Apical plasma membrane-bound enzymes of rabbit uterine epithelium

Summary In order to monitor changes in the apical cell membrane of rabbit uterine epithelium which are postulated to be a precondition for trophoblast attachment, the marker enzymes: alkaline phosphatase, aminopeptidase M, -glutamyl transferase and dipeptidyl peptidase IV were investigated during the periimplantation phase. Endometrium of early pregnancy (implantation chamber, interblastocyst endometrium; 5–8 days post coitum, d p.c.) was compared with specimens obtained at hCG-induced pseudopregnancy (p. hCG) to distinguish between membrane changes regulated by maternal plasma steroid hormones and such which might be induced locally by blastocyst-derived signals.All enzymes tested showed their main activity at 5 d p.c./p. hCG. The weakest reaction in this series of stages was generally found at 8 d p.c. (interblastocyst segments) or at 8 d p. hCG. In contrast to the rest of the epithelium, the implantation chamber retained high activity of dipeptidyl peptidase IV, and the activity of alkaline phosphatase even raised here again at 7 and 8 d p.c. indicating a direct local influence of the blastocyst on the luminal epithelium. The results suggest that 1) considerable changes occur in the composition of the apical plasma membrane of the uterine epithelium when the endometrium enters the receptive state, 2) the overall trend is towards a loss of apical-type characteristics of this membrane domain and 3) the changes are modulated both systemically (by plasma steroid hormone levels) and locally by signals from the implanting blastocyst.Abbreviations d p.c. days post coitum - d. p. hCG days post hCG injection - hCG human chorionic gonadotropin - aP alkaline phosphatase - ATPase adenosine triphosphatase - Ca²⁺-ATPase Ca²⁺-activated adenosine triphosphatase - APM aminopeptidase M - GGT -glutamyl transferase - DPP IV dipeptidyl peptidase IV - PCMB p-chloromercuric benzoate - DFP di-isopropylfluorophosphate - DMF dimethylformamide     

Alkaline phosphatase distribution in the plasma membrane of uterine epithelial cells is markedly altered during early pregnancy in the rat

Ultrastructural and light microscopic cytochemical methods were used to study the distribution and changes in distribution of alkaline phospatase in the apical plasma membrane of rat uterine epithelial cells during different stages of early pregnancy up to the time of attachment of the blastocyst. Reaction product generated by alkaline phosphatase (AP) was located along the apical plasma membrane at each stage investigated. However, a very different organization of reaction product was observed depending on the time during early pregnancy with a continuous pattern appearing all along the microvilli on day 1. This pattern was subsequently converted into a clumped and highly ‘patchy’ appearance around the time of blastocyst attachment by day 6 of pregnancy. This change in pattern and distribution was only seen on the luminal epithelial cells with glandular epithelial cells and blood vessels displaying an unchanging distribution.     

Stage-specific expression of the intermediate filament protein cytokeratin 13 in luminal epithelial cells of secretory phase human endometrium and peri-implantation stage rabbit endometrium

In preparation for blastocyst implantation, uterine luminal epithelial cells express new cell adhesion molecules on their apical plasma membrane. Since one mechanism epithelial cells employ to regulate membrane polarity is the establishment of specific membrane-cytoskeletal interactions, this study was undertaken to determine if new cytokeratin (CK) intermediate filament assemblies are expressed in endometrial epithelial cells during developmental stages related to blastocyst implantation. Type-specific CK antibodies were used for immunocytochemical and immunoblot analyses of 1) intermediate filament networks of the endometrial epithelium during embryo implantation in rabbits and 2) proliferative and secretory phases of the human menstrual cycle. CK18, a type I CK found in most simple epithelia, was expressed in all luminal and glandular epithelial cells of both the human and rabbit endometrium at all developmental stages analyzed; it was also strongly expressed in trophectoderm of the implanting rabbit blastocyst. In contrast, CK13, another type I cytokeratin, exhibited a regulated expression pattern in luminal, but not glandular, epithelial cells of secretory phase human and peri-implantation stage rabbit endometrium. Furthermore, in the rabbit implantation chambers, CK13 was predominantly localized at the cell apex of luminal epithelial cells, where it assembled into a dense filamentous network. These data suggest that the stage-specific expression of CK13 and a reorganization of the apical intermediate filament cytoskeleton of uterine luminal epithelial cells may play important functions in preparation for the implantation process.     

