Evaluation of Strain Transition Properties between Cast-In Fibre Bragg Gratings and Cast Aluminium during Uniaxial Straining.

Current testing methods are capable of measuring strain near the surface on structural parts, for example by using strain gauges. However, stress peaks often occur within the material and can only be approximated. An alternative strain measurement incorporates fibre-optical strain sensors (Fiber Bragg Gratings, FBG) which are able to determine strains within the material. The principle has already been verified by using embedded FBGs in tensile specimens. The transition area between fibre and aluminium, however, is not yet properly investigated. Therefore, strains in tensile specimens containing FBGs were measured by neutron diffraction in gauge volumes of two different sizes around the Bragg grating. As a result, it is possible to identify and decouple elastic and plastic strains affecting the FBGs and to transfer the findings into a fully descriptive FE-model of the strain transition area.We thus accomplished closing the gap between the external load and internal straining obtained from cast-in FBG and generating valuable information about the mechanisms within the strain transition area.It was found that the porosity within the casting has a significant impact on the stiffness of the tensile specimen, the generation of excess microscopic tensions and thus the formation of permanent plastic strains, which are well recognized by the FBG. The knowledge that FBG as internal strain sensors function just as well as common external strain sensors will now allow for the application of FBG in actual structural parts and measurements under real load conditions. In the future, applications for long-term monitoring of cast parts will also be enabled and are currently under development.

Divergent regulation of angiopoietin-1 and -2, Tie-2, and thrombospondin-1 expression by estrogen in the baboon endometrium.

Estrogen has an important role in the reconstruction of a new vascular network in the endometrium during each menstrual cycle; however, the underlying mechanisms are incompletely understood. Angiopoietin-1 (Ang-1) promotes vessel assembly, whereas Ang-2 and thrombospondin-1 (TSP-1) cause vessel breakdown. To determine the potential effect of estrogen on the expression of these angioregulatory factors in the endometrium, Ang-1, Ang-2, TSP-1, and Tie-2 receptor mRNA levels were assessed by real-time reverse transcriptase polymerase chain reaction in glandular epithelial and stromal cells isolated from the endometrium of ovariectomized baboons treated acutely with estradiol. Corresponding protein expression was assessed by immunocytochemistry and the proximity ligation assay (PLA) during advancing stages of the baboon menstrual cycle. Serum estradiol levels in ovariectomized baboons were 400 pg/ml within 4-6 hr of estradiol treatment. Ang-1 mRNA levels in glandular epithelial cells increased threefold (P < 0.01) within 4 hr of estradiol administration. In contrast, TSP-1 mRNA levels decreased four- to fivefold (P < 0.01) in endometrial glandular epithelial and stromal cells 4-6 hr after estradiol, whereas Ang-2 and Tie-2 expression was unaltered. Immunostaining for Ang-1 increased, TSP-1 decreased, and Ang-2 and Tie-2 were unaltered in the endometrium during the secretory compared with the proliferative phase of the cycle. Endometrial Ang-1 protein expression, quantified by PLA, increased 10-fold (P < 0.05) between the early proliferative and late proliferative/mid-secretory phases of the menstrual cycle in association with the rise in estrogen. In summary, estrogen induced a rapid, divergent, and cell-specific change in expression of angiostimulatory and angioinhibitory growth factors in the endometrium of the nonhuman primate.

Phase Transition Kinetics in Austempered Ductile Iron (ADI) with Regard to Mo Content.

The phase transformation to ausferrite during austempered ductile iron (ADI) heat treatment can be significantly influenced by the alloying element Mo. Utilizing neutron diffraction, the phase transformation from austenite to ausferrite was monitored in-situ during the heat treatment. In addition to the phase volume fractions, the carbon enrichment of retained austenite was investigated. The results from neutron diffraction were compared to the macroscopic length change from dilatometer measurements. They show that the dilatometer data are only of limited use for the investigation of ausferrite formation. However, they allow deriving the time of maximum carbon accumulation in the retained austenite. In addition, the transformation of austenite during ausferritization was investigated using metallographic methods. Finally, the distribution of the alloying elements in the vicinity of the austenite/ferrite interface zone was shown by atom probe tomography (APT) measurements. C and Mn were enriched within the interface, while Si concentration was reduced. The Mo concentration in ferrite, interface and austentite stayed at the same level. The delay of austenite decay during Stage II reaction caused by Mo was studied in detail at 400 °C for the initial material as well as for 0.25 mass % and 0.50 mass % Mo additions.

