ultrastructural morphometry of gap ...cap junctions in epithelium 69 no. of animals 5 table no. of...

J. Cell Set. 69, 67-85 (1984) 67Printed in Great Britain © The Company of Biologists Limited 1984

ULTRASTRUCTURAL MORPHOMETRY OF GAP

JUNCTIONS DURING DIFFERENTIATION OF

STRATIFIED SQUAMOUS EPITHELIUM

F. H. WHITEDepartment of Anatomy and Cell Biology, University of Sheffield, Sheffield S10 277VU.K.D. A. THOMPSON AND K. GOHARIDepartment of Oral Pathology, University of Sheffield, Sheffield S102TA, U.K.

SUMMARY

The presence of gap junctions in stratified epithelia has now been extensively documented, butthere have been few attempts to quantify them. In the present report, samples of hamster cheekpouch mucosa were processed for electron microscopy and electron micrographs from defined basal,spinous and granular layers were obtained. Using a combination of direct measurement andstereological intersection counting techniques, the relative surface areas of peripheral gap junctions(i.e. those in direct contact with the epithelial plasma membrane) and annular gap junctions (i.e.those present as complete, approximately circular profiles within the epithelial cell cytoplasm) weredetermined. Following estimation of the plasma membrane surface area of 'average' epithelial cellsfrom each of the defined strata, relative values were transformed into absolute data. Data fromperipheral and annular junctions were pooled to provide an estimate of total gap junctional area.

Relative surface area estimates were similar for peripheral, annular and total gap junctions, in thatvalues were invariably highest in the spinous layer and lowest in the granular layer. Absolute dataindicate that there is more than a threefold increase in the area of membrane differentiated into gapjunctions in the average spinous cell when compared with the average basal cell. Values for total gap-junctional areas in the average granular cell are reduced somewhat with respect to the averagespinous cell and this is effected by a decrease in the area of peripheral gap junctions. We concludethat there is synthesis of gap junctions between basal and spinous cells, which is followed by evidenceof degradation between spinous and granular cells. The magnitude of the estimates of area iscomparable to those obtained from other stratified and non-stratified epithelia and it would thusappear that gap junctions may play a significant role in cellular control processes in all viableepithelial strata.

INTRODUCTION

In excitable tissues such as nerve and muscle the gap junction is an area of low-resistance electrotonic coupling, i.e. it serves as an electrical as opposed to a chemicalsynapse (Bennett, Aljure, Nakajima & Pappas, 1963; Bennett, 1973; McNutt &Weinstein, 1973). However, the gap junction is so ubiquitous that its absence in aparticular tissue could almost be considered exceptional. The widespread distributionof this junction suggests that it must have a role in non-excitable as well as excitabletissues, but so far this role has remained elusive. Whilst electrical charges have beenshown to pass between cells of the nervous system known to be attached by gapjunctions (see Bennett & Goodenough, 1978), substances of low (<1000) molecularweights such as fluorescein (Furshpan& Potter, 1968; Pitts &Finbow, 1977), procion

68 F. H. White, D. A. Thompson and K. Gohari

yellow (Loewenstein, 1975) and fluorescein-labelled maltotetriose (Simpson, Rose &Loewenstein, 1977) also pass between coupled cells. Although it is now generallyaccepted from experimental studies that gap junctions represent the morphologicalspecializations responsible for ionic and metabolic coupling between cells, there is stillonly circumstantial evidence as to the nature and function of molecules that mightpass through them in vivo.

Gap junctions have been described in stratified squamous epithelia (Hashimoto,1971; Breathnach, Stolinski & Gross, 1972; Orwin, Thomson & Flower, 1973;Shimono & Clementi, 1976; Caputo & Peluchetti, 1977; Archard & Denys, 1979;White & Gohari, 1981a), in which their distribution appears less frequent than thatof the desmosome, a specialization that is particularly prominent in this tissue. Inaddition to conventional gap junctions situated on the peripheral plasma membranes,another type of junction, the annular gap junction, has been described in theseepithelia (Shimono & Clementi, 1976; Caputo & Peluchetti, 1977; Archard & Denys,1979; White & Gohari, 1981a). Gap junctions have been described between nucleatedcells of stratified squamous epithelia, and in view of the complex cellular interactionsthat occur during epithelial differentiation and keratinization, their presence suggeststhat they may play a significant role in these processes. The aim of the present reportis to use well-established quantitative morphological techniques (Weibel, 1979) todetermine the relative surface area of plasma membrane occupied by gap junctions indefined epithelial strata and to transform these relative estimates to data characteriz-ing the absolute area of gap junctions present on the 'average' cell, using methods thatwe have described previously (White, Mayhew & Gohari, 1982; White & Gohari,1982). A preliminary account of this work has been published elsewhere in abstractform (Thompson, White & Gohari, 1981).