Release of alkaline phosphodiesterase I from rat kidney plasma membrane produced by the phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis

The release of plasma-membrane-bound enzymes by phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis was investigated. Among the ectoenzymes of plasma membrane tested, alkaline phosphodiesterase I was released markedly from rat kidney cortex slices, in addition to alkaline phosphatase and 5'-nucleotidase. Other membrane-bound enzymes; alanine aminopeptidase, leucine aminopeptidase, dipeptidyl peptidase, leucine aminopeptidase, dipeptidyl peptidase IV, esterase and gamma-glutamyl transpeptidase could not be liberated from the treated slices. Alkaline phosphodiesterase I was released linearly from rat kidney slices with the concentration of phosphatidylinositol-specific phospholipase C, but little enzyme was released from rat liver slices. Alkaline phosphodiesterase I separated from kidney tissue with n-butanol still retained phosphatidylinositol and was transformed into a lower molecular weight form by phosphatidylinositol-specific phospholipase C. This suggests an important function for phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membrane of rat kidney cells. The alkaline phosphodiesterase I released from rat kidney had a molecular weight of about 240,000 and an isoelectric point (pI) of 5.4. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9 and had a Km value of 0.3 mM. The enzyme was activated by Mg2+ and Ca2+, but was inhibited by EDTA. Strong inhibition took place on the addition of adenosine 5'-phosphosulfate or the nucleotide pyrophosphates, i.e., UDP-galactose and alpha, beta-methylene ATP.     

Biosynthesis and degradation of altered immature forms of intestinal dipeptidyl peptidase IV in a rat strain lacking the enzyme.

We have used a strain of rat (Fischer 344) lacking brush border membrane dipeptidyl peptidase IV activity to examine its effect on the intestinal assimilation of prolyl peptides. In addition, we have examined the biochemical basis for the enzyme deficiency. An analysis of several brush border membrane hydrolases in different regions of the small intestine demonstrates that these rats lack only dipeptidyl peptidase IV. They also have a greatly reduced ability to hydrolyze and absorb in vivo peptides of the NH2-X-Pro-Y type which are known substrates for the enzyme. Immunoblot analysis with polyclonal and monoclonal antibody indicates that the animals lack an identifiable dipeptidyl peptidase IV protein in intestinal epithelial cells. Levels and types of dipeptidyl peptidase IV mRNA were analyzed in several tissues and found to be similar to that of control animals. Biosynthetic labeling of intestinal explants revealed that two distinct forms (102 and 108 kDa) of dipeptidyl peptidase IV are initially synthesized by deficient rats, in contrast to the single protein (106 kDa) observed in normal animals. Pulse-chase labeling experiments (+/- endoglycosidase H) show that these two altered forms of dipeptidyl peptidase IV, although initially glycosylated with N-linked high mannose carbohydrate, fail to be processed to the mature complex glycosylated form and undergo intracellular degradation.     

Histochemistry of some proteases in the normal rabbit, pig and ox corneas

The distribution of activities of membrane aminopeptidases (aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), gamma-glutamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II)) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4 degrees C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4 degrees C) for the demonstration of lysosomal enzymes. In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand, APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity. Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.     

Regulation of gap junction connexins in the endometrium during early pregnancy

In mammalian species embryo implantation into uterine tissue is restricted to a limited time period, the receptive phase. For successful implantation appropriate differentiation of the receptive endometrium is under the control of ovarian steroid hormones. In addition, locally acting embryonic signals are needed to modulate the maternal environment before invasion of the trophoblast is permitted. The expression pattern of gap junction channel proteins, connexins (cx), is directly related to this process. In rodents as well as in rabbit and humans the receptive endometrium is characterized by a lack of such cell-to-cell communication channels. In the rat endometrium cx26 is suppressed in the epithelium and cx43 in the stromal compartment by maternal progesterone, a phenomenon that can be observed similarly in human endometrium. Experimental approaches revealed that both connexin genes react very sensitively to progesterone and estrogen treatment. In rat and rabbit connexin expression is induced locally in the endometrium in response to the implanting blastocyst. In both species this induction of connexins can be mimicked by a traumatic stimulus. In conclusion, suppression of connexin expression in the endometrium is a characteristic cell biological indication for receptivity in different species. The limited induction of direct cell-to-cell communication properties, probably due to locally acting blastocyst signals, seems to be a precondition for embryo implantation.     

