Indrani Bagchi - US grants

The long-term goal of this proposal is to explore how the steroid hormone estrogen (E) regulates growth and differentiation processes in the endometrium during early pregnancy (preimplantation period), which lead to acquisition of the receptive state that allows blastocyst implantation. E exerts its cellular effects by regulating the expression of specific target genes. The identity, profile of expression, and function of the E-regulated genes at various stages of the reproductive cycle and pregnancy, however, remain largely unknown. The specific aims of this proposal are to: 1. Isolate and identify genes that are regulated in response to nidatory E in rat uterus during delayed implantation. The messenger RNA differential display method will be used to isolate and identify the cDNAs representing mRNAs whose expression is induced or repressed in rat uterus in response to an implantation-inducing dose of E. To determine whether the newly identified E-regulated genes are potential modulators of implantation, their spatio-temporal expression in rat uterus during early pregnancy will be analyzed by Northern blotting, in situ hybridization, and immunohistochemistry. 2. Assess the functional roles of the newly identified cDNAs in implantation. A recently developed antisense technology will be used to regulate specific gene expression in intact rat uterus. This will involve administration of antisense oligodeoxynucleotides targeted against mRNA transcript of each candidate gene into the preimplantation uterus to suppress the steady state level of that mRNA . If this intervention also leads to an impairment in implantation, it will allow the establishment of a functional link between this gene and the implantation process. 3. Analyze the function of a novel E-regulated gene, ERG1, in early pregnancy. A novel gene (ERG1) that is tightly regulated by E in two key reproductive tissues, the uterus and oviduct, has recently been isolated. ERG1 is expressed in the surface epithelium of the uterus in a highly stage-specific manner during the ovarian cycle and early pregnancy. To determine the functional role of ERG1 during early pregnancy, mice harboring a targeted germ line mutation of ERG1 will be developed and analyzed for potential reproductive phenotypes, such as impairment in embryo transport through the oviduct, defects in epithelial cell morphology and function, and lack of uterine receptivity for implantation. The proposed research will help us to identify molecules that mediate E regulation of critical events during early pregnancy.

DESCRIPTION (provided by applicant): The steroid hormone progesterone (P) profoundly influences the function of the uterus during establishment and maintenance of pregnancy. The cellular actions of P are mediated through intracellular progesterone receptor (PR) isoforms, PR-A and PR-B, which are well-known transcription factors. It is postulated that hormone-occupied PR triggers the expression of specific gene networks in different cell types within the uterus and the products of these genes mediate the hormonal effects. The long-term goal of this proposal is to identify and functionally characterize the PR-regulated pathways, which are critical mediators of P response within the uterus during early pregnancy. The specific aims of this study are to: 1. Analyze PR isoform-specific regulation and expression of DNA microarray-derived genes in the preimplantation mouse uterus. Oligonucleotide microarrays were utilized to identify several genes whose expression is markedly down regulated in pregnant uterus at the time of implantation in response to a PR antagonist. The PR isoform-specific gene knock-out (KO) mouse models, PRAKO and PRBKO, will be employed to identify the genes that are potentially important for implantation. The spatio-temporal expression of these genes in the pregnant uterus will be analyzed. 2. Determine the functional roles of microarray-derived genes in the preimplantation uterus. Using a newly developed methodology, antisense oligodeoxynucleotides directed against mRNA transcripts of selected candidate genes will be administered into the preimplantation uterus to block specific gene expression during implantation. The functional effects of this intervention will be determined. In preliminary studies, antisense ODN-induced blockade of the expression of Irgl in the surface epithelium results in a severe impairment of implantation. The molecular target(s) of Irgl in the pregnant uterus will be identified by yeast two-hybrid approach. 3. Investigate the functional role of the PR-regulated protease inhibitor p12 in the decidual uterus, p12 is a serine protease inhibitor induced by P during trophoblast invasion and decidualization. The target protease(s) of p12 in the pregnant uterus will be identified by in vitro protein interaction methods and proteomics. Additionally, a p12 KO mouse will be developed and analyzed for potential reproductive defects. The proposed study will help us to identify molecules that are critical mediators of P regulation of embryo-uterine interactions during early pregnancy.

