the maturatio onf cortisone-treated embryonic duodenum in ... · length of 670 m/x. conversion of...

J. Embryol. exp. Morph., Vol. 14, Part 2, pp. 169-179, October 1965Printed in Great Britain

The maturation of cortisone-treated embryonicduodenum in vitro. II. The striated border

by R A Y M O N D L. H A Y E S J r . 1

From the Department of Anatomy, Harvard Medical School, Boston

WITH FOUR PLATES

R E G U L A T I O N of the morphological and physiological maturation of embryonicduodenal mucosa by adrenocorticoid hormones has been demonstrated with avariety of techniques. Single injections of cortisone acetate (Moog & Richardson,1955; Moog & Thomas, 1957); multiple daily administrations of cortisone(Moog & Ford, 1957; Moog, 1959); or in vitro maintenance of duodenalfragments with adrenal steroids (Moog & Nehari, 1954; Moog & Kirsch, 1955)all accelerate morphogenesis and the onset of alkaline phosphatase activity inthis tissue.

The differentiation of the duodenal mucosa is not only responsive to hormonalapplication but is dependent on it. Hinni & Watterson (1963) have ablatedpituitary glands from chick embryos, hence interrupting the hypophyseal-adrenal axis and arresting adrenal function. Following this procedure, morpho-genesis is retarded and alkaline phosphatase activity in the epitheliumdiminished. Release from adrenocortical influence does not modify the courseof differentiation, but does affect the rate of cellular maturation.• Implicit in the hormone-dependent quality of duodenal maturation is theexistence of a delicate balance between the rate of tissue differentiation and thehormonal exposure. The present investigation has been conducted to examinethis relationship between the concentration of hormone applied to explants invitro and the responses induced in the epithelial striated border. This relationshiphas been studied in embryos representing a series of incubation ages to observedifferential responsiveness of the tissue when explanted at various stages.

MATERIALS AND METHODS

Duodenal loops from White Leghorn chicken embryos of stages 37-45(Hamburger & Hamilton, 1951) were removed aseptically and minced intofragments 1-2 mm3 in size. These explants were maintained as organ cultures

Author's address: Department of Anatomy and Cell Biology, University of Pittsburgh,School of Medicine, Pittsburgh, Pennsylvania 15213, U.S.A.

170 R. L. HAYES, JR.

aboard a Millipore filter disc supported by a stainless steel grid (after Trowell,1959). The nutritive medium was #199 (Morgan, Morton & Parker, 1950)supplemented with 10 per cent, calf serum. Cortisone acetate was added in thefollowing concentrations: 0-01, 0-025, 0-05, 0-075, 0 1 , 0-25, 0-5, and 1-0/xg./ml.

All explants were fixed after 48 hr. of incubation at 37° C. They, as well asthe unincubated control tissues, were fixed in 2 per cent, glutaraldehyde bufferedwith sodium cacodylate (280-320 mOs; pH 7-2-7-6) for 1 hr. at 4° C. and sub-sequently washed overnight in an isotonic solution of sucrose in cacodylate buffer(Gordon et al. 1963). For detection of alkaline phosphatase activity, the fixedtissues were incubated in toto for 1 hr. at 37° C. in the presence of sodiumj8-glycerophosphate at pH 9 • 4. They then were reacted according to Gomori(1941) to produce a visible precipitate of cobalt sulphide. Sample tissues wereincubated in media without substrate (glycerophosphate) to test for specificityof the reaction.

Quantification of enzyme activity was achieved by a procedure reported byDoyle (1950). After wet weights were obtained, controls and cultures werereacted for alkaline phosphatase by a modified Gomori procedure to yield adeposit of lead sulphide at sites of enzymatic activity. This deposit was extractedand reacted with N,N-dimethyl-p-phenylenediamine sulphate to synthesizemethylene blue. After oxidation of this solution with ferric chloride, the contentof methylene blue was determined by spectrophotometric transmission at a wavelength of 670 m/x. Conversion of these optical readings to values per milligramof tissue yielded a series of quantitative estimates of enzyme activity (Table).

Tissues were prepared for histological observation by post-fixation in 1 percent, osmium tetroxide in cacodylate buffer for 1 hr. at 4° C. Following alcoholicdehydration, they were embedded in Epon 812 (Luft, 1961) and sectioned on aPorter-Blum microtome. For light microscopic observation, 1 • 5 [x thick sectionswere visualized by staining with 1 per cent, toluidine blue. Thin sections forelectron microscopy were cut from the same blocks and stained with lead citrate(Venable & Coggeshall, 1965).

