John E. Morris

Estradiol induces E-cadherin degradation in mouse uterine epithelium during the estrous cycle and early pregnancy

Mouse uterine epithelium is a tissue that undergoes cyclic endocrine‐regulated cell dissociation and regeneration. It shows a dramatic cell loss following normal estrus. If pregnancy ensues, cell loss is averted during the first 2.5–3.5 days. However, this is followed by a precipitous loss of basal‐lateral cell adhesion and apoptosis in preparation for blastocyst invasion. By comparing epithelia isolated by protease treatment, we show that a reduction of lateral cell adhesion is a primary event in these instances of normal tissue loss. It was readily induced in ovariectomized adult and immature mice by injections of estradiol (E2), and to some extent also by progesterone (P4). The reduction of lateral adhesion induced by including ethylene glycol‐bis (β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid (EGTA) in the isolation medium mimicked and was additive to the effect of E2 injection. However, the E2 effect was different in not being prevented by adding Ca2+. The E2 effect also was mimicked by the action on isolated epithelium of monoclonal antibody against the calcium‐dependent cell adhesion molecule, E‐cadherin, suggesting that inactivation of E‐cadherin was induced by E2. In detergent extracts of estrous and metestrous epithelium there was an increase in 80‐kDa extracellular domain of E‐cadherin relative to the intact 120‐kDa molecule. The loss of adhesion between 3.5 and 4.5 days of pregnancy was associated with a loss of both intact membrane‐associated 120‐kDa E‐cadherin and cleavage products. Cleavage of 80‐kDa E‐cadherin was uniquely induced by E2 in ovariectomized adult and immature mice; P4 was without effect. The cleavage of E‐cadherin correlated with increased basal accumulation of E‐cadherin antigen in estrous and E2‐injected mice and a loss of both basal and lateral antigen at 4.5 days of pregnancy. Only the E‐cadherin antigen within junctional complexes appeared unaffected. The data are consistent with the hypothesis that the cyclic and pregnancy‐dependent disruption of uterine epithelial integrity are promoted by E2‐dependent modification of E‐cadherin, including its extracellular cleavage.

Steric exclusion of cells: A mechanism of glycosaminoglycan-induced cell aggregation

Abstract The mechanism by which chondroitin sulfate enhances both the self-aggregation and the concanavalin A (ConA)-induced agglutination of trypsin-dissociated embryonic chick retina cells was investigated. Studies with fluorescently labeled ConA showed no influence of chondroitin sulfate on patching or capping. When 3H- or 35SO4-labeled glycosaminoglycans or proteoglycans from retinas were added to freshly dissociated cells, an average of less than 2% of the label became associated with the unwashed cell pellet, and most of this was removed from the cells by a single wash. The presence of ConA did not alter the amount of binding by this criterion. Rapid cell aggregation in the absence of ConA was promoted by a number of natural and synthetic polymers. Aggregation rate bore a direct relationship to polymer viscosity at low viscosities and was inhibited at high viscosities, apparently due to reduced cell collision frequency. For any given polymer, aggregation was directly related to its molecular weight and concentration. Linear polymers were more effective than branched ones. Neutral polymers were as effective as those which were strongly polyanionic. Stable aggregates of formalin-fixed cells were not promoted by polymers. All of these observations are consistent with the hypothesis that enhancement of retina cell aggregation by physiological concentrations of glycosaminoglycans is due largely to steric exclusion of the cells by the polymer mesh. Although others have shown that glycosaminoglycans probably interact specifically with some cells, the evidence presented here suggests that these macromolecules by virtue of their excluded volumes could also have important non-specific influences on cell migration and cell reorganization during morphogenesis.