The overall goal of this proposal is to analyze the functional role of bone morphogenetic protein-2 (BMP2), a pleiotropic signaling molecule, and its downstream pathways in the control of uterine function during implantation. Our recent studies revealed that mice deficient in uterine BMP2 are infertile and exhibit a severe defect in stromal decidualization, a prerequisite for successful pregnancy. We also found that siRNAmediated downregulation of BMP2 expression in primary cultures of stromal cells isolated from pregnant mouse uterus efficiently blocked the differentiation process. To analyze the role of BMP2 during decidualization, we formulated the following specific aims: 1. Analyze the mechanisms by which the BMP2 signaling pathway controls stromal differentiation. Using microarray-based gene expression profiling, we identifed potential downstream targets of BMP2 in the uterus during decidualization. We will evaluate the functional contribution of selected BMP2 target genes in stromal cell differentiation by siRNA-mediated blockade of their expression and elucidate the signaling pathways of BMP2 in primary stromal cultures. 2. Investigate the function of Wnt 4 in murine uterine function. We identified Wnt 4 as a downstream target of BMP2 regulation in stromal cells undergoing decidualization. Attenuation of Wnt 4 expression by siRNAs greatly reduced stromal differentiation in vitro, suggesting that it is a candidate mediator of BMP2-induced decidualization. We will generate a conditional knockout of Wnt 4 to confirm our in vitro findings, and to analyze the functional consequences of this loss-of-function mutation during implantation. 3. Investigate the function of the BMP2 pathway in human decidualization. The expression of BMP2 is markedly induced in human endometrial stromal cells undergoing decidualization in vitro in response to steroid hormones and cAMP. Addition of exogenous BMP2 to these cultures also led to a robust enhancement of Wnt 4 expression and stimulated the differentiation process. We will evaluate the role of Wnt 4 in human stromal cell differentiation and identify additional gene pathways mediating BMP2 function in human stromal cells using microarray analysis. Of particular interest would be the factors regulated by BMP2 in both mouse and human endometrium. Collectively, the proposed experiments will test the hypothesis that BMP2 and its downstream signaling pathways play critical and conserved roles during decidualization and implantation.

DESCRIPTION (provided by applicant): Concerted actions of the ovarian steroid hormones estrogen (E) and progesterone (P), acting via their cognate receptors in uterine epithelial and stromal compartments, determine the maternal competency for embryo implantation. A clear understanding of the molecular pathways via which these hormone receptors regulate uterine functions during the reproductive cycle and pregnancy would require a definition of their cell type-specific roles in the uterus. Until recently, it was thought that estrogen receptor alpha (ER? present in the epithelial cells drives the E-induced proliferation of these cells during the reproductive cycle. Generation of a conditional knockout of ER? in uterine epithelium has revealed the surprising fact that E-induced proliferation of uterine epithelial cells is independen of the epithelial ER?. This finding has led to the hypothesis that E may act via the stromal ER? to control uterine epithelial proliferation by paracrine mechanisms. To test this new paradigm, it is necessary to create a conditional knockout mouse model in which ER? is deleted specifically in the uterine stromal cells. A major objective of this R21 application is to develop the Hand2-Cre transgenic mice in which Cre recombinase expression, under the control of an 11-kb regulatory region of the Hand2 gene, will be induced exclusively in the uterine stromal cells in response to P, thereby ablating the floxed ER? gene in these cells. This mouse model will provide novel insights into the role of ER? in directing stromal-epithelial dialogue during the establishment of pregnancy and would serve as an extremely valuable tool for researchers in the field of uterine biology.

