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E Winterhager

Publications and source records attributed to E Winterhager.

At least 55 records · Page 3Linked to original sources
PubMed1996

Connexins and E-cadherin are differentially expressed during trophoblast invasion and placenta differentiation in the rat.

We have characterized the spatial and temporal expression pattern of six different connexin genes and E-cadherin during trophectoderm development in the rat. During the initial phase of trophoblast invasion at 6 days postcoitum (dpc), the trophoblast expressed E-cadherin but no connexin expression could be observed. With progressing invasion of the polar trophoblast into the maternal decidua, from 7 dpc onwards E-cadherin expression in the ectoplacental cone cells was lost and was now restricted to the extraembryonic ectoderm. In the ectoplacental cone and extraembryonic ectoderm instead connexin31 mRNA and protein could be found. This pattern was maintained up to day 10 postcoitum. The start of labyrinthine trophoblast differentiation from day 11 postcoitum onwards was characterized by persisting expression of E-cadherin in the extraembryonic ectoderm and its derivative, the chorionic plate. In addition to E-cadherin, from 10 dpc onwards, connexin26 started to be expressed in the chorionic plate, and both molecules remained coexpressed in the labyrinthine trophoblast of the mature placenta. During this differentiation process connexin31 remained expressed mainly in the proliferating spongiotrophoblast. From day 14 postcoitum onwards, the expression of connexin31 in the spongiotrophoblastic cells decreased, and in parallel they started to express connexin43. The trophoblastic giant cells, first characterized by connexin31, lost all of the investigated connexins during midgestation on day 12 postcoitum but started to express connexin43 from day 18 postcoitum onwards. Our studies suggest that loss of E-cadherin and induction of connexin31 expression is correlated with the proliferative and invasive stages of the ectoplacental cone, whereas appearance of connexin26, E-cadherin and connexin43 reflects the switch to the differentiated phenotypes of the mature placenta.

Animals↗
PubMed1996

Properties of connexin40 gap junction channels endogenously expressed and exogenously overexpressed in human choriocarcinoma cell lines.

We have investigated the properties of gap junction channels of three human malignant trophoblast (choriocarcinoma) cell lines: BeWo, Jeg-3 and JAr, as well as in Jeg-3 cells stably transfected with rat connexin40 (Cx40). Reverse-transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis and immunostaining demonstrated expression of Cx40 in BeWo and JAr cell lines. JAr cells also expressed minor amounts of Cx43. Very low levels of Cx40 transcripts were revealed by RT-PCR in parental Jeg-3 cells, but Cx40 protein was not detected. To compare properties of endogenously and exogenously expressed Cx40 channels we have transfected Jeg-3 cells with rat Cx40. Recordings with dual whole-cell methods were used to determine the junctional conductance (gj) in the various cell lines and transfectants. Cx40 channels exogenously expressed in Jeg-3 cells demonstrated steep voltage sensitivity in the transjunctional voltage range of +/-30 to +/-40 mV and a unitary mainstate conductance of 175 pS, values which are similar to the data obtained from endogenously expressed Cx40 in BeWo cell pairs. In addition, greater driving forces resulted in a lower unitary conductance of about 30 pS, exclusively in BeWo cells. Between JAr cell pairs we determined a gj of 10 nS and unitary conductances were predominantly 100 and 152 pS. Voltage dependence was less sensitive in JAr cells compared to Cx40 transfectants and BeWo cells. Thus, coexpression of Cx43 and Cx40 leads to a macroscopic conductance with a mixture of properties expected for each connexin, whereas single-channel properties of each connexin type are maintained.

Animals↗
PubMed1996

Gap junction and tissue invasion: a comparison of tumorigenesis and pregnancy.

