Molecular characterization of human impacted third molars: diversification of compartments.

To gain more insight into the development of human teeth, we characterized different compartments of impacted third molars at two developmental stages by assessing expression levels of a set of genes. We considered genes known to be essential for the development of teeth and ectomesenchyme as well as genes covering characteristic features of stemness. Molars were divided into the operculum, periodontal ligament, developing pulp and, using a new approach, the pad-like tissue beneath the developing pulp. Markers for ectomesenchyme and tooth development known from rodents were assayed by semiquantitative PCR and every compartment was assigned its own signature of gene expression. The expression of markers characteristic of stem cells pointed to multipotent features. The expression patterns found shift in the course of development underscoring the relevance of these genes involved in human tooth development. The results suggest an inherent asymmetry between the developing pulp and pad-like tissue established early in tooth development. A microarray analysis of cells derived from pad-like tissue and pulp proper was performed to obtain cues regarding the consequences of tissue diversification. Both sets of data support the validity of our new approach to the subdivision of the developing tooth, by indicating a compartment-dependent commitment of isolated cells probably due to the postulated asymmetry within the developing tooth germ.

The regulatory function of the uterine epithelium for trophoblast attachment: experimental approaches.

Embryo implantation in the mammalian uterus is initiated by the formation of a direct cell-to-cell contact between the trophoblast of the blastocyst and the uterine epithelium. This process is far from trivial since apical plasma membranes of epithelial cells are normally non-adhesive. The uterine epithelium has the remarkable ability to enter, under steroid hormone control, a specific state ("receptivity") at which it can down-regulate this repellent property and can finally become apically adhesive for trophoblast (probably aided additionally by local paracrine signals). Experimental data from recent years are beginning to shed some light on the involved cell biological/molecular events. They will be discussed on the basis of concepts concerning the regulation of epithelial cell polarity and with side views on epithelial-mesenchymal transformation. Recently developed experimental in-vitro systems have allowed to detect a remarkable degree of selectivity in the interaction of trophoblast and uterine epithelium, in contrast to stroma invasion. A new approach enables us to determine actual adhesive forces between living trophoblast and uterine epithelial cells with a special modification of the atomic force microscope (force spectroscopy). The potential use of such an approach is discussed.

Calcium influx in human uterine epithelial RL95-2 cells triggers adhesiveness for trophoblast-like cells. Model studies on signalling events during embryo implantation.

RL95-2 is a human uterine epithelial cell line that exhibits adhesion competence on its apical surface for trophoblast-like JAR cells. Using confocal microscopy and an adhesion assay we have found that changes in intracellular free calcium ([Ca(2+)](i)) in RL95-2 cells are involved in binding of JAR spheroids. Impact of spheroids upon, and movement of spheroids across, monolayers of RL95-2 cells produced a transient increase in [Ca(2+)](i). Pretreatment of RL95-2 cells with the Ca(2+) channel inhibitor, diltiazem, reduced the [Ca(2+)](i) increase. Interestingly, resting of JAR spheroids on RL95-2 cells caused no detectable alterations in [Ca(2+)](i) although cell-cell bonds were formed during prolonged contact. However, separation of established bonds did produce an increase in [Ca(2+)](i) which could be reduced by the Ca(2+) channel blocker, SKF-96365, but not by diltiazem. SKF-96365 also reduced adhesion of JAR spheroids to RL95-2 cells. In all experiments, the increase in [Ca(2+)](i) was due to influx from the external medium, as it could be blocked both by removing extracellular Ca(2+) and by nickel. These results suggest that the plasma membrane of uterine RL95-2 cells contains two types of Ca(2+) channels that are involved in trophoblast adhesion, i.e. diltiazem-sensitive channels contributing to initiation of JAR cell binding and SKF-96365-sensitive channels participating in a feedback loop that controls the balance of bonds.

Interactions between trophoblast and uterine epithelium: monitoring of adhesive forces.

