Glucose transporter expression in rat embryo and uterus during decidualization, implantation, and early postimplantation.

Efficient transfer of glucose from the mother to the embryonic compartment is crucial to sustain the survival and normal development of the embryo in utero, because the embryo's production of this primary substrate for oxidative metabolism is minimal. In the present study, the temporal sequence of expression of the sodium-independent facilitative glucose transporter isoforms GLUTs 1, 3, 4, and 5 was investigated in the developing rat uteroembryonic unit between conception and Gestational Day 8 using immunohistochemistry. The GLUTs 1, 3, and 4 were expressed in the embryonic tissues after the start of implantation, being colocalized in the parietal endoderm, visceral endoderm, primary ectoderm, extraembryonic ectoderm, and the ectoplacental cone. In the uterus, a faint GLUT1 labeling emerged, but not until Gestational Day 3, in the luminal epithelium, endometrial stroma, and decidual cells. The intensity of GLUT1 staining increased in the latter population with progressing decidualization. Endometrial glands and myometrial smooth muscle cells stained neither for GLUT1 nor for GLUT3 until postimplantation. During all developmental stages examined, GLUT4 was visualized throughout the pregnant rat uterus, as was GLUT3 (with the above-mentioned exceptions). The density of GLUT5 was generally less than the sensitivity of the immunohistochemical detection method in all tissues investigated. In conclusion, the data point to a significant expression of the high-affinity glucose transporters GLUTs 1, 3, and 4 in the rat uteroembryonic unit, providing supportive evidence for an important role of facilitative glucose diffusion during peri-implantation development.     

MicroRNA expression and regulation in mouse uterus during embryo implantation

MicroRNAs (miRNAs) are 21-24-nucleotide non-coding RNAs found in diverse organisms. Although hundreds of miRNAs have been cloned or predicted, only very few miRNAs have been functionally characterized. Embryo implantation is a crucial step in mammalian reproduction. Many genes have been shown to be significantly changed in mouse uterus during embryo implantation. However, miRNA expression profiles in the mouse uterus between implantation sites and inter-implantation sites are still unknown. In this study, miRNA microarray was used to examine differential expression of miRNAs in the mouse uterus between implantation sites and inter-implantation sites. Compared with inter-implantation sites, there were 8 up-regulated miR-NAs at implantation sites, which were confirmed by both Northern blot and in situ hybridization. miR-21 was highly expressed in the subluminal stromal cells at implantation sites on day 5 of pregnancy. Because miR-21 was not detected in mouse uterus during pseudopregnancy and under delayed implantation, miR-21 expression at implantation sites was regulated by active blastocysts. Furthermore, we showed that Reck was the target gene of miR-21. Our data suggest that miR-21 may play a key role during embryo implantation.     

High expression of heat shock protein 90 alpha and its significance in human acute leukemia cells

This study investigated the expression of heat shock protein 90 alpha (Hsp90α) in acute leukemia cells. The expression of Hsp90α was investigated in leukemia cell lines and human bone marrow mononuclear cells derived from acute leukemia patients and from healthy individuals using polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Compared with cells from healthy individuals, the expression of Hsp90α in the untreated patients was higher. Similarly high levels were observed in remission patients. Significantly higher expression levels were observed in all the tested cell lines, and in cells from refractory and relapsed patients. No obvious relationship was observed between the occurrence of graft versus host disease and the expression of Hsp90α. The untreated patients showing higher expression levels of Hsp90α had lower complete remission rates. During remission of untreated patients, the expression of Hsp90α decreased and reached the lowest level after transplantation, but the expression increased again before relapse. Hsp90α was highly expressed in leukemia cells. The expression level of Hsp90α was associated with leukemia prognosis. However, no obvious relationship was observed between the occurrence of graft versus host disease and the expression of Hsp90α.     

