a study of the intestinal epithelium of the goldfish …...a total of 200 goldfish were used, of...

93

A study of the intestinal epithelium of the goldfishCarassius auratus: its normal structure, the dynamics of

cell replacement, and the changes induced by salts ofcobalt and manganese

ByT. VICKERS

(From the Physiological Laboratory, Downing Place, Cambridge)

With 2 plates (figs, i and 2)

SummaryThe normal structure of the intestine of the goldfish is described, with special atten-tion to the character of the mucosal epithelium.

The rate of epithelial replacement has been determined by the use of colchicine, andfound to vary between 6 days for the upper intestine and 9 days for the lower parts.The colchicine data have also been used to determine the duration of a normalmitosis, which has been found to be between 2 and 3 h.

Cobaltous and manganous salts have been shown to cause a considerable buttransient increase in the level of mucification which can be ascribed to the formationof new mucous cells; although there sometimes appears to be formation of mucoidsubstances in the principal cells, there is no evidence that these cells undergo trans-formation into goblet cells or that goblet cells ever become principal cells after theyhave lost their secretion.

It is suggested that mitosis in the intestine produces cells initially capable of bothmodes of differentiation, and that the effect of the metal ions is to increase temporarilythe likelihood of mucous differentiation.

IntroductionCOBALTOUS salts have been shown by Van Campenhout and Cornelis (1951)to cause degenerative changes in the a-cells of the islets of Langerhans in thepancreas of rabbits. In a study of the effects of hypertonic sodium chloridesolutions on fish, similar changes were observed in the islet cells, and the effectof cobaltous salts on the reaction to the salt load was therefore thought worthinvestigating. It soon became apparent that the effects on the a-cells werenon-specific; many forms of stress proved capable of causing degranulationof the cells, which seem, in the fish, to be extremely labile. A simultaneousaction of cobalt on the alimentary canal was, however, noted, and that actionis the subject of the work here described.

The effect, at first sight extremely variable in its intensity, was an increasein the level of mucification in the intestinal mucosa; in some cases the changewas a very dramatic one. The phenomenon seemed worthy of attentionbecause the intestinal epithelium, in mammals at any rate, has the property,shown to a comparable extent only by bone-marrow, of both having anextremely high rate of replacement of cells and at the same time maintaining

[Quarterly Journal of Microscopical Science, Vol. 103, part 1, pp. 93-110, March 1962.]

94 Vickers—Intestinal epithelium of the goldfish

remarkably constant the number, and the relative proportions, of the differentcells. The mechanisms underlying this dynamic equilibrium are still obscure,partly because in the mammalian intestine, although the degree of activity ofan individual cell can be altered, the balance of the cell population as a wholeis not readily susceptible to experimental changes. Here was apparently ameans by which to cause profound changes.

The first necessity was to show that the dynamic equilibrium present inmammalian intestine is also present in the fish; this condition is not entirelyfulfilled in that, although the population shows a relatively rapid renewal, itscomposition is much more variable than is that of the mammal. But it hasproved possible to relate the changes noted to the actions of the metallic ions.

In the first part of this work, the structure of the intestine of the normalfish is described, since there appears to be no recent account of its structurein the species used, the goldfish, Carassius auratus (Lin.). Secondly, the rateof epithelial replacement is assessed using the colchicine technique of Leblondand Stevens (1948); the alkaloid colchicine blocks cell-division in metaphaseand thus enables the determination of the number of cells entering mitosisover a known period. Thirdly, the actions of cobaltous salts, and those ofsome other heavy metals, are described.

Methods and materialsA total of 200 goldfish were used, of which 130 provided material for histo-

logy. Goldfish were purchased locally and were normally kept for about3 weeks in the laboratory before use so that they might become adapted tothe conditions there. During this period approximately 10% died, but afterthis the death-rate was very low among the normal fish. The stock of fish waskept in accumulator jars holding about 30 1. of water and 4-5 fish of averageweight 5 g each.

Experimental fish were kept, generally in pairs, in smaller tanks holding5 1. of water. All the tanks were continuously aerated and were filled withCambridge tap-water which was vigorously aerated for 48 h before the fishwere added. During the greater part of the work the water temperature wasuncontrolled until fluctuations in temperature were realized to be a cause ofinconsistent controls. Thereafter, the temperature was held between 17-200 Cthermostatically, and no experiments were done in hot weather if the watertemperature rose above 200 C.

The usual form of experiment to test the effects of agents on the level ofmucification in the intestine was to keep a group of fish in a known concentra-tion of the agent, and to take fish for histological study at intervals, usuallyincreasing geometrically, e.g. after 1, 2, 4, 8, . . . days. Controls were takenat intervals from the stock tanks, and any changes produced by the experi-mental agents defined by comparison with these controls and with referenceto the kinetics of epithelial replacement as determined by colchicine.

Colchicine was given by intraperitoneal injection of aqueous solutions.Using a 20-gauge needle carefully inserted between the scales at the posterior

Vickers—Intestinal epithelium of the goldfish 95

end of the lateral wall of the abdominal cavity so that the needle was runninganteriorly, injections of up to 0-2 ml could be given without leakage (checkedwith strongly coloured injections) or any apparent harm to the fish. The areaof injection was previously painted with tincture of iodine, which seemed toassist by causing a plug of mucus to be formed. Injections were done with thefish out of water, after immobilization with water chilled to about 40 C.

