Segregation during cleavage in the mammalian embryo? A critical comparison of whole-mount/CLSM and section immunohistochemistry casts doubts on segregation of axis-relevant leptin domains in the rabbit.

Segregation of certain cytoplasmic molecules during cleavage and blastocyst formation that was previously reported to occur in the human and the mouse (Antczak and Van Blerkom Mol Hum Reprod 3:1067-1086, 1997; Antczak and Van Blerkom Hum Reprod 14:429-447, 1999) has been reinvestigated in the rabbit model. Additional methodology was used and two approaches were compared: (1) whole-mount immunohistochemistry followed by confocal laser scanning microscopy (WM-IHC/CLSM) versus (2) IHC performed on histological sections of resin-embedded material (S-IHC). This study concentrates on leptin and cytoskeletal proteins (actin and cytokeratins). With S-IHC, leptin was localized predominantly on the surface of blastomeres which is facing the perivitelline space, and in the extracellular embryonic coats, without any polar asymmetry being detectable along (presumptive) embryonic axes. A polar distribution of leptin with a pattern that could be interpreted as predictive of the prospective embryonic-abembryonic axis was seen only with WM-IHC/CLSM, not with S-IHC, although the latter gave excellent resolution. With both techniques, no differences between blastomeres were detected with respect to actin and cytokeratin patterns, an increased expression of cytokeratin in trophoblast cells occurring no earlier than at blastocyst formation. Artifacts that can occur with the two methodological approaches are critically discussed, as is the possible significance of the findings for theories on the differentiation of trophoblast versus embryoblast and on axis formation in early mammalian development. It is concluded that these data call for cautioning when studying distribution patterns of diffusible molecules with WM-IHC/CLSM technology, whereas patterns obtained with S-IHC are more reliable. Specifically these data cast doubts on previous claims that leptin IHC would allow to monitor cytoplasmic domain segregation occurring during cleavage as an element of early embryonic pattern/axis formation.

Influences of extracellular matrix and of conditioned media on differentiation and invasiveness of trophoblast stem cells.

Embryo implantation in the human and rodents relies on the trophoblast's ability to invade into the uterine stroma, partly depending on proteinases degrading components of basement membrane and underlying extracellular matrix (ECM). We have utilized mouse trophoblast stem (TS) cells (Science, 1998, 282:2072) to study trophoblast invasion and trophoblast-ECM interactions in vitro. On plastic in fibroblast-conditioned medium containing fibroblast growth factor (FGF)-4 and heparin, the cells remain proliferative but display increased differentiation in media without these components. Marker gene expression (Eomes, Pl-1, Tpbp) and invasion assays showed that TS cells exhibit increased invasive capacity when differentiating into giant cells and spongiotrophoblasts in unconditioned media without FGF-4 and heparin. Concomitantly, an up-regulation of matrix metalloproteinases (MMP)-9 and -14 was observed. Culture on gels of the basement membrane-like Matrigel resulted in striking changes in morphology and gene expression. Differentiating TS cells invaded into this ECM in a three-dimensional culture, while in turn ECM contact enhanced differentiation of TS cells and up-regulated the expression of MMP-9 and its tissue inhibitor (TIMP)-3. These findings implicate that the TS cell culture system used in this study can be utilized as a model for studying the regulation of trophoblast-ECM interactions, differentiation, and invasion in vitro.

A clock network for geodesy and fundamental science.

Leveraging the unrivalled performance of optical clocks as key tools for geo-science, for astronomy and for fundamental physics beyond the standard model requires comparing the frequency of distant optical clocks faithfully. Here, we report on the comparison and agreement of two strontium optical clocks at an uncertainty of 5 × 10(-17) via a newly established phase-coherent frequency link connecting Paris and Braunschweig using 1,415 km of telecom fibre. The remote comparison is limited only by the instability and uncertainty of the strontium lattice clocks themselves, with negligible contributions from the optical frequency transfer. A fractional precision of 3 × 10(-17) is reached after only 1,000 s averaging time, which is already 10 times better and more than four orders of magnitude faster than any previous long-distance clock comparison. The capability of performing high resolution international clock comparisons paves the way for a redefinition of the unit of time and an all-optical dissemination of the SI-second.