At embryo implantation, it is postulated that the initial contact between blastocyst and maternal tissues is by adhesion of the trophoblast to the uterine epithelium. This cell-to-cell interaction is thought to be critical for implantation, although the actual adhesive forces have never been determined. In the present study, the atomic force microscope (AFM) was used to study the adhesion between human uterine epithelial cell lines (HEC-1-A; RL95-2) and human trophoblast-type cells (JAR). Specific interaction forces of these epithelia via their apical cell poles were determined on the basis of approach-and-separation cycles. For this purpose, the AFM tip was functionalized with JAR cells, then brought to the surface of uterine epithelial monolayers and was kept in contact for different periods of time (ms, 1, 10, 20, 40 min). The approach force curves displayed repulsive interactions for both HEC-1-A and RL95-2 cells. However, RL95-2 cells (with a smooth surface structure and a thin glycocalyx) showed lower values of the repulsive regime than HEC-1-A cells (with a rough surface structure and a thick glycocalyx). After having overcome repulsive interactions, the initial contact was followed by adhesive interactions. For contact times of 20 and 40 min, RL95-2 cells, but not HEC-1-A cells, showed specific JAR binding, i.e. the separation force curves displayed repeated rupture events in the range of 1-3 nN with a distance between 7-15 microm and, thereafter, a final rupture event at a distance of up to 45 microm. These features point to the formation of strong cell-to-cell bonds. Collectively, these studies provide the first definition of interaction forces between the trophoblast and the uterine epithelium, and are consistent with the hypothesis that an RL95-2-like architecture of uterine epithelial cells, i.e. an non-polarized phenotype, is essential for apical adhesiveness for the human trophoblast.

Adhesiveness of the free surface of a human endometrial monolayer for trophoblast as related to actin cytoskeleton.

Adhesiveness of the apical (free) plasma membrane of uterine epithelial cells for trophoblast is essential for the process of human embryo implantation. As epithelial cells are normally repellent, i.e. apically non-adhesive, we argue that a remodelling of the epithelial organization from a polarized to a non-polarized phenotype might prepare the apical pole for cell-cell adhesion during the so-called receptive phase. To identify details of apical adhesiveness we examined human epithelial RL95-2 cells (RL cells) which, in contrast to other cell lines, allow trophoblast to adhere to their apical plasma membrane. To determine whether the cytoskeletal structure is functionally critical for adhesiveness for trophoblast, RL cells were treated with actin depolymerizing cytochalasin D, i.e. 0.4 microM for 120 min. Changes in adhesiveness for trophoblast were monitored with a centrifugal force-based adhesion assay. Moreover, ultrastructural features, organization of the actin network and expression of integrins, i.e. alpha 6, beta 1, beta 4, were studied using electron microscopy, confocal laser scanning microscopy and cell surface immunogold-labelling techniques. Changes in transmission of mechanical signals via integrins into uterine cells were examined using a magnetic drag force device, thereby monitoring intracellular calcium responses. The results suggest that adhesiveness of the free surface of RL cells for human trophoblast requires an intact but non-polarized actin cytoskeleton, apically localized integrins linked to actin, and calcium signalling originating at the free surface.

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.

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.

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.

Differential expression and localization of integrins and CD44 in the membrane domains of human uterine epithelial cells during the menstrual cycle.

Human uterine epithelium displays a distinctly polarized organization with basal, lateral, and apical plasma membrane domains. Although nonadhesive throughout most of the menstrual cycle, uterine epithelial cells allow attachment of trophoblast cells to their apical pole during embryo implantation. Development of the receptive state might involve expression of cell adhesion molecules and/or redistribution of such molecules with respect to their localization at the basal, lateral, and apical membrane domains of cells. Expression and distribution of alpha 1-, alpha 3-, alpha 5-, alpha 6-, beta one-, beta 3- and beta 4-integrin subunits as well as of CD44 were examined in the luminal epithelium of human endometrium by immunohistochemistry in different phases of the menstrual cycle. The luminal epithelium was found to express alpha 1-, alpha 3-, alpha 6-, beta 1-, beta 4-integrin subunits and CD44. alpha l6-Integrin subunits and CD44 displayed cycle dependency. The alpha 6-integrin subunits were detected in the basal membrane domains in all phases. However, in correlation with increasing expression during the secretory phase of cycle, these subunits newly appeared in the lateral membranes of epithelial cells. CD44 showed increased expression in the secretory phase but was always restricted to the lateral membranes. The conspicuous behavior of alpha 6-integrin subunits and CD44 is discussed with respect to its possible functional significance for embryo implantation, and in relation to a hypothesis postulating that steroid-controlled master genes direct the acquisition of the receptive state of the luminal uterine epithelium by changing elements of the apicobasal polarity of these cells.