The intestine was removed intact, complete with the associated liver,pancreas, and spleen, and fixed without further dissection. The final paraffinblock was sectioned in the horizontal plane, and by taking sections at variouslevels through the block a representative series of points along the length ofthe intestine could be studied.

Good fixation of the tissues of the goldfish was extremely difficult to achieve:in general, any fixative gave results considerably worse than it usually giveswith mammalian tissues. Helly's fluid was finally chosen for routine use, withthe sodium sulphate of the original formula reduced by 25 % and with theformalin neutralized with magnesium carbonate. Fixation for 24 h wasfollowed by thorough washing in running water. Some tissues were post-chromed, either after Helly's fluid or after Regaud's.

Tissues were dehydrated in alcohol and cleared, at first, in benzene orcedar-wood oil; but later a modification of a w-butyl alcohol technique recom-mended by Gatenby and Beams (1950) for difficult tissues was used. Thisclearing agent has the advantage that it will tolerate up to 4% of water; itcauses little shrinkage and hardening itself, and it reduces the subsequentdamage caused by the paraffin bath. By using a brief rinse in benzene toremove the butyl alcohol, the prolonged infiltration in butyl alcohol-paraffinmixtures of the original technique becomes unnecessary and the method moresuitable for routine use.

The structure and morphology of the alimentary tractAs might be expected from other studies of the anatomy of the fish alimen-

tary tract (e.g. Al Hussaini, 1949; Rogick, 1932), the goldfish, as a predomin-antly vegetarian feeder, has a gut of considerable length, approximately 6 cmin fish weighing 5 g. Although the well-marked regional differentiation ofstructure shown by the guts of tetrapods is absent, certain segmental transi-tions can be distinguished.

The pharynx, longitudinally ridged and lined with a stratified, heavily-mucified epithelium, opens directly, via a large sphincter, into the intestine;no stomach, in the sense of a cytologically differentiated structure, is present.The intestine immediately dips down ventrally behind the heart and greatveins and then runs caudally along the ventral wall of the abdominal cavity.A well-developed system of honeycomb-like modifications of the mucosa lies,predominantly, caudal and dorsal to the junction of the pharynx and theintestine, but, unlike the pyloric caecae of some other species of fish, thesestructures are contained within the muscular coats, which are continuous overthe pharynx and intestine.


Below the caecae, the bile duct enters the intestine, the muscular coatsserving as a sphincter. Below the entry of the bile duct, the muscle-coats aremuch reduced; the striated muscle, which is a specially prominent feature ofthe two sphincters, eventually disappears and is replaced with a double layerof smooth muscle of which the inner, circular, layer is the better developed.

The section of intestine supplied with striated muscle constitutes theequivalent of the so-called intestinal bulb of other species, and below it thereare no marked changes in the macroscopic appearance of the gut. There is,however, a gradual reduction in calibre which is paralleled by a progressivereduction in the degree and amplitude of folding shown by the mucosa, whichis thrown into a system of reticular or zigzag folds or rugae along the wholelength of the tract; the long axis of these folds lies predominantly longitudin-ally in the upper intestine; lower down the predominant axis is transverse.No villi are present, and at no point are any true glandular developments ofthe mucosa present. The epithelial lining of the caecae is identical with thatof the associated intestine; there are no cytological modifications for specializedactivity.

The basic arrangement of the intestine in the abdominal cavity is a longi-tudinal and vertical coil, and in the young fish may be seen as such. Viewedfrom the animal's left side, the sense of coiling is anticlockwise beginning fromthe intestinal bulb, but superimposed on this simple coiling is a variabledegree of doubling and folding of the loops of the coil. Some arrangement ofthis kind is obviously necessary to accommodate within reasonable dimen-sions increases in the length of a coil with fixed ends, and since, as the animalgrows, the length of the intestine increases out of proportion to the lineardimensions of the abdominal cavity, the folding of the coil is greater in olderanimals. There is also a good deal of variation in the length of the intestine inanimals of the same size; this seems to be associated with the chronic stateof nutrition of the animal.

The epithelial lining of the intestine is composed mainly of two cell-types,which are indeed the only cells described by previous workers (McVay andKaan, 1940; Curry, 1939): ordinary columnar cells, the principal cells; andgoblet cells. The mucosa, especially in the upper intestine, is often heavilyinfiltrated with cells, presumably lymphocytes, which have small, dark nucleiand little cytoplasm.

The columnar epithelium is simple in nature, except that at the bases of therugae it may be pseudo-stratified; normally all the mitotic activity lies in thecells near the boundary between the simple and pseudo-stratified zones. Theother cell types are also not uniformly distributed; in the control animals thereare progressively fewer mucous cells towards the apex of a ruga, while in thebottoms of the folds mucous cells may predominate.

This distribution of the two cell-types parallels that found on a mammalianvillus (Moe, 1955), and the restriction of division potential to a limited partof the epithelium is also characteristic of a mammal (Leblond and Stevens,1948). The cytology of both the columnar cells and the goblet cells is


apparently similar to that of the same cell types as found in a mammalianintestine.

Two other cell types are variably present; the first is similar in appearanceto the goblet cell but with a much smaller enlargement, or theca, in which,again in contrast to the goblet cell, the presumed secretion remains afterfixation in the form of discrete granules. The granules themselves are acido-phil and stain only feebly with the periodic acid/Schiff reaction but morestrongly with Gomori's aldehyde/fuchsin. They thus resemble the zymogengranules of pancreatic acini rather than the stored secretion of the goblet cells,which is strongly basophil, gives a strong periodic acid/Schiff reaction butusually only a weak staining with aldehyde/fuchsin. These cells, which areoften apparently absent, and whose number is never great, are characteristicof the upper and middle intestine rather than of the lower parts. Theirdistribution is therefore not the same as the mucous goblet cells, whichbecome progressively more frequent lower down the intestine. They arepresumably enzyme-secreting cells, comparable perhaps with the Panethcells of the mammalian intestine.