Estrogen rapidly activates the PI3K/AKT pathway and hypoxia-inducible factor 1 and induces vascular endothelial growth factor A expression in luminal epithelial cells of the rat uterus.

We have previously shown that 17beta-estradiol (E(2)) increases vascular endothelial growth factor A (Vegfa) gene expression in the rat uterus, resulting in increased microvascular permeability, and that this involves the simultaneous recruitment of hypoxia-inducible factor 1 (HIF1) and estrogen receptor alpha (ESR1) to the Vegfa gene promoter. Both events require the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. However, those studies were carried out using whole uterine tissue, and while most evidence indicates that the likely site of E(2)-induced Vegfa expression is luminal epithelial (LE) cells, other studies have identified stromal cells as the site of that expression. To address this question, the pathway regulating Vegfa expression was reexamined using LE cells rapidly isolated after E(2) treatment. In addition, we further characterized the nature of the receptor through which E(2) triggers the signaling events that lead to Vegfa expression using the specific ESR1 antagonist ICI 182,780. In agreement with previous results in the whole uterus, E(2) stimulated Vegfa mRNA expression in LE cells, peaking at 1 h (4- to 14-fold) and returning to basal levels by 4 h. Treatment with E(2) also increased phosphorylation of AKT in LE cells, as well as of the downstream mediators FRAP1 (mTOR), GSK3B, and MDM2. The alpha subunit of HIF1 (HIF1A) was present in LE cells before E(2) treatment, was unchanged 1 h after E(2), but was >2-fold higher by 4 h. Chromatin immunoprecipitation analysis showed that HIF1A was recruited to the Vegfa promoter by 1 h and was absent again by 4 h. The E(2) activation of the PI3K/AKT pathway, HIF1A recruitment to the Vegfa promoter, and Vegfa expression were all blocked by ICI 182,780. In summary, the rapid E(2)-induced signaling events that lead to the expression of Vegfa observed previously using the whole uterus occur in LE cells and appear to be initiated via a membrane form of ESR1.

Estrogen-induced activation of hypoxia-inducible factor-1alpha, vascular endothelial growth factor expression, and edema in the uterus are mediated by the phosphatidylinositol 3-kinase/Akt pathway.

Vascular endothelial growth factor (VEGF) plays an essential role in normal uterine physiology and function as well as endometrial cancer and other uterine disorders. Recently we showed that estrogen regulation of VEGF expression in the rat uterus involves rapid recruitment of both estrogen receptor (ER)-alpha and hypoxia-inducible factor (HIF)-1alpha to the VEGF promoter. Estrogen is known to stimulate both the MAPK and phosphatidylinositol 3-kinase (PI3K) pathways, which have been linked to the activation of both of these transcription factors. Therefore, the involvement of these pathways in estrogen-induced VEGF expression was investigated. Inhibitors of the MAPK (U0126) or PI3K pathways (wortmannin or LY294002) were administered ip to immature female rats 1 h before 17beta-estradiol (E(2)) treatment. E(2) activation of both pathways occurred and was completely inhibited by the appropriate antagonist. Only PI3K inhibitors, however, blocked E(2) stimulation of VEGF mRNA expression and E(2)-induced uterine edema. In vivo chromatin immunoprecipitation analysis showed that this was associated with a failure of both HIF-1alpha and ERalpha to bind to the VEGF promoter. To determine whether inhibiting the PI3K pathway affected ERalpha induction of other estrogen target genes, the expression of creatine kinase B and progesterone receptor A/B was also examined. The expression of each was also inhibited by wortmannin, as was ERalpha binding to the creatine kinase B promoter. In conclusion, although estrogen activates both the MAPK and PI3K pathways in the rat uterus, activation of HIF-1alpha and ERalpha, and therefore regulation of VEGF gene expression is dependent only on the PI3K/Akt pathway. Furthermore, activation of the PI3K pathway appears to be a common requirement for the expression of estrogen-induced genes. These findings not only shed light on estrogen action in normal target tissues but also have important implications for cancer biology because excessive PI3K, HIF-1alpha, and VEGF activity are common in estrogen-dependent tumors.