The MAL proteolipid is necessary for the overall apical delivery of membrane proteins in the polarized epithelial Madin-Darby canine kidney and fischer rat thyroid cell lines

The MAL proteolipid has been recently demonstrated as being necessary for correct apical sorting of the transmembrane influenza virus hemagglutinin (HA) in Madin-Darby canine kidney (MDCK) cells. The fact that, in contrast to MDCK cells, Fischer rat thyroid (FRT) cells target the majority of glycosylphosphatidylinositol (GPI)-anchored proteins to the basolateral membrane provides us with the opportunity to determine the role of MAL in apical transport of membrane proteins under conditions in which the majority of GPI-anchored proteins are (MDCK cells) or are not (FRT cells) targeted to the apical surface. Using an antisense oligonucleotide-based strategy to deplete endogenous MAL, we have observed that correct transport of apical transmembrane proteins associated (HA) or not (exogenous neurotrophin receptor and endogenous dipeptidyl peptidase IV) with lipid rafts, as well as that of the bulk of endogenous apical membrane, takes place in FRT cells by a pathway that requires normal MAL levels. Even transport of placental alkaline phosphatase, a GPI-anchored protein that is targeted apically in FRT cells, was dependent on normal MAL levels. Similarly, in addition to the reported effect of MAL on HA transport, depletion of MAL in MDCK cells caused a dramatic reduction in the apical delivery of the GPI-anchored gD1-DAF protein, neurotrophin receptor, and the bulk of membrane proteins. These results suggest that MAL is necessary for the overall apical transport of membrane proteins in polarized MDCK and FRT cells.     

A comparison of membrane enzymes of human and pig oesophagus; the pig oesophagus is a good model for studies of the gullet in man

Summary The distribution and relative catalytic activities of five plasma membrane enzymes (alkaline phosphatase, dipeptidyl peptidase IV, γ-glutamyl transpeptidase, microsomal alanyl aminopeptidase and glutamyl aminopeptidase) were examined in human and pig oesophagus. In both species, alkaline phosphatase activity occurred in basal and suprabasal cells of the epithelium and in capillaries. Stromal cells in the human submucosa were particularly reactive. Dipeptidyl peptidase IV was present in blood vessels and capillaries in man and pig and in submucous glands in the pig. The enzyme was also present in both species in the lamina propria cells immediately adjacent to the epithelial basal lamina. In the human, γ-glutamyl transpeptidase occurred in the epithelial basal cells and in isolated basal and lower prickle cells in the pig. Stromal cells in the human submucosa were strongly reactive and capillaries in the muscularis propria in both species moderately active. Microsomal alanyl aminopeptidase was detected in lamina propria cells adjacent to the epithelial basal cell layer in man and pig and at the apices of mucous cells in pig submucous glands. Weak glutamyl aminopeptidase activity was confined to capillaries in both species. The findings of this study, along with the ready availability of pig oesophagus, suggest that the pig may be a suitable model for studies of the gullet in man.     

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.     

Extraenzymatic functions of the dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration and apoptosis