Aortic smooth muscle cells in a three-dimensional collagen lattice culture. Evidence for posttranslational regulation of collagen synthesis.

Aortic smooth muscle cells were cultivated as monolayers on plastic or within collagen lattices with low- and high-serum supplementation, and the expression of mRNAs specific for pro alpha 1 (I) and pro alpha 1 (III) collagen were studied by slot blot hybridization. The steady-state levels of pro alpha 1 (I) and pro alpha 1 (III) collagen mRNA of cells within collagen lattices were found to be higher than those grown on plastic, although the production of collagen was lower. The degradation of pro alpha 1 (I) and pro alpha 1 (III) collagen mRNAs as revealed in the presence of actinomycin D was not affected by culturing the cells within a collagen lattice. In vitro translation assays of mRNAs of monolayer- and lattice-cultured cells showed no differences in translatability. These data suggest the involvement of posttranslational control of collagen production in collagen lattice-cultured smooth muscle cells.

Responsiveness of aortic smooth muscle cells to soluble growth mediators is influenced by cell-matrix contact.

Excessive proliferation and overexpression of collagens by smooth muscle cells (SMCs) are important features of atherogenesis. To understand the role of the extracellular matrix in the regulation of these processes, we examined proliferation and protein/collagen synthesis of SMCs in contact with a collagen matrix. Adult pig SMCs were isolated from the aortic media by collagenase digestion, subcultured as monolayers, and then embedded into a three-dimensional network of type I collagen, ie, a collagen lattice. Cells were subsequently exposed to growth-promoting media, and their behavior was observed in comparison with monolayer cultures on plastic. Treatment of monolayers with increasing concentrations of fetal calf serum resulted in activation of the cell cycle, onset of cell proliferation, and increased protein/collagen synthesis. In contrast, similar treatment of collagen lattice-cultured SMCs failed to influence cell proliferation and protein/collagen synthesis. However, stimulation of proliferation of lattice-cultured SMCs by platelet-derived growth factor-A/B was feasible; nevertheless, the rate of proliferation was modest compared with monolayers. In addition, the onset of proliferation was accompanied by a decrease in collagen synthesis of the cells. Thus, a collagenous matrix appears to suppress the responsiveness of SMCs to soluble growth mediators. It is speculated that interactions between SMCs and the extracellular matrix may modify proliferation and protein/collagen synthesis of cells not only in vitro but also in vivo during atherogenesis by making and breaking binding sites between extracellular collagen and matrix receptors.

Differential regulation of protein synthesis in smooth muscle cells from normotensive and spontaneously hypertensive rats by a three-dimensional matrix of type I collagen.

OBJECTIVE: The objective of this study was to gain insight into the metabolic and morphological properties of smooth muscle cells (SMC) from spontaneously hypertensive rats (SHR) when cultured in vivo under similar conditions. DESIGN: Three-dimensional cell-collagen systems represent living tissue equivalents in vitro and simulate natural conditions more closely than conventional monolayer cultures. METHODS: The effect of a three-dimensional matrix of type I collagen on ultrastructure, total protein and collagen synthesis and cell cycle distribution of SMC from SHR and normotensive Wistar-Kyoto (WKY) rats was studied. RESULTS: Collagen lattice-cultured SMC from SHR and WKY rats showed the synthetic phenotype, i.e. the cytoplasm was filled with organelles characteristic of secretory protein synthesis. There was a decrease in the percentage of cells in the 5 phase compared with monolayer cultures. Total protein synthesized by SMC from SHR and WKY rats in lattices was lowered compared with monolayer cultures. However, reduction of protein synthesis in SMC from SHR was less than in SMC from WKY rats. Differences in the proportion of collagen in SMC from SHR and WKY rats were not demonstrable in collagen lattice cultures. CONCLUSION: The present study suggests that a three-dimensional matrix of type I collagen may modulate total protein synthesis in SMC from SHR and WKY rats. However, cells from SHR react less to this matrix than those from WKY rats.

Collagen synthesis in cultured aortic smooth muscle cells. Modulation by collagen lattice culture, transforming growth factor-beta 1, and epidermal growth factor.