Al Hussaini (1949) in his study of fish intestines (not including that of thegoldfish) considered that zymogen and mucus are produced by one type ofcell at different times in its life-history. This seems unlikely as a generalphenomenon, although the possibility is difficult to eliminate as an abnormaloccurrence; in my view, there is certainly no invariable occurrence of sucha stage in the life-history of the mucous cell.

The second cell-type previously undescribed in the goldfish has also beennoted by Al Hussaini in several other species, although apparently not by anyother worker. Al Hussaini calls it a 'pear-shaped' cell and considers it to bea stage in the life-cycle of the goblet cell. I sympathize with that statement,but with several reservations: under experimental conditions pear-shapedcells may occur in the bile duct, where goblet cells are never found; if a pear-shaped cell is related to any other type of cell, it is clearly the terminal cell-state, since what appear to be the remains of its characteristic 'strings' areoften to be found lying between the other epithelial cells of the mucosa. Thereseems to be no simple explanation of the pear-shaped cell's peculiar structure(fig. 2, A) ; the number of strings (9-12) varies little and the staining properties(table 1) of the strings and their beading are relatively constant. The cyto-logical appearance is substantially the same with each of the fixatives used,and the strings therefore seem unlikely to be a gross artifact. The brush-border of the columnar epithelial cells is interrupted where these cells projectto the surface, and it is commonly impossible to see any boundary membraneat those points in the free border of the epithelium. The pear-shaped cellis clearly not an inevitable stage in the cycle of the goblet cell although, sinceit is most common under experimental conditions which also favour theproduction of goblet cells, some relationship is not unlikely.

No argentaffin cells are demonstrable by either the Fontana or the diazomethods (Pearse, 1953).


The other histological differentiation which is of interest is the demarcationof the terminal segment of the intestine by the presence of a heavy granulationin the distal parts of all, or a large proportion, of the columnar epithelial cells.Under experimental conditions granules with the same staining propertiesoccur in other parts of the alimentary canal but, in contrast to McVay andKaan (1940), I find this abnormal; there is normally a sharp transition pointbetween non-granular and granular cells which marks off the final section of

T A B L E I

Staining properties of the 'pear-shaped' cells

Reaction

Gram method (Pearse, 1953)Periodic acid/SchiffAldehyde/fuchsin .Azan . . . .Iron haematoxylin.Feulgen (nucleus + )Sudan black (at 6o° C) .Osmiophilia .

Beads

blue

Strings

red

the intestine, presumably the rectum, although the presence in the goldfishof a true rectum is doubtful; the criteria for this segment given by Barrington(1957) are not satisfied: ileocaecal valve, greater development of the muscle-coats, and increased mucification. Although the level of mucification rises alongthe length of the intestine in the goldfish, the rise is gradual and without anysharply marked transitions. The granules are unlikely to be of 'zymogen', asMcVay and Kaan suggest, since the length of the intestine involved is short(about 6 mm) and the functional value of enzyme secretion at that level mustbe very small.

Curry (1939) described a terminal segment with stratified keratinous epi-thelium which he regarded as ectodermal in origin. If such a segment occursin the goldfish it cannot be more than 1 mm long, representing an ano-rectaljunction and being that length of gut remaining attached to the anus when thealimentary tract was dissected out.

Groups of nerve-cells (presumably parasympathetic neurones) are commonin the wall of the intestinal bulb of the goldfish, lying within the outer layersof muscle, especially in association with the two sphincters. This appears tobe an example of autonomic motor-nerves to striated muscle; where thestriated muscle disappears farther along the intestine, the nerve-cells are farfewer, and in the lower half of the intestine they are rare or perhaps evenabsent. No mucosal plexus appears to be present in any region; since thereis little or no intestinal secretory activity requiring neural control, this is notparticularly surprising, and large-scale movements of the intestine wouldsimilarly not be expected, except at the sphincters, since the loops of intestineare closely bound together, and no movements except spasm of the circularmuscle are seen in isolated pieces.


All the epithelial cells of the mucosa, except cells in mitosis, are attachedto a well-developed basement membrane. There is no muscularis mucosa,and smooth muscle seems to be absent from the lamina propria, whichextends from the basement membrane to the muscle-coats. The laminapropria is of loose areolar tissue often containing large numbers of granulatedcells which are considered by some workers to be mast cells. Their stainingproperties, in the goldfish at any rate, do not seem to support this suggestion;the granules neither show strong metachromatic staining properties, nor givea positive reaction with the periodic acid/Schiff method, nor stain withaldehyde/fuchsin: all characteristic of the mammalian mast cell. The numberof granulated cells is extremely variable and not obviously correlated with anyother factor.

The rate of epithelial replacement in the intestinal mucosaThe restriction of division potential to the cells of the crypts of Lieberkiihn

in the mammalian intestine was first noted by Bizzozero (1892), and is nowusually held to indicate the replacement of the cells of the villus by themigration of new cells from the crypts. The speed of this 'escalator' wasdetermined by Leblond and Stevens (1948) in the rat, and shown to besurprisingly high; the life of a cell on the villus must be of the order of 2 daysonly.