Expression of nidogens in rat uterus and embryo during decidualization and implantation

The purpose of this study was to demonstrate the expression of nidogen-1 and nidogen-2 and their possible role in decidualization and implantation events during early pregnancy in rats. The tissue samples were examined from pregnant animals between gestational days 1-8 using immunocytochemistry. The uterine luminal epithelium, the glandular epithelium, and the myometrial smooth muscle cells stained strongly from gestational days 1-8 with both nidogen antibodies. At day 4 the decidual reaction areas began to appear in the stromal matrix and immunostaining of both nidogens revealed that the basement membrane of the surface epithelium was discontinuous. The differentiation of stromal cells into decidual cells was seen at gestational day 5 and both nidogens were weakly expressed in the decidualizing cells. At day 6, nidogen-2 immunoreactivity was higher in the primary decidual cells close to the embryo than nidogen-1, and during development of the decidual tissue both nidogens appeared in the endometrial stromal cells. At day 7, while expression of both nidogens declined in the primary decidual cells, their expression was markedly observed in the secondary decidual cells close to the myometrium. At day 8, expression of both nidogens was also observed to increase in the primary decidual cells. While nidogen-2 expression was seen in the parietal endoderm and primary ectoderm of the rat embryos at this developmental stage, nidogen-1 expression was only detected in the parietal endoderm. These results indicate that nidogen-1 and nidogen-2 could play important roles during embryogenesis, decidualization, and implantation in the endometrium of rat uterus.     

Increased expression of collagen-binding heat shock protein 47 in murine bleomycin-induced pneumopathy

The 47-kDa heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been shown to play a major role during the processing and/or secretion of procollagen. Expression of HSP47 has been reported to increase in parallel with expression of collagens during the progression of various fibrosis models. The aim of the present study was to investigate the association between HSP47 expression and collagen accumulation in bleomycin (BLM)-induced murine fibrosis. We investigated the expression of HSP47 protein and mRNA using immunohistochemical analysis and semi-quantitative RT-PCR in murine BLM-induced pulmonary fibrosis. Immunohistochemical analysis showed that higher expression of HSP47 protein was present in BLM-induced pulmonary fibrosis compared with controls. HSP47 was localized predominantly in alpha-smooth muscle actin-positive myofibroblasts, F4/80 negative, surfactant protein-A-positive type II pneumocytes, and F4/80-positive macrophages. RT-PCR also demonstrated an increase of HSP47 mRNA expression in BLM-treated lungs. Moreover, the relative amounts of HSP47 mRNA correlated significantly with the lung hydroxyproline content as an indicator of pulmonary fibrosis in BLM-treated lungs (r = 0.406, P <0.05). Our results suggest that these cells may play a role in the fibrotic process of BLM-treated lungs through upregulation of HSP47.     

Characterization of cold-induced heat shock protein expression in neonatal rat cardiomyocytes

Cardiac surgery is usually performed under conditions of cardioplegicischemic arrest. To protect the heart during the ischemic period, themyocardium is exposed to varying degrees of hypothermia. Althoughhyperthermia is known to induce the heat shock response, the moleculareffects of hypothermia on the myocardium have not been investigated. We havestudied the effect of hypothermia on the induction of heat shock proteins inprimary cultures of neonatal cardiomyocytes. Cold stress in cardiomyocytesinduced a 6 fold increase in the heat shock protein HSP70 as compared tocontrol. Increased HSP70 protein levels correlated with induction of HSP70mRNAs. Maximal levels of HSP70 protein appeared 4-6 h following recoveryfrom cold shock, indicating the transient nature of the response. Inductionof HSP25 mRNA was also observed in cold-shocked cardiomyocytes, even thoughincreased HSP25 protein levels were not detected. Our results indicate thathypothermia is capable of inducing the heat shock response in neonatalcardiomyocytes.     

GRP78 expression and regulation in the mouse uterus during embryo implantation

The aim of this study was to investigate the spatiotemporal expression and regulation of GRP78 in the mouse uterus during the peri-implantation period. The GRP78 protein was mainly detected in the luminal and glandular epithelia on days 1–4 of pregnancy. On day 5 of pregnancy, the GRP78 protein was more highly observed around the implanted embryo at the implantation site. There was no detectable GRP78 protein signal on day 5 of pseudopregnancy. GRP78 mRNA and protein levels gradually increased on days 6–8 of pregnancy, and the expression pattern was also expanded, coinciding with the development of decidua. Similarly, GRP78 expression was also strongly expressed in decidualised cells following artificial decidualisation. Compared with the results obtained with the delayed uterus, a high level of GRP78 expression was detected in the implantation-activated uterus. In the uteri of ovariectomised mice, GRP78 expression increased and reached its highest level after injection of oestrogen, and progesterone seemed to have an antagonistic effect on oestrogen up-regulation of GRP78 expression. Our data indicate that GRP78 might play an important role during the process of mouse embryo implantation, and GRP78 expression was mainly regulated by active blastocysts and maternal oestrogen.     