We investigated the effects of transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the protein synthesis and production of collagen in cultured smooth muscle cells (SMCs) from the aortic media of pigs. SMCs were cultured as monolayers on plastic as well as in three-dimensional collagen lattices to gain some information about the influence of a preexisting collagenous matrix on the growth factor-induced effects. A 48-hour exposure of SMCs to TGF-beta 1 at concentrations of 5 ng/ml in the presence of 1% serum caused a marked enhancement of the production of collagen and noncollagen proteins. The rate of net collagen production by SMCs exposed to TGF-beta 1 was approximately threefold higher than that of control cells. Moreover, TGF-beta 1 specifically stimulated collagen synthesis, resulting in a greater proportion of collagen in total proteins synthesized compared with controls. The preexisting matrix of collagen lattices affects the response of SMCs to TGF-beta 1 and EGF. In monolayer cultures the collagen proportion increased twofold under the influence of TGF-beta 1, whereas in collagen lattices the specific stimulation of collagen synthesis was lower. We found that EGF enhanced TGF-beta 1-induced protein production in collagen lattices but not in monolayer cultures. In addition, the protein production by SMCs was influenced differently by EGF in these culture systems. Taken together, these data show a mutual influence of growth factors and extracellular matrix components on collagen production in SMCs, thus indicating that TGF-beta 1 may be an important pathophysiological regulator of collagen metabolism in the vessel wall.

Aortic smooth muscle cells in collagen lattice culture: effects on ultrastructure, proliferation and collagen synthesis.

Adult pig smooth muscle cells (SMC) were isolated from the aortic media by collagenase digestion, subcultured as monolayer, and then re-integrated into a three-dimensional network of type I collagen. The contractile state characteristic for resident arterial wall SMC changed to the synthetic, fibroblast-like state. The cells reorganized the randomly orientated collagen fibrils causing the lattice to shrink. The influence of the extracellular matrix on the ultrastructure, the proliferation, and the collagen synthesis of these SMC embedded in the collagen lattice was investigated and compared to cells cultured in monolayer. The amount of total protein and collagens synthesized by SMC embedded in lattices was lowered as compared to monolayer cultures. Whereas total protein synthesis decreased continuously during the culture period, the proportion of collagen synthesis remained at a constant level. Although cells proliferated in lattices, proliferation was clearly slowed down as compared to monolayer cultures. The ultrastructure of entrapped synthetic state SMC was comparable to that of monolayer-cultured cells. Their cytoplasm was largely filled by elements of the endoplasmic reticulum, Golgi complexes and abundant mitochondria. With prolonged culture time, electron-dense granules as well as bodies containing whorled membranes could be found in the cytoplasm. These results indicate that synthetic state SMC can exhibit differential biosynthetic activity dependent on the actual matrix environment; cells seem to be able to sense the macromolecular composition of the extracellular matrix and to modify their production of matrix components accordingly.

[Ultrastructural and proliferation studies of human, catheter atherectomy extracted plaque material]. / Ultrastruktur- und Proliferationsuntersuchungen an humanem, durch Katheteratherektomie gewonnenem Plaquematerial.

Atherectomy with a Simpson catheter enables a detailed characterization of percutaneously removed human atheromatous plaque material. Cell biological investigations (flow-cytometric analyses, cell type-specific antibodies, immunohistological labelling methods of selected extracellular matrix proteins) are aimed at a comparative characterization of excised material from primary lesions and restenoses to yield further insight into the mechanisms of plaque formation. First results on 17 specimens would like to contribute to a better understanding of the structure and composition of the extracellular matrix and their interaction with cells in atherosclerotic plaques.

Deposition and ultrastructural organization of collagen and proteoglycans in the extracellular matrix of gel-cultured fibroblasts.

