Rita A. Meyer

Intercellular junction development in maturing rat seminiferous tubules.

Sertoli cells of the seminiferous tubules in immature rats (1–20 days) contain gap and tight junctions in different stages of development as identified in freeze-fracture replicas. Typical gap junctions and gap junction formation were observed between Sertoli cells. Tight junctions were observed to assume a variety of configurations including linear, macular, and extensive occluding complexes. Tight junction formation was also observed. A decrease in the frequency of gap junctions and a corresponding increase in the number of tight junctions was quantitatively shown.

Enhanced gap junction formation with LDL and apolipoprotein B

Gap junctions are plasma membrane specializations involved in direct cell-cell communication. Intercellular communication is dependent upon the assembly of gap junction structures and would be influenced by agents which alter the assembly process. We investigated the effects of low density lipoprotein (LDL) on gap junction assembly between cultured Novikoff cells using quantitative dye transfer and freeze-fracture electron microscopic methods. We observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms/ml) and a sixfold increase in the number of aggregated gap junction particles per cell. Immunoblots of Novikoff cells probed with anti-connexin43 antibody revealed no detectable increase in gap junction protein (connexin) levels. The influence of the different components of LDL on junction formation was also examined. First, we treated cells with cholesterol (0-150 microM) in serum-free BSA media and observed a decrease in junction assembly. Second, we added apolipoprotein-B (apo-B) in phosphatidyl choline vesicles to the cells and observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms protein/ml) and a fivefold increase in the number of aggregated gap junction particles per cell. The addition of phosphatidyl choline vesicles without apo-B had no effect on gap junction formation. Thus, we demonstrated that gap junction assembly can be modulated by LDL and apo-B treatments.

Analysis of gap junctions and formation plaques between reaggregating Novikoff hepatoma cells

Freeze-fracture techniques have been used to study gap junction formation between Novikoff hepatoma cells reaggregated for 0–60 min. Quantitative data have been obtained for the proportion of membrane “interfaces” containing formation plaques with and without gap junctions [FP(+)s and FP(−)s, respectively], for the areas and numbers of FP(+)s and FP(−)s, and for the numbers and densities of 9- to 11-nm particles in FPs. Methods have been devised to correct these data for FP(+)s carried over from dissociation. Comparisons have also been made with undissociated Novikoff cells. The results show that new FP(−)s and FP(+)s first appear between 5 and 15 min of reaggregation, and increase gradually between 15 and 30 min. Interfaces with FP(−)s develop more rapidly than those with FP(+)s. Between 30 and 60 min, FP(−)s begin to disappear while FP(+)s continue to increase. The results suggest that many FP(−)s are converted into FP(+)s, but some FP(−)s may “abort.” The mean areas of FP(−)s and FP(+)s, either per plaque or per interface, show no significant changes with reaggregation time, although there is an increased average number of FPs per interface at later times. There is a progressive increase with time in the number of total 9- to 11-nm particles in FPs per plaque and per interface, with densities of 9- to 11-nm particles also generally increasing. However, FP(+)s, especially in 15-min samples, have a relatively constant density of unaggregated 9- to 11-nm particles, in spite of a large variation in aggregated particle density. A number of related analyses have led us to suggest that the 9- to 11-nm particles in FP(+)s behave as though they are in a saturated solution, with the aggregated particles being analogous to a precipitate. Particle aggregation in FP(−)s is considered analogous to precipitation from a supersaturated solution. The relation of the results to previous physiological data is discussed.

Effects of aspirin on tight junction structure of the canine gastric mucosa

The effects of aspirin on the canine gastric mucosal barrier were examined using the freeze-fracture and extracellular tracer techniques. Aspirin treatment (3, 20, or 40 min) resulted in alterations in tight junction complex morphology and permeability. Discontinuities in the apical occluding complex, hyperplastic tight junctions (extensions of the apical tight junction strands radiating over the lateral plasma membrane), and a variability in the number of strands (1-20) comprising the complex were observed. A concurrent increase in lanthanum permeability between nonnecrotic surface mucous epithelial cells was also demonstrated. The results of these experiments may suggest that aspirin-induced impairment of the tight junction complexes between viable gastric mucosal epithelial cells may be a major contributing factor in the etiology of stomach disorders.