Increased appearance of inducible nitric oxide synthase in the uterus and embryo at implantation.

The aim of this study was to investigate the presence of iNOS in the murine uterus and embryo at implantation. Western blot analysis showed the presence of a 130-kDa band with strong reactivity to anti-iNOS antibody in the pre- and peri-implantation stage uteri. This band was faint in the postimplantation uteri. Immunocytochemical studies showed a heavy localization of iNOS specifically on the apical cells of the uterine endometrium in the pre- and peri-implantation stages. But the postimplantation uteri showed resorbed endometrium showing weaker expression of iNOS. The iNOS was induced by estrogen and the induction was intensified when progesterone was given along with estrogen. This truly mimics the in vivo situation since implantation in mice occurs when an estrogen surge occurs on a background of progesterone. The embryos too express iNOS at the peri-implantation stage. We suggest that iNOS expressed at peri-implantation would lead to enhanced NO production, which could act as a vasodilator and an angiogenic mediator. These effects could promote the attachment of the blastocyst to the uterus.     

Initiation of embryo implantation and maintenance of early pregnancy in the rat by chlordecone (Kepone).

The effect of chlordecone (Kepone), an insecticide/fungicide with reproductive toxicity, on the early stages of pregnancy in the rat was studied. Intraperitoneal injection of chlordecone into adult virgin female Holtzman strain rats before mating, in doses as high as 80 mg/kg, did not prevent fertilization, early development of the embryo to the blastocyst stage, transport of the embryo through the oviduct, or its implantation into the uterus. However, a single dose of 60 or 80 mg/kg, but not 20 or 40 mg/kg, before mating significantly reduced the concentration of progesterone in the serum of rats undergoing normal embryo implantation 5 days later. A dose of 80 mg/kg of chlordecone reduced progesterone levels in the serum by more than 50% within 48 hr in ovariectomized rats with Silastic tubing implants containing crystalline progesterone. This dose of chlordecone induced deciduomata formation in progesterone-primed ovariectomized rats to the same extent as 1 microgram of estradiol benzoate. The minimal effective single dose of chlordecone to initiate implantation of blastocysts in the uteri of hypophysectomized progesterone-primed rats, and to maintain embryo development for at least 5 days, was 50 mg/kg. Daily doses of 20 mg/kg for 3 or 5 days were effective at initiating implantation but did not maintain pregnancy. The latter treatment, however, did not prevent initiation of implantation or embryo development induced by subsequent administration of estrone. The results are consistent with the view that chlordecone is a weak estrogen that has both nongenomic and genomic estrogenic actions.     

Regulation of heat shock protein 70 synthesis by heat shock in the preimplantation murine embryo

Induced thermotolerance in murine embryos occurs at the 8-cell stage when embryos are maintained in vitro but not until the blastocyst stage if development proceeds in vivo. Present results indicate that ability of embryos to undergo induced thermotolerance is not limited by heat shock protein 70 (HSP70) synthesis. Exposure of 8-cell embryos to 40 degrees C enhanced synthesis of 2 constitutive HSP70 proteins (HSC70 and HSC72) and induced another protein, HSP68; exposure of 43 degrees C was required to induce similar responses in expanded blastocysts. Unlike induced thermotolerance, increased synthesis of HSP70 molecules did not depend on whether embryos were cultured or developed in vivo. Thus, other biochemical mechanisms in addition to HSP70 confer thermotolerance in the preimplantation-stage murine embryo. The observation that the temperature threshold for induction of HSP70 synthesis increased from the 8-cell to the blastocyst stage is indicative of these other biochemical processes.     

Enzymic activity in rat uterus during early pregnancy.