MATERIALS AND METHODS

Samples of mucosa were removed from the medial aspects of cheek pouches of five male Syriangolden hamsters. The mucosa was immediately covered in a freshly prepared glutaraldehyde/formaldehyde fixative in phosphate buffer (White & Gohari, 1981a) with a total osmolality of1060 mosM. The tissue was cut into 1 mm wide slices, immersed in fixative for 2 h and subsequentlydiced into cubes of <1 mm3. Following phosphate buffer rinses, the tissue was postfixed in 2%aqueous osmium tetroxide, dehydrated in successively higher concentrations of ethanol commenc-ing at 70%, and flat-embedded in rubber moulds in Araldite. A minimum of 20 blocks was em-bedded for each animal. Five blocks were obtained by lottery from the pool available for each animaland these were cut with glass knives on a Cambridge Huxley Mark 2 ultramicrotome. Sections (1 ftm)were stained with toluidine blue in borax in order to establish whether blocks were being sectionedperpendicular to the epithelial surface. Blocks not sectioned in this way were discarded and areplacement obtained from the pool.

Ultrathin (approx. 50-70 nm) sections were prepared from appropriately orientated blocks,mounted on Formvar-coated copper grids and stained with methanolic uranyl acetate and leadcitrate. Sections were viewed on an AE1 EM6B electron microscope at an accelerating voltage of60kV. Micrographs were recorded at X7500 and photographically enlarged to 18750. The micro-scope was calibrated after every 100 exposures using a diffraction grating replica (2160 lines/mm).

Quantitative methodsThe sampling strategy for the quantitative analysis is summarized in Table 1. Eight micrographs

were recorded at random from each of the basal, spinous and granular layers of the epithelium,

Cap junctions in epithelium 69

No. ofanimals

5

Table

No. ofblocks/animal

5

1. Summary of stereological

No. ofsections/block

1

No. oflayers/block

3

sampling design

No. of micro-graphs/layer

8

Total no. ofmicrographs/layer

200

defined according to White & Gohari (1982, 1983). This provided a stratified random sample of 200micrographs for each cellular stratum.

The stereological analysis was performed by superimposing a transparent test lattice containingstraight parallel lines over each micrograph. The lines were separated by intervals of 5 mm and everyfifth line was thickened. Intersections of the thick lines with the plasma membranes (/PM) and of anyline with gap junctions (/cj) were counted.

In random plane sections, a substantial proportion of the plasma membranes are cut tangentially,and although their position is clearly visible, this is not the case for gap junctions. These structuresare found differentiated within and between adjacent plasma membranes, and contain no cytoplas-mic specialization that might provide an indication of their presence in oblique sections, such as isusually possible with desmosomes. It is sometimes possible to distinguish gap-junctional areas incertain oblique planes of section, since the intercellular component forms hexagonal arrays in enfaceviews (Revel & Karnovsky, 1967), but this is not a reliable method for quantification (White,unpublished data). Consequently, the approach adopted in this study was that only membrane andgap-junctional profiles that had been sectioned perpendicularly, and which were therefore unam-biguously identifiable, were quantified. Gap-junctional profiles were further classified into twocategories. Peripheral gap junctions (PGJ) were those junctions that were situated on the plasmamembrane and were observed to be in direct continuity with it (Figs 1-10). Annular gap junctions(AGJ) were present as approximately circular profiles within the cytoplasm of epithelial cells anddemonstrated no direct continuity with the remainder of the cell plasma membrane (Figs 11-16).Therefore differential intersection counting yielded separate counts for peripheral (/PGJ) andannular (/ACJ) junctions.

The intersection counts were used to determine the relative surface area of gap junctions presentper unit surface of plasma membrane (5s) according to the relationship:

5 S = /, (Weibel, 1979).

Thus for peripheral gap junctions, the relevant relationship is given by:

and for annular gap junctions, by:

SSACJ = /AGj/ /pM •

In addition, it was possible to pool the intersection counts in order to determine a similar parameterfor the total gap junctions (TGJ) present, i.e.:

Minimal sample size was determined for each animal using the accumulative mean plot technique(Schroeder & Miinzel-Pedrazzoli, 1970).