If the cells of the ruga move as a sheet, all at the same rate, then the timetaken for complete renewal may be defined as the period necessary for theoccurrence of a number of cell-divisions equal to the total number of cells onthe ruga; colchicine can be used to determine the number of cells which entermitosis per unit time and, from that figure and from a knowledge of thenumber of cells in the population to be renewed, the renewal time can becalculated. The situation is complicated, however, by the fact that not all thecells of the ruga will move at the same rate; so far as can be judged histo-logically, cell loss takes place almost exclusively from the peaks of the rugae,so that at the base of each ruga there is a population lying up-stream of thedivision zone and therefore moving only when a cell death takes place. Therate of movement must increase in the division zone itself, and in the epi-thelium above the division zone cells are usually assumed to move at a uniformspeed.

The most meaningful estimate of renewal time will thus be obtained byconsidering only the epithelium above the division zone, that is, the simpleepithelium; the pseudo-stratified epithelium will be static apart from itsborder, containing the dividing cells, and except for unorganized movementscaused by random cell deaths.

The optimum dose of colchicine for mammalian tissues has been found byLeblond and Stevens to be of the order of 0-2 mgper 100 g body-weight; theyused 6 h for blockade. In the goldfish, this dose does not cause any obviousincrease in the numbers of cells in division until after 12 h blockade, and after24 h a few cells are beginning to break free: stages later than metaphase begin

ioo Vickers—Intestinal epithelium of the goldfish

to appear. With doses of 0-4 mg per 100 g this waning of the colchicine effectdoes not appear. The best way of determining the number of cells enteringdivision seems to be to subtract the number of blocked metaphases after 12 hfrom the number after 24 h; this gives the number of cells entering divisionin the second period of 12 h and avoids complications due to the apparentdelay before colchicine begins to act.

TABLE 2

Frequency of cell-division in the intestinal epithelium

Fish

ControlControli2

345678

Dosage of colchicine

—0-2 mg per 100 g

,,

O'4 mg per 100 g

Average values for:Controlsi, 3, 4, and 75, 6, and 8

Replacement times

Time ofblockade

—12 h24 h12 h12 h24 h24 h12 h24 h

—

12 h24 h

Number of mitoses per

Upperintestine

1 8262 4743 02 4

1 0 49 02 49 0

2 2

2 41 2 4

6 days

Middleintestine

1 610r44 02 0187 27 2

1 464

1316

949 days

,000 cells

Lowerintestine

1610

1 44 2161 26 0601 0 *

54*

13H8af

9 days

* These fish were treated with cobalt and showed a high level of mucification.f Fish 2 is omitted because it showed a substantial number of mitotic stages later than

metaphase.

Four fish were killed 12 h after receiving 0-2 mg colchicine per 100 g body-weight, and 4 were killed after 24 h at the higher dose. In each fish thenumber of cells blocked in metaphase was counted on a total of 5 rugae ateach of three levels in the intestine; the number of blocked metaphases in thedivision zones of the same rugae was also determined. The figures for the8 fishes expressed as the number of mitoses per 1,000 cells are given in table 2.The considerable range of variation will be noted, but since only the correctorder of magnitude is important, it seems reasonable to proceed.

If #]2 = number of blocked metaphase per 1,000 cells after 12 h,and #24 = the number after 24 h,then x2i—x-,2 = the number of cells entering division per 1,000 cells in

12 h = X.

Therefore the number entering division in one hour

= ^X per 1,000 cells= the number of new cells formed per hour per 1,000 old cells.


Therefore the time taken to form 1,000 new cells, that is to completelyrenew the simple columnar epithelium,

xApplied to the figures in the table, this argument gives renewal times for

the upper intestine of approximately 6 days, and for the lower and middleintestine of approximately 9 days.

Colchicine can also be used to determine the duration of a normal mitosis;all the dividing cells in the population in the control fish must be counted( = xc per i.ooo cells).

Then if -^X cells/1,000 cells enter mitosis per hour and xc cells/1,000 cellscan be seen to be in mitosis at any one time, the duration of a normal mitosismust be

XI2

x h-The figures for two control fish, injected with saline only, are included intable 2; the time for cell-division in the goldfish, calculated from these figures,is between 2 and 3 h.

The effects of the experimental agentsThe experimental observations on the animals treated with metallic ions are

presented in table 3; to correspond with the different renewal rates, the levelof mucification is separately expressed for the upper intestine and for themiddle and lower intestine. Three degrees of mucification were recognized:high (H), normal (N), and low (L); the level in the simple epithelium is givenseparately from that in the pseudo-stratified zone. Only considerable differ-ences from the control levels were classified as being H or L.

Both cobaltous and manganous salts in the experimental concentrationsstimulated the formation of mucous cells in the intestine; this effect firstshowed in the division zone and then spread up the walls of the rugae. Thisprovided a further control for the experiments, since the composition of newlyformed epithelium could be compared with the remains of the population asit was before the experiment began. Cases in which the entire rugae werefound to be heavily mucified after short treatment could usually be explainedby reference to some other cause rather than on the assumption that mucifica-tion was stimulated at all points on the rugae. Two such conditions could bedefined from the occasional controls which showed high level of mucification:high environmental temperature and overcrowding. The latter condition wasovercome, or at least standardized, by allowing z\ to 3 1. of aerated water toeach fish of about 5 g: the former by regular checks of the water temperatureand by thermostatic control. With these precautions the range of variationshown by the control fish was much reduced.

