Environmental Signaling in Reproduction and Pregnancy

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

Women’s health and quality of life are impacted by the status of their reproductive tract. The female reproductive tract consist of the ovary , oviduct, uterus, cervix and vagina. A healthy female reproductive tract is critical for the ability of a women to bare children. The function of the female reproductive tract is regulated by steroid hormones, progesterone and estrogen. These hormones act through their receptors, the estrogen receptor (ESR1) and the progesterone receptor, PGR. Environmental mimics of these hormones can impair the function and health of the female reproductive tract resulting in infertility, preterm birth, preeclampsia and chronic diseases such as endometriosis and uterine fibroids. Therefore, the environment impacts a women’s overall health and the ability of women to give birth to healthy children. The goal of this project is to investigate the molecular mechanisms governing reproductive tract function and health. Over the last year we have focused on the role of the steroid hormone receptors, ESR1, PGR and the Vitamin D receptor, VDR in reproductive tract function. We have accomplished this by investigating: 1. The role of these receptors in endometrial stroma cells and smooth muscles cells of the myometrium in the uterus. 2. The regulation of steroid hormone receptors in stroma cells of the endometrium and myometrium, and 3. The identification of coregulators of ESR1 and PGR, in the uterus. In order to accomplish these goals we combine the use of in vivo and in vitro approaches. For the in vivo approach we employ genetically engineered mouse models which allows us the alteration of gene expression in specific cell types of the uterus. The in vitro approaches employ: human clinical tissues, primary endometrial stroma, primary epithelial organoid culture and the use of human transformed cell lines. This approach allows the in vivo analysis of gene function in pregnancy and the validation of the clinical relevance in the human samples. During the last year we focused on the role of the PGR in the endometrial stroma cells. We first examined the role of the progesterone receptor in mouse endometrial stroma cell in vivo using two mouse models. The first mouse model was ablating the progesterone receptor in endometrial stroma using the Foxl2Cre mouse model. Ablation of the PGR using this model resulted in mouse sterility and demonstrated the stroma PGR regulated the ability of the uterus to support embryo implantation by controlling the ability of the stroma cell to transform during pregnancy to allow the embryo to develop. This result was predictable however the unique finding of this research was that the stroma PGR regulated the function and differentiation of the uterine glands. This was unexpected and we are in the process of using single cell RNA sequencing to determine how the stroma PGR regulates gland differentiation. The second model we used to investigate the role of progesterone receptor in endometrial stroma function is a novel model we generated over the last year. This model consists of ablating gene expression using a Tamoxifen regulated Cre recombinase inserted into the PGR locus. This model has been published in Biology of Reproduction. As a proof of principle we demonstrated that ablation of the PGR in the adult uterus renders the female infertile. The strength of this model is we can now investigate the uterine factors regulating pregnancy in the post implantation period of pregnancy which was a deficit in the field. We now are ablating the PGR in the uterus after the embryo implants . We are investigating the impact of post embryo implantation ablation of the PGR on placental development. This research will explore the understudied role of the uterine cells in the placental development. In addition to investigating the role of PGR in the endometrial stroma cells we investigate the role of the PGR in the regulation of the smooth muscle compartment of the uterus, the myometrium. The myometrium is the outer layer of the uterus and plays a critical role during pregnancy allowing the uterus to expand during pregnancy and contract during the birth process. Emerging studies in mice indicate a dynamic change of the myometrial gene expression and chromatin structure during pregnancy to ready the contractile machinery for parturition. We investigated human term pregnant nonlabor myometrial biopsies for gene expression and the structure of the myometrial chromatin. We demonstrated that where PGR to the myometrial DNA these sites were enriched of other transcription factors that are important for regulating inflammation such as AP-1, STAT, NFkB. In human myometrial specimens, inferred PGR activities are positively correlated with a signaling molecule that regulates muscle contraction, phospholipase C like 2 (PLCL2) mRNA levels, Using CRISPR activation, we assessed the functionality of a PGR putative distal region of the PLCL2 gene, 35 kilobases upstream of the contractile-restrictive gene PLCL2. In summary, the results of this study serve as a resource to study gene regulatory mechanisms in the human myometrium at the term pregnancy stage for further advancing women's health research. This work was published in eLife. Studying the role of the ESR1 in human uterine stroma cells have been hampered by the fact that the current cell lines have very low levels of estrogen receptor. In order to rectify this need we used CRSPR technology to activate the expression of the endogenous human ESR1 in transformed human endometrial stroma cells. The generation of the estrogen receptor in these cells allowed us to define not only which genes were regulated by ESR1in stroma cells but we were able to identify where ESR1 bound to the DNA to regulate the expression of these genes. By combining the RNA expression analysis, the binding of the ESR1 to the DNA and the changes in the genomic architecture revealed the role of the estrogen receptor in in regulating stroma cell differentiation and inflammation-related gene networks, with relevance to endometrial pathologies