Epithelial cell polarity and embryo implantation in mammals.

At embryo implantation we are confronted with the fact that uterine and trophoblast epithelium make contact via their apical cell membranes. This epithelium-epithelium adhesion leading to definitive attachment of the embryo to the uterine wall, however, is far from being trivial and has been called a cell biological paradox. It has been proposed that some of the molecular events involved in epithelium-to-mesenchyme transformation might play a role in the interaction between uterine cells and trophoblast. As a mechanism to achieve uterine epithelium adhesiveness for trophoblast it is postulated that uterine cells partially modulate their epithelial phenotype. Data from recent in vitro experiments give support to this hypothesis and suggest that loss of apical-basal cell polarity might prepare the apical cell pole of uterine epithelium for cell-to-cell contact with trophoblast in vivo.

Adhesiveness of the apical surface of uterine epithelial cells: the role of junctional complex integrity.

Embryo implantation necessitates that the apical plasma membrane of uterine epithelial cells acquires adhesiveness. Recent studies have indicated that modulation of a major element of the epithelial phenotype, i.e. apical-basal cell polarity, might be critical in this respect. Here, we analyze polar characteristics of nonadhesive vs. adhesive uterine epithelial cell lines focusing on cytoskeletal-junctional interactions that may play a role in regulating adhesiveness of the apical plasma membrane. HEC-1-A is a human uterine epithelial cell line exhibiting nonadhesive properties of its apical surface for trophoblast, whereas RL95-2 represent another such cell line exhibiting adhesive properties enabling trophoblast attachment. Homotypic intercellular contacts and functionally related proteins, i.e. ZO-1, E-cadherin, alpha-catenin, beta-catenin, plakoglobin, and desmoplakin 1, were examined by transmission electron microscopy, immunocytochemistry, confocal laser scanning microscopy, and immunoprecipitation techniques. In addition, details of actin filament architecture were studied after phalloidin labeling. While nonadhesive HEC-1-A exhibited the well-known pattern of cell-to-cell contacts of polarized epithelial cells, adhesive RL95-2 showed a lack of ZO-1 expression, tracer leakiness of the paracellular pathway, and atypical features in adherens junctions: E-cadherin, alpha-catenin and plakoglobin were colocalized in all plasma membrane domains and beta-catenin was localized in lateral membrane domains. Immunoprecipitations showed in both cell lines the presence of two different E-cadherin-catenin complexes, one composed of E-cadherin, alpha-catenin and beta-catenin, and the other of E-cadherin, alpha-catenin and plakoglobin. Concerning RL95-2 these data indicate that E-cadherin/plakoglobin complexes are randomly distributed, whereas E-cadherin/beta-catenin complexes are laterally localized in these cells. Additionally, the actin-based cytoskeleton of RL95-2 lacked a polar organization. With respect to the intermediate filament-desmosome system, both cell types expressed desmoplakin I, but the vast majority of RL95-2 lacked well-formed desmosomes as demonstrated by electron microscopy. It is concluded that modulation of tight junctions and/or remodelling of adherens junctions, e.g. differential distribution of E-cadherin/plakoglobin complexes and E-cadherin/beta-catenin complexes, are correlated with the development of apical adhesiveness of human uterine epithelial cells. This model system should allow to test experimentally whether this correlation is due to any causal function in the development of epithelial cell polarity.

Cell adhesion to the apical pole of epithelium: a function of cell polarity.

