acting element for this action. In summary, our findings reveal a role of the NR2F2-HAND2 regulatory axis that determines the uterine transcriptomic pattern in preparation for the endometrial receptivity. This work has been published in Frontiers in Endocrinology.