including endometrial cancer. This model serves as a powerful tool to study not only estrogen signaling in endometrial stromal cell biology but can be used as a tool to investigate the impact of environmental endocrine disruptors on human endometrial biology. This work was published in Fertility and Sterility Sciences. One environmental factor that impacts female reproduction is Vitamin D. Epidemiological studies demonstrate Vitamin D influences female reproduction efficiency. Vitamin D and its cognate receptor, VDR, are recognized for their role in calcium homeostasis. Over the last year we continued to investigate the role of Vitamin D and its Receptor in female reproduction. We demonstrate that mice fed a Vitamin D deficient diet have an reduced implantation, decidual, response when treated with estrogen and progesterone. To translate these findings to human endometrial reproduction we demonstrate that human endometrial stromal cell line, THESC, have decreased VDR expression during in vitro decidualization. Knockdown of the VDR in T-HESC enhanced decidualization while overexpression of VDR inhibited decidualization. We demonstrated that VDR regulates the accessible of chromatin to transcription factors and acts as a repressor of transcription and stroma cell differentiation. We then demonstrate that the Vitamin D treatment of these cells alleviates the repression and allows full differentiation of these stroma cells. These findings identify VDR as a key regulator of transcriptional and chromatin landscapes in endometrial stromal cells, offering novel insights into vitamin D signaling in reproduction. This study highlights the potential of targeting vitamin D pathways to treat uterine disorders associated with impaired decidualization. Having investigated the role and regulation of the steroid receptors in endometrial biology, we next investigated the role of transcription factors that work in concert with these receptors in the regulation of endometrial function and health. We focused on two transcription factors, Zinc Finger MIZ-Type Containing 1 (Zmiz1) and Serum Response Factor (SRF) in regulating endometrial function and health. We utilized in vivo analysis in genetically engineered mice and in vitro analysis in human endometrial stroma cells to decipher the function of these transcription factors. ZMIZ1 was identified by analysis of ESR1 binding to chromatin. ESR1 binding to chromatin identified a extended region or potential regulatory factors called Super Enhancers. ZMIZ1 was co-localized adjacent to an ESR1-binding super-enhancer. ZMIZ1 mutations are found in endometrial cancer. ZMIZ1 is dynamically expressed in human endometrial tissues during the menstrual cycle. Disrupting ZMIZ1 in cultured human endometrial stromal cells resulted in impaired cell proliferation and decidual differentiation. Ablation of Zmiz1 in PGR expressing cells in the mouse resulted in infertility and accelerated age-dependent uterine fibrosis. The uterus of Zmiz1d/d mice was unable to undergo a hormonally induced decidual response, had decreased expression of stromal PGR and decreased stromal and epithelial cell proliferation. Analysis of the genes regulated by Zmiz1 in the mouse uteri showed decreased transcription factors regulating proliferation such as E2F, CCNA2 and FOXM1. We then demonstrated that ablation of ZMIZ1 resulted in an impairment of the uterus to respond to an estrogen stimulation. Our findings demonstrate the importance of ZMIZ1 as an ESR1 co-regulator in uterine biology and pathology and may be integral in modulating the response of the uterus to environmental estrogens. In our analysis of the binding of the PGR to the chromatin of uterine myometrial cells we identified an enhancement of binding sites for the transcription factor Serum response factor (SRF). SRF is a widely expressed transcription factor essential for mesenchymal cell growth and differentiation with noted roles in hormonal regulation of muscle tissues but little characterization in reproductive organs. Here, we reveal that endometrial SRF is dysregulated in infertility-associated human endometrial pathology and is critical for female reproductive success in mice through regulation of endometrial stromal and epithelial cells. Immunohistochemical analysis identified decreased endometrial SRF expression in infertile endometriosis patient tissues. RNAi-based SRF knockdown in human endometrial stromal cells resulted in disrupted cytoskeletal structure, viability, and decidual response. SRF was then ablated in the PGR expressing cells of the mouse uterus. Uterine ablation of SRF resulted in mice that were infertile due to multiple reasons. Embryo delivery to the uterus from the oviduct was impaired. Embryos failed to implant . The uterine stroma cells failed to under go a hormonally induced differentiation and the mice development of severe endometrial fibrosis with age. Single cell RNA-sequencing was conducted to identify the cell specific changes in RNA due to ablation of SRF. This analysis identified numerous changes in cell specific gene expression including large scale myeloid immune cell infiltration, stromal fibroblasts displaying aberrant cytoskeletal and extracellular matrix gene expression and downregulation of genes important for decidual growth response. The most novel finding was that SRF deficient epithelial cells displayed the most prominent dysregulation, strongly overexpressing estrogenic innate inflammatory genes including C3 and Lcn2 and fibrogenic genes Mmp7 and Fbln1, all of which paralleled patterns in human endometriosis patient data that we identified through comparative analysis using a published single cell atlas. These results demonstrate the profound impact of SRF on endometrial homeostasis with relevance to human endometriosis-related infertility. In summary this research has developed novel tools for the investigation of the role of steroid hormones in uterine biology. This research has identified novel pathways governed by these hormones in the regulation of uterine health. All of the research involving model organisms has been evaluated in human samples to ensure clinical relevance.