Proteoglycans synthesized by embryonic chicken retina in culture: Composition and compartmentalization

Characteristics of the chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs) and heparan sulfate proteoglycans (HSPGs) from retinas of 14-day chicken embryos were examined following specific lyase digestion of the HSPG and CS/DSPG glycosaminoglycans, respectively. On the basis of gel exclusion chromatography the prevalent CS/DSPGs in the tissue were above Mr 400 X 10(3) with two or three glycosaminoglycan chains of Mr 60-70 X 10(3). The HSPGs existed in two distinct populations in the tissue. Those in the dominant population appeared to be in the range of Mr 250-300 X 10(3) with 9 to 12 glycosaminoglycan chains of Mr 15-25 X 10(3). The other population consisted of free heparan sulfate chains of Mr 15-25 X 10(3). The HSPGs in the medium tended to be intermediate in size. To examine the distribution of proteoglycans, tissues were sequentially homogenized and extracted in saline and reextracted with 4 M guanidine HCl (GdnHCl) and Triton X-100 (TX), or they were washed in heparin solution and dissociated to single cells with trypsin before sequential extraction in saline and GdnHCl with TX. Through comparison of the results of these two extraction methods, CS/DSPGs were found to be almost entirely within the medium or matrix or loosely associated with the cell surface, and most HSPGs were associated with either the basal lamina or the plasma membrane. The single heparan sulfate glycosaminoglycan chains appeared to be intracellular degradation products. These results support reports that CS/DSPGs may be present in the retina interphotoreceptor matrix and that HSPGs may be present in regions of synaptogenesis, associated with cell membranes.

A comparison of developmental changes in surface charge in mouse blastocysts and uterine epithelium using DEAE beads and dextran sulfate in vitro

The ability of 3.5-day mouse blastocysts and vesicles prepared from maternal uterine epithelium to adhere to surfaces by charge interactions was compared by observing their adhesion to DEAE-Sephadex beads in the presence of increasing concentrations of dextran sulfate. The adhesion frequency for both the blastocysts and epithelium declined in a manner suggesting that predominantly ionic sites were being titrated, but differences between the two tissues in characteristics of the titration curve and susceptibility to neuraminidase digestion indicated that nonionic interactions were relatively more important for blastocysts. Because the threshold concentration of dextran sulfate required to initiate displacement of uterine epithelium from the DEAE beads was at least 4X that required to initiate the displacement of blastocysts, we argue that the uterine epithelium had at least 4X more interactive charged groups on its surface than the blastocysts. These differences were even more pronounced 4.5 days after mating, a time when attachment to the uterine epithelium is normally first seen in vivo. Blastocysts isolated at this time showed a marked increase in resistance to polyanion competition, but the epithelium showed a nearly 50% decline in surface negative charge that was not compensated by nonionic mechanisms. These observations support the conclusion that the initial adhesion of blastocysts in vivo is accompanied by a reduction in negativity of the uterine epithelial surface and by the formation of new trophoblast cell surfaces that adhere by nonelectrostatic interactions.

Histogenesis and dependent glutamine synthetase inducibility in embryonic neural retina. Irreversible inhibition of differentiation by 5-bromodeoxyuridine

Abstract Young, mitotically active neural retinas from 7-day chick embryos were cultured with 5-bromodeoxyuridine (BrdU) for 8 hr or more. After this treatment, they failed to differentiate beyond the stage at which they were explanted; there was no histogenesis or increase in glutamine synthetase (GS) inducibility in intact tissues or in aggregates of dissociated cells. Normally GS can be induced in the retina with hydrocortisone as the cells cease to be mitotically active and begin showing histological organization after day 7. This inhibition by BrdU was irreversible even in the presence of excess thymidine. Overall incorporation of 14C-amino acids into protein was only slightly inhibited, and the ability of cells from treated tissue to aggregate and sort out from nonneural cell types was unaffected. Control cultures without BrdU showed considerable histogenesis and a parallel increase in enzyme inducibility. Postmitotic 10-day retinas appeared to be unaffected by BrdU. The incorporation rates of both tritiated BrdU and thymidine (dT) into DNA were 14× higher in 7- than in 10-day retinas. Simultaneous addition of excess unlabeled dT with either of the labeled nucleosides reduced their incorporation and reduced the inhibitory action of BrdU on differentiation. It is concluded that BrdU irreversibly inhibits the differentiation of retina cell surface properties involved in histogenesis and dependent cytodifferentiation without affecting already differentiated properties of the cell surface. The results support the hypothesis that histogenesis is directed by genes affecting specific cell surface properties.