Nickel and zinc ions were only slightly effective, if at all, at the lower of theconcentrations used; at the higher concentration they were obviously very toxic.

Vickers—Intestinal epithelium of the goldfish

TABLE 3

Data from the experiments involving treatment with salts of heavy metals

Fish

9I Oi i

2

3456789012

4567S9

3i3233343536373839404.142434445464748495°515253545556575859606162

Treatment

Co++ M/600

CO++'M/3OOCO++ M/1,200CO++ M/300CO++ M/600CO++ M/I.2OOCo++ M/600Co++ M/600, M/300Co++ M/600Co-1"1- M/1,200Co"1"1" M/600

"

Co++M/300Co-1-1- M/600

lt

Co++M/600, H2O

Co"^ M/600, H A Co+ + M/600

Mn + + M/600?1

Mn++>M/i,2OO

Mn++ M/300Mn++ M/600

,,

Mn++

Zn++

M/300

vr/i,2ooZn++ M/600N1++ M/1,200Ni++ M/600Kept in water at between 25 "-30° C

Duration oftreatment

12 h1 day2 days4 days5 daysS days5 days5 days6 days6 days6 days7 days4 + 3 days8 days8 days

10 days10 days13 days13 days18 days18 days31 days31 days36 days36 days38 days40 days45 days

2 + 2 days2 + 6 days2 + 6 days6 4-11 4-1 days6 4 - i t + 6 days1 day2 days2 days2 days2 days2 days2 days4 days4 days

12 days17 days17 days30 days42 days42 days

5 days1 day5 days2 days

10 days10 days

~,evel of mucification

Upper intestine

D

NNHHHNHNNNNLLLNLNNNNNHHNHHHHHLLNHNHNNNNHHNNNNNNNN—N—•HH

R

NNNHHHHNHNNNNLNLNLNNNHHNNHHHHLLNHNNNNNNNHHNNNNNHN—N—HH

Middle and lowerintestine

D

NNHHHHHNHHNNNHNLLNNNNHHNNHHHHLLNHNHHHNNHHHNNNHHHN—H—HH

R

NNNHHHHNHHNNHHHNNLNNNHNNNHHHHLLNHNNNNNNNHHNNNNHHN—N—HH

D = the division zone of the ruga; R = the epithelium above the division zone of the ruga.Cobaltous ions were present as either the nitrate or the sulphate; manganous, nickel, and zinc ions

vere present as sulphates.


The importance of the time-scale and the spatial distribution of the effectswas not realized in the early experiments, and conflicting results wereobtained until the stimulatory effects of cobalt and manganese were realizedto be transient rather than permanent; thus in fish taken after 5 to 6 days inthe solutions of the metallic salts the level of mucification might be, in aggre-gate, high or low or normal.

The characteristic levels of mucification (fig. 1, A-F), expressed in terms ofthe days of exposure to the experimental solutions, were as follows:

after 1 day: normal;after 2 days: heavy mucification in the pseudo-stratified zone,

especially in the division zone;after 4 days: heavy mucification on the walls of the rugae (in the

simple epithelium);after 6 days: highest mucification levels on the rugae, reduced in

the pseudo-stratified zone;after 8 days: the level of mucification falling on the rugae;and after 16 days: apparently normal again.

The middle and lower intestine have both a higher control level of mucifica-tion than the upper section and a wider range of normal variation, and theylagged behind the upper intestine in the sequence of the changes, presumablybecause of their lower rate of cell replacement. This again resulted in whatat first seemed to be anomalies, especially in the period between the 4th and10th days when a high level of mucification might be found in one section ofthe intestine and a very low level in another. When the factors listed abovewere considered, however, almost all the experimental animals conformed tothe basic pattern.

The transience of the effects of the metal ions was further defined in experi-ments where brief exposure to the metal was followed by return to water, andfish again killed at intervals (table 3: fish 37, 38, 39). A period of between1 and 2 days in cobalt produced the same series of changes as did continuousexposure; a wave of heavy mucification which travelled up the rugae, followedby a period of greatly reduced mucification.

All these descriptions apply only to the simple columnar epithelium and tothe division zone; at the base of the rugae in both the control and experimentalanimals at all stages, there was usually a group of mucous cells which I inter-pret as being independent of the cell escalator and therefore having a long life.This point will be discussed later.

Simultaneously with the formation of goblet cells in the generative zone inresponse to the metals, granulations often appeared in the principal cells of thesimple epithelium. These granules have been previously referred to. Unlikethe goblet cells, they tended to disappear in the animals returned to purewater after immersion in cobalt and to persist for as long as the cobalt treat-ment continued, even increasing in amount.

During the period of cobalt treatment from the 8th day or so onwards, in


addition to the restoration of the normal population of mucous cells, pro-gressively greater numbers of the cells described in the previous section, andnamed by Al Hussaini 'pear-shaped' cells, began to appear. The number ofsuch cells was not sufficient for a definite site of origin to be decided, and theyappeared on all parts of the rugae, especially in the upper parts of the intestine.It is tempting to suggest that they represented an attempt to form anotherwave of mucous cells but that, with the prolonged cobalt treatment, the syn-thetic potential of the cell was altered to produce these 'strings' instead.