Human uterine epithelium displays a distinct polarized organization with apical, lateral, and basal plasma membrane domains. Although non-adhesive throughout most of the menstrual cycle, epithelial cells allow attachment of trophoblast cells to their apical pole during embryo implantation. A recent hypothesis postulates that epithelial cells turn off genes for apical-basal polarity and turn on genes for a more mesenchyme-like phenotype allowing cell-cell interaction with trophoblast. Using an in vitro assay human uterine cell lines (RL95-2, HEC-1-A, AN3-CA) were selected on the basis of adhesiveness for trophoblast-type cells (JAR). Subsequently, uterine cells were examined for epithelium-specific ultrastructure using transmission electron microscopy, and for the expression of E-cadherin, alpha 6-, beta 1-, beta 4-integrin subunits and cytokeratin using immunocytochemistry, confocal laser scanning microscopy, and surface replication technique. HEC-1-A monolayers are non-adhesive for JAR cells and appear highly polarized expressing E-cadherin, alpha 6-, beta 1-, beta 4-integrin subunits, and cytokeratin. Both, integrins and E-cadherin, are present at the lateral membrane. RL95-2 monolayers which are adhesive for JAR cells appear non-polarized. Like HEC-1-A cells, RL95-2 cells express E-cadherin, alpha 6-, beta 1-, and beta 4-integrin subunits, and cytokeratin. In contrast to HEC-1-A cells, integrins and E-cadherin are distributed at the entire cell surface. AN3-CA monolayers are non-adhesive for JAR cells and appear non-polarized. Cells lack epithelial-specific markers such as keratin and E-cadherin. They show only low expression of alpha 6-, beta 1-integrin subunits and lack beta 4-integrin subunit. Conversely, they express vimentin. Thus, modulation of the epithelial phenotype of uterine cells, i.e. loss of apical-basal polarity, might prepare the apical cell pole for cell-cell interaction with trophoblast. However, loss of cell polarity would not lead to enhancement of adhesiveness for trophoblast if accompanied by a loss of epithelium-specific adhesion molecules.

The role of matrix contact and of cell-cell interactions in choriocarcinoma cell differentiation.

Cell differentiation is supported much better by gels of extracellular matrix than by the same matrix provided as a rigid substrate. Many cell types including normal and malignant trophoblast cells, however, form multicellular multilayered aggregates on matrix gels with increased cell-to-cell contacts as compared to regular monolayers on rigid matrix substrates. In such cultures, it remained open, so far, whether stimulated expression of differentiation markers is caused by enhanced cell-to-cell communication or is displayed only by cells in direct contact to the gel. Therefore, choriocarcinoma cells (BeWo) were grown as aggregates: (a) on gels of the basement membrane-like Matrigel, (b) on plastic coated with poly-HEMA, or (c) as aggregates (spheroids) in suspension culture. Production of the differentiation marker chorionic gonadotropin was stimulated significantly in aggregates attached to gels of Matrigel or to the poly-HEMA substrate but not in suspended spheroids. With respect to cell-cell communications, however, expression of E-cadherin mRNA was not altered in any type of aggregates, as compared to control cultures on plastic. The expression of connexin43 mRNA (not of connexin26) was increased only in suspended spheroids, while microinjection of the fluorescent dye Lucifer Yellow suggested that cell communication via gap junctions was absent from cells grown as monolayers and was not induced in any type of aggregate. When cells were grown on gels of Matrigel, the relevance of direct cellular contact to the substrate for differentiation was analyzed by immunohistochemistry. Trophoblastic differentiation markers (chorionic gonadotropin, placental lactogen, placenta-type alkaline phosphatase, and pregnancy-specific glycoprotein beta 1) as well as the proliferation marker Ki-67 were not preferentially expressed in cells that were in contact with the gel. Similar random distributions of all these markers were also observed in spheroids cultured in suspension. The distributions of several matrix molecules and of different integrins were comparable between aggregates on matrix gels and those in suspension culture. According to these data, cell-cell communication appears to play a subordinate role for cytodifferentiation in cell aggregates on matrix gels, so that substrate anchorage and physical properties of the substrate may be the decisive factors. Interestingly, however, direct contact to the substrate does not seem to be essential for the stimulation of differentiation in cells on matrix gels. The results are discussed in the context of the "tensegrity"-model for cell-matrix interactions in which proper mechanical properties of the substrate are important for the regulation of cell differentiation by allowing a balanced integrity of external and cell-internal tensile forces.

Identification of differentially expressed genes in human trophoblast cells by differential-display RT-PCR.