Isolation of the major chondroitin sulfate/dermatan sulfate and heparan sulfate proteoglycans from embryonic chicken retina

A technique is presented for the preparation of three major proteoglycans from 14-day embryonic chicken retinas following their culture overnight with [35S]sulfate and either [3H]glucosamine or [3H]serine. Homogenization of the tissue in saline permitted extraction of heterogeneous soluble proteoglycans separately from most of the heparan sulfate proteoglycans. The latter were extracted from the 140,000g pellet with 0.5% Triton X-100 in 8 M urea. The medium plus the saline and urea-detergent extracts were separated from low-molecular-weight contaminants, and fractionated into two peaks of radioactivity on Sephacryl S-300 in saline with 3 M urea and 0.5% Triton X-100. The proteoglycans were isolated directly from these fractions on DEAE-Sephacel, and subjected to ultrafiltration concentration and then further purification on cesium chloride density gradient centrifugation in 4 M guanidine hydrochloride. A further step involving cetylpyridinium chloride precipitation was examined, but it resulted in essentially no further purification. The fractionations separated a large chondroitin sulfate/dermatan sulfate proteoglycan from the culture medium that was excluded from S-300 and of low buoyant density; a large heparan sulfate proteoglycan from the urea-detergent extract that was also excluded from S-300 and of low buoyant density; and two smaller and possibly related heparan sulfate proteoglycans. One was found in the medium and showed low to intermediate buoyant density; the other was isolated from the urea-detergent extract and showed a significantly higher buoyant density, associated with a lower protein content. The saline extract contained both of the two larger proteoglycans and only minor amounts of the smaller molecules.

Comparison of Proteoglycans Extracted by Saline and Guanidinium Chloride from Cultured Chick Retinas

Abstract: To compare the loosely associated sulfated proteoglycans with those tightly bound to membranes, retinas from 14‐day chick embryos were subjected to progressively disruptive techniques. The most easily removed proteoglycans were isolated from the medium in which the tissue was labeled with [35S]sulfate. On the average, 25% of the glycosaminoglycans were in the labeling medium, 39% were in proteoglycans extracted from the tissue in the balanced salt solution, 32% were in a 4 m‐guanidinium chloride (GuCl) fraction, and 4% remained unextracted. These glycosaminoglycans contained, respectively, 28, 28, 40, and 4% of the incorporated [35S]sulfate. On the basis of electrophoretic mobility and TLC of chondroitinase digests, the ratio of 35S in chondroitin sulfate to that in heparan sulfate was 4–7 times higher in the medium and balanced salt extracts than in the GuCl extracts. In both extracts there was more 35S in chondroitin‐6‐sulfate than in chondroitin‐4‐sulfate. Dialysis of the extracts against 0.5 M‐NaCl resulted in the precipitation of about 12% of the glycosaminoglycans in the saline extracts and about 40% in GuCl extract. These subfractions, which were relatively enriched in heparan sulfate, were largely soluble in dithiothreitol in 8 m‐urea (DTT). Similarities between the proteoglycans in the medium and those extracted by balanced salt solutions suggest that the saline‐extracted proteoglycans were for the most part loosely associated with cell surfaces or extracellular matrices, whereas the GuCl‐extracted proteoglycans probably were bound to membranes.

Estradiol-17 beta stimulates proliferation of uterine epithelial cells cultured with stromal cells but not cultured separately.