Effects of ERalpha overexpression on female reproduction in mice.

Recently, we generated transgenic mice in which ERalpha can be inducibly overexpressed in reproductive tissues (ERalpha overexpressors). These mice were used to test the hypothesis that prenatal and postnatal ERalpha overexpression reduces female fertility. To do so, litter sizes, ovulation, follicle numbers, uterine histology, implantation sites, and hormone levels were compared in ERalpha overexpressors and controls. The data indicate that ERalpha overexpressors have reduced fertility compared to controls and that the reduced fertility is not due to reduced ovulatory capacity, altered levels of estradiol, FSH, and LH, or impaired follicular growth. ERalpha overexpressors, however, had a higher number of apoptotic cells in the endometrial epithelium and a reduced number of implantation sites compared to controls. Thus, the increased number of apoptotic cells and reduced number of implantation sites observed in ERalpha overexpressing uteri compared to controls may, in part, account for the reduced litter size produced by ERalpha overexpressing females.

Methoxychlor induces atresia of antral follicles in ERalpha-overexpressing mice.

Methoxychlor (MXC) is a pesticide that is known to bind to estrogen receptor alpha (ERalpha) and to induce atresia of antral ovarian follicles. Although studies have shown that MXC is toxic to the ovary, we hypothesize that perturbation to the estrogen-signaling system (i.e., increase or decrease in estrogen sensitivity) might alter ovarian responsiveness to MXC. Thus, we examined whether ERalpha overexpression alters the ability of MXC to increase follicle atresia. To do so, we employed a transgenic mouse model in which ERalpha can be inducibly overexpressed in animal tissues (ERalpha overexpressors). We dosed female controls and ERalpha overexpressors with sesame oil (vehicle control) or MXC (32 and 64 mg/kg/day) for 20 days. After dosing, the ovaries were collected for histological evaluation of follicle numbers and follicle atresia, while blood was collected for measurements of hormones. Estrous cycles were determined in all animals to ensure that all were terminated during estrus. Although there were no significant effects of MXC on the numbers of primordial, primary, and preantral follicles in both controls and ERalpha overexpressors, there was an effect on antral follicles. Specifically, our data indicate that 32 and 64 mg/kg MXC increased the percentage of atretic follicles compared to vehicle in both control and ERalpha overexpressor groups. Moreover, there was a clear trend toward greater sensitivity to 64 mg/kg MXC in ERalpha-overexpressing mice compared to control animals. Specifically, at the 64-mg/kg MXC dose, ERalpha-overexpressing mice had a significantly higher percentage of atretic follicles compared to control animals (controls = 21.5 +/- 3%, n = 5; ERalpha overexpressors = 37 +/- 23%, n = 9, p < or = 0.05 vs. controls). After 20 days of dosing, there were no differences in estradiol levels between controls and ERalpha-overexpressing mice in all treatment groups. Follicle-stimulating hormone (FSH) levels were similar in sesame oil-treated control mice and control mice treated with 32 mg/kg MXC, while control mice treated with 64 mg/kg MXC had significantly lower levels of FSH compared to sesame oil-treated controls (sesame oil = 4.31 +/- 0.7, MXC [64 mg/kg/day] = 1.89 +/- 0.4, n = 3, p < or = 0.02 vs. sesame oil). ERalpha-overexpressing mice treated with sesame oil, 32 or 64 mg/kg MXC, had similar FSH levels. Thus, we observed an increased percentage of atretic antral follicles in ERalpha-overexpressing mice treated with MXC compared to control mice treated with the same compound, suggesting that the ERalpha-signaling pathway plays an important role in MXC-induced atresia. The trend toward greater sensitivity to MXC in ERalpha-overexpressing mice compared to control animals cannot be explained by alterations in estradiol and/or FSH levels.

Chromatin immunoprecipitation analysis of gene expression in the rat uterus in vivo: estrogen-induced recruitment of both estrogen receptor alpha and hypoxia-inducible factor 1 to the vascular endothelial growth factor promoter.

