Over the last year we have made significant progress investigating the molecular mechanisms regulating the ability of the uterus to support pregnancy as maintain pregnancy. First we investigated the role of SOX17, a member of the SOX (SRY-related HMG-box) transcription factor family, regulates embryonic development as well as cell fate in various tissues. Recent studies have identified SOX17 as a novel gene targeted by progesterone receptor (PgR), and demonstrated that haploinsufficiency and ablation of SOX17 affects female fertility. However, the underlying mechanism remains unknown. Over the last year we demonstrated that that loss of SOX17 in the uterine epithelia is critical for female fertility via the Indian hedgehog (Ihh) pathway-mediated epithelial transition between periods of proliferation and quiescence during the window of implantation. SOX17 was expressed in uterine blood vessels, luminal (LE) and glandular epithelia (GE). We initially ablated SOX17 in the whole uterus using PgRCre mouse model, which resulted in epithelial stratification, and therefore, subfertility. However, pre-pubertal inhibition of gland development occurred in this model. To exclude any off-target effects caused by such developmental defect, we ablated SOX17 in post-pubertal uterine epithelia using LtfiCre mouse model. The delay of Sox17 ablated showed that gland development was normal, but uterine epithelia underwent hyperproliferation, and again, resulted in subfertility. Based on analyses of microarray and ChIP-seq in female mice at Day 3.5 of pregnancy, we have validated that SOX17 maintains PgR but inhibits ESR signaling pathways, promotes Ihh-COUPTFII-HAND2-FGF signal transduction between LE and stroma, and therefore, inhibits hyperproliferation in uterine epithelia. Interestingly, SOX17 disruption eliminated FOXA2 in structurally normal uterine glands, whereas loss of SOX17 down-regulated ARID1A (modulating epithelial proliferation and lower in the eutopic endometrium of women with endometriosis) and not vice versa, indicating the hierarchy of SOX17 in uterine function. Furthermore, in vivo deletion of Ihh upstream SOX17-binding sites resulted in its loss of response to progesterone stimulation. Our results suggest that SOX17 has a critical role in maintaining epithelial quiescence from hyperproliferation during the window of implantation via Ihh pathway-mediated reduction of FGF paracrine signaling. This analysis shows that Sox17 may be a critical regulator of the response of the uterus to hormones and may protect the uterus from environmental endocrine disruptors. Next we investigated the role of progesterone in regulating human myometrial function. The myometrium maintains the uterine integrity and provides contractile force in response to hormone regulation. During pregnancy, myometrium undergoes extensive remodeling via smooth muscle proliferation and hypertrophy to accommodate structural alterations of the uterus. Functionally, contraction of myometrium is under the control of progesterone signaling mediated by the progesterone receptor (PGR). To explore molecular mechanisms that modulate structural and functional changes of myometrium, we examine transcriptome of human myometrial tissues by RNAseq. Results from comparing between three non-pregnant and three full-term pregnancy samples reveal differential expression in 2862 genes, of which 1004 genes have higher mRNA levels in the non-pregnant group and 1858 genes exhibit increased expression in the full-term pregnancy samples. Gene ontology analyses demonstrate the differences in characteristics of these two groups showing that the non-pregnant group has a molecular profile enriched of transcription regulation and histone modification while full-term pregnancy samples express genes associated with immune responses and carbohydrate metabolism. Using all 2862 genes as reporters, Ingenuity Pathway analysis predicts classic smooth muscle regulators serum response factor(SRF), transforming growth factor , platelet derived growth factor and PGR as potential upstream regulators that mediate the change of myometrial molecular profiles. Importantly, the PGR cistrome in non-pregnant myometrial samples also exhibits enriched SRF-binding motif in the PGR occupying sites, suggesting a potential interaction between PGR and SRF in regulation of this hormone-responsive smooth muscle tissue. In addition, PGR occupancy is found in the SRF locus of both human and mouse myometrial tissues, which provides implications on a conserved PGR-SRF regulatory axis for myometrial remodeling. In summary, our findings identified association between SRF and PGR, two master regulators for smooth muscle homeostasis and hormonal response, respectively. Understanding how Progesterone regulates the maintainance of pregnancy may allow us to determine how environmental factors cause complications in pregnancy such as preterm birth. We then investigated using ultrasonography as a tool to non-invasively monitor in real time the development of the human fetus in utero. Although genetically engineered mice have served as valuable in vivo models to study both embryo implantation and pregnancy progression, such studies usually require sacrifice of parous mice for subsequent phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction in silico of high-frequency ultrasound (HFUS) imaging data for early detection and characterization of murine embryo implantation sites and their development in utero. With HFUS imaging followed by 3-D reconstruction, we were able to precisely quantify embryo implantation site number and embryonic developmental progression in pregnant C57BL6J/129S mice from as early as 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. using a VisualSonics Vevo 2100 (MS550S) transducer. In addition to measurements of implantation site number, location, volume and spacing, embryo viability via cardiac activity monitoring was also achieved. A total of 12 dams were imaged with HFUS with approximately 100 embryos examined per embryonic day. For the post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in mesh or solid overlay format enabled visual representation in silico of implantation site location, number, spacing distances, and site volume within each uterine horn. Therefore, this short technical report describes the feasibility of using 3-D HFUS imaging for early detection and analysis of post-implantation events in the pregnant mouse with the ability to longitudinally monitor the development of these early pregnancy events in a non-invasive manner. As genetically engineered mice continue to be used to characterize female reproductive phenotypes, we believe this reliable and non-invasive method to detect, quantify, and characterize early implantation events will prove to be an invaluable investigative tool for the study of female infertility and subfertility phenotypes based on a defective uterus. This technique will also help reduce animal numbers required to investigate the impact of factor on pregnancy. This work was published in PLoS One
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