Role of Msx homeobox genes in uterus during embryo implantation

Implantation of the embryo into the wall of the uterus is a crucial event in mammalian embryogenesis. This complex event involves a series of interactions between the developing embryo and the receptive endometrium ultimately leading to successful establishment of pregnancy. The sequential events of implantation include apposition of the blastocyst to the uterine luminal epithelium followed by adhesion to the epithelium and then penetration through the epithelium and basal lamina into the uterine stroma. Uterine sensitivity with respect to implantation has been classified as prereceptive, receptive, and nonreceptive phases. In the mouse, day 1–3 of pregnancy constitutes the prereceptive phase and day 4 is considered receptive. The window of uterine receptivity is transient and lasts for a limited time. On day 5 of pregnancy, the uterus is nonreceptive or refractory. Studies over the past decade have identified a variety of molecules including growth factors, cytokines, transcription factors, and extracellular matrix proteins as potential regulators of this complex process. This dissertation work investigates the critical role of Msx homeobox genes in the uterus during embryo implantation. The mammalian Msx homeobox genes, Msx1 and Msx2, encode transcription factors that control organogenesis and tissue interactions during embryonic development. Uterine specific deletion of Msx1 and Msx2 resulted in female infertility due to a failure in implantation. Further analysis indicated that mice lacking uterine Msx1 and Msx2 (Msx1d/dMsx2d/d) exhibited a failure in uterine receptivity due to enhanced estrogen signaling in the luminal epithelium, failure of microvilli remodeling, sustained epithelial cell polarity and persistent proliferative activity of luminal and glandular epithelium. More recent studies revealed that canonical Wnt/ β-catenin signaling were upregulated in the Msx1Msx2-null uteri, which in turn stimulated the production of a subset of fibroblast growth factors (FGFs) in the stromal cells. The FGFs subsequently activate FGFR-ERK-MAP kinase signaling pathway in the luminal epithelium resulting in sustained epithelial cellular proliferation. These results uncovered a unique signaling network, involving Msx1/2, Wnts, and FGFs, which regulate stromal-epithelial cross talk in the mouse uterus at the time of receptivity. The last chapter addresses the role of Msx homeobox genes during uterine stromal cell decidualization. As the embryo attaches to the uterine wall and invades into the stromal bed, the stromal cells surrounding the implanting blastocyst differentiate into decidual cells in a process known as decidualization. This process is critical for embryo survival, angiogenesis and successful establishment of pregnancy. We previously reported that Bone morphogenetic protein 2 (BMP2) regulates uterine stromal cell differentiation in the mouse and the human. Subsequent studies revealed that the expressions of Msx1 and Msx2 were markedly altered in response to exogenous BMP2. Functional studies performed using Msx1d/dMsx2d/d mice revealed that mouse uteri lacking Msx1 and Msx2 fail to elicit a decidual response, indicating a critical role of these homeobox genes in stromal cell differentiation. Further studies revealed that the addition of BMP2 stimulated MSX1 and MSX2 expression in human endometrial stromal cell cultures and enhanced the differentiation process. Silencing of MSX1 or MSX2 expression by siRNAs severely impaired human stromal differentiation indicating that MSX1 and MSX2 are key regulators of BMP2-mediated decidualization in the mouse and the human

Renormalized and Entropy Solutions of Tumor Growth Model with Nonlinear Acid Production

This paper establishes the existence of renormalized and entropy solutions for a system of nonlinear reaction-diffusion equations which describes the tumor growth along with acidification and interaction. Under the assumptions of L1 data and no growth conditions with zero Dirichlet boundary conditions, we prove the existence of renormalized and entropy solutions for the considered mathematical model

Msx homeobox genes critically regulate embryo implantation by controlling paracrine signaling between uterine stroma and epithelium.

The mammalian Msx homeobox genes, Msx1 and Msx2, encode transcription factors that control organogenesis and tissue interactions during embryonic development. We observed overlapping expression of these factors in uterine epithelial and stromal compartments of pregnant mice prior to embryo implantation. Conditional ablation of both Msx1 and Msx2 in the uterus resulted in female infertility due to a failure in implantation. In these mutant mice (Msx1/2(d/d)), the uterine epithelium exhibited persistent proliferative activity and failed to attach to the embryos. Gene expression profiling of uterine epithelium and stroma of Msx1/2(d/d) mice revealed an elevated expression of several members of the Wnt gene family in the preimplantation uterus. Increased canonical Wnt signaling in the stromal cells activated β-catenin, stimulating the production of a subset of fibroblast growth factors (FGFs) in these cells. The secreted FGFs acted in a paracrine manner via the FGF receptors in the epithelium to promote epithelial proliferation, thereby preventing differentiation of this tissue and creating a non-receptive uterus refractory to implantation. Collectively, these findings delineate a unique signaling network, involving Msx1/2, Wnts, and FGFs, which operate in the uterus at the time of implantation to control the mesenchymal-epithelial dialogue critical for successful establishment of pregnancy

The mechanical response of the mouse cervix to tensile cyclic loading in term and preterm pregnancy: Data

These files contain mechanical data from a study evaluating the cyclic tensile loading properties of mouse cervices in normal gestation and 2 models of preterm birth. The exp_data folder contains a .xlsx files corresponding to each tested sample. Each file includes the experimental cyclic tensile data. The name of the file indicates the sample ID, which includes the the gestation day (NP = nonpregnant, d6 = gestation day 6, d12 = gestation day 12, d15 = gestation day 15, d18 = gestation day 18, d15LPS = infection PTB model on day 15, d15LPSsham = sham procedure of the infection model on day 15, d15RU486 = hormone withdrawal PTB model on day 15) and the sample number identifier. For example, “NP_1_raw_data” corresponds to the experimental data for nonpregnant sample #1 and “d6_3_raw_data” corresponds to the experimental data for gestation day 6 sample #3. In these files, the columns correspond to: the time [s], force [N], averaged cervical opening [mm], standard deviation of the cervical opening [mm], average width [mm], standard deviation of the width [mm], average length [mm], standard deviation of the length [mm], average height [mm], standard deviation of the height [mm], of the cervical sample. The cervical opening, average and standard deviation of the width, length, height, are determined via camera images. The time and force measurements are data from the universal testing machine

Ulipristal blocks ovulation by inhibiting progesterone receptor-dependent pathways intrinsic to the ovary

Ulipristal acetate (UPA), a progesterone receptor (PR) modulator, is used as an emergency contraceptive in women. Here, using a mouse model, we investigated the mechanism of action of UPA as an ovulation blocker. In mice, ovulation is induced ~12 hours following the treatment with exogenous gonadotropins, including human chorionic gonadotropin (hCG), which mimics the action of luteinizing hormone (LH). When administered within 6 hours of hCG treatment, UPA is a potent blocker of ovulation. However, UPA’s effectiveness declined significantly when it was given at 8 hours post hCG. Our study revealed that, when administered within 6 hours of hCG, UPA blocks ovulation by inhibiting PR-dependent pathways intrinsic to the ovary. At 8 hours post hCG, when the PR signaling has already occurred, UPA is unable to block ovulation efficiently. Collectively, these results indicated that UPA, when administered within a critical time window following the LH surge, blocks PR-dependent pathways in the ovary to function as an effective antiovulatory contraceptive

Ablation of uterine <i>Msx</i>1 and <i>Msx</i>2 leads to female infertility.

<p>The results of a six-month breeding study are shown.</p

Enhanced proliferation in the uterine epithelium and lack of receptivity in <i>Msx1^d/dMsx2^d/d</i> mice.

<p>A. Immunohsitochemical localization of Ki67 in the uterine sections of <i>Msx</i>1<i>^f/fMsx</i>2<i>^f/f</i> (left panel, a and c) and <i>Msx</i>1<i>^d/dMsx</i>2<i>^d/d</i> (right panel, b and d) mice on day 4 of pregnancy. Panels a and b indicate lower magnification (20×) and c and d indicate higher magnification (40×). L and G indicate luminal epithelium and glandular epithelium respectively. B. Real-time PCR was performed to analyze the expression of glandular factors, <i>Lif</i>, <i>Foxa2</i> and <i>Spink3</i> in uteri of <i>Msx</i>1<i>^f/fMsx</i>2<i>^f/f</i> and <i>Msx</i>1<i>^d/dMsx</i>2<i>^d/d</i> mice on day 4 of pregnancy. The level of <i>Ck18</i> was used as internal control to normalize gene expression. The data are represented as the mean fold induction ± SEM, ***p<0.0001. C. Transmission electron microscopy of uterine sections obtained from <i>Msx1^f/f Msx2^f/f</i> (left panel, a and b) and <i>Msx1^d/dMsx2^d/d</i> (right panel, c and d) mice on day 4 of pregnancy. Panels a and c indicate lower magnification (5Kx) and b and d indicate higher magnification (30Kx). D. Immunohistochemical analysis of Muc-1 expression in the uterine sections of <i>Msx</i>1<i>^f/fMsx</i>2<i>^f/f</i> (upper panel) and <i>Msx</i>1<i>^d/dMsx</i>2<i>^d/d</i> (lower panel) mice on day 1 (a and d), day 4 (b and e) and day 5 (c and f) of pregnancy. L indicates luminal epithelium.</p

Enhanced FGFR signaling in the epithelium of <i>Msx1^d/dMsx2^d/d</i> uteri.

<p>A. The level of p-FRS2 was examined in the uterine sections of <i>Msx1^f/fMsx2^f/f</i> (upper panel) and <i>Msx1^d/dMsx2^d/d</i> (lower panel) mice on day 4 of pregnancy by immunohistochemistry. Magnification: a and d: 10×, b and e: 20×, c and f: 40×. B. The level of p-ERK was examined in the uterine sections of <i>Msx1^f/fMsx2^f/f</i> (upper panel) and <i>Msx1^d/dMsx2^d/d</i> (lower panel) mice on day 4 of pregnancy by immunohistochemistry. Magnification: a and d: 10×, b and e: 20×, c and f: 40×. L, G and S indicate luminal epithelium, glandular epithelium, and stroma respectively. C. FGFR-specific inhibitor PD173074 was applied to one uterine horn of <i>Msx1^d/dMsx2^d/d</i> (n = 3) mice on day 3 of pregnancy. The other horn served as vehicle-treated control. Uterine horns were collected on day 4 morning and sections were subjected to immunohistochemistry to detect p-FRS2, Ki67, and Muc-1.</p

Lack of uterine <i>Msx</i>1 and <i>Msx</i>2 causes implantation failure.

<p>A. Embryo implantation sites were examined in <i>Msx1^f/fMsx2^f/f</i> and <i>Msx1^d/dMsx2^d/d</i> mice by the vascular permeability assay, which can be scored as distinct blue bands (red arrows) following an injection of Chicago blue dye on day 5 of pregnancy (D5, n = 6) or direct eye-visualization of implanted embryo on day 6 (D6, n = 4) and on day 7 (D7, n = 4) of pregnancy. The graph represents the quantification of implantation sites in <i>Msx1^f/fMsx2^f/f</i> and <i>Msx1^d/dMsx2^d/d</i> mice on day 5 of pregnancy. B. Failure of embryo attachment in <i>Msx1^d/dMsx2^d/d</i> uteri. Histological analysis of uterine sections obtained from <i>Msx1^f/fMsx2^f/f</i> (a) and <i>Msx1^d/dMsx2^d/d</i> (b) mice on day 5 (n = 3) of pregnancy by Hematoxylin and Eosin staining. Note the intimate contact between embryo and luminal epithelium in <i>Msx</i>1<i>^f/fMsx</i>2<i>^f/f</i> mice and the free floating embryo in the uterine lumen of <i>Msx</i>1<i>^d/dMsx</i>2<i>^d/d</i> mice. L and E indicate luminal epithelium and embryo respectively.</p