The gastric mucosal barrier: structure of intercellular junctions in the dog

The canine gastric mucosa consists of two regions, the surface mucous cells and gland area cells including parietal, chief, and mucous-containing cells. We have used quantitative freeze-fracture methods in conjunction with thin-section extracellular tracers to document and correlate tight junction morphology with epithelial permeability. The number of strands in the tight junction complexes of the surface cells and gland cells is the same, but differences in strand arrangement exist. The surface cells have an interwoven tight junction configuration which is impermeable to extracellular tracers. The gland cell junctions are regularly arranged and often permeable to extracellular lanthanum. The possibility that the observed difference in permeability between the tight junctions of the surface mucous cells and those of the gland cells is related to their structural configuration is discussed.

The gastric mucosal barrier: tight junction structure in gastritis and ulcer biopsies.

Tight junctions of the human gastric mucosa were examined using quantative freeze-fracture methods. Biopsies examined were from patients with gastric diseases including gastritis, ulcers, and pernicious anemia. No significant differences were seen in strand number or tight junction complex depth among the biopsies analyzed, however, anomalous tight junction structures were observed. Discontinuities in the tight junction complex and hyperplastic tight junctions (extensions of the apical tight junction strands radiating over the lateral plasma membrane) were seen. These alterations were not associated exclusively with either the diagnosis of gastritis or ulcers. However, a higher frequency of tight junction breaks was seen in stomach biopsies diagnosed as gastritis while those diagnosed as ulcers displayed a higher occurrence of hyperplastic tight junctions.

The effects of follicle-stimulating hormone (FSH) on sertoli cell junctions in vitro: A freeze-fracture study

The mechanism(s) for the control of Sertoli cell tight junction development and orientation has not been established. We have studied the effect of FSH (5 U/ml) on tight junction formation using an organ culture technique that allows maintenance of tubular structure and viability of Sertoli cells, without the other influencing factors that are encountered in vivo . Tight junctions developed as the control organ cultures aged and consisted of extensive junctional complexes by 30–40 days in culture. Yet their development did not correspond to that seen in the maturing animal at 30 days, remaining basically unoriented. In the 30- to 40-day FSH-treated cultures the tight junctions were not only aligned parallel to one another, but were also condensed in bands at the basal part of the Sertoli cell and seemed to be oriented parallel to the myoid cell layer. These observations suggest that FSH affects the tight junctions of Sertoli cells by influencing their overall orientation. Additionally, the occurrence and possible significance of a septate-like character of the Sertoli junctions that was occasionally noted is discussed.

Development of sertoli cell junctions in vitro—A freeze-fracture study

SummarySeminiferous tubules of 1-day-old rats were maintained in organ culture for up to 40 days. Five classes of intercellular junctions between Sertoli cells were observed by the freeze-fracture method as the tissue aged: (a) typical gap junctions; (b) focal tight junctions; (c) macular tight junctions; (d) meandering tight junctions; and (e) extensive tight junctions. The relative proportions of these types of Sertoli cell junctions were quantitated as the organ cultures progressed. The junctional structures observed and classified in organ culture were identical to those seen in vivo, but the timing of their appearance and/or disappearance, as well as their relative proportions, was different from that observed in the developing animal. Extensive tight junctions, with numerous parallel strands, were observed in the 40-day cultures; however, their oblique orientation with respect to the myoid layer was in contrast to the parallel orientation observed in vivo.

The effects of 16,16-dimethyl prostaglandin E2 + aspirin on the canine gastric mucosal barrier.

The canine gastric epithelium was exposed to solutions containing 20 mM aspirin and 20 mM aspirin + 30 µg/kg 16,16-dimethyl prostaglandin E2 (dmPGE2) for periods of three and forty minutes. No macroscopic hemorrhagic lesions were seen. Light microscopically, surface lesions were reduced from 10 percent (aspirin alone) to 2.5% (aspirin+dmPGE2). However, dmPGE2 does not appear to attenuate aspirin induced tight junction alterations. Discontinuities in the apical occluding complexes, hyperplastic tight junctions and stand number variability were documented in freeze frature replicas of aspirin as well as aspirin+ dmPGE2 treated dog stomachs. The results of these experiments would seem to suggest that 30 µg/kg dmPGE2 does not prevent aspirin induced damage to the tight junctions of the canine gastric epithelium or enhance their repair.