RESULTS

The localization of alkaline phosphatase activity

Inspection of thick sections of explants treated for alkaline phosphatase bythe Gomori technique reveals no reaction product in 11-, 13- and 15-day controltissues. When cultivated without hormone, no change in activity is observedin these tissues. A positive reaction, however, is observed in 17-day controls.Also, the 19-day control is positive (Plate 2, Figs. E & F) and although thisactivity diminishes during cultivation without hormone, the reaction is not lost(Plate 2, Fig. G).

Addition of cortisone to the nutritive medium does not induce a positive

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reaction for alkaline phosphatase in the 11-day culture (Plate 1, Fig. A), but doesyield a reaction in the 13- and 15-day explants (Plate 1, Figs. B & C). The 17-daytissues continue to demonstrate positive activity after hormone treatment(Plate 1, Fig. D). Also, the 19-day tissue treated with hormone is positive forenzymatic activity (Plate 2, Figs. H & I).

No precipitate is found on those tissues incubated in medium lacking j8-glycero-phosphate but otherwise treated by the Gomori method. This is true even whencontrols or cultures of the same age are positive for alkaline phosphatase.

Where a positive reaction is obtained, the precipitated sulphide is locatedinvariably on the luminal surface of the duodenal epithelium. This deposit isscattered irregularly over the surface of villi on isolated cells as well as smallgroups of cells. Specifically, the reaction product is localized on the microvillousmembrane and along the apical cell membrane separating microvilli (Plate 3,Figs. J & K). Negatively-staining cells are found adjacent to positive ones, inwhich case the reaction product ends abruptly at the intercellular junction (Plate3, Fig. J). Whenever the microvilli are positive, the apical cell membrane is also.

Quantification of alkaline phosphatase activity in embryonic duodenal tissue

Results obtained from application of the Doyle procedure for quantifyingenzyme activity are presented in the Table. Spectrophotometric readings werecorrected for variation in luminal surface area of duodenal explants by con-version to values per milligram of tissue. These values are significant within anyone age group but are not comparable between age groups because of differencesin thickness of the duodenal wall.

Alkaline phosphatase activity reaches a maximum at a definite hormonalconcentration regardless of tissue age (Text-fig. 1). Increasing cortisone levelsbeyond this optimal concentration reduces the enzymatic response of the tissue.Moreover, the amount of cortisone necessary to induce maximal activity induodenal explants decreases with increasing incubation age of the tissue. Inother words, the older the tissue, the greater its sensitivity to the hormone(Text-fig. 2).

Morphological alterations in the striated border

The only dramatic change in morphology of the striated border occurs in the19-day tissue following hormonal exposure. Compared to that of the untreated19-day culture (Plate 2, Fig. G), the striated border of the hormone-treatedepithelium is higher (Plate 2, Figs. H & I). Electron micrographs of this cortisone-treated epithelium reveal that the apparent increase in height of the border isdue to the apposition of a layer of vesicles onto the microvilli (Plate 3, Fig. M;Plate 4). The thickness of the vesicular layer is 2-3 times the height of themicrovilli (Plate 4, Fig. N). The combined height of the vesicular and micro-

J. Embryo!, e.xp. Murph. Vol. 14, Part 2

PLATE 1

Hormone-treated cultures reacted for alkaline phosphatase by the Gomori technique.

FIG. A. 11-day duodenum cultivated with 0-25 jixg./ml. cortisone. No visible enzymaticactivity is observed. Toluidine blue, x 1400.

FIG. B. 13-day duodenum cultivated with 0-5 /ng./ml. of cortisone. An irregular phosphatasereaction is localized on the lateral sides of villi. x 1400.

FIG. C. 15-day duodenum cultivated with 0-5 /Ag./ml. of cortisone. Alkaline phosphataseactivity is shown on sides of villi. x 2000.

FIG. D. 17-day duodenum cultivated with 0-25 ju.g./ml. of cortisone. Individual cells andisolated groups of cells stain positively for phosphatase. x 1400.