The strings, especially in their early stages, had staining properties whichseem to be consistent with their representing the Golgi network of the cellwith associated secretory material. The staining properties are shown intable i, with the observation of Al Hussaini that they are osmiophil included.As previously pointed out, whatever its nature, the pear-shaped cell is clearlya terminal stage since its stages of regression can be traced; it does not seemto be derived from a normal mucous cell since the terminal states of that cellcan also be readily defined, at any rate in the cobalt-treated animal. Itsoccurrence in the control animals is only infrequent and it may well representan abnormal cell. Pear-shaped cells may also appear in the bile duct, wherethere are normally no goblet cells even under the experimental conditions;in this site too, the pear-shaped cells are far more common after treatmentwith the metals.

In fish transferred from cobalt to water and then, after a time presumedto be sufficient to allow return to normal, re-exposed to cobalt, a second waveof mucification of the same proportions as before was produced (table 3 :fish 40 and 41), and this perhaps supports the view that the long-term con-tinuous treatment induced further changes in the cells. Variations in theconcentrations of the metals did not alter the time-course of the wave ofmucification; the lowest of the three concentrations did not produce anyeffect distinguishable from the control; the other two concentrations hadapparently identical effects so far as the extent and magnitude of the wave wasconcerned, but the higher concentration seemed to be more effective in theproduction of the string cells. Manganese, which could be used at higher

FIG. I (plate). Goldfish tissue stained by the periodic acid / Schiff method.A, upper intestine of control fish. Goblet cells at the bases of the rugae show supranuclear

granules of mucus, i.e. only these cells are engaged in active mucigenesis. Other cells showa positive reaction in the theca only.

B, upper intestine after 5 days of cobalt treatment. There is a high level of mucification.Cells at all points on the rugae show signs of mucigenesis.

C, upper intestine after 10 days of cobalt treatment. There is an unusually low level ofmucification. In the original slide it could be seen that the cells at the base of the ruga con-tained granules giving a positive reaction.

D, lower intestine of control fish. Few signs of mucigenesis.E, lower intestine after 5 days of cobalt treatment. A wave of newly formed goblet cells is

leaving the division zone.F, lower intestine after 8 days of cobalt treatment. There is a moderately high level of

mucification. Mucigenesis is visible at all levels on the ruga. Faint signs of granulation canbe detected in the principal cells.

Fie. i

T. VICKERS

FIG. 2

T. VICKERS


concentrations than cobalt without toxic side-effects, was extremely effectiveat those concentrations.

During the phase when the cobalt-induced mucous cells were spreading upthe rugae, two other cell types could be distinguished in greater numbers thanusual. One was a thin, strongly siderophil, cell without a brush-border, andthe other was represented by a nucleus with only a thin thread of residualcytoplasm reaching up to the free border of the epithelium. These I interpretas representing the normal terminal stages of the goblet cell; they were presentpredominantly near the tops of the rugae at the advancing front of mucouscells.

Cobalt, in addition to stimulating the formation of new mucous cells, oftenseemed to stimulate mucigenesis in those formed earlier; thus, cells on therugae, which do not normally contain mucigen granules in the supra-nuclearzone, often did so after exposure to cobalt, and sometimes the newly formedgoblet cells had the bulge or theca in that place rather than in the more usualposition. Sections stained with toluidine blue showed that many of the cobalt-induced mucous cells had relatively weak metachromatic staining properties;these two observations seem to imply that the action of cobalt is more likelyto be on the early stages of mucous formation than on the later ones.

Long-term experiments with cobalt (table 3: fish 30 to 36) showed that,after periods of about 3 weeks, symptoms most easily interpreted as represent-ing general toxicity began to appear: the rugae were greatly reduced in height,and the cells of the liver and pancreas became vacuolated and filled with fat.From this time onwards, the mortality rate increased markedly.

A consequence of the reduction in the number of cells on the rugae wasthat, since the reduction appeared to affect the principal cell population most,the level of mucification appeared to rise, but this I interpret as representingthe differential persistence of the goblet cells in a dying epithelium rather thanthe favouring of mucous cell-formation. During this period, mitoses werevery rare in the epithelium; presumably this was the reason for the reductionof the number of cells.

DiscussionThe relationships between the various cells of the intestinal epithelium have

been well reviewed recently by Moe (1955), and there is a classical review ofthe subject by Macklin and Macklin (1932).

Since the increased mucification began in the division zone of the ruga and

FIG. 2 (plate). Goldfish tissue stained with iron-haematoxylin and periodic acid/Schiff.A, bile duct after 30 days of manganese treatment. There are several 'pear-shaped' cells.

Although the typical structure can only be envisaged by focusing, most of the characteristicsof the cell can be seen here.

B, kidney after 18 days of cobalt treatment. There are densely-stained granules (proteinabsorption droplets) in the proximal tubule.

C, upper intestine showing a very high level of mucification. The common bile duct is alsoshown, and it can be seen that, although there is some positive reaction for mucus in the cells,there are no goblet cells present. Nor, at this time, have any 'pear-shaped' cells been formed.


since its later appearance over the remainder of the ruga was consistent withmigration of cells from the division zone, the entire increase in mucificationis ascribable to the formation of new mucous cells from undifferentiated cellsrather than to a transformation of already differentiated principal cells. Themucoid granulation, which was restricted to the cells above the generative zone,may appear to represent a partial transformation, but whether this is the caseor not, these granules never got beyond the stage of being a mucoprotein;sulphation never occurred, and the brush-border of the cell was never lost,so that there is no reason to believe that these cells were undergoing a genuinetransformation.