Vascular endothelial growth factor (VEGF) plays a pivotal role in the regulation of microvascular permeability and angiogenesis, processes essential for normal endometrial growth and implantation. Estrogen [17beta-estradiol (E2)], via its receptor (ER alpha), rapidly stimulates VEGF expression in the uterus at the transcriptional level. The VEGF gene promoter, however, lacks a consensus estrogen response element (ERE), and attempts to identify the site through which E2 induces VEGF expression have yielded contradictory results. To address this question, we modified the chromatin immunoprecipitation method to identify transcription factors that interact with the VEGF promoter in the rat uterus in response to E2. Chromatin immunoprecipitation showed that both Sp1 and Sp3 were associated with a proximal, GC-rich region of the promoter before E2 treatment. E2 induced an increase in Sp1 binding and the recruitment of ER alpha, and the coactivator p300 to this region. The association of ER alpha persisted, however, after VEGF mRNA levels had declined again (at 4 h), indicating that other factors might be involved in that expression. Western analysis showed that both the alpha- and beta-subunits of the transcription factor hypoxia-inducible factor 1 (HIF-1), which regulates VEGF expression in response to hypoxia and several hormones and growth factors, were present in the uterus. Furthermore, E2 rapidly induced their recruitment to the -944 to -611 bp region of the VEGF promoter, which contains the hypoxia response element to which HIF-1 binds. This binding was transient, matching the pattern of E2-induced VEGF expression. These results indicate that HIF-1 is an important mediator of E2-induced VEGF expression in the uterus. In addition, E2 also induced a later increase in HIF-1alpha mRNA and protein expression in the uterus, suggesting that it may be required for longer term effects of E2 on the uterus as well. In contrast to the uterus, HEC1A cells cultured in 95% air-5% CO2 (and therefore 20% O2) contained no HIF-1alpha, consistent with the inability of E2 to stimulate the expression of VEGF by these and other cell types in vitro.

New insight into the transcriptional regulation of vascular endothelial growth factor expression in the endometrium by estrogen and relaxin.

Increased uterine capillary permeability, which can be induced by both estrogen and relaxin, is required for endometrial growth and implantation. This effect is mediated in both cases by estrogen receptors (ERs), via stimulation of vascular endothelial growth factor (VEGF) expression. The sites on the VEGF promoter through which induction occurs, however, are completely unclear. We have used the technique of chromatin immunoprecipitation in vivo to localize the site of ER action and identify other transcription factors that are involved. We have found that ERa associates with Sp1/Sp3 at a GC-rich region of the promoter. More interesting, however, is the observation that estrogen also induces rapid, transient binding of hypoxia-inducible factor 1 (HIF-1), which mediates VEGF transcription in response to hypoxia, to the promoter. The estrogen-induced HIF-1 binding closely matches the estrogen-induced pattern of VEGF expression in the uterus, suggesting that HIF-1 is involved in that induction, and probably that of many other genes as well (HIF-1 is now known to regulate the expression of more than 40 genes). It is likely that studies now under way will also link relaxin-induced VEGF expression to HIF-1. This is based on the similarities in the effects of the two hormones on VEGF expression and on their shared ability to activate the PI3K and MAPK pathways, both of which can activate HIF-1.

Minireview: Putting physiology back into estrogens' mechanism of action.

After decades of research, the mechanism by which estrogens stimulate the proliferation of epithelial cells in the endometrium and mammary gland, and in the carcinomas that arise in those tissues, is still not understood. Cells do not proliferate in response to 17ß-estradiol (E2) alone, and although it is widely recognized that growth factors play a role in E2's proliferative effect, exactly how they are involved is unclear. It has long been known that the proliferation of endometrial epithelial cells is preceded by dramatic increases in blood flow and microvascular permeability, filling the subepithelial stroma with plasma and the proteins it contains, such as IGF-I, which is known to synergize with E2 in the induction of cell proliferation. The hyperpermeability is caused by vascular endothelial growth factor (VEGF), which is rapidly induced by E2, via the transcription factors hypoxia-inducible factor 1 and estrogen receptor α, in luminal epithelial cells in vivo. As we recently showed, VEGF is also strongly induced in endometrial cancer cells in vitro when excessive degradation of hypoxia-inducible factor 1α, caused by the abnormally high oxygen level to which cultured cells are exposed, is prevented. Putting these facts together, we now propose a new model of E2-induced proliferation in which VEGF-induced vascular hyperpermeability plays an essential role. E2 first induces the expression by endometrial epithelial cells of VEGF, which then acts in a paracrine manner to induce interendothelial cell gaps in subepithelial blood vessels, through which plasma and the proteins therein enter the adjacent stroma. Plasma carries even more E2, which circulates bound to proteins, and IGF-l, which together drive epithelial cells completely through the cell cycle.