Since gap junctions are not found on the basal plasma membrane (i.e. that part of the basal cellin contact with the basal lamina complex) nor at the granular cell-keratin interface, intersectionsof the lattice with these membranes were excluded from the analysis.

In previous reports, we have suggested ways in which absolute data can be arrived at for the'average' epithelial cell in each stratum (White & Gohari, 1982, 1983, 19846; White et al. 1982).While the relative estimates described above are useful in a comparative sense, it would obviouslybe advantageous if we could determine the absolute surface area of the average cell in each stratumand then transform the relative areas into absolute areas. This provides additional information and,for example, can tell us whether gap-junctional specializations are undergoing synthesis or degrada-tion between different epithelial strata.


Briefly, the approach adopted to determine cellular areas was as follows. Measurements of majorand minor nuclear axes were performed, and these were transformed to obtain the diameter of acircle of equivalent area (White & Gohari, 1982). This enabled nuclear volume to be estimated.Following point-counting procedures, the nuclear/cytoplasmic ratio was determined (White &Gohari, 19816) and by combining these data, the absolute volume of the average cell in each stratumcan be estimated. Subsequently, an interrupted multipurpose test lattice (Weibel, 1979) can beapplied to micrographs in order to determine the cellular volume-to-surface ratio, using the methodof Chalkley, Cornfield & Park (1949). This then enables the generation of estimates of cell surfacearea, since cell volume has already been derived. Differential intersection counting of profiles ofplasma membrane that are not required in the analysis (e.g. the basal plasma membrane, and thegranular cell-keratin interface) enabled absolute estimates of these surfaces to be obtained and thesewere subtracted from the cell surface estimates. Thus the final estimate of average cell area (SCELL)excluded areas of membrane in which gap junctions were absent. The total area of gap junctionspresent on the average cell from each stratum was obtained by multiplying SCELL by the appropriaterelative surface estimate, Ss .

Means were calculated for each animal in each cellular layer and the results are presented as thefinal means of the five animals used. Means of individual animals were used for statistical analysis.A one-way analysis of variance was performed using a magnetic card program on a Hewlett-Packarddesk-top calculator. If the test proved significant, data for individual strata were compared usingStudent's J-test.

RESULTS

Qualitative observations. At high magnifications, the preparative procedures used

in this s tudy resulted in a pentalaminar appearance for gap junctions of all types. T h i s

morphology was due to the apparent fusion of the outer membrane leaflets contr ibut-

ing to the junction, each of which possessed a tri laminar appearance.

Fig. 1. Spinous layer. Two peripheral gap junctions (arrows) can be seen differentiatedon the membranes between two spinous cells. They are both relatively straight and one isclosely associated with a desmosome (d). X 39 000.

Fig. 2. Granular layer. At high magnification the pentalaminar structure of the gapjunction (gj) is apparent. This peripheral junction is linear. X 78 000.

Fig. 3. Basal layer. The gap junction (gj) is becoming curved. The epithelial cellcytoplasm adjacent to the concavity (arrow) is highly electron-lucent. X65 000.

Fig. 4. Spinous layer. Two peripheral gap junctions in close proximity are slightly morecurved than that present in Fig. 3. Electron-lucent peripheral cytoplasm is present adja-cent to each concavity, being separated by a central region of normal density and contain-ing a few ribosomal particles. X65 000.

Fig. S. Basal layer. The degree of curvature of the peripheral gap junction is increasing,as is the adjacent area of pale peripheral cytoplasm. X65 000.

Fig. 6. Spinous layer. The peripheral gap junction profile is approximately semicircular.Adjacent to the light cytoplasmic zone is cytoplasm of normal density, which contains twomembrane-coating granules (m) and a multivesicular body (mv). X65 000.

Fig. 7. Spinous layer. Some parts of the gap-junctional profiles are curved (c) whereasothers are more angular (a). Much of the immediately adjacent cytoplasm is structureless(arrows). Between the two profiles ( a t / ) intermediate filaments (tonofilaments) arepresent. X72000.

Fig. 8. Spinous layer. This almost circular gap-junctional profile (gj) is attached to adja-cent non-specialized membrane at its lower aspect. Note the presence of an electron-lucenthalo adjacent to the concavity of the gap-junction profile and within this the presence ofa more organized region with some ribosomes. X72 000.

