Hugh S. Taylor

HOXA10 is expressed in response to sex steroids at the time of implantation in the human endometrium.

Hox genes are well-known transcriptional regulators that play an essential role in directing embryonic development. Mice that are homozygous for a targeted disruption of the Hoxa10 gene exhibit uterine factor infertility. We have recently demonstrated that HOXA10 is expressed in the adult human uterus. To examine expression of HOXA10 during the menstrual cycle, Northern blot analysis and in situ hybridization were performed. Expression of HOXA10 dramatically increased during the midsecretory phase of the menstrual cycle, corresponding to the time of implantation and increase in circulating progesterone. Expression of HOXA10 in cultured endometrial cells was stimulated by estrogen or progesterone. Stimulation of HOXA10 by progesterone was concentration-dependent within the physiologic range, and the effect of estrogen was inhibited by cycloheximide. These results identify sex steroids as novel regulators of HOX gene expression. HOXA10 may have an important function in regulating endometrial development during the menstrual cycle and in establishing conditions necessary for implantation in the human.

In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing müllerian system

Diethylstilbestrol (DES) was widely used to treat pregnant women through 1971. The reproductive tracts of their female offspring exposed to DES in utero are characterized by anatomic abnormalities. Here we show that DES administered to mice in utero produces changes in the expression pattern of several Hox genes that are involved in patterning of the reproductive tract. DES produces posterior shifts in Hox gene expression and ho‐meotic anterior transformations of the reproductive tract. In human uterine or cervical cell cultures, DES induces HOXA9 or HOXA10 gene expression, respectively, to levels approximately twofold that induced by estradiol. The DES‐induced expression is not inhibited by cyclohexamide. Estrogens are novel morphogens that directly regulate the expression pattern of posterior Hox genes in a manner analogous to retinoic acid regulation of anterior Hox genes. Alterations in HOX gene expression are a molecular mechanism by which DES affects reproductive tract development. Changes in Hox gene expression are a potential marker for the effects of in utero drug use that may become apparent only at late stages of development.—Block, K., Kardana, A., Igarashi, P., Taylor, H. S. In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing mullerian system. FASEB J. 14, 1101–1108 (2000)

Bisphenol-A exposure in utero leads to epigenetic alterations in the developmental programming of uterine estrogen response

Bisphenol‐A (BPA) is a nonsteroidal estrogen that is ubiquitous in the environment. The homeobox gene Hoxa10 controls uterine organogenesis, and its expression is affected by in utero BPA exposure. We hypothesized that an epigenetic mechanism underlies BPA‐mediated alterations in Hoxa10 expression. We analyzed the expression pattern and methylation profile of Hoxa10 after in utero BPA exposure. Pregnant CD‐1 mice were treated with BPA (5 mg/kg IP) or vehicle control on d 9–16 of pregnancy. Hoxa10 mRNA and protein expression were increased by 25% in the reproductive tract of mice exposed in utero. Bisulfite sequencing revealed that cytosine‐guanine dinucleotide methylation was decreased from 67 to 14% in the promoter and from 71 to 3% in the intron of Hoxa10 after in utero BPA exposure. Decreased DNA methylation led to an increase in binding of ER‐α to the Hoxa10 ERE both in vitro as and in vivo as determined by EMSA and chromatin immunoprecipitation, respectively. Diminished methylation of the ERE‐containing promoter sequence resulted in an increase in ERE‐driven gene expression in reporter assays. We identify altered methylation as a novel mechanism of BPA‐induced altered developmental programming. Permanent epigenetic alteration of ERE sensitivity to estrogen may be a general mechanism through which endocrine disruptors exert their action.—Bromer, J. G., Zhou, Y., Taylor, M. B., Doherty, L., Taylor, H. S.. Bisphenol‐A exposure in utero leads to epigenetic alterations in the developmental programming of uterine estrogen response. FASEBJ. 24, 2273–2280 (2010). www.fasebj.org

Implantation failure: molecular mechanisms and clinical treatment

BACKGROUND Implantation is a complex initial step in the establishment of a successful pregnancy. Although embryo quality is an important determinant of implantation, temporally coordinated differentiation of endometrial cells to attain uterine receptivity and a synchronized dialog between maternal and embryonic tissues are crucial. The exact mechanism of implantation failure is still poorly understood. METHODS This review summarizes the current knowledge about the proposed mechanisms of implantation failure in gynecological diseases, the evaluation of endometrial receptivity and the treatment methods to improve implantation. RESULTS The absence or suppression of molecules essential for endometrial receptivity results in decreased implantation rates in animal models and gynecological diseases, including endometriosis, hydrosalpinx, leiomyoma and polycystic ovarian syndrome. The mechanisms are diverse and include abnormal cytokine and hormonal signaling as well as epigenetic alterations. CONCLUSIONS Optimizing endometrial receptivity in fertility treatment will improve success rates. Evaluation of implantation markers may help to predict pregnancy outcome and detect occult implantation deficiency. Treating the underlying gynecological disease with medical or surgical interventions is the optimal current therapy. Manipulating the expression of key endometrial genes with gene or stem cell-based therapies may some day be used to further improve implantation rates.

Experimental Murine Endometriosis Induces DNA Methylation and Altered Gene Expression in Eutopic Endometrium

Abstract The eutopic endometrium in women with endometriosis demonstrates diminished endometrial receptivity and altered gene expression. It is unknown if the endometrium being defective gives rise to a predisposition toward endometriosis and infertility or, alternatively, if endometriosis causes the altered endometrial receptivity. Here we created experimental endometriosis in mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. Methylation of Hoxa10 was also evaluated as a potential mechanism responsible for altered gene expression. Expression of each gene was measured using quantitative real-time RT-PCR at 14 wk after induction of endometriosis. Expression of Hoxa10 and Hoxa11, which are necessary for endometrial receptivity, were decreased in the endometriosis group. Insulin-like growth factor binding protein-1 (Igfbp1) mRNA was decreased in the endometriosis group. However, there was no change in Integrin beta3 (Itgb3) mRNA expression. Total progesterone receptor (Pgr-AB) was increased in the endometriosis group and the ratio of Pgr-B to Pgr-AB was increased, indicating a shift from Pgr-A to Pgr-B expression. Basic transcription element-binding protein-1 (Bteb1), official symbol and name Klf9, Kruppel-like factor 9, which functionally interacts with Pgr in endometrium, was also decreased in the endometriosis group. In addition, hypermethylation of Hoxa10 in the endometriosis group was shown by methylation-specific PCR and confirmed by bisulfite sequencing. These findings demonstrate that normal endometrium, when placed in an ectopic location to create experimental endometriosis, led to characteristic changes in gene expression in eutopic endometrium. These data suggest the existence of a signal conduction pathway from endometriosis that alters endometrial gene expression through altered Pgr signaling and epigenetic programming.

Endometrial stem cell transplantation restores dopamine production in a Parkinson’s disease model

Parkinson’s disease (PD) is a neurodegenerative disorder caused by the loss of dopaminergic neurons. Adult human endometrial derived stem cells (HEDSC), a readily obtainable type of mesenchymal stem‐like cell, were used to generate dopaminergic cells and for transplantation. Cells expressing CD90, platelet derived growth factor (PDGF)‐Rβ and CD146 but not CD45 or CD31 were differentiated in vitro into dopaminergic neurons that exhibited axon projections, pyramidal cell bodies and dendritic projections that recapitulate synapse formation; these cells also expressed the neural marker nestin and tyrosine hydroxylase, the rate‐limiting enzyme in dopamine synthesis. Whole cell patch clamp recording identified G‐protein coupled inwardly rectifying potassium current 2 channels characteristic of central neurons. A 1‐methyl 4‐phenyl 1,2,3,6‐tetrahydro pyridine induced animal model of PD was used to demonstrate the ability of labelled HEDSC to engraft, migrate to the site of lesion, differentiate in vivo and significantly increase striatal dopamine and dopamine metabolite concentrations. HEDSC are a highly inducible source of allogenic stem cells that rescue dopamine concentrations in an immunocompetent PD mouse model.

Transcriptional Repression of Peri-Implantation EMX2 Expression in Mammalian Reproduction by HOXA10

ABSTRACT HOXA10 is necessary for mammalian reproduction; however, its transcriptional targets are not completely defined. EMX2, a divergent homeobox gene, is necessary for urogenital tract development. In these studies we identify and characterize the regulation of EMX2 by HOXA10. By using Northern analysis and in situ hybridization, we found that EMX2 is expressed in the adult urogenital tract in an inverse temporal pattern from HOXA10, suggestive of a negative regulatory relationship. Constitutive expression of HOXA10 diminished EMX2 mRNA, whereas blocking HOXA10 through the use of antisense resulted in high EMX2 mRNA expression. Deletional analysis of the EMX2 5′ regulatory region revealed that a 150-bp element mediated transcriptional repression when cotransfected with pcDNA3.1/HOXA10 in transient-transfection assays. Binding of HOXA10 protein to this element was demonstrated by electrophoretic mobility shift assay and further localized to a consensus HOXA10 binding site within this element by DNase I footprinting. Site-directed mutagenesis abolished binding, as well as the negative transcriptional regulation. Transcriptional activation of empty spiracles, the Drosophila ortholog of EMX2, by Abdominal-B (HOXA10 ortholog) has been previously demonstrated. These findings demonstrate conservation of the transcription factor-target gene relationship, although the direction of regulation is reversed with possible evolutionary implications.

Demonstration of Multipotent Stem Cells in the Adult Human Endometrium by In Vitro Chondrogenesis

Stem cells are defined by their unique capacity for self-renewal and multilineage differentiation. Stem cells have been obtained from multiple extramedullary tissues. Recently, a population of progenitor cells have been identified in the endometrium. However, multilineage differentiation of endometrial stem cells has not been reported.Endometrial tissue was obtained from reproductive-aged women undergoing surgery for benign disease, from which monolayer endometrial stromal cell (ESC), myometrial, fibroid, fallopian tube, and uterosacral ligament tissue cultures were generated. Once confluent, cells were trypsinized and centrifuged in conical tubes to form a cell pellet. Cell pellets were cultured in a defined chondrogenic media (CM) containing dexamethasone and transforming growth factor (TGF)—β2 or TGF-β3 for 3 to 21 days. Samples were analyzed for markers of human articular cartilage, including sulfated glycosaminoglycans and expression of type II collagen. ESC pellets cultured in CM were found to contain cells that resemble chondrocytes. These cells expressed sulfated glycosaminoglycans and type II collagen typical of human articular cartilage. Myometrial, fibroid, fallopian tube, and uterosacral ligament cells were unable to undergo chondrogenic differentiation using the pellet culture method. Cells derived from the endometrium were able to differentiate into a heterologous cell type: chondrocytes, thus demonstrating the presence of multipotent stem cells. Endometrium is a potential source of multipotent stem cells.

Derivation of Insulin Producing Cells From Human Endometrial Stromal Stem Cells and Use in the Treatment of Murine Diabetes

Pancreatic islet cell transplantation is an effective approach to treat type 1 diabetes, however the shortage of cadaveric donors and limitations due to rejection require alternative solutions. Multipotent cells derived from the uterine endometrium have the ability to differentiate into mesodermal and ectodermal cellular lineages, suggesting the existence of mesenchymal stem cells in this tissue. We differentiated human endometrial stromal stem cells (ESSC) into insulin secreting cells using a simple and nontransfection protocol. An in vitro protocol was developed and evaluated by assessing the expression of pan β-cell markers, followed by confirmation of insulin secretion. PAX4, PDX1, GLUT2, and insulin, were all increased in differentiated cells compared to controls. Differentiated cells secreted insulin in a glucose responsive manner. In a murine model, differentiated cells were injected into the kidney capsules of diabetic mice and human insulin identified in serum. Within 5 weeks blood glucose levels were stabilized in animals transplanted with differentiated cells, however those treated with undifferentiated cells developed progressive hyperglycemia. Mice transplanted with control cells lost weight and developed cataracts while those receiving insulin producing cells did not. Endometrium provides an easily accessible, renewable, and immunologically identical source of stem cells with potential therapeutic applications in diabetes.

Xenoestrogen exposure imprints expression of genes (Hoxa10) required for normal uterine development

The developing reproductive tract is sensitive to endocrine perturbation. Bisphenol A (BPA), a xenoestrogen, is a common component of food storage plastics and dental composites. We tested the ability of BPA to alter expression of HOXA10, a gene necessary for uterine development. A dose‐response increase in HOXA10 mRNA expression was demonstrated in Ishikawa cells treated with 0.1 nM to 25 µM BPA. To determine whether in utero BPA exposure resulted in a lasting alteration of uterine HOXA10 expression, mice were treated with 0.5–5.0 mg/kg BPA on gestational days 9–16. A dose‐responsive increase was seen in stromal cell HOXA10 expression in 2‐and 6‐week‐old mice exposed in utero. To discern the mechanism of BPA action, the HOXA10 estrogen response element (ERE) and autoregulatory element (ARE) were tested for BPA responsiveness. BPA drove luciferase expression from HOXA10‐ERE and ARE reporter constructs. HOXA10 ERE mediated induction was blocked by ER antagonist ICI, while HOXA10 ARE induction was blocked by either ICI or HOXA10 antisense. BPA affects HOXA10 expression through the HOXA10 ERE and indirectly through the ARE. BPA initially alters HOXA10 expression through the ERE, however, the response is imprinted and uncoupled from estrogen stimulation in the adult. Several xenoestrogens alter HOX gene expression, indicating that HOX genes are a common target of endocrine disruption. In utero exposure to a xenoestrogen produces reproductive tract alterations by imprinting essential developmental regulatory genes. Smith, C. C., Taylor, H. S. Xenoestrogen exposure imprints expression of genes (Hoxa10) required for normal uterine development. FASEB J. 21, 239–246 (2007)