Dexamethasone enhances fertility and preovulatory serum prolactin levels in eCG/hCG primed immature rats.

Glucocorticoids have heterogeneous effects on reproductive function. We used a gonadotropin-primed, immature rat model to study the influence of dexamethasone (1 mg/kg), given during the latter stages of follicular development, on litter size, the number of oocytes released, and pituitary hormone levels. Dexamethasone-treated females released a larger number of oocytes at ovulation and gave birth to larger litters indicating the oocytes were viable. Survival to weaning age was not affected but average weight at weaning was lower for pups born to DEX-treated females. Serum FSH and LH were assayed at 12, 24 and 48 h following eCG and did not differ between dexamethasone-treated and control animals, but prolactin showed a prolonged pattern of elevation in DEX-treated females. Prolactin, which normally exhibits an elevation on proestrous, may modulate follicular development. Dexamethasone enhances fertility and fecundity possible through an effect of prolactin on follicle development, or by other direct effects on the ovary. These results may improve our understanding of the usefulness of DEX in assisted reproductive therapies for women.

Inhibition of oxygen-induced hypoxia-inducible factor-1alpha degradation unmasks estradiol induction of vascular endothelial growth factor expression in ECC-1 cancer cells in vitro.

Estradiol (E(2)) rapidly and strongly induces vascular endothelial growth factor (VEGF) transcription in uterine endometrial epithelial cells in vivo. We have shown that this is mediated by both the estrogen receptor-alpha and hypoxia-inducible factor (HIF)-1alpha. By contrast, E(2) induces little or no VEGF expression in cultured breast or endometrial cancer cells, which lack HIF-1alpha due to the abnormally high concentration of oxygen ( approximately 20%) to which they are exposed. To test the hypothesis that restoring HIF-1alpha in cultured cells would restore the ability of E(2) to induce VEGF expression, we treated human endometrial cancer cells (ECC-1) with cobalt chloride (CoCl(2);100 microm), which prevents oxygen-induced HIF-1alpha degradation. HIF-1alpha was absent in untreated ECC-1 cells but detectable by 4 h after treatment with CoCl(2) alone, as was a significant increase in VEGF mRNA. E(2) plus CoCl(2) induced detectable HIF-1alpha expression at 2 h and an even higher level than that induced by CoCl(2) alone at 4 h; this HIF-1alpha was localized in the nuclei. This was accompanied by increasing VEGF expression, with the increase at 4 h severalfold higher than that induced by CoCl(2) alone and was concurrent with recruitment of both HIF-1alpha and estrogen receptor-alpha to the VEGF promoter. These results confirm that HIF-1alpha plays an essential role in E(2)-induced expression of VEGF. Through the induction of increased microvascular permeability and the consequent exudation of plasma growth factors, VEGF in turn may play an essential role in cancer cell proliferation in vivo.

Treatment of rats with 17beta-estradiol or relaxin rapidly inhibits uterine estrogen receptor beta1 and beta2 messenger ribonucleic acid levels.