Cap junctions in epithelium n

Figs 1-4

F. H. White, D. A. Thompson and K. Gohari

Figs 5-8. For legend see p. 70.

Gap junctions in epithelium 73

Figs 9-12. For legend see p. 75.


Figs 13-16


Peripheral gap junctions were seen distributed in an apparently random manner onall plasma membranes, with the exception of those of the keratin squames and be-tween granular cells and the deepest keratinized cells. The majority were linear (Figs1, 2) or slightly curved (Figs 3, 4). They were not present on the basal plasmamembrane, i.e. that membrane forming the epithelial-connective tissue junction,although they were sometimes present on lateral plasma membranes adjacent to thisjunction. The peripheral junctions appeared to be more numerous in lower strata thanin the granular layer. A common observation was the presence of curved or loop-likejunctions, which were obviously directly related to the remainder of the plasmamembrane. The degree of curvature varied (Figs 3, 4, 5, 6) but, this notwithstanding,there was a definite tendency for the cytoplasm situated immediately adjacent to theconcave surface of the curved junctions to possess a relatively electron-lucentcytoplasm devoid of intracellular organelles. Occasional areas of junctional specializa-tion were present that demonstrated more angular, rather than curved, profiles (Fig.7). Other peripheral gap junctions were observed with profiles that were almostcircular (Fig. 8) or elliptical (Figs 9, 10). Many of these junctions were closely relatedto desmosomes (Figs 1, 3, 4, 5, 6, 9).

Annular gap junctions were usually more frequently observed in spinous andgranular cells. They were generally approximately circular in profile (Figs 11, 12, 13,14), although occasional elongated forms were observed (Figs 15, 16). Annular junc-tions contained cytoplasm that resembled that of the surrounding cell, but they

Fig. 9. Spinous layer. A small curved gap junction (a) and a larger ellipsoidal one (b) arepresent. The concave surface of gap junction b is associated with areas of cytoplasm ofnormal morphology (arrow) whereas other areas are pale and structureless (arrowhead).Vesicular material, ribosomes and fine filaments can be seen partially enclosed by theellipsoidal junction. X390OO.

Fig. 10. Basal layer. This extensive peripheral gap junction is ellipsoidal in shape and isin direct contact with unspecialized regions of the plasma membrane (pm). Note that theelectron-lucent zone is absent from most of the junctional concavity, with the exceptionof a small localized area (arrow). X66500.

Fig. 11. Basal layer. This annular gap junction appears as a circular profile and does notseem to be directly related to the plasma membrane. The convex junctional membrane isassociated with localized bundles of cytoplasmic filaments (/) whereas the concavity isrelated to a narrow zone of structureless cytoplasm (arrow). The pentalaminar structureof the contributing junctional membranes is evident. X123 000.

Fig. 12. Granular layer. The convex surface of the annular gap junction is related tocytoplasmic filaments over much of its area. The subjacent cytoplasmic haloing isprominent on one side only (arrow). X116000.Fig. 13. Granular layer. The annular gap junction contains a complete electron-lucenthalo and a membrane-coating granule (m) is present in the central zone. X114000.

Fig. 14. Basal layer. This annular gap junction is situated adjacent to the nucleus (n).X62000.Fig. 15. Spinous layer. The annular gap junction is ellipsoidal, and located centrallywithin the cytoplasm. X102000.

Fig. 16. Basal layer. The ellipsoidal annular gap junction lies within the peripheral partof the cytoplasm, but no direct continuity between junctional and non-specialized mem-branes is apparent, ics, intercellular space. X78 000.


usually possessed a structureless, electron-lucent peripheral zone in which organelleswere absent. This zone formed a halo immediately adjacent to the concavity of thejunctional plasma membrane. Within the more central region, a number of organelleswere observed. These included ribosomes, which were invariably present (Figs11—16), small vesicles, membrane-coating granules (Fig. 13) and mitochondria.Cytoplasmic tonofilaments were often associated with the convex outer perimeter ofthe junctions. These were seen in large dense bundles localized to a particular area of theperimeter (Fig. 11), whereas in other profiles a less-dense zone of tonofilamentssurrounded the junctional convexity (Fig. 12). The annular junction showed nopreferential distribution within the cytoplasm, many beingsituated close to the nucleus(Fig. 14) whereas others were found lying in the peripheral cytoplasm (Fig. 16).