1. Trophoblast invasion during embryo implantation in some aspects resembles tumour cell invasion but, unlike tumour cells, trophoblast cells are able to differentiate and establish a placenta. Because direct cell-cell communication is believed to be involved in growth control and differentiation, we have investigated connexin (cx) gene expression during trophoblast development. 2. Pre-implantation embryos expressed cx43 as well as cx31 proteins from the 8-cell stage onwards. Following implantation, compartmentalization of both connexins occurred: cx31 expression was restricted to the invasive trophoblast cell population, whereas the embryo proper was characterized by cx43. Trophoblast differentiation was indicated by induction of cx26 in the labyrinth and cx43 in the spongiotrophoblast accompanied by a disappearance of cx31. Comparison with trophoblast cell lines revealed that rat trophoblast HRP-1 cells express connexin43, while malignant choriocarcinoma cells express cx31. Treatment with retinoic acid led to a disappearance of cx31 in the choriocarcinoma. Both cell lines reduced their invasion properties after retinoic acid treatment, but growth retardation was only observed in the malignant trophoblast. 3. It seems that the cx31 channel is needed for trophoblast cell populations to maintain the highly proliferative properties but does not alter their invasion properties.

Animals↗
PubMed1996

Expression of the gap junction proteins connexin31 and connexin43 correlates with communication compartments in extraembryonic tissues and in the gastrulating mouse embryo, respectively.

We have characterized the pattern of connexin expression in embryonic and extraembryonic tissues during early mouse development. In the preimplantation blastocyst, at 3.5 days post coitum (dpc), immunofluorescent signals specific for connexin31 and connexin43 proteins were present in both the inner cell mass and the trophectoderm, as shown by confocal laser scan microscopy. Immediately after implantation at 6.5 dpc, however, we find complete compartmentation of these two connexins: connexin31 mRNA and protein are expressed exclusively in cells derived from the trophectoderm lineage, whereas connexin43 mRNA and protein are detected in cells derived from the inner cell mass. This expression pattern of connexin31 and connexin43 is maintained at 7.5 dpc when the axial polarity of the mouse embryo is established. It correlates with the communication compartments in extraembryonic tissues and the gastrulating mouse embryo, respectively. The communication boundary between those compartments may be due to incompatibility of connexin31 and connexin43 hemichannels, which do not communicate with each other in cell culture.

Animals↗
PubMed1996

Expression pattern of different gap junction connexins is related to embryo implantation.

Successful implantation in mammals requires a close interaction between the embryo and the uterus. Direct cell-cell communication via gap junctions seems to play an important role in the preparation of the uterus for embryo implantation and in the regulation of trophoblast invasion. During preimplantation in the rat the gap junctional proteins connexin (cx) 26 and cx43 are suppressed. This loss of cell-cell communication seems to be important for transformation of the endometrium into the receptive phase. The suppressive effect is mediated by progesterone as demonstrated by the application of antigestagens. At implantation, however, a spatial and temporal pattern of connexin expression is induced in response to embryo recognition. cx26 is locally expressed in the uterine epithelium of the implantation chamber, cx43 in the surrounding decidua prior to invasion. With progressing invasion, the decidual cells surrounding the invading trophoblast in addition to cx43 reveal cx26. In this phase, the invasive partner, the blastocyst, is characterized by coexpression of cx43 and cx31. During trophoblast invasion however, cx31 becomes restricted to the cells of the invasive ectoplacental cone, cx43 to the embryo proper. It seems that compartmentalization of the trophoblast and the inner cell mass is established by two different connexins. During placental differentiation connexin expression switches from cx31 to cx26 and cx43, indicating the end of the invasive phase. The highly regulated pattern of connexin expression in the endometrium as well as in the trophoblast suggests a key role of this different intercellular pathways in regulating the invasion process of the trophoblast into its host tissue, the endometrium.

Animals↗
PubMed1996

Induction of connexin 32 expression by potential embryonic signals in rabbit uterine epithelium.