The dipeptidyl peptidase IV gene family contains the four peptidases dipeptidyl peptidase IV, fibroblast activation protein, dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Dipeptidyl peptidase IV and fibroblast activation protein are involved in cell-extracellular matrix interactions and tissue remodeling. Fibroblast activation protein is upregulated and dipeptidyl peptidase IV is dysregulated in chronic liver disease. The effects of dipeptidyl peptidase 8 and dipeptidyl peptidase 9 on cell adhesion, cell migration, wound healing and apoptosis were measured by using green fluorescent protein fusion proteins to identify transfected cells. Dipeptidyl peptidase 9-overexpressing cells exhibited impaired cell adhesion, migration in transwells and monolayer wound healing on collagen I, fibronectin and Matrigel. Dipeptidyl peptidase 8-overexpressing cells exhibited impaired cell migration on collagen I and impaired wound healing on collagen I and fibronectin in comparison to the green fluorescent protein-transfected controls. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 enhanced induced apoptosis, and dipeptidyl peptidase 9 overexpression increased spontaneous apoptosis. Mechanistic investigations showed that neither the catalytic serine of dipeptidyl peptidase 8 or dipeptidyl peptidase 9 nor the Arg-Gly-Asp integrin-binding motif in dipeptidyl peptidase 9 were required for the impairment of cell survival, cell adhesion or wound healing. We have previously shown that the in vitro roles of dipeptidyl peptidase IV and fibroblast activation protein in cell-extracellular matrix interactions and apoptosis are similarly independent of catalytic activity. Dipeptidyl peptidase 9 overexpression reduced beta-catenin, tissue inhibitor of matrix metalloproteinases 2 and discoidin domain receptor 1 expression. This is the first demonstration that dipeptidyl peptidase 8 and dipeptidyl peptidase 9 influence cell-extracellular matrix interactions, and thus may regulate tissue remodeling.     

Histochemistry of some proteases in the normal rabbit,pig and ox corneas

Summary The distribution of activities of membrane aminopeptides (aminopeptidases M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), -gluamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4°C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4°C) for the demonstration of lysosomal enzymes.In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity.Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.     

Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV

Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 respectively. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar K(i) values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.     

Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen

Dipeptidyl peptidase IV is an exopeptidase found in the serum and in plasma membranes of most animal tissues. The role of this enzyme in cell-matrix interaction of BHK cells and hepatocytes grown on collagen-coated surfaces was investigated by three different approaches. 1) Glass surfaces were derivatized with bovine serum albumin which resulted in a cell-repulsing substratum. When it was further modified with Gly-Pro-Ala tripeptide, which is a substrate for dipeptidyl peptidase IV, BHK fibroblasts spread on it rapidly. The spreading could be inhibited by addition of free Gly-Pro-Ala or other substrates of the enzyme as well as by an inhibitor peptide Val-Pro-Leu. It was not influenced by tripeptides which were neither substrates nor inhibitors of dipeptidyl peptidase IV. 2) The addition of Gly-Pro-Ala to seeded cells slowed down the initial process of cell spreading on denatured collagen in the presence of fibronectin. The presence of both collagen and fibronectin was a necessary precondition for the spreading of cells in a manner sensitive to Gly-Pro-Ala. 3) Antiserum raised against mouse liver dipeptidyl peptidase IV added to the medium delayed the spreading of rat hepatocytes on denatured collagen in the presence of fibronectin in a manner similar to when Gly-Pro-Ala was added to the medium. These observations lead to the conclusion that plasma membrane dipeptidyl peptidase IV may be involved in the initial phase of fibronectin-mediated cell spreading on collagen.     

Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions

Using a mutant hepatocyte cell line in which E-cadherin and beta-catenin are completely depleted from the cell surface, and, consequently, fail to form adherens junctions, we have investigated adherens junction requirement for apical-basolateral polarity development and polarized membrane trafficking. It is shown that these hepatocytes retain the capacity to form functional tight junctions, develop full apical-basolateral cell polarity, and assemble a subapical cortical F-actin network, although with a noted delay and a defect in subsequent apical lumen remodeling. Interestingly, whereas hepatocytes typically target the plasma membrane protein dipeptidyl peptidase IV first to the basolateral surface, followed by its transcytosis to the apical domain, hepatocytes lacking E-cadherin-based adherens junctions target dipeptidyl peptidase IV directly to the apical surface. Basolateral surface-directed transport of other proteins or lipids tested was not visibly affected in hepatocytes lacking E-cadherin-based adherens junctions. Together, our data show that E-cadherin/beta-catenin-based adherens junctions are dispensable for tight junction formation and apical lumen biogenesis but not for apical lumen remodeling. In addition, we suggest a possible requirement for E-cadherin/beta-catenin-based adherens junctions with regard to the indirect apical trafficking of specific proteins in hepatocytes.     