Quantitative observations (Tables 2, 3, 4)

Relative surface estimates for peripheral, annular and total gap-junctional profilesshowed similar trends, with values being highest in the spinous layer and lowest in thegranular layer. The relative estimates for peripheral gap junctions were higher thanthose for annular junctions by a factor of at least 3. When data were transformed torepresent gap-junctional surfaces present on the average cell, a shift in theirdistribution was detected. In all junctional categories, the basal cells possessed the

Table 2. Relative surface estimates of gap junctions in cheek pouch epithelium

Granular

Spinous

Basal

12345

S.D.

12345

XS.D.

12345

XS.D.

Peripheral

0-01080-00810-00690-00740-0134

0-009300027

0-01560-01790-0143002190-0280

0-01950-0055

0-02210-01080-00850-01580-0072

001280-0061

Annular

0-00220-00420-00260-00240-0073

0-00370-0021

0-00090-00530-00510008200063

0-00520-0027

0-00320-00190-00120-00560-0072

0-00380-0025

Total

0-01300-01230-00950-00980-0207

0-01310-0045

0-01650-02320-01940-03010-0343

0-02470-0074

0-02530-01270-00970-02140-0144

0-01670-0064


Table 3. Absolute surface area of estimates of gap junctions in cheek pouch epithelium(pen2)

Peripheral Annular Total

Granular

Spinous

Basal

12345

XS.D.

12345

XS.D.

12345

35-1719-5421-7629-2128-81

26-906-28

30-6226-5835-2135-6345-16

34-646-95

28-226-775-54

18-586-32

7-1610138-209-47

15-69

10-133-31

1-777-87

12-5613-3412-99

9-704-96

4-081-190-786-586-32

42-3329-6729-9638-6844-50

37 026-91

32-3934-4547-7748-9758-15

44-3510-78

32-307-966-32

25-1612-64

x 13-09S.D. 10-02

Table 4. Results of the

SSPGJ 5s A G J S3T,

3-792-74

statistical analysis

3J -Spcj •SAGJ

1611

•88•35

STGJ

B versus SB versus GS versus G

N.S.N.S.

/><0-05

N.S.N.S.N.S.

N.S.N.S.

P<005

P<0-01P<005

N.S.

P<0-05P<0-05

N.S.

P<0-05P<0-05

N.S.

B, basal; S, spinous and G, granular cells.N.S., not significant.

lowest, and spinous cells the highest, values, whereas granular-cell junctional areaswere intermediate between those obtained for basal and spinous cells.

DISCUSSIONThe presence of gap junctions in the stratified squamous epithelium of the oral

cavity has been documented previously (Barnett & Szabo, 1973; Shimono &Clementi, 1976; Archard & Denys, 1979). Before the report of Barnett & Szabo, itwas postulated that the intercellular space of gingival masticatory mucosa was sub-divided into a number of separate compartments by the presence of tight-junction


Table 5. Some relative surface estimates of gap junctions published in the literature(expressed as %)

Author/Species

Orwin et al. (1973)Sheep

Coons &Espey (1977)Rabbit

Gabbiani et al. (1978)Rat

Yee& Revel (1978)Rat

Mtdaetal. (1980)Rat

Campbell & Albertini (1981)Rat

Meyer & Overton (19836)Chick

White et al. (1984)Hamster, present study

Site

Liver

Ovarian follicle

Epidermis

Liver

Pancreas /3 cell

Ovarian follicle

Liver

Cheek pouch epithelium

SSQ]

