Members of the Wnt family of signaling molecules are important in cell specification and epithelial-mesenchymal interactions, and targeted gene deletion of Wnt-7a in mice results in complete absence of uterine glands and infertility. To assess potential roles of the Wnt family in human endometrium, an endocrine-responsive tissue, we investigated in the proliferative and secretory phases of the menstrual cycle, endometrial expression of several Wnt ligands (Wnt-2, Wnt-3, Wnt-4, Wnt-5a, Wnt-7a, and Wnt-8b), receptors [Frizzled (Fz)-6 and low-density lipoprotein receptor-related protein (LRP)-6], inhibitors [FrpHE and Dickkopf (Dkk)-1], and downstream effectors (Dishevelled-1, glycogen synthase kinase-3beta, and beta-catenin) by RT-PCR, real-time PCR and in situ hybridization. No significant menstrual cycle dependence of the Wnt ligands (except Wnt-3), receptors, or downstream effectors, was observed. Wnt-3 increased 4.7-fold in proliferative compared with secretory endometrium (P < 0.05). However, both inhibitors showed dramatic changes during the cycle, with 22.2-fold down-regulation (P < 0.05) of FrpHE and 234.3-fold up-regulation (P < 0.001) of Dkk-1 in the secretory, compared with the proliferative phase. In situ hybridization revealed cell-specific expression of different Wnt family genes in human endometrium. Wnt-7a was exclusively expressed in the luminal epithelium, and Fz-6 and beta-catenin were expressed in both epithelium and stroma, without any apparent change during the cycle. Both FrpHE and Dkk-1 expression were restricted to the stroma, during the proliferative and secretory phase, respectively. These unique expression patterns of Wnt family genes in different cell types of endometrium and the differential regulation of the inhibitors during the proliferative and secretory phase of the menstrual cycle strongly suggest functions for a Wnt signaling dialog between epithelial and stromal components in human endometrium. Also, they underscore the likely importance of this family during endometrial development, differentiation and implantation.

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Background: The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1 -/-embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation.

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To test the hypothesis that the cytochrome P-450-inhibiting antiepileptic drug (AED) stiripentol (STP) can reduce the incidence of phenytoin (PHT) induced congenital malformations, we chronically administered the AEDs to three inbred mouse strains. The frequency of congenital defects in PHT-treated animals was dosage dependent, ranging from 7 to 55%. When STP was coadministered orally with PHT, there was a significant reduction in fetal defects in SWV and C57BL/6J mouse strains. A replication of the experiment was conducted with addition of a seventh group of mice that received the high dosage of PHT together with STP. In the replicate, all three strains demonstrated a significant reduction in fetal defects in fetuses exposed to PHT (60 mg/kg) and STP (200 mg/kg) as compared with PHT (60 mg/kg) monotherapy. These results strongly suggest that oxidative metabolites activated by cytochrome P-450 are the primary teratogenic molecules involved in PHT-induced teratogenesis and that clinical management of pregnant epileptic patients may be improved through heightened awareness of these drug interactions.

The present study was undertaken to examine the role of heat shock response in the development of tolerance and cross-tolerance in an in vivo murine model of teratogen-induced neural tube defects. The experimental paradigm designed to address this question was to utilize inbred mouse strains that differed in their sensitivity to hyperthermia and valproic acid induced neural tube defects, subjecting the dams to subteratogenic pretreatments with either heat or valproic acid at two different timepoints during development prior to the administration of the teratogenic insult. A statistically significant reduction in the frequency of neural tube defects and/or embryolethality following a pretreatment in dams subsequently exposed to a teratogenic treatment was considered evidence for the induction of tolerance. This was observed in the SWV embryos exposed to the 38 degrees C pretreatment at 8:06 and to embryos exposed to either pretreatment temperature at 8:10 prior to a teratogenic heat shock at 8:12. In the LM/Bc embryos, only the 41 degrees C pretreatment at 8:06 induced thermotolerance. There was no evidence of tolerance induced in either mouse strain using valproic acid. On the other hand, cross-tolerance was clearly demonstrated in this study, with a low temperature (41 degrees C) pretreatment successfully protecting SWV fetuses from a subsequent teratogenic treatment with valproic acid, while valproic acid (200 mg/kg) was effective in reducing the risk of hyperthermia-induced neural tube defects in the LM/Bc fetuses. In all instances, tolerance was induced in the absence of significant induction of hsp synthesis. The lack of concordance between hsps and thermotolerance suggests that some other factor(s) is involved in conferring thermotolerance on developing murine embryos.

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Microphthalmia-associated Transcription Factor (Mitf) is expressed in neural crest cell-derived melanocytes, and in the retinal pigment epithelium (RPE) during ocular development. Mutations in Mitf are associated with auditory/visual/pigmentary syndromes in humans. Mitf mi/mi mouse mutants lack pigmentation, and are microphthalmic, while Mitf vit/vit mouse mutants display abnormal RPE pigmentation, and progressive retinal degeneration. Microarray analysis was used to identify novel downstream gene targets/pathways in the RPE that are altered by mutations in the transcription factor Mitf. Using the Affymetrix platform, gene expression profiles were generated using the eyes of E13.5 mouse fetuses that were wildtype, heterozygous, or homozygous for the Mitf mi mutation. In a separate experiment, eyes from E13.5 mouse fetuses homozygous for the Mitf vit mutation were compared to eyes from the C57BL/6 control background strain. Statistical analyses were performed using Robust Multiarray Average, mixed-effects ANOVA and random-variance t-tests. Altered expression of genes involved in pigment formation, melanosome biogenesis/transport, and redox homeostasis were observed. 12 genes were commonly mis-regulated in the eyes of both Mitf mutants: 10 of these genes were downregulated in both mutants relative to controls, while 2 of the genes (Nramp1 (Slc11a1) and epoxide hydrolase) were downregulated in Mitf mi/mi mutants, and conversely, upregulated in Mitf vit/vit mutants. Quantitative RT-PCR and immunohistochemistry were used to confirm altered gene/protein expression. RPE expression of the Fe +2 iron transporter Nramp1 (Slc11a1) has not previously been reported. Fe +2 is an important co-factor utilized by the iron-dependent isomerohydrolase RPE65 in the retinoid visual cycle. However, excess accumulation of Fe +2 in the RPE has recently been associated with oxidative damage and age-related macular degeneration. Abnormal pigmentation and increased activity of Slc11a1 in the RPE of Mitf vit mice may contribute to the pathology and progressive retinal degeneration observed in these mutants.