It is possible to identify and define the terminal stages of the mucous cellfirst as a cell staining heavily with iron haematoxylin, and later as a cell soshrunken as to be visible after staining with routine haematoxylins only asa nucleus lying between two principal cells. There is little evidence that thegoblet cell of the fish intestine is capable of more than one cycle of synthesisand discharge; signs of mucigenesis were usually restricted to the lower partsof the rugae.

Neither is there any hint that the discharged goblet cell becomes a principalcell. This denial can be supported by the distribution of alkaline phos-phatase; activity is present in the brush-border of the principal cells andbecomes progressively more intense from the division zone to the peaks of therugae. The only interruptions in this gradation are recognizable as eithergoblet cells or the cells which I have suggested represent the terminal stagesof goblet cell life-history; I find it difficult to believe that a discharged gobletcell could so rapidly assume an activity of enzyme as to take its place in thegradation shown by the principal cells. The principal cells at the level on theruga where goblet cell discharge is most obvious have presumably takenseveral days to form their own enzyme. If there were a significant number oftransformations of goblet cells into principal cells, one would expect to findconsiderable variation in the brush-border staining from cell to cell, depend-ing on the length of the life of each cell as a principal cell. I have made similarobservations on the rat intestine and, although the possibility of such trans-formations cannot be denied, they are obviously not normal phenomena.

The effects of cobalt and manganese varied with the time after the initialexposure to the metal, and they are most easily considered in terms of thekinetics of cell replacement as determined by the use of colchicine. Theeffects can be briefly summarized as follows:

a, after a short lag period, a wave of increased mucification spread up therugae from the division zone;

b, the phase of increased mucification was followed by a phase of greatlyreduced mucification; and

c, the population finally returned to within the normal range of variation.

The rate of spread of the wave of mucification is of the same order as thatexpected from the colchicine data but, even allowing for the lag period,


appreciably lower. The mitotic rate after cobalt was much the same as in thecontrols (fishes 7 and 8 in table 2 were cobalt-treated and apparently at thepeak of mucous cell-formation) and so an action on cell-division is probablynot the explanation. Three explanations for the discrepancy may be advanced:the rate of movement of the escalator as determined by colchicine will be anaverage value, and different types of cells and individual members of thepopulation may move at different rates. For reasons stated below, goblet cellsseem likely to be more closely adherent to the basement membrane initiallythan the principal cells and will therefore represent the more slowly movingcomponent. On this hypothesis, the advancing wave of goblet cells wouldmake up a relatively small proportion of the total population, with a fewgoblet cells caught up in the flow of principal cells and carried to the top of therugae.

Alternatively, the advancing front of mucification, representing the oldercells, may be retarded by the loss of mucus so that the cells are effectivelydisappearing at two points on the escalator. Either of these hypotheses mayalso provide an explanation of the way in which the proportion of goblet cellsdecreases towards the tops of the rugae. But the latter hypothesis alone seemsquantitatively inadequate; the number of discharged goblet cells, if my identi-fication was correct, was not sufficiently great.

In the rat intestine there are no signs of loss of discharged goblet cells; allthe loss is at the tops of the villi. Since there is no evidence for a transforma-tion of goblet cells into principal cells in that species, the first hypothesisseems the better: that above the division zone the principal cells move at auniform rate, while the goblet cells are almost static except for a small numberwhich get caught up in the stream of principal cells and accelerate away. Ifthis explanation is correct, the renewal rate of the principal cells must behigher than expected on the assumption that all the cells move as a continuoussheet, and the renewal of goblet cells may be very much slower. This hypo-thesis simplifies the problem of the maintenance of an epithelium of constantcomposition, because the bulk of formation of new cells can be of one sort: theprincipal cell. The formation of the comparatively small number of newgoblet cells may then be explicable as a fortuitous process.

This hypothesis, as applied to the goldfish intestine, may be stated in thefollowing terms: since the division zone is not at the base of the ruga, theremust be two components: one, above the division zone, moving relativelyrapidly; the other, basal to the division zone, moving slowly, if at all. Newlyformed cells must, therefore, differ in the rates at which they move, accordingto whether they originate near the top or the bottom of the division zone; sincethere are normally far more goblet cells below the division zone than above it,the implication is that the more slowly moving cells are those most likely tobecome goblet cells. If the essential difference between the goblet cell andthe principal cell is considered, it will be seen that the goblet cell is concernedinitially with protein metabolism and that the basal pole of the cell will be ofprimary importance; there is a growing body of evidence to indicate that cells


involved in this way in the formation of protein secretory products do notnecessarily synthesize the protein de novo from amino-acids, but usuallyabsorb as precursors proteins from the blood (Fisher, 1954). Such absorptionmay be assumed more likely in a cell which can establish a relationship withthe basement membrane and with the local blood-supply than in a cell whichis moving rapidly. I suggest therefore that the essential requirement for theformation of a new goblet cell is that an undifferentiated cell should have anopportunity to take up protein at its basal surface. Any factor which stimu-lates or facilitates this initial uptake of protein may then tend to increase thenumber of cells becoming goblet cells.

Both the formation of the granulation in the principal cells and the forma-tion of new goblet cells may be taken to represent an increased passage ofprotein into the cell, whether from the intestinal lumen or from the blood.This view of the effect of cobalt is supported by the fact that, simultaneouslywith the changes in the intestinal principal cells, there was also a great forma-tion of protein absorption droplets in the cells of the proximal tubule of thekidney (fig. 2, B). HOW cobalt brings about these changes is not clear: perhapsthere is some change in the permeability of the cell membrane; or perhapscobalt interferes with the processes responsible for the normal catabolism ofabsorbed protein, which then accumulates in the cell; or possibly the effectis elsewhere, by promoting the formation of blood-proteins of a kind morereadily absorbed into the cells than normally. As yet no decision is possible.