1-3

4-13

005-0-18

1-3

1-9

9

0-2-5-0

1-2

networks (Schroeder & Theilade, 1966; Thilander & Bloom, 1968). This postulateis no longer valid, since it was based on the erroneous identification of tight junctions,which subsequently have been shown to be gap junctions. In the report of Barnett &Szabo, no mention is made of the annular gap junction, although this structure hasbeen identified in rat buccal and lingual epithelium (Shimono & Clementi, 1976) andguinea-pig lingual, palatal and buccal epithelium (Archard & Denys, 1979). They arealso present in micrographs of hamster cheek pouch epithelium published by Listgar-ten, Albright & Goldhaber (1963), in which they were considered to be unusual'vacuoles'. Annular gap junctions have been observed in the epidermis (Orwin et al.1973; Elias & Friend, 1976; Caputo, Innocenti, Gasparini & Peluchetti, 1978;Mahrle, 1978; Archard & Denys, 1979) and cervical epithelium (McNutt & Wein-stein, 1970; McNutt, Hershberg & Weinstein, 1971) as well as in corpus luteum(Adams & Hertig, 1969), ovarian follicles (Byskov, 1969; Merk, Botticelli & Albright,1972; Merk, Albright & Boticelli, 1973; Espey & Stutts, 1972; Albertini, Fawcett &Olds, 1975; Coons & Espey, 1977), enamel organ (Reith, 1970; Garant, 1972), liver(Perissel, Charbonne, Chessebeuf & Malet, 1976) and between amphibian neuroglialcells (Decker, 1976). In all reports, including the present one, the cytoplasm withinthe annular junctions contained ribosomal particles and the inclusion of largerorganelles such as membrane-coating granules (Archard & Denys, 1979) has also beendescribed. Both Merk et al. (1973) and Perissel et al. (1976) have reported multiplemitochondria within a single gap-junctional profile, and Merk et al. (1973) andShimono & Clementi (1976) have described annular gap junctions within annular gapjunctions. These appearances were occasionally seen in our material, but were muchmore common in cheek pouch epithelium treated with the carcinogen 7,12 dimethyl-benz(a)anthracene (White, Thompson, Cameron & Gohari, unpublished).


There are some reports of a close association between developing gap-junctionalparticles and tight-junctional elements (Revel, Yip & Chang, 1973; Decker & Friend,1974; Albertini e* a/. 1975; Montesano, Friend, Perrelet & Orci, 1975; Yee & Revel,1978). In hamster cheek pouch epithelium, associations between gap junctions anddesmosomes, rather than tight junctions, were commonly observed and this relation-ship has been reported previously (Barnett & Szabo, 1973; Orwinei al. 1973; Archard& Denys, 1979). Furthermore, tight junctions were never observed between spinouscells in which the most active gap-junctional synthesis was detected (Table 3). In thefreeze-fracture study of Shimono & Clementi (1976), tight junctions were observedonly in the upper granular layer of rat oral epithelium. Thus it would appear that nofunctional relationship between gap and tight junctions exists in stratified squamousepithelium. The occasional presence of tonofilaments with annular gap junctionsobserved in the present work (see Fig. 11) and illustrated by other workers alsosuggests an association with desmosomes.

The present report has demonstrated the potential of using quantitative morpho-logical techniques to study intercellular junctions on sectioned tissues. We sampledonly those junctions and membranes that were sectioned perpendicularly to obtainrelative estimates. Such sampling should not introduce systematic errors of gap-junctional relative surface areas, since there is no reason to suppose that junctionalareas in obliquely sectioned membranes are any different from those sectioned per-pendicularly. In estimates of plasma membrane areas for the average cell, both per-pendicularly and obliquely sectioned membranes were quantified. In grossly obliquesections, membrane profiles are not always morphologically distinct entities and thusthe data for absolute areas of gap junctions are likely to be underestimates. Neverthe-less, the results obtained are valuable for comparing individual epithelial strata.

During epithelial differentiation, all three relative surface estimates exhibit similartrends, with the highest values being found in spinous cells and the granular layerhaving the lowest values (Table 2). These data suggest that the basal cell, the leastdifferentiated epithelial cell, is capable of producing gap junctions. In subsequentepithelial cell migration, the relative surface estimates differ markedly, suggestingthat metabolic turnover of either the gap-junctional elements or the plasma mem-branes is occurring. This suggestion becomes more clear when the absolute data areconsidered, since the possibility of synthesis of areas of unspecialized membrane,which can obviously affect relative surface estimates, is taken into account. FromTable 3 there is substantial evidence that active synthesis of gap junctions is takingplace between basal and spinous layers, whereas subsequently reductions in total gap-junctional area occur, from which we conclude that gap junctions are being degraded.Alterations occurring in total gap-junctional estimates are paralleled by those for bothperipheral and annular junctions. Consequently, it becomes necessary to suggest waysin which gap-junctional turnover might be effected in stratified epithelia.