Estrogen regulates the growth and differentiation of the uterus via binding to estrogen receptors (ERs), members of the nuclear receptor family of transcription factors. Two forms of ER exist: ERalpha and ERbeta. The former is a well-characterized mediator of estrogen-induced transcription, but the function of the latter is unclear. Recent in vitro studies suggest that both splicing forms of ERbeta expressed in rat tissues, beta1 and beta2, may function as inhibitors of ERalpha transcriptional activity. To gain insight into the role of ERbeta in estrogen action, we examined the effects of estrogen and relaxin, a ligand-independent activator of ERs, on the expression of ERbeta1 and ERbeta2 mRNA in the uterus in vivo. Eighteen-day-old female rats were ovariectomized and, after recovery, treated with 17beta-estradiol, relaxin, or vehicle. Quantitative reverse transcription-polymerase chain reaction analyses of uterine RNA from estrogen-treated animals revealed marked decreases in the steady-state levels of the mRNAs for both ERbeta1 and ERbeta2 at 3, 6, and 24 h after treatment. Relaxin induced a similar effect. Neither hormone had any significant effect on ERalpha mRNA levels. To determine if endogenous estrogen exerts this effect, we examined the expression of ERbetas in the uterus during the estrous cycle. Levels of both isoforms were highest at diestrus (low estrogen), were significantly lower at early proestrus (rising estrogen), reached a nadir during late proestrus (peak estrogen), and rebounded at estrus (declining estrogen). These data suggest that down-regulation of ERbeta expression may be required for estrogen to exert its full trophic effects on the uterus.

Inhibition of vascular endothelial growth factor/vascular permeability factor action blocks estrogen-induced uterine edema and implantation in rodents.

Estrogen induces a rapid increase in microvascular permeability in the rodent uterus, leading to stromal edema and a marked increase in uterine wet weight. This edema is believed to create an environment optimal for the growth and remodeling of the endometrium in preparation for implantation and pregnancy. Increased endometrial microvascular permeability also occurs in conjunction with implantation. Estrogen-induced uterine edema is immediately preceded by an increase in the expression of vascular endothelial growth factor (VEGF), a potent stimulator of microvascular permeability. The objective of this study was to determine to what degree immunoneutralization of VEGF would interfere with a) estradiol-induced uterine edema and b) pregnancy. In the first set of experiments, immature female rats were injected with either VEGF antiserum or normal rabbit serum (NRS) prior to 17beta-estradiol treatment. Rats treated with estradiol alone showed a 57% increase in uterine wet weight at 6 h compared with controls. Injection of 200 or 300 micro l of VEGF antiserum reduced the response to only 20% and 10% above controls, respectively. In the second set of experiments, young adult female mice were treated with 100 micro l of either VEGF antiserum or NRS at 1200 h on the fourth day after mating. NRS-treated mice had normal pregnancies. VEGF antiserum, however, completely blocked pregnancy. When VEGF antiserum-treated females were examined on Day 5 for the presence of implantation sites, none were found. These results show that a) VEGF is the major mediator of estrogen-induced increase in uterine vascular permeability and b) VEGF-induced edema is absolutely essential for implantation to take place.

Conditional over-expression of estrogen receptor alpha in a transgenic mouse model.

Attempts to delineate the mechanisms of estrogen action have promoted the creation of several estrogen receptor alpha (ERalpha) mouse models in the past decade. These traditional models are limited by the fact that the receptors are either absent or present throughout all stages of development. The purpose of this work was to develop a conditional transgenic model that would provide an in vivo method of controlling the spatial and temporal regulation of ERalpha expression. The tetracycline responsive system was utilized. Three lines of transgenic mice carrying a transgene composed of the coding sequence for murine ERalpha placed under the regulatory control of a tet operator promoter (tet-op) were generated. These three lines of tet-op-mERa mice were each mated to an established line of transgenic mice expressing a tetracycline-dependent transactivator protein (tTA) from the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Double transgenic MMTV-tTA/tet-op-mERalpha mice were produced. All three lines demonstrated dominant gain of ERalpha shown by RT-PCR, immunoprecipitation, and immunohistochemistry. Transgene-specific ERalpha was expressed in numerous tissues including the mammary gland, salivary gland, testis, seminal vesicle, and epididymis. Expression was silenced by administration of doxycycline in the drinking water. This model can be utilized to evaluate the consequences of ERalpha dominant gain in targeted tissues at specific times during development. In this study dominant gain of ERalpha was associated with a reduction in epididymal/vas deferens and seminal vesicle weights consistent with the proposed action of ERalpha on fluid transport in the male reproductive tract. Combining this model with other dominant gain and gene knockout mouse models will be useful for testing effects of ERalpha action in combination with specific gene products and to evaluate if developmental and stage-specific expression of ERalpha can rescue identified phenotypes in gene knockout mice.