Environmental Signaling in Reproduction and Pregnancy

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

The environment impacts the ability of women to give birth to healthy children. Theo 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 into the role of progesterone receptor isoforms in regulating the function of the uterine musculature, myometrium, during pregnancy. 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. We have also initiated investigations into how modifiers of Progesterone Receptor action and the role of Vitamin D impact reproduction. Preterm birth is a serious health issue that impacts 10% of pregnancies in the United States. One of the critical regions regulating the duration of pregnancy is the musculature surrounding the uterus, the myometrium. The myometrium must relax during growth of the fetus and then at the timing of birth contract to expel the fetus. Problems with this contraction process will leads to pregnancy complications such as preterm birth and delayed birth. One of the critical regulators of myometrial function is the hormone progesterone. Progesterone regulates the ability of the myometrium to grow and expand during development of the fetus. At birth it is thought that changes in the uterine sensitivity or progesterone is one of the triggers for pregnancy. Progesterone acts through its receptor the progesterone receptor, PGR. The PGR is a transcription factor that binds the hormone progesterone and regulates the expression of genes in responsive tissues that contain this molecule. The PGR exists as two isoforms, a longer B isoform, PGR-B, and a shorter, A isoform, isoform PGR-A. The B isoform contains signals that enhance its ability to regulate gene expression however little is known about the distinct function of these two isoforms. In humans, it is hypothesized that PGR-B promotes a relaxed state of the myometrium, and PGR-A facilitates uterine contraction. In order to test this hypothesis in a living pregnancy we generated two lines of genetically engineered mice that expressed the PGR-A and PGR-B isoform in the myometrial smooth muscle cells. These studies shed light on the distinct role of these isoforms in birth. Mice expressing the PGR-B transgene showed a marked increase in gestational length compared to control mice. Measuring muscle contraction in vivo using telemetry and ex vivo using muscle strips we showed that PGR-B overexpression leads to a significant decrease in uterine contractility, and a high incidence of labor dystocia. Conversely, PGR-A overexpression leads to an increase in uterine contractility without a change in gestational length. To determine how PGR-B and PGR-A regulated uterine muscle contraction we determined the impact of these isoforms on gene expression using RNA sequencing at midpregnancy. We identified 1,174 isoform-specific downstream targets and 424 genes that are commonly regulated by both PGR isoforms. Gene signature analyses further reveal that PGR-B regulated genes involved in muscle relaxation and PGR-A regulated genes involved in proinflammatory. Specifically elevated PGR-B abundance reduces the sensitivity of the myometrium to the hormone oxytocin by reducing the expression of its receptor. Oxytocin is a hormone that stimulates muscle contraction at birth. We also identified changes in the expression of two molecules that mediate oxytocin signaling Trpc3 and increases Plcl2 expression demonstrating compromised oxytocin signaling. We showed in vitro that both endogenous PLCL2 and its paralog PLCL1 can inhibit uterine muscle cell contraction in a CRISPRa-based assay system. These findings provide in vivo support that PGR isoform levels determine distinct transcriptomic landscapes and pathways in myometrial function and labor, which may help further the understanding of abnormal uterine function in the clinical setting. Progesterone is the only approved molecule for the treatment of preterm birth. However the effectiveness of this therapy is variable. Our research demonstrated that this molecule action depends on the ratio of the PGR isoforms. PGR-A activation stimulates inflammation which intern can trigger parturition while PGR-B causes muscle relaxation and can prevent preterm birth. Moving forward, understanding the expression of the PGR isoforms in patients undergoing preterm birth and the development of isoform specific therapies may be the most effective way to treat preterm birth. This work was published in the Proceedings of the National Academy of Sciences USA. Our Investigations into the role of modifiers of PGR action have identified two transcriptional regulatory proteins TRIM28 and SRF1 as critical regulators of PGR action. Using genetically modified mice and primary human endometrial cells we have shown that both of these proteins are critical regulators of female reproduction. Female mice with TRIM28 deletion in the reproductive tract are sterile. The cause of this sterility appears to be the inability of the uterus to response to steroid hormones. Impairment of TRIM28 expression in human endometrial stroma cells in vitro impairs the ability of these cells to differentiate in response to hormone signaling. Female mice with ablation of the transcription factor SRF in the reproductive tract are sterile. This sterility are due to multiple defects in the ovary and uterus. Alteration of SRF expression in human endometrial stroma cells show defects in many regulatory pathways. Over the next year we will investigate the molecular mechanism by which these molecules regulate female reproduction. In order to investigate the impact of Vitamin D on female reproduction we have initiated a study to examine the reproductive capacity of mice with dietary Vitamin D deficiency. Preliminary data shows that these mice have a defect in hormonal responsiveness in the uterus. Human endometrial stromal cells also show that Vitamin D and its receptor the Vitamin D receptor are critical regulators of their ability to differentiate in vitro in response to hormones. We are in the process of confirming these results and investigating the role of Vitamin D receptor in mice by ablating the gene for this molecule in the Reproductive tract. In summary, over the last year we have made strides in determining the role of the PGR isoforms in the regulation of the ability of the uterus to support pregnancy. We have identified two new molecules that are critical regulators of uterine function and we have initiated studies into the role of Vitamin D in female reproduction.