Total protein, RNA and DNA content and the activity of acid and alkaline phosphatases, 5'-nucleotidase and isocitrate dehydrogenase were studied in rat uterus during the first 8 days of pregnancy. Isocitrate dehydrogenase activity showed marked fluctuations from day to day. Nucleotidase and acid phosphatase activities showed a significant increase on day 8. The most marked change in activity was that of alkaline phosphatase which showed a 10-fold increase between days 6 and 8, due largely to an increase in the activity of this enzyme in the decidual nodule. The rise in alkaline phosphatase activity did not occur in rats ovariectomized on days 1, 2 or 4 of pregnancy and was markedly decreased in those ovariectomized on day 6. [3H]-uridine incorporation into RNA showed a significant increase between days 2 and 6 whereas [3H]-thymidine incorporation into DNA showed a significant increase on day 6.     

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.     

Alterations in protein kinase C type III-alpha during heat shock of rat embryo fibroblasts.

Heat shock treatment of rat embryo fibroblasts resulted in a 60% increase in cytosolic protein kinase C activity, in contrast to phorbol ester-induced translocation to the membrane. During reversal of the cells back to the normal temperature a decrease in cytosolic PKC activity was observed and paralleled by an increase in protamine kinase activity. Cell lysates prepared from heat shock-treated cells show a marked calcium/phospholipid-dependent phosphorylation of several endogenous PKC substrate proteins, while the 28-kDa stress protein was shown to be a PKC substrate. These cells express the TYPE III-alpha isoform of PKC and, thus, the alterations induced within cells exposed to hyperthermic treatment may reflect a functional significance with regard to the regulation of this specific isoform.     

Differential expression of prostaglandin E receptor subtype EP2 in rat uterus during early pregnancy

PGE2 is essential for mammalian female reproduction. This study was to examine the expression of EP2 gene in the rat uterus during early pregnancy, delayed implantation and artificial decidualization by in situ hybridization and immunohistochemistry. There was no detectable EP2 mRNA expression in the uterus from days 1 to 4 of pregnancy (day 1 = day of vaginal sperm). A low level of EP2 immunostaining was observed in the luminal and glandular epithelium from days 1 to 4 of pregnancy. Both EP2 mRNA and protein expression were highly detected in the luminal epithelium at implantation sites on day 6 of pregnancy. EP2 expression decreased from day 7 of pregnancy and was undetectable on days 8 and 9 of pregnancy. After delayed implantation was terminated by estrogen treatment and the embryo implanted, both EP2 mRNA and protein expression were strongly observed in the luminal epithelium at the implantation site. There was no detectable EP2 expression in both control and decidualized uteri. In conclusion, these data suggest that EP2 expression at implantation site may play an important role during embryo implantation in rats.     

Heat shock and thermotolerance during early rat embryo development

Effects of heat shock on the development of early pre-somite embryos have been studied using cultured rat embryos. The results illustrate the sensitivity of the developing head and brain to elevated temperatures prior to neural tube closure and the capacity of embryos to acquire thermotolerance. Embryos exposed briefly to an elevated temperature (43 degrees C for 7.5 min) developed severe craniofacial defects including microphthalmia, microcephaly, gross reduction of the forebrain region, and open neural tubes. In contrast, a nonteratogenic heat shock (42 degrees C for 10 min) caused embryos to acquire thermotolerance during a 15-min recovery period at 38.5 degrees C. Acquired thermotolerance was effective in protecting embryos from a subsequent more severe heat treatment which would have been teratogenic in an unprotected embryo. Recovering embryos mounted a heat shock response as evidenced by the induction of a 71 kilodalton heat shock protein. Activation of the heat shock response was not a teratogenic event in the developing embryo.     

Cell signaling and heat shock protein expression

Exposure of cells and organs to heat shock is associated with numerous changes in various cellular metabolic parameters and overexpression of proteins collectively known as heat shock proteins (HSP). In this communication we review the cell-signaling events that are altered in response to heat shock as they relate to the subsequent induction of HSP 70 kd (HSP-70) expression. We also review the mechanisms by which HSP-70 is involved in conferring cytoprotective effects. The possibility of altering HSP expression through manipulations of the cell-signal process has clinical importance.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or Department of Defense.