SummaryThere is indirect evidence that the in vivo proliferative response of rodent uterine epithelium to estrogen requires interaction with the underlying stroma in pre- and post-pubescent animals. To examine this potential requirement directly, the proliferative response of epithelium to 17β-estradiol in the presence or absence of stroma was measured in vitro. Uterine epithelial and stromal cells were isolated separately from immature or adult mice, and were maintained as monocultures or cocultures in defined, serum-free medium with or without 8 × 10−9M 17β-estradiol. Incorporation of bromodeoxyuridine into the DNA was determined by immunolabeling to assay proliferation in individual cells. Cell morphology and immunolabeling of cytokeratin were used to distinguish epithelial from stromal cells. Treatment of cocultures with 17β-estradiol for 24 h increased the proliferation of epithelial cells relative to controls approximately threefold, whereas, in monocultures of epithelial or stromal cells 17β-estradiol decreased the number of bromodeoxyuridine-incorporating cells by approximately half. Furthermore, cell contact between epithelial and stromal cells was important for the effects of 17β-estradiol on cells in cocultures. Approximately three quarters of the 17β-estradiol-induced proliferation of epithelial cells in cocultures was produced by epithelial cells within colonies that were also contacting stromal cells. These results are consistent with the hypothesis that stromal cells mediate the estrogenic proliferative response, and provide evidence that this mediation involves cell contact or stroma-mediated changes in the microenvironment immediately around the epithelial cell.

Heparan sulfate in the inner limiting membrane of embryonic chicken retina binds basic fibroblast growth factor to promote axonal outgrowth.

During neural development retinal ganglion cell axons migrate over the retinal basal lamina (inner limiting membrane, ILM) in directed growth toward the optic nerve. We found that both growth rate and distribution density of the ganglion cell axons on isolated cell-free ILM was greatly inhibited by pretreatment with heparitinase but not with chondroitinase ABC. The persistence of radioactively labeled proteoglycans added to the culture medium eliminated residual heparitinase as an explanation for the inhibition. A cell binding assay showed that heparitinase acted on the ILM to influence axonal behavior without apparent inhibition of cell adhesion. These results indicated that the neurite outgrowth promoting activity of the ILM depended on the heparan sulfate (HS) side chains of its proteoglycans. Basic fibroblast growth factor (bFGF) stimulated additional neuronal sprouting and neurite elongation on the ILM. This neurotropic activity of bFGF was inhibited by heparitinase pretreatment of the ILM, suggesting that bFGF bound to HS on the ILM. The activity of bFGF was enhanced by exogenous heparin added to the culture medium; although heparin alone failed to stimulate either neurite extension or neuronal cell sprouting. These results demonstrate that HS in the ILM possesses neurotropic activity for axons of the ganglion cells by binding bFGF for presentation to cell-surface receptors and may, therefore, play a significant role in stimulating axonal outgrowth during development.

Low buoyant density proteoglycans from saline and dissociative extracts of embryonic chicken retinas

Abstract: Retinas were labeled in culture with [3H]glucosamine or [3H]leucine and [35S]sulfate and extracted sequentially with physiologically balanced saline and 4 M guanidine HCl. They were dialyzed into associative conditions (0.5 M NaCl) and chromatographed on agarose columns. Under these conditions, some of the proteoglycans were associated in massive complexes that showed low buoyant densities when centrifuged in CsCl density gradients under dissociative conditions (4 M guanidine HCl). Much of the label in these complexes was in molecules other than proteoglycans. Most of the proteoglycans, however, were included on the agarose columns, where they appeared to be constitutionally of low buoyant density. They resisted attempts to separate potential low buoyant density contaminants from the major proteoglycans by direct CsCl density gradient centrifugation or by the fractionation of saline or 8 M urea extracts on diethylaminoethyl‐Sephacel. The diethylaminoethyl‐Sephacel fractions were either subjected to CsCl density gradient centrifugation or were chromatographed on Sephacryl S‐300, in both cases before and after alkaline cleavage, to confirm the presence of typical O‐linked glycosaminoglycans. The medium and balanced salt extracts were enriched in chondroitin sulfate and other sul‐fated macromolecules, possibly highly sulfated oligosaccharides, that resisted digestion by chondroitinase ABC but were electrophoretically less mobile than heparan sulfate. Guanidine HCl or urea extracts of the residues were mixtures of high and low density proteoglycans that were enriched in heparan sulfate.