Similarly, the reasons why stress stimuli such as heat cause the formationof more goblet cells are still obscure; all that can be said with confidence isthat it is unlikely that cobalt and manganese, at least in the early stages of theiraction, are acting as non-specific causes of stress.

The phase of reduced mucification which follows the initial increase maybe explicable in the following way. During the period of heavy mucification,the formation of new cells seems to proceed unchanged, and, since the slowlymoving goblet cells then form a much higher proportion of the population, theother cells must move more quickly than is normal. If rapid movement isinconsistent with the initiation of mucous differentiation, few of these cellsare likely to become goblet cells, and the level of mucification must conse-quently fall below normal. Since the effect of cobalt on the intestine seemsonly transient, these cells and those formed subsequently are not abnormallystimulated to form mucus, and the sequence ends with restoration of thenormal picture.

A second hypothesis is that there may be two distinct populations of undif-ferentiated cells in the intestine, one dividing freely to produce principal cells,the other dividing much less often to produce goblet cells. On this argumentcobalt would be regarded as favouring the division of the second group, andthe waves of reduced mucification could be explained by a compensatorydecrease in mitotic activity, perhaps as a result of temporary exhaustion of thepopulation's reserve of division potential. I regard this explanation as lesslikely, since it assumes that the process of mitosis in the two populations


would not only react in opposite ways to the stimulus of cobalt, but wouldalso be affected to such a degree as to cause no significant change in the totalmitotic activity.

I therefore favour the first hypothesis, which has the virtue of simplicity inthat it does not require the existence of two distinct types of undifferentiatedcells; a uniform totipotent population, or perhaps more likely, a populationshowing a normal distribution of potential, is all that is required.

In summary, the hypothesis postulates:

a, a population of totipotent cells dividing in a limited area only;b, loss of cells at one point only, with the exception of occasional cell deaths

randomly distributed;c, as a consequence of a and b, a cell escalator moving from the source of

the cells to the point at which they are shed;d, a range of speeds of movement of the escalator in the division zone and

hence of the undifferentiated cells;e, the necessity for a cell, before it can become a goblet cell, to establish

contact with the basement membrane in such a way that protein for theinitiation of mucus formation can be readily absorbed;

/ , that as a consequence of e the more slowly moving cells are most likelyto become goblet cells;

g, that as a consequence of / goblet cells are likely to be more closelyadherent to the basement membrane than are the principal cells andhence will move more slowly;

h, that some of the goblet cells are caught in the stream of principal cellsand carried up the sides of the rugae;

/, that to maintain the population only a relatively small number of gobletcells need be formed: perhaps one goblet cell for every fifty principal cells(equalling the ratio of the two types of cells near the tops of the rugae).

It is worth noting that the progressively higher level of muciflcation in thelower intestine follows by this hypothesis from the lower rate of replacementof the epithelium.

Although these effects of cobalt and manganese should ideally be explainedin terms of their biochemical actions, there is so little relevant evidence avail-able at present that speculation in this direction is unjustified; especially sosince I have found no evidence that these metals have any marked effect onthe rat intestine. It is impossible to say whether the stimulatory effect of themetal ions on the formation of mucus is peculiar to the fish intestine, orwhether the mechanisms governing the relative proportions of the cell typesin the rat intestine are more stable and less liable to interference by externalfactors.

As it stands, this hypothesis is incomplete in that it does not offer anyexplanation for the restriction of the division potential to a part of the mucosa,but I hope to consider that and other questions in a paper dealing with theprocess of differentiation in the rat intestine.

i io Vickers—Intestinal epithelium of the goldfish

This work was partly carried out during the tenure of a Medical ResearchCouncil Scholarship. I should like to acknowledge the advice and helpfulcriticism I have received from Dr. E. N. Willmer.

ReferencesAL HUSSAINI, A. L., 1949. Quart. J. micr. Sci., 90, 323.BARRINGTON, E. J. W., 1957. In The physiology of fishes, edited by M. E. Brown. New York

(Academic Press).BIZZOZERO, G., 1892. Arch. mikr. Anat., 40, 325.CURRY, E., 1939. J. Morphol., 65, 53.FISHER, R. B., 1954. Protein metabolism. London (Methuen).GATENBY, J. B., and BEAMS, H. W., 1950. The microtomist's vade-mecum, n th ed. London

(Churchill).LEBLOND, C. P., and STEVENS, C. E., 1948. Anat. Rec, 100, 357.MACKLIN, C. C , and MACKLIN, M. T., 1932. In Special cytology, vol. 1, edited by E. V.

Cowdry. New York (Hoeber).MCVAY, J. A., and KAAN, H. W., 1940. Biol. Bull. Wood's Hole, 78, S3-MOE, H., 1955. Int. Rev. CytoL, 4, 299.PEARSE, A. G. E., 1953. Histochemistry, theoretical and applied. London (Churchill).ROGICK, M. D., 1932. J. Morphol., 52, 1.VAN CAMPENHOUT, E., and CORNELIS, G., 1951. C.R. Soc. Biol. Paris, 145, 933.

a study of the intestinal epithelium of the goldfish …...a total of 200 goldfish were used, of...

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