Connexin 32 induction is found in rabbit uterine epithelium as a response to embryo recognition. Here we have chosen this connexin 32 expression as a cell biological marker to define the type of a locally acting embryonic signal. 17 beta-estradiol, onapristone, catechol estrogen (4-hydroxy-estradiol), prostaglandins E2 and F2 alpha, db-cAMP, and glass beads as mechanical stimuli were given to pseudopregnant animals on day 4, 5 or 6 posthuman chorionic gonadotropin (hCG). The induction of connexin 32 corresponded to the time of implantation at days 6-8 post-hCG by immunohistochemistry and Northern blot analysis. Untreated pseudopregnant animals started to express connexin 32 on day 8 post-hCG. In animals treated with 4-hydroxy-estradiol, 17 beta-estradiol or prostaglandins, connexin 32 expression started 1 day earlier (day 7 post-hCG) and led to an enhanced connexin 32 expression on day 8 post-hCG compared to control animals. The antigestagen, onapristone, as well as cAMP did not alter the endogenous program. Mechanical stimuli led to a high expression of connexin 32 starting at day 7 post-hCG whereas in pregnancy the blastocyst induces connexin 32 expression from day 6 postcoitum onwards. Combination of mechanical stimuli with 17 beta-estrogen advanced the induction to day 6 post-hCG. We conclude that a mechanical stimulus in combination with 17 beta-estradiol induces connexin 32 synthesis in a similar manner as compared to the blastocyst during pregnancy.

Animals↗
PubMed1995

Expression of gap junction genes, connexin40 and connexin43, during fetal mouse development.

The expression patterns of the gap junction genes connexin40 and connexin43 have been analyzed during late mouse fetal development, i.e., at embryonic days 14.5 and 16.5, by in situ hybridization and immunofluorescence. Connexin40 was found in endothelial cells of vessels, cardiomyocytes and in developing myoblasts and myotubes. Expression of connexin40 in developing muscle fibers was strong in the back muscles and weaker in the muscles of the limbs. The number of labeled cells in the back muscle decreased with ongoing differentiation of myoblasts, in accordance with the idea that connexin40 is only expressed in the early stages of muscle cell differentiation. Within a muscle bundle, connexin40 expression was predominantly found at the outermost side where myoblasts fuse to multinucleated myotubes. In contrast, connexin43 exhibits a wide and complex pattern of expression in fetal mouse development. It is found in organs originating from all three germ layers, such as epidermis, heart, lung, muscle, kidney and gut. Connexin43 transcript and protein were very abundant in tissues that had been undergoing inductive interactions, e.g., the inner enamel epithelium of the teeth, the glomeruli of the kidneys and the infundibulum forming the neural part of the pituitary gland. Very high connexin43 expression was found in the embryonic meninges (dura mater) and in the fetal adrenal cortex. During keratinocyte differentiation connexin43 mRNA expression decreased, being much stronger in the stratum basale than in stratum granulosum. No obvious discrepancy between the amount of mRNA and protein of either connexin was noticed, suggesting that there is no specific translational regulation at these developmental stages.

Animals↗
PubMed1995

Expression of gap junction connexins in the human endometrium throughout the menstrual cycle.

Expression of connexins, the proteins which comprise gap junction channels, is regulated by ovarian hormones in the female reproductive tract of rodents. In order to determine if these hormones also affect connexin expression in the human uterus, the distribution patterns of different connexins (cx26, cx32, cx43) were investigated by immunohistochemistry in human endometrial tissue collected throughout the menstrual cycle. During the early proliferative phase of the cycle extremely low staining for connexin 43 was observed and connexin 26 antigens could not be detected. An increase in the amount of connexin 43 in stromal cells and of connexin 26 in glandular and luminal epithelial cells was seen from days 11-15 of the cycle. Following ovulation, the expression of both connexins was suppressed and was completely abolished in the late secretory phase. Weak staining for connexin 32 was found mainly in the late proliferative and the early secretory phase and was restricted to the basal membrane region of the glandular cells. These results suggest that the different connexins could represent cell biological markers for the proliferation and differentiation of the human endometrium throughout the menstrual cycle.