There are several freeze-fracture studies that describe the formation of gap junc-tions either in vivo or in vitro (Revel et al. 1973; Albertini & Anderson, 1974;Benedetti, Dunia & Bloemendal, 1974; Decker & Friend, 1974; Johnson, Hammer,Sheridan & Revel, 1974; Decker, 1976; Yee & Revel, 1978). Initially, formation


plaques appear in areas of non-specialized membrane. This is followed by the appear-ance of large 'precursor' intramembranous particles with a concomitant reduction inthe width of the intercellular space. The precursor particles are subsequently replacedby smaller 'junctional' particles in characteristic polygonal arrangements. The junc-tion then increases in size by the insertion of additional junctional particles. Thesestages are difficult to identify in thin sections since little intra- or extracellularmodification of the contributing membranes is apparent. At present little is knownabout the factors that control gap-junctional synthesis, but there are reports of in-creased synthesis during epithelial regeneration following wounding (Gabbiani,Chaponnier & Huttner, 1978; Yee & Revel, 1978), in ependymoglial cells in responseto thyroid hormones (Decker, 1976), in pancreatic ^3-cells following stimulation ofinsulin secretion by both glucose and glibenclamide (Meda, Denef, Perrelet & Orci,1980), following infection of skin by Molluscum contagiosum virus (Caputo, Gas-parini & Innocenti, 1980), in the ovarian follicle in response to hormonal stimulation(Albertini & Anderson, 1974) and during the course of mucous metaplasia inducedby vitamin A (Elias & Friend, 1976).

Mechanisms of gap-junctional removal have been suggested by Epstein, Sheridan& Johnson (1977), who described dispersion of gap-junctional particles followinginterference with protein synthesis. Drugs such as concanavalin A (Meller, 1979),which bind to cell surface receptors, and colchicine (Meller, 1981), which disruptsmicrotubules (Wilson et al. 1974), have been shown to interfere with the assembly ofgap-junctional intramembranous particles. Gap-junctional disruption has also beeninduced by perfusion with hypertonic dissaccharide (Goodenough & Gilula, 1974),combinations of enzymes and divalent-cation chelators (Metz, Forssman & Ito, 1977)or by using chelating agents and sucrose (Campbell & Albertini, 1981). The depen-dence of junction formation on the presence of isotonic Ca2+-containing salt solutionshas also been demonstrated (Campbell & Albertini, 1981).

Annular variants have been interpreted as being truly intracellular and consequentlyhave been termed 'sphaerae occlusae' (Espey & Stutts, 1972). These authors sugges-ted that the interiorization of gap junctions might be a means of transferringcytoplasm from one cell to another, whereas Merk et al. (1973) suggested that theymay represent a means of removing immobile or adhesive junctions from the surfaceof ovarian granulosa cells. In relation to the present report, the latter would providea means of permitting movement of epithelial cells relative to each other during thecontinual migration of cells towards the surface during differentiation. There is noconclusive evidence that annular gap junctions in stratified squamous epithelia areactually interiorized. Their annular appearance could be produced by transversesections through the loop-like peripheral junctions that are in contact with the plasmamembrane (see Figs 8, 9, 10). The presence of the intercellular tracer lanthanumwithin the intercellular component of these junctions (Orwin et al. 1973), includingthose situated deep within the cytoplasm (Shimono & Clementi, 1976), has beendescribed and it is therefore possible that many of these annular variants are not trulyintracellular but are found on very long invaginations of the cytoplasm of one cell intothat of an adjacent cell. Since we have not observed any such extensive invaginations


in longitudinal section, this possibility would seem more likely for annular junctionssituated in the peripheral, rather than the central, parts of the cytoplasm. Further, ifannular junctions represent gap junctions present on long cylindrical extensions,random plane sectioning should generate a significant number of profiles that areelliptical, with substantially different axial lengths. In all annular junctions observedin the present study, axial lengths differed only slightly, suggesting that they representprofiles of an essentially spherical component in central parts of the cytoplasm, or ofa curved junction in the peripheral cytoplasm.

If we assume that annular variants represent a stage in the process of degradationof peripheral junctions, it might be expected that the annular junctions would increasein area in proportion to a decrease in the area of peripheral junctions. From Table 3,however, we see that the absolute surface areas of both peripheral and annular junc-tions increase approximately threefold between basal and spinous layers, suggestingsimultaneous formation and degradation. While this might seem an inefficient biologi-cal mechanism, it should be borne in mind that the cells in this tissue are undergoingan orderly migration during differentiation and that the making and breaking ofintercellular junctions might be a necessary requirement for the structural and func-tional integrity of this epithelium. We have made a similar suggestion with regard todesmosomal junctions in this tissue (White & Gohari, 1984a,6). The turnover ofdesmosomes can be induced following incubation with extrinsic proteases (Overton,1968; Fukuyama, Black & Epstein, 1973) or chelating agents (Borysenko & Revel,1973), and these studies have indicated that desmosomal plaques undergo endo-cytosis. However, in our in vivo studies we have not detected endocytotic vacuolescontaining desmosomal or gap-junctional remnants, and if the annular junctionrepresents a stage in the turnover of gap junctions, its subsequent fate remains un-known. There is a need for a precise evaluation of events concerned with junctionaldegradation. At present, quantitative morphological studies are hampered by theproblems in determining whether an annular junction is intracellular or not. Tracerstudies are not reliable since the problems associated with penetration do not guaran-tee homogeneous 'labelling' of all extracellular spaces. The only available alternativeis that of serial sectioning, which is difficult to perform on a large scale.