Molecular mechanisms controlling human trophoblast invasiveness are still poorly understood. In the present investigation, mRNA patterns of trophoblast cells isolated from first trimester (high invasiveness) and term placentae (no/low invasiveness) were compared by differential-display reverse transcriptase polymerase chain reaction (DDRT-PCR) revealing differential expression of numerous genes. Of 18 differentially expressed DDRT-PCR products analysed, 11 were unknown, four showed homologies with expressed sequence tag sequences, and three others were homologous to integrin-beta1, ATP-synthetase U6 (both showing higher expression in first trimester) or to aldose-reductase (higher expression at term), respectively. One of the unknown transcripts (PBK1, accession number: AJ007398) was cloned from a first trimester placenta cDNA library and was characterized. The 1908-bp gene fragment contains an open reading frame of 1551 bp and an Alu-sequence in the 3' non-coding region. According to Northern blot analysis on JAr choriocarcinoma cells, the fragment is close to full-length cDNA. By in situ hybridization, PBK1 was detected only in first trimester but not term placentae in the proximal parts of cell islands and in closely adjacent villous cytotrophoblast. This expression pattern suggests that the newly identified molecule, PBK1, could be involved in the regulation of proliferation/ differentiation and potentially in invasion of trophoblast cells.

Retinoic acid decreases attachment of JAR choriocarcinoma spheroids to a human endometrial cell monolayer in vitro.

The ability of retinoic acid (RA) to modulate attachment of JAR choriocarcinoma multicellular spheroids to monolayers of a human uterine epithelial cell line (RL95-2) was examined using a centrifugal force-based adhesion assay. Exposure of choriocarcinoma spheroids to RA (10(-7) to 10(-5) M) over a 3-day culture period resulted in a dose-dependent decrease of attachment. Significant decreases in attachment were detected after 30 min (75 per cent versus 25 per cent) and 1 h (92 per cent versus 26 per cent) of confrontation-culture between choriocarcinoma and uterine cells for control versus 10(-5) M RA; by 5 h 100 per cent spheroid attachment was detected in all treatment groups. RA had no effect on cell proliferation in JAR spheroids, but 10(-5) M RA treatment induced a fivefold increase in secretion of human chorionic gonadotrophin (hCG), a known marker of conversion of cytotrophoblast to syncytiotrophoblast-like cells. These findings demonstrate that RA modulates cellular attachment and differentiation in choriocarcinoma spheroids in vitro.

Adhesion and invasion of three human choriocarcinoma cell lines into human endometrium in a three-dimensional organ culture system.

A novel in vitro model was developed to study attachment and invasion of choriocarcinoma cell spheroids using pre-cultured secretory phase human endometrium as a host tissue. During pre-culturing in shaker culture human endometrium had regenerated a complete epithelial covering and had shed cells damaged during explantation. Spheroids of three human choriocarcinoma cell lines (BeWo, Jeg-3, JAr) which displayed linear growth in culture and produced placental hormones were used in this study as models for trophoblast behaviour. Morphological differences were noted in the spheroids from the three choriocarcinoma cell lines; BeWo and Jeg-3 spheroids exposed flattened and more differentiated cells on their surfaces while superficial cells in JAr spheroids maintained their cytotrophoblast-like morphology. Spheroids from all three cell lines were proven to be invasive in a general invasion assay using embryonic chick heart fragments, with JAr spheroids being the most aggressive. When spheroids were confronted with pre-cultured re-epithelialized endometrial fragments, however, Jeg-3 spheroids showed the highest incidence of attachment (52%) and the greatest amount of invasion into the underlying stroma. BeWo spheroids also attached (37%) and penetrated the epithelium, but did not invade into the stroma. JAr spheroids showed a minor degree of attachment (12%) and little or no invasion into the stroma. These results show that the three choriocarcinoma cell lines, although all invasive in a general invasion assay, differ in adhesion to uterine epithelium and invasion into endometrial stroma. This model offers opportunities for studying mechanisms of trophoblast adhesion and invasion, using human endometrium as the natural host tissue.

The dynamic structure of rabbit blastocyst coverings. II. Ultrastructural evidence for a role of the trophoblast in neozona formation.