Connexin 26↗
PubMed1994

Morphological and immunohistochemical differentiation patterns of rabbit uterine epithelium in vitro.

We describe morphological and immunohistochemical changes of uterine epithelium from immature rabbits in vitro in response to hormonal treatments, using a matrix-coated semipermeable filter. These investigations were compared to in vivo studies of uterine epithelium from immature rabbits treated with estrogen and/or progesterone. In vitro, polarization of the epithelium seems to be best developed under progesterone dominance, and the pattern of cell organelles is similar to those seen in vivo. Two types of apical protrusions could be observed in cultures treated with progesterone, some shaped like domes, containing cell organelles, and some irregular in shape with small lucent vesicles. Both types of apical differentiation are typical for the in vivo situation. In vitro, estrogen leads to a more pseudostratified growth pattern of the cells. They develop apical protrusions with big vesicles probably containing mucin, as in vivo. Treatment with both steroid hormones leads to a heterogeneous response of the uterine epithelial cells in culture, some cells responding more to the estrogen, others to the progesterone whereas in vivo the progesterone-dominant features are obvious. Immunohistochemistry of uteroglobin in monensin-treated cultures gives evidence for uteroglobin secretion in all cultures, but to a lesser extent in the untreated, and this is strongly increased in cultures treated with estrogen and progesterone. These results correspond to observations made in vivo. This in vitro cell culture method seems therefore to provide a useful model for investigating the regulatory mechanisms of sexual steroid hormones and the cell biology of uterine receptivity.

Animals↗
PubMed1994

Regulation of connexin26 and connexin43 expression in rat endometrium by ovarian steroid hormones.

A distinct spatial and temporal pattern of connexin26 and connexin43 (cx26 and cx43) expression was observed in the rat endometrium in response to embryo implantation; however, connexin expression was suppressed during the preimplantation period. Pseudopregnant rats did not show connexin mRNA, while artificial decidualization induced by a scratch led to a strong expression of cx26 and cx43 in the endometrium of these animals. In order to examine the regulatory effects of ovarian steroid hormones on connexin expression, ovariectomized rats were treated with progesterone (P) and/or estradiol-17 beta (E2). Untreated, ovariectomized animals expressed mRNA for cx43, but not for cx26. Endometrial expression of mRNA for both connexins was strongly enhanced by E2 treatment; immunolabeling revealed protein for cx26 in the uterine luminal epithelial cells and for cx43 in the uterine stromal cells. P treatment, either alone or in combination with E2, suppressed expression of connexin mRNA. P suppression in the presence of E2 was reversible when P was withdrawn. When administered on Days 0-2 of pregnancy, the antiprogestin onapristone inhibited the effect of P and gave rise to strong expression of both connexin transcripts. These results demonstrate that expression of cx26 and cx43 in the rat uterine endometrium is differentially regulated by E2 and P during early pregnancy.

Animals↗
PubMed1993

Spatial and temporal expression of connexin26 and connexin43 in rat endometrium during trophoblast invasion.

The expression of two different gap junction transcripts, connexin26 and connexin43, has been investigated in rat endometrium during implantation and early chorionic-allantoic placentation. An induction of connexin26 protein is detected locally in the epithelium of the implantation chamber prior to trophoblast invasion. At the same stage connexin43 is found exclusively in the primary decidual zone. By the sixth day of pregnancy the uterine epithelium of the implantation chamber has degenerated and the invading trophoblast is directly confronted with the developing decidua. Connexin26 is then detected in decidual cells accompanying the invading trophoblast, whereas connexin43 has spread through the decidual tissue up into the myometrial layers. This distribution pattern of connexin43 and connexin26 is maintained until at least Day 9 of pregnancy. In decidual cells adjacent to the trophoblast both connexins seem to be coexpressed in the same gap junctional plaque. In these early stages of pregnancy a clear correlation exists between levels of connexin43 mRNA and protein expression. In regard to connexin26 mRNA a similar correlation is found from Day 5 of pregnancy onward. The amount of mRNA on Day 4, however, does not correspond to the amount of connexin26 antigen in the endometrium at that stage. The spatial and temporal expression pattern of connexin26 accompanies the process of embryo implantation in the surrounding endometrium, pointing to a specialized function of these cells.