Blastocyst implantation in the Chinese hamster (Cricetulus griseus)

Embryonic development of the Chinese hamster (Cricetulus griseus) was studied from the onset of implantation to the formation of the parietal yolk sac placenta. Implantation began on day 6 of pregnancy, when the embryo became fixed to the uterine luminal epithelium. At this time there was no zona pellucida, and microvilli of the trophoblast and uterine epithelium were closely apposed. Stromal cells immediately adjacent to the implantation chamber began to enlarge and accumulate glycogen. By day 7 the mural trophoblast penetrated the luminal epithelium in discrete area. The trophoblast appeared to phagocytize uterine epithelial cells, although epithelium adjoining the points of penetration was normal. In other areas nascent apical protrusions from the uterine epithelium indented the surface of the trophoblast. The epiblast had enlarged and both visceral and parietal endoderm cells were present. The well-developed decidual cells were epithelioid and completely surrounded the implantation chamber. On day 8 the uterine epithelium had disappeared along the mural surface of the embryo. The embryonic cell mass was elongated and filled the yolk sac cavity. Reichert's membrane was well developed. The uterine epithelial basal lamina was largely disrupted, and the trophoblast was in direct contact with decidual cells. Primary and secondary giant trophoblast cells were present and in contact with extravasated maternal blood. The mural trophoblast formed channels in which blood cells were found in close proximity to Reichert's membrane. Decidual cells were in contact with capillary epithelium and in some cases formed part of the vessel wall. Structural changes occurring in the embryo and endometrium during implantation in the Chinese hamster are described for the first time in this report and are compared to those described for some other myomorph rodents.     

子宫内膜接受性的建立和分子调控

胚泡着床是一个复杂的生理过程,依赖于胚泡发育和子宫内膜获得接受能力的同步进行.着床只发生在具有接受性的子宫内膜,而子宫内膜只在很短的时间内具有接受性.被称为"着床窗口".子宫内膜接受性的建立涉及子宫腔上皮的形态学改变,以及甾类激素和许多细胞因子复杂的调控作用.本文综述了子宫内膜接受性的建立及其分子调控.     

Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters

Reabsorption of filtered solutes from the glomerular filtrate and excretion of waste products and xenobiotics are the main functions of the renal proximal tubular (PT) epithelium. A human PT cell line expressing a range of functional transporters would help to augment current knowledge in renal physiology and pharmacology. We have established and characterized a conditionally immortalized PT epithelial cell line (ciPTEC) obtained by transfecting and subcloning cells exfoliated in the urine of a healthy volunteer. The PT origin of this line has been confirmed morphologically and by the expression of aminopeptidase N, zona occludens 1, aquaporin 1, dipeptidyl peptidase IV and multidrug resistance protein 4 together with alkaline phosphatase activity. ciPTEC assembles in a tight monolayer with limited diffusion of inulin-fluorescein-isothiocyanate. Concentration and time-dependent reabsorption of albumin via endocytosis has been demonstrated, together with sodium-dependent phosphate uptake. The expression and activity of apical efflux transporter p-glycoprotein and of baso-lateral influx transporter organic cation transporter 2 have been shown in ciPTEC. This established human ciPTEC expressing multiple endogenous organic ion transporters mimicking renal reabsorption and excretion represents a powerful tool for future in vitro transport studies in pharmacology and physiology.     

Rabbit endometrium in organ culture: Morphological evidence for progestational differentiation in vitro

Summary This communication describes conditions for long-term organotypic culture of rabbit endometrium allowing progesterone-induced transformation, as typcial 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.Abbreviations d.p.c. dies post coitum - d p. hCG days after injection of hCG (dies post injectionem hCG) - FBS fetal bovine serum - hCG human chorionic gonadotropin Preliminary results were presented by Denker et al. (1984) and by Hohn et al. (1984)     

Induction of trophinin in human endometrial surface epithelia by CGbeta and IL-1beta

During embryo implantation, trophinin mediates cell adhesion by homophilic binding at the apical surfaces of trophectoderm and endometrium. Trophinin is expressed on the human endometrial epithelia in rare occasions. We developed hCG-coated agarose beads that mimic the physical and physiological features of an implantation-stage human blastocyst. When hCG-coated beads were applied to human endometrial epithelial cells in the presence of IL-1beta, endometrial cells acquired strong trophinin expression and the ability for apical cell adhesion with trophinin-expressing human trophoblastic cells. These results provide a mechanism for trophinin-mediated adhesion of human blastocyst to endometrium by a spatially and temporally restricted paracrine effect of hCG derived from the blastocyst.