The neozona is the innermost layer of the complex blastocyst coverings of the rabbit and is located between the mucoprotein layer and the trophoblast. As shown previously the neozona begins to replace the zona pellucida from the 4th day post coitum (d p.c.) on (Denker and Gerdes 1979). In the present study, rabbit blastocyst coverings were checked for regional differences in their composition, comparing the embryonic and the abembryonic pole of the blastocyst, at 5 and 6 d p.c. These two stages were chosen because at 5 d p.c. a complete trophoblast layer is still present at both the embryonic disc (Rauber's layer) and the extraembryonic regions (mural and abembryonic pole trophoblast), whereas at 6 d p.c. Rauber's layer has largely degenerated. Correlation of regional differences in blastocyst coverings structure with presence or absence of an intact trophoblast is taken as suggestive evidence for a role of the trophoblast in the formation or the structural modification of blastocyst coverings components. Blastocysts of both stages were fixed in glutaraldehyde with and without ruthenium red and processed for TEM. The neozona was found to be almost equally well developed in all regions at 5 d p.c. On contrast, at 6 d p.c. (Rauber's layer defective) the neozona is consistently found to be much thinner at the embryonic disc than in the extraembryonic regions where the trophoblast is still intact. This is the first report on regional differences of the structural composition of blastocyst covering within the same blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

The dynamic structure of rabbit blastocyst coverings. III. Transformation of coverings under non-physiological developmental conditions.

Under physiological conditions the zona pellucida disappears in the rabbit between Day 3 and early Day 4 post coitum (p.c.) and is replaced by a new layer, the neozona. The dissolution of the zona pellucida and the formation of the neozona was investigated in three different experimental approaches, all of them characterized by non-physiological developmental conditions for the embryo: Prevention of embryo migration from the oviduct into the uterus by postcoital (48 h p.c.) tubal ligation, in vitro culture, and asynchronous embryo transfer into uteri of recipient rabbits. Embryos of age 2 1/2, 3, 4 and 4 1/2 days p.c. were cultured for 12 to 72 h. The media used for in vitro culture were supplemented with BSA, serum or with uterine secretions that were collected either synchronously or asynchronously to the developmental stage of the cultured embryos. Three-day-old embryos were transferred into uteri of pseudopregnant foster rabbits of either synchronous (Day 3) or asynchronous stages (Day 0, 2, 4, 5, 6) and were recovered 24 to 72 h after transfer. The transformation of the coverings was evaluated by light and transmission electron microscopy. The dissolution of the zona pellucida was greatly disturbed in tube-locked embryos, and in cultured embryos if standard protein supplements (BSA or serum) had been used for in vitro culture. In many cases the zona was still completely preserved after 2 or 3 days in culture, at a time when it normally would have already been replaced by the neozona in vivo. The dissolution in vitro, however, progressed incomparably better if the culture medium had been substituted with synchronous or asynchronous uterine secretions. The formation of the neozona could not be verified in cultured blastocysts. After embryo transfer, the dissolution of the zona pellucida was completed in most cases by 2 days after transfer, irrespective of the recipients' progestational stage. Present results indicate that uterine components are essential for the dissolution of the rabbit zona pellucida. These components appear to be present in the uterine cavity constitutively, i.e. independently of the uterine progestational transformation, and need not be in synchrony with the embryo's developmental stage for dissolution of the zona. Normal formation of the neozona does not take place under the non-physiological developmental conditions of in vitro culture.

Quantitation of human choriocarcinoma spheroid attachment to uterine epithelial cell monolayers.