Implantation is initiated when the blastocyst attaches to the uterine luminal epithelium and subsequently penetrates into the underlying stroma to firmly embed into the endometrium. While many aspects of implantation remain unclear, emerging evidence indicates that intercellular communication between endometrial epithelial and stromal cells is vital for successful establishment of pregnancy. The paracrine signals that mediate this crosstalk remain poorly understood. Our recent studies revealed that the transcription factor, hypoxia-­?inducible factor 2 alpha (HIF2?), is induced selectively in endometrial stromal cells subjacent to the luminal epithelium at the time of implantation. This finding is significant because it is known for a long time that the maternal environment during implantation is hypoxic. To address the mechanisms of maternal adaptation to hypoxia during implantation, we generated Hif2?d/d mice that conditionally lack HIF2? in the endometrium. In these mice, the blastocysts are closely apposed to the uterine epithelium but fail to sustain firm attachment to it, resulting in implantation failure. The uterine stromal cells of Hif2?d/d mice exhibited downregulation of metabolic factors, such as lactate and a subset of amino acids, and a distinct set of Rab GTPases that regulate secretion of extracellular vesicles (EVs), including microvesicles and exosomes. The EVs are known to mediate a novel mechanism of cell-cell communication. That hypoxia promotes EV secretion by certain cells support the concept that HIF2?-driven EV secretion by the endometrial stromal cells represents a critical adaptive response that promotes intercellular communication during implantation. In the proposed study, we will employ the Hif2?d/d mouse model to test the hypothesis that HIF2? regulates a novel paracrine signaling mechanism under the hypoxic conditions of early pregnancy by controlling EV trafficking in the maternal tissue and that it directs transfer of key signaling molecules and metabolites from the stromal to luminal epithelial cells to influence epithelial functionality during implantation. We have proposed three specific aims to test this hypothesis. In Aim 1, we will investigate HIF2?-mediated EV trafficking in endometrial stromal cells and identify the resident molecular cargo in EVs. In Aim 2, we will investigate the effects of EVs and metabolic factors secreted from endometrial stromal cells on the functionality of epithelial cells. In Aim 3, we will investigate the role of HIF2? in regulating the secretory function of human endometrial stromal cells. A multi-pronged approach, utilizing a combination of (i) a unique animal model harboring a specific defect in adaptive response (ii) functional analyses using mouse and human primary endometrial cells, and (iii) genomic and proteomic analyses to understand cell-to-cell trafficking mechanisms, will enable us to provide answers to important unresolved questions regarding the impact of hypoxia on stromal-epithelial communication controlling endometrial function during early pregnancy.

Phthalates are ubiquitous synthetic chemicals used as plasticizers and stabilizers in a myriad of consumer products. People are ubiquitously exposed to phthalates on a daily basis, and gender- and sex-specific differences exist in phthalate exposures and toxicity. However, few studies have examined the gender- and sex-specific effects of prenatal exposure to phthalates on the reproductive health of the offspring. The reproductive system is complex and requires a timeline of normal development and function of the reproductive tissues (i.e., brain, pituitary, ovary, testes, and uterus), key reproductive hormones (i.e., estrogens, androgens, progesterone, luteinizing hormone, and follicle-stimulating hormone), and reproductive sex behaviors (i.e., male and female sexual interest, behavioral responsiveness, and partner preference), which may differ by gender and sex. Therefore, the parent studies test the hypothesis that exposure to an environmentally relevant phthalate mixture causes earlier subfertility in female offspring than male offspring through mechanisms that involve gender- and sex-specific effects on the brain, pituitary, gonads, uterus, key hormone pathways, and reproductive behaviors. To test this hypothesis, the parent studies will determine: 1) the effects of prenatal exposure to an environmentally relevant phthalate mixture on the morphology of key reproductive tissues, 2) the effects of prenatal exposure to an environmentally relevant phthalate mixture on hormone production and responsiveness, and 3) the impact of prenatal exposure to an environmentally relevant phthalate mixture on reproductive behaviors and sexual attractiveness. Collectively, the parent studies will determine the mechanisms by which prenatal phthalate exposure causes sex-specific and gender- specific subfertility and reproductive behavior in the offspring. A better understanding of the mechanisms of action of phthalates may lead to the development of novel targets for the treatment of phthalate-induced diseases. It also may lead to the identification of factors that cause sex-specific and gender-specific adverse reproductive outcomes. This could have a major impact on improving the health of women and men, particularly because it currently is not possible to eliminate phthalate exposure.