Animals↗
PubMed1993

Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-sensitive v-SRC gene.

Loss of histotypic organization of epithelial cells is a common feature in normal development as well as in the invasion of carcinomas. Here we show that the v-src oncogene is a potent effector of epithelial differentiation and invasiveness. MDCK epithelial cells transformed with a temperature-sensitive mutant of v-src exhibit a strictly epithelial phenotype at the nonpermissive temperature for pp60v-src activity (40.5 degrees C) but rapidly loose cell-to-cell contacts and acquire a fibroblast-like morphology after culture at the permissive temperature (35 degrees C). Furthermore, the invasiveness of the cells into collagen gels or into chick heart fragments was increased at the permissive temperature. The profound effects of v-src on intercellular adhesion were not linked to changes in the levels of expression of the epithelial cell adhesion molecule E-cadherin. Rather, we observed an increase in tyrosine phosphorylation of E-cadherin and, in particular, of the associated protein beta-catenin. These results suggest a mechanism by which v-src counteracts junctional assembly and thereby promotes invasiveness and dedifferentiation of epithelial cells through phosphorylation of the E-cadherin/catenin complex.

Animals↗
PubMed1992

Characterization of gap junctions between osteoblast-like cells in culture.

The structure of gap junctions in osteoblast-like cells (OBs) and the connexins (cx) that build up these structures were characterized by ultrastructural, immunocytochemical, and molecular techniques. Ultrastructural studies revealed numerous gap junctions which were mostly located on processes of neighboring cells. Immunofluorescence labeling using two different antibodies (specific to mouse live cx26 and cx32 and to a peptide-specific rat heart gap junction protein cx43) gave evidence that in OBs, gap junctions consist mainly of cx43. The presence of cx43 in cultured OB was also confirmed by Western blot analysis. Dye-coupling with Lucifer yellow led to a staining of up to 30 neighboring cells. Parallel intracellular recordings showed that membrane potential amplitude changes (4-5 mV) are typically related to those in the coupled cells. Thus, there is morphological and functional evidence for intercellular communication between OB in culture. OBs in culture express the same connexins as observed in vivo and may serve as a model to investigate electrophysiological events in response to different stimulation signals.

Animals↗
PubMed1991

Expression of different connexin genes in rat uterus during decidualization and at term.

The expression of different connexin genes (cx26, cx32, cx37, cx43) that code for the protein subunits of gap junctions, was investigated in various uterine tissues during the estrous cycle of nonpregnant rats, in pregnant rats at decidualization and at term. Connexin gene expression was studied at the mRNA level by Northern blot hybridization and at the protein level by immunocytochemistry. In gap junctions from uterine epithelium, stroma, or myometrium, connexin 26 and/or connexin 43 are much more abundant than connexins 32 and 37. The expression of connexin 26 and 43 appears to be modulated by maternal steroid hormones. High expression of these connexins is found in developing decidual cells by day 7 to 8 post coitum; furthermore, coexpression of connexins 26 and 43 in myometrium is observed just before delivery on day 21 post coitum. In both the decidua and the myometrium, the connexin 26 protein appears to be distributed in lower abundance than connexin 43. In uterine epithelium only connexin 26 is expressed throughout all of the reproductive phases investigated. The enhanced expression of this gene correlates with higher levels of maternal estrogen both in the proestrus/estrus phase and at term. The distinct spatial and temporal pattern of expression of connexins 26 and 43 in different uterine tissues suggests a physiological role for these proteins during embryo implantation and subsequent contraction of the uterus at birth.

Animals↗
PubMed1989

Rabbit endometrium in organ culture: morphological evidence for progestational differentiation in vitro.