Adhesive interactions of trophoblast cells with the endometrium are essential for embryo implantation in the uterus. Choriocarcinoma cells, the malignant counterpart of trophoblast, show pronounced invasiveness and are of interest for model studies. We describe here an in vitro model system for the study of adhesion of human JAR choriocarcinoma multicellular spheroids to different human endometrial epithelial cell lines (RL95-2, HEC-1A, KLE, AN3-CA) grown as monolayers. Cell characterization showed JAR spheroids to secrete the placental hormones human chorionic gonadotropin and progesterone into the culture medium; distinct patterns of keratin, vimentin, and uvomorulin expression were seen in the endometrial cell lines. Spheroid attachment to endometrial monolayers was quantified using a centrifugal force-based adhesion assay, and morphology was examined by light and electron microscopy. Results showed the JAR spheroids to attach to three of the endometrial monolayers (RL95-2, HEC-1A, KLE) progressively over a 24-h period (by which time ≥80% of the spheroids attached). Significant differences in spheroid attachment were most pronounced at 5 h (RL95-2 > HEC-1A > KLE and poly-D-lysine control, i.e. 90:45:17:17% attached). JAR spheroids did not attach to the endometrial cell line AN3-CA. Morphology revealed choriocarcinoma cells to begin to intrude between the uterine RL95-2 epithelial cells at 5 h. At 24 h, this intrusive type of penetration continued to be seen only with the RL95-2 monolayer. The assay system thus identifies differences in attachment properties between choriocarcinoma cells and various endometrial cell lines and forms the basis for further studies on the molecular interactions involved.

Quantitation of human choriocarcinoma spheroid attachment to uterine epithelial cell monolayers.

Adhesive interactions of trophoblast cells with endometrium are essential for embryo implantation in the uterus. Choriocarcinoma cells, the malignant counterpart of trophoblast, show pronounced invasiveness and are of interest for model studies. We describe here an in vitro model system for the study of adhesion of human JAR choriocarcinoma multicellular spheroids to different human endometrial epithelial cell lines (RL95-2, HEC-1A, KLE, AN3-CA) grown as monolayers. Cell characterization showed JAR spheroids to s++ecrete the placental hormones human chorionic gonadotropin and progesterone into the culture medium; distinct patterns of keratin, vimentin, and uvomorulin expression were seen in the endometrial cell lines. Spheroid attachment to endometrial monolayers was quantified using a centrifugal force-based adhesion assay, and morphology was examined by light and electron microscopy. Results showed the JAR spheroids to attach to three of the endometrial monolayers (RL95-2, HEC-1A, KLE) progressively over a 24-h period (by which time > or = 80% of the spheroids attached). Significant differences in spheroid attachment were most pronounced at 5 h (RL95-2 > HEC-1A > KLE and poly-D-lysine control, i.e. 90:45:17:17% attached). JAR spheroids did not attach to the endometrial cell line AN3-CA. Morphology revealed choriocarcinoma cells to begin to intrude between the uterine RL95-2 epithelial cells at 5 h. At 24 h, this intrusive type of penetration continued to be seen only with the RL95-2 monolayer. The assay system thus identifies differences in attachment properties between choriocarcinoma cells and various endometrial cell lines and forms the basis for further studies on the molecular interactions involved.

A novel artificial substrate for cell culture: effects of substrate flexibility/malleability on cell growth and morphology.

Gels of glyoxyl agarose (GA) are evaluated as a novel flexible substrate for cell culture with physical properties comparable to extracellular matrix (ECM) gels. We show here that cells adhere well to pure GA gels; in addition, specific interactions involving matrix receptors can be studied when individual matrix molecules are bound to the gel covalently. When cells are grown on such substrates, morphology is comparable to that observed on "natural" matrix gels (reconstituted gels of collagen type I or of Matrigel): rather than being flattened as in monolayer cultures on tissue culture plastic the cells assume a rounded morphology and tend to form tissue-like aggregates. The effects of the artificial matrix gels are discussed in the context of previous publications on cell interactions with the extracellular matrix, suggesting that in addition to specific recognition of matrix molecules the physical properties of ECM by themselves can be decisive for cell differentiation. We conclude that gels of glyoxyl agarose a) provide a useful model to mimic the physical properties of matrix gels without the presence of specific adhesion factors; b) may be useful as a general, non-specific ECM allowing cells to be cultured in vitro under conditions favorable for differentiation; and c) allow to design a variety of "synthetic" ECM models composed of a chemically defined gel matrix, which can be supplemented with covalently bound molecules to be recognized by cell surface receptors.