Morphometry has been used in several previous studies to estimate the relativesurface areas of gap junctions on plasma membranes. The results (Table 5) indicatethat these cells have between 1 and 10% of their membranes occupied by gap junc-tions. Only in the ovarian follicle are values higher than this found (Coons & Espey,1977), whereas Gabbiani, Chaponnier & Huttner (1978) reported that epidermalplasma membranes have less than 1 % of their surface area differentiated into gapjunctions. There are several other studies in which gap junctions have beenquantified using methods that are not directly comparable to those of the presentreport (McNutt et al. 1971; Sheridan, Hammer-Wilson, Preus & Johnson, 1978;Hooper & Parry, 1980). Since gap-junctional densities are similar in cheek pouchepithelium and liver, a tissue of high metabolic activity, one may reasonably suggestthat the gap junctions play a significant role in the functional activity of cheek pouchepithelial cells.


The gap junction represents the structural component responsible for electro-chemical communication between cells, enabling direct transmission of electricaland chemical signals between adjacent cells. Perissel et al. (1976) have suggestedthat gap junctions are indispensible for cellular coupling and are necessary tomaintain the synchronous differentiation of hepatic tissue. Hooper & Parry (1980)have described a reduced area of gap junctions per unit cell volume in a metaboliccooperation-defective embryonal carcinoma cell variant when compared with normalcells. Although experimental studies have indicated that molecules of different sizeand structure can pass through gap-junction channels in vitro (Bennett &Goodenough, 1978), little is known about the type of molecules involved inintercellular communication in vivo. There is obviously a requirement for theorderly regulated differentiation of cells in normal epithelium and the presence ofgap junctions occupying a substantial area in basal, spinous and granular cellsprovides the structural pathway for such regulatory homeostatic control. Theobservation by Gabbiani et al. (1978) that wounding of rat skin induced anapproximately threefold increase in gap-junctional surface area suggests that whenthe normal state is disturbed, the structural pathway for intercellular communicationis increased. In this particular case, cytoplasmic factors responsible for the controlof both cell division and cell movement might pass across the gap-junctionalchannels.

Although structural evidence for the presence of gap junctions exists, there isno certainty that all the channels are patent and some aspects of this problem havebeen discussed by Bennett & Goodenough (1978). Thus large, extensive junctionsmay be relatively impermeable whereas small junctions might provide a rapidcommunication pathway. At present our knowledge of the structure—functionrelations of gap junctions is limited but possibilities for significant advances in ourunderstanding of these membrane components might be provided by combinedmorphometric and electrophysiological approaches (Sheridan et al. 1978). From apurely morphological viewpoint, much may still be learned by applying stereologicaltechniques to investigate differences in gap-junctional area and frequency inepithelial tissues subjected to adverse chemical and/or mechanical stimuli, in whichmorphological changes in the constituent cells are induced. These should includeinflammatory and hyperplastic conditions as well as benign and malignant neoplasticconditions. Indeed, Meyer & Overton (1983a,b) have used a morphometricapproach to investigate changes in gap junctions during embryonic chick liverdevelopment and its modulation by inhibition of cell proliferation or by promotionof cell maturation. The more widespread application of quantitative morphologicaltechniques to both thin sections and freeze-fracture replicas could provide a morecomplete knowledge of the structure and function of these important intercellularjunctions.

This work was funded in part by grants from the Medical Research Council (F.H.W.), theWellcome Trust (F.H.W.) and the Yorkshire Branch of the Cancer Research Campaign (K.G.). Thesupport and facilities provided by Professors C. J. Smith and R. Barer are gratefully acknowledged,as is the technical assistance of Mr M. A. Turton, Mr N. J. Smith and Ms M. Tune.


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{Received 18 January 1984-Accepted 13 February 1984)

ultrastructural morphometry of gap ...cap junctions in epithelium 69 no. of animals 5 table no. of...

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