This communication describes conditions for long-term organotypic culture of rabbit endometrium allowing progesterone-induced transformation, as typical for early pregnancy, to continue in vitro. This system appears to compare favorably with in vitro models so far proposed for the study of hormonal control of uterine function or for the investigation of cell-biological aspects of embryo implantation. The specific aim in the presented system is to provide approximate normal epithelium-stroma interrelationships. Fragments of endometrium consisting of epithelium and stroma were obtained during early pseudopregnancy and cultured on a gyratory shaker. Morphology was investigated by light microscopy, transmission and scanning electron microscopy. During the first two days the epithelium grows over the exposed stroma regenerating a complete epithelial lining. No central necrosis is found in the stroma for up to 6 days, and the tissue keeps its organotypic architecture although certain morphological differences can be observed between regenerated versus original epithelium. In the regenerating portion a stage-specific cell differentiation and the reformation of a basal lamina are missing. Progesterone substitution preserves cell morphology and allows to maintain, in vitro, the stage-specific pattern of cell organelles. Most characteristic is the induction of extensive fusion of epithelial cells. These large symplasms are comparable in size and structure to those formed in pregnancy in the implantation chamber in vivo. Only the superficial parts of the original (not the regenerated) epithelium are capable of progesterone-induced large-scale fusion. This organotypical culture system appears to be of potential value for in vitro studies on hormone action and on endometrial receptivity for embryo implantation.

Animals↗
PubMed1988

Changes in proteins synthesized by rabbit endometrial epithelial cells following primary culture.

Morphological and biochemical changes occurring in rabbit endometrial epithelial cells when placed in culture were investigated. Cells were examined by scanning- and transmission electron microscopy and freeze-fracture. Morphologically, cultured cells are shorter and broader than the columnar epithelial cells in vivo, but retain their polarity as indicated by the presence of apical microvilli and a well-developed junctional belt. To study changes in biochemical function, proteins synthesized by cells in primary culture were analyzed by two-dimensional gel electrophoresis. Proteins were labeled during a 24-h incubation with 35S-methionine and gels examined by fluorography. The pattern of proteins changed after cells had been in culture for 48 h. On day 3 new proteins were synthesized and several protein species labeled during days 1 or 2 of culture, including uteroglobin, no longer appeared. On days 3-8 of culture the protein patterns were similar. Addition of progesterone, estradiol, prolactin, or combinations of these hormones to the culture medium for 24-144 h failed to elicit consistent changes in the pattern of labeled proteins established after 3 days of culture. Minor differences in protein patterns among unrelated cultures appear to have been derived from the original cells of the culture. These results indicate that after 48 h in primary culture, cells grown in vitro resemble endometrial epithelial cells morphologically, but no longer reflect functionally the character of epithelial cells in the uterus.

Animals↗
PubMed1988

Gap junction formation in rabbit uterine epithelium in response to embryo recognition.

Gap junction formation was studied in the uterine epithelium of nonpregnant, pregnant, and pseudopregnant rabbits in the periimplantation phase (6, 7, 8 days post coitum/post human gonadotropin injection) using freeze-fracture and immunocytochemistry as well as intracellular Lucifer yellow injection. At implantation (7 days post coitum) the uterine epithelial cells of the implantation chamber become junctionally coupled as evidenced by all three methods used. Gap junction protein (26K) becomes detectable immunocytochemically with a monoclonal antibody at 6 days post coitum in the epithelium surrounding the blastocyst, i.e., in the forming implantation chamber. The same sequence of events, starting with the presence of the gap junction protein before cell-to-cell coupling becomes evident, was observed in the blastocyst-free segments 1 day later. In contrast, uterine epithelium of nonpregnant and pseudopregnant animals in comparable phases shows an extremely low degree of coupling. The presence of the blastocyst is a necessary condition for the induction of gap junctions as demonstrated by unilateral pregnancy produced by tubal ligation. Thus, gap junction formation is one of the first maternal responses to a locally acting signal of the blastocyst.

Animals↗