Human skin fibroblasts were cultivated within the three-dimensional space of polymerized alginate and collagen, respectively. The in vitro synthesis of collagens and proteoglycans was measured during the first 3 days of culture, and the deposition as well as the ultrastructural organization of newly synthesized extracellular matrix components were examined by electron microscopy. The amount of collagens and proteoglycans synthesized by fibroblasts, embedded in calcium alginate gels as well as in collagen lattices, was lowered as compared to monolayer cultures. Furthermore, it was found that collagen synthesis was reduced to a greater extent in alginate gels than in collagen lattices. On the contrary, total proteoglycan biosynthesis was similarly reduced either in alginate gels or in collagen lattices. At the end of a 3-day-culture period, filamentous material as well as cross-striated banded structures were found extracellularly in the alginate gel. According to their periodicity, their banding pattern, their association with polyanionic matrix components and their sensitivity towards glycosaminoglycan-degrading enzymes we could distinguish (1) sheets of amorphous non-banded material consisting of irregularly arranged filaments and containing dermatan sulfate-rich proteoglycans (type I structures), (2) sheets of long-spacing fibrils consisting of parallel orientated filaments and containing chondroitin sulfate-rich proteoglycans (= zebra bodies; type II structures), and (3) fibrillar structures with a complex banding pattern different from that of native collagen fibrils (type III structures). In fibroblasts cultured in collagen lattices, we only sporadically found depositions which are identified as type I structures. Using indirect immunoelectron microscopy and monospecific polyclonal antibodies, we localized type VI collagen in type I structures and type II structures. Type III structures can be identified as type I collagen derived as becomes obvious by comparison with segment long spacing crystallites of type I collagen.

Active immunization of rabbits against progesterone: increase in hormone levels, and changes in metabolic clearance rates and in genital tract tissues.

11 alpha-Hemisuccinyl progesterone was coupled to rabbit serum albumin and injected into intact male rabbits, and into intact ovariectomized female rabbits, for a period of 52 weeks. The ovariectomized females and the intact males showed better immune responses than the intact females. Specificity of the antisera was tested against both progesterone and the carrier protein, and against 19 other selected steroids. Antibody titres and serum levels of progesterone in all groups, and testosterones in the males, showed characteristic changes with a first maximum 12-18 weeks after the beginning of immunization, a decrease despite booster injections and a second increase after 35-40 weeks. Hormone levels were 5-12 times higher by the end of immunization than before. High antibody titres were correlated with decreased metabolic clearance rates for progesterone, reflecting the binding of the steroid to the antibody. Increased production rates of progesterone in the immunized females and of testosterone in the immunized males showed that the antibody-bound hormone was not available for feedback control. Absence of primordial follicles, hyperplasia of the Leydig cells, decreased spermatogenesis and involution of the seminal vesicle epithelium were interpreted as direct or indirect effects of the removal of free progesterone.

Mechanical confinement inhibits collagen synthesis in gel-cultured fibroblasts.

Collagen synthesis in fibroblasts cultivated in collagen lattices is known to be repressed compared to synthesis in monolayer cultures on plastic. Inhibition of synthesis is supposed to be due to interactions between the plasma membrane and adjacent collagen fibrils. To evaluate how collagen synthesis is regulated in gel-cultured cells we cultivated fibroblasts within gels of polymerized alginate in which preexisting extracellular matrix components, e.g., type I collagen fibrils, were lacking. When alginate gels were examined at the ultrastructural level, normal collagen fibrils were not observed. However, broad sheets of microfibrillar material and so-called zebra bodies were found. The amount of collagen synthesized by fibroblasts in calcium alginate gels remained constant during the entire culture time and was about 70% of that produced in monolayer-cultured cells. This value corresponded to levels found in fully retracted collagen lattices on day 7 of culture. Our data suggest that interactions between the plasma membrane and adjacent collagen fibrils are not necessary for the inhibition of collagen synthesis. Thus, we present data that mechanical confinement is capable of inhibiting collagen synthesis in fibroblasts.

Uteroglobin as progesterone-binding protein in the preimplantation uterine epithelium of the rabbit: histochemical studies.

[3H] progesterone was injected into the uterine lumen of rabbits toward the end of preimplantation period (162 h post coitum). Light-microscopic autoradiography showed accumulation of label in single cell groups of the uterine epithelium. Fluorographs of thin layer chromatograms of steroid extracts indicated the metabolization of progesterone in the uterine tissue. Incubation of uterine sections with fluorescein isothiocyanate-conjugated progesterone-rabbit serum albumin revealed binding sites for this reagent: 162 h post coitum, staining was also localized in single cell groups of the uterine epithelium. Pretreatment with a monospecific antiserum showed uteroglobin to be the binding protein.