RAYMOND L. HAYES Jr. {Facing page 172)

/ . Embryol. exp. Morph. Vol. 14, Part 2

PLATE 2

19-day controls and cultures stained for alkaline phosphatase.FIG. E. Control tissue. Striated border (sb) stains positively, x 1400.

FIG. F. Control tissue. Note irregular enzymatic activity, which changes abruptly at inter-cellular junctions, x1400.

FIG. G. 19-day duodenum cultivated 48 hr. with no hormone, x 1400.FIG. H. 19-day duodenum cultivated 48 hr. with 0 01 /xg./ml. of cortisone. The striated

border is thicker and stains heavier than in (B) and more cells stain positively, x 1400.FIG. I. 19-day duodenum cultivated 48 hr. with 0-5 ftg./ml. of cortisone. The striated border

is thicker than in G. x 2000.

RAYMOND L. HAYES Jr.

J. Embryo/, cxp. Morph. Vol. 14, Part 2

mv

PLATE 3

FIG. J. Electron micrograph of 19-day tissue cultivated for 48 hr. with no hormone and stainedfor alkaline phosphatase. Enzyme activity is localized on microvillous membrane andapical cell membrane between microvilli. Reaction ends at intercellular junction (arrow).x 24,100.

FIG. K. 19-day explant with 0 1 /xg./ml. of cortisone and stained for alkaline phosphatase.Note phosphatase-positive vesicles and unstained hirsute coating (he) of membranes.FIG. L. 19-day control tissue showing absence of vesicular layer on microvilli. x 7000.

FIG. M. 19-day culture exposed to 0-1 /xg./ml. of cortisone. Notice development of terminalweb (tw) and presence of vesicular layer (ves) on luminal surface of microvilli (mv). x 6300.

RAYMOND L. HAYES Jr.

/ . Embryol. exp. Morph. Vol. 14, Part 2

PLATE 4

Electron micrographs of 19-day explants treated with 0.5 /xg./ml. of cortisone for 48 hr.

FIG. N. Vesicular layer (ves) is 2-3 times thicker than the height of the microvilli. x 12,100.Insert shows lack of internal structure in vesicles.

FIG. O. Apical extensions or distortions on microvilli might represent developing vesicles(arrows), x 17,800.

RAYMOND L. HAYES Jr. {Facing page 173)

Cortisone-treated embryonic duodenum 173

Alkaline phosphatase activity in 48-hour culturesof embryonic chick duodenum treated with cortisone

15 day

0 0-2 0-4 0-6 0-8 10

19 day

0 0-2 0-4 0-6 0-8 1-0

0 0-2 0-4 0-6 0-8 1-0 0 0-2 0-4 0-6 0-8 10Cortisone acetate (y/ml.)

TEXT-FIG. 1. Alkaline phosphatase activity in the striated border of 48-hr, cultures of embryonicchick duodenum treated with cortisone. Induction of enzyme activity reaches a maximumat a definite hormonal concentration. Greatest response to the hormone is shown by 15-and 17-day tissues.

Cortisone inducing maximal alkaline phosphatase activityembryonic chick duodenum, 48-hour cultures

1)

8

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13 15 17Tissue age (days)

19

TEXT-FIG. 2. Cortisone inducing maximal alkaline phosphatase activity in embryonic chickduodenum maintained in vitro. Tissue sensitivity to the hormone increases as tissue ageincreases.


villous layers is comparable to the height of the border as viewed in light micro-graphs. The spheroid units comprising the vesicular layer are slightly smaller indiameter than the microvilli, are membrane-bound, and their external surfacesare covered with a hirsute coating as characterizes the surface of enteric microvilli(Ito, 1964). When stained for alkaline phosphatase, these extra-cellular spherulesdemonstrate positive enzymatic activity (Plate 3, Fig. K). Both the hirtellouscoat and the phosphatase-positivity suggest that these vesicles are derived fromthe cell membrane.

Further indication of the origin of these vesicles is provided in micrographsshowing apical distentions of the microvilli (Plate 4, Fig. O). These protrusionsof the apical membrane of the microvillus suggest that the gemmules are pinchedoff from the tips of the microvilli. The surface filamentous coat retains these budsin contact with one another and with the cell surface. Hence, a definite layeringof these vesicles on the luminal surface of the epithelium results. As hormoneconcentration is augmented, the striated border increases in height through agreater degree of vesiculation. During this process, the microvillus does notdecrease noticeably in height, suggesting