Fibroblast growth factor receptor two (FGFR2) regulates uterine epithelial integrity and fertility in mice.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate luminal epithelial (LE) cell proliferation in the adult mouse uterus. This study tested the hypothesis that FGFR2 has a biological role in postnatal development and function of the uterus by conditionally deleting Fgfr2 after birth using progesterone receptor (Pgr)-Cre mice. Adult Fgfr2 mutant female mice were initially subfertile and became infertile with increasing parity. No defects in uterine gland development were observed in conditional Fgfr2 mutant mice. In the adult, Fgfr2 mutant mice possessed a histologically normal reproductive tract with the exception of the uterus. The LE of the Fgfr2 mutant uterus was stratified, but no obvious histological differences were observed in the glandular epithelium, stroma, or myometrium. Within the stratified LE, cuboidal basal cells were present and positive for basal cell markers (KRT14 and TRP63). Nulliparous bred Fgfr2 mutants contained normal numbers of blastocysts on Day 3.5 postmating, but the number of embryo implantation sites was substantially reduced on Day 5.5 postmating. These results support the idea that loss of FGFR2 in the uterus after birth alters its development, resulting in LE stratification and peri-implantation pregnancy loss.

WNT4 acts downstream of BMP2 and functions via ß-catenin signaling pathway to regulate human endometrial stromal cell differentiation.

Differentiation of endometrial stromal cells into decidual cells is a prerequisite for successful embryo implantation. Our previous studies in the mouse have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFß superfamily, is essential for this differentiation process. BMP2 is markedly induced in human primary endometrial stromal cells (HESCs) as they undergo differentiation in response to steroid hormones and cAMP. The present study was undertaken to identify the BMP2-mediated molecular pathways in primary cultures of HESCs during decidualization. Using gene expression profiling, we identified wingless-related murine mammary tumor virus integration site 4 (WNT4) as a target of BMP2 regulation during decidualization. Attenuation of WNT4 expression in HESCs by small interfering RNA administration greatly reduced BMP2-induced stromal differentiation. Additionally, adenovirus-mediated overexpression of WNT4 in HESCs markedly advanced the differentiation program, indicating that it is a key regulator of decidualization. The stimulatory effect of WNT4 was accompanied by the accumulation of active ß-catenin in the nuclei of decidualizing stromal cells, indicating the involvement of the canonical WNT signaling pathway. Functional inhibition of WNT4/ß-catenin pathway by Dickkopf-1, an inhibitor of the canonical WNT signaling, or small interfering RNA-mediated silencing of ß-catenin expression, greatly reduced the BMP2- and WNT4-induced decidualization. Gene expression profiling revealed that Forkhead box protein O1, a forkhead family transcription factor and previously reported regulator of HESC differentiation, is a common downstream mediator of both BMP2 and WNT4 signaling. Taken together, these studies uncovered a linear pathway involving BMP2, WNT4/ß-catenin, and Forkhead box protein O1 that operates in human endometrium to critically control decidualization.

Postnatal deletion of Wnt7a inhibits uterine gland morphogenesis and compromises adult fertility in mice.

The success of postnatal uterine morphogenesis dictates, in part, the embryotrophic potential and functional capacity of the adult uterus. The definitive role of Wnt7a in postnatal uterine development and adult function requires a conditional knockout, because global deletion disrupts müllerian duct patterning, specification, and cell fate in the fetus. The Wnt7a-null uterus appears to be posteriorized because of developmental defects in the embryo, as evidenced by the stratified luminal epithelium that is normally found in the vagina and the presence of short and uncoiled oviducts. To understand the biological role of WNT7A after birth and allow tissue-selective deletion of Wnt7a, we generated loxP-flanked exon 2 mice and conditionally deleted Wnt7a after birth in the uterus by crossing them with Pgr(Cre) mice. Morphological examination revealed no obvious differences in the vagina, cervix, oviduct, or ovary. The uteri of Wnt7a mutant mice contained no endometrial glands, whereas all other uterine cell types appeared to be normal. Postnatal differentiation of endometrial glands was observed in control mice, but not in mutant mice, between Postnatal Days 3 and 12. Expression of morphoregulatory genes, particularly Foxa2, Hoxa10, Hoxa11, Msx1, and Wnt16, was disrupted in the Wnt7a mutant uteri. Conditional Wnt7a mutant mice were not fertile. Although embryos were present in uteri of mutant mice on Day 3.5 of pregnancy, blastocyst implantation was not observed on Day 5.5. Furthermore, expression of several genes (Foxa2, Lif, Msx1, and Wnt16) was reduced or absent in adult Wnt7a-deleted uteri on Day 3.5 postmating. These results indicate that WNT7A plays a critical role in postnatal uterine gland morphogenesis and function, which are important for blastocyst implantation and fertility in the adult uterus.

Progesterone resistance in PCOS endometrium: a microarray analysis in clomiphene citrate-treated and artificial menstrual cycles.

CONTEXT: Polycystic ovary syndrome (PCOS), the most common endocrinopathy of reproductive-aged women, is characterized by ovulatory dysfunction and hyperandrogenism. OBJECTIVE: The aim was to compare gene expression between endometrial samples of normal fertile controls and women with PCOS. DESIGN AND SETTING: We conducted a case control study at university teaching hospitals. PATIENTS: Normal fertile controls and women with PCOS participated in the study. INTERVENTIONS: Endometrial samples were obtained from normal fertile controls and from women with PCOS, either induced to ovulate with clomiphene citrate or from a modeled secretory phase using daily administration of progesterone. MAIN OUTCOME MEASURE: Total RNA was isolated from samples and processed for array hybridization with Affymetrix HG U133 Plus 2 arrays. Data were analyzed using GeneSpring GX11 and Ingenuity Pathways Analysis. Selected gene expression differences were validated using RT-PCR and/or immunohistochemistry in separately obtained PCOS and normal endometrium. RESULTS: ANOVA analysis revealed 5160 significantly different genes among the three conditions. Of these, 466 were differentially regulated between fertile controls and PCOS. Progesterone-regulated genes, including mitogen-inducible gene 6 (MIG6), leukemia inhibitory factor (LIF), GRB2-associated binding protein 1 (GAB1), S100P, and claudin-4 were significantly lower in PCOS endometrium; whereas cell proliferation genes, such as Anillin and cyclin B1, were up-regulated. CONCLUSIONS: Differences in gene expression provide evidence of progesterone resistance in midsecretory PCOS endometrium, independent of clomiphene citrate and corresponding to the observed phenotypes of hyperplasia, cancer, and poor reproductive outcomes in this group of women.

Complex cardiac Nkx2-5 gene expression activated by noggin-sensitive enhancers followed by chamber-specific modules.

We previously reported that an Nkx2-5-GFP bacterial artificial chromosome in transgenic mice recapitulated the endogenous gene activity in the heart. Here, we identified three additional previously uncharacterized distal enhancer modules of Nkx2-5: UH6, which directed transgene expression in the right ventricle, interventricular septum, and atrial ventricular canal; UH5, which directed expression in both atria; and UH4, which directed transgene expression in tongue muscle. Nkx2-5 enhancers drive cardiogenic gene activity from the earliest progenitors to the late-stage embryonic heart, reside within its 27 kb of 5' flanking sequences, organized in a tandem array. Nkx2-5 enhancers involved with stomach-, tongue-, and chamber-restricted expression displayed lacZ transgene activity and chromatin histone acetylation patterns consistent with tissue-specific expression. An examination of Nkx2-5 gene activity in murine embryonic stem cells converted to beating embryoid bodies showed that only the proximal active region 2 and GATA-Smad enhancers were chromatin-remodeled. Chromatin remodeling of active region 2 and GATA-Smad enhancers were blunted by noggin coexpression, which indicated dependence on bone morphogenetic protein signaling for their chromatin activation during activation of Nkx2-5 expression.

Mouse lung neuroendocrine carcinomas: distinct morphologies, same transcription factors.

Constitutive expression of human achaete-scute homolog-1 (hASH-1) in combination with simian virus large Tantigen under the Clara cell 10-kDa secretory protein (CC10) promoter results in adenocarcinomas with focal neuroendocrine (NE) differentiation. Mice carrying conditional alleles for both Rb-1 and p53 in lung epithelial cells develop aggressive lung tumors with similarities to human small cell lung cancers, including high level expression of ASH-1, NE markers, and extra-pulmonary metastases. Tumors in both models originate from bronchiolar epithelium, reveal a range of premalignant changes, express thyroid transcription factor-1 (TTF-1), a marker of peripheral airway cell lineage, and display varying degrees of bidirectional epithelial/NE differentiation.