Implantation is a complex event requiring synchronization between a developing embryo and receptive endometrium. This process is governed by molecular mechanism mediated by homeobox (Hox) genes, which encode transcription factors. These factors guide embryologic development as well as regulate differential gene expression within the endometrium with each menstrual cycle. Spatiotemporal aberrations in HOX gene expression as seen with polycystic ovarian syndrome, endometriosis, hydrosalpinges, and endocrine disrupters compromise implantation. The role of HOX genes and their products continues to be explored as animal models that demonstrate implantation-specific infertile phenotypes continue to be investigated.
HOXA10 encodes a transcription factor required for endometrial receptivity and embryo implantation. The objective of this study was to identify and to characterize those molecular markers regulated by HOXA10 expression. The authors have identified putative HOXA10 target genes identified by microarray analysis employing a murine model of transient HOXA10 expression during the anticipated implantation window. Microarray analysis identified 40 statistically significant genes regulated by HOXA10 overexpression of which 31 genes were downregulated greater than 2-fold over control and 9 genes were upregulated. Cellular ontogenies of differentially expressed genes include cell adhesion molecules, signal transduction factors, and metabolic regulators. Semiquantitative real-time reverse transcriptase polymerase chain reaction confirmed regulation of selected candidate genes. Examples included clusterin (Clu), phoshoglycerate 3-dehydrogenase (3-Pgdh), and tumor-associated calcium signal transducer 2 (Tacstd2). Elucidation of these pathways will allow further characterization of the molecular mechanisms governing endometrial development, which also may function to enhance uterine receptivity. KeywordsImplantation; HOXA10; 3-phosphoglycerate dehydrogenase; microarray; endometrium The ability of the blastocyst to implant into a receptive endometrium governs reproductive success. Acute changes within this environment are driven by molecular events essential to the implantation process. Thus, the cascade of signaling events that occur in both fetal and maternal tissues at the time of implantation establishes an appropriate milieu critical to the development and survival of the fetus. Defects in the formation of this network and the inability to sustain this cross talk are believed to result in various pregnancy-associated complications that may manifest throughout the pregnancy. 1,2The homeobox (HOX) genes encode transcription factors that guide embryologic development as well as regulate differential gene expression within the endometrium with each menstrual cycle. 3 These regulators, first associated with directing axial patterning 4 through activation and repression of downstream targets, since have been demonstrated to be conserved across species and are essential to metazoan existence. [5][6][7] Although there are no known human mutations in HOXA10, women with decreased secretory phase HOXA10 expression have lower implantation rates. 13 Animal models generated to harbor selected gene knockouts demonstrate infertility phenotypes and continue to provide insight into the potential biomarkers that are involved in regulating the molecular implantation window. In particular, targeted disruption of the HOXA10 gene in mice results in a transformation of the upper uterine segment into an oviduct-like structure and inhibits implantation, even when embryos are transferred to the grossly unaffected lower uterine segment. 13,14 HOXA10-null mice produce normal numbers of embryos that are able to implant in wild-type surrogate ...
A model system to examine the expression and antiviral activity of trans-acting ribozymes in mammalian cells has been developed and evaluated. Hairpin ribozymes were engineered to cleave a specific site, identified by a combinatorial activity-based selection method, within genomic and subgenomic RNA species of Sindbis virus. Transiently transfected cells expressed moderate levels of ribozyme (ϳ50,000 molecules/cell) with predominant nuclear localization and a short halflife (23 min). Stable cell lines expressed ribozymes at modest levels (ϳ2,000 molecules/cell). Ribozyme-mediated RNA cleavage activity was detected in cell extracts. Clonal cell lines were challenged with recombinant Sindbis virus, and viral replication was examined using plaque formation and green fluorescent protein assays. Significant inhibition of viral replication was observed in cells expressing the active antiviral ribozyme, and lower levels of inhibition in control cells expressing inactive or irrelevant ribozymes. These findings are consistent with a model in which inhibition of viral replication occurs via ribozyme cleavage of viral RNAs, suggesting that ribozymes may represent useful antiviral agents.Small trans-acting ribozymes such as the hammerhead and hairpin can function as highly selective endoribonucleases, the specificity of which can be manipulated in the laboratory (for review, see Ref. 1) (2-3). This property has led to an exploration of the potential utility of ribozymes for the targeted inactivation of cellular and viral RNAs. Indeed, there is an urgent need for the development of novel and effective measures to combat existing and emerging viral pathogens. Several groups (4 -7) have described attempts to achieve ribozyme inhibition of viral replication, but success has been limited. In some cases, there remains uncertainty as to whether viral inhibition results from ribozyme cleavage of the targeted RNA, because other mechanisms of inhibition, such as antisense effects and interference with the expression of cellular genes that support viral replication, have not always been ruled out.Our laboratory has recently addressed two major issues that may limit RNA inactivation by engineered ribozymes: (i) intracellular catalysis (8, 9) and (ii) target site accessibility (13,14). Results have shown that hairpin ribozymes can catalyze efficient cleavage of RNA substrates within the cytoplasm of mammalian cells both in cis (8) and in trans (9). These studies were undertaken systematically to monitor intracellular ribozymemediated RNA cleavage and were accompanied by controls that included inactive ribozyme, uncleavable substrates, and active ribozymes with irrelevant substrate specificity. We concluded that the intracellular selectivity and biochemical requirements of the hairpin ribozyme appear to parallel closely those that have been elucidated in the test tube. The same conclusion has recently been shown to be true for cleavage and ligation of the hairpin ribozyme in yeast (11,12).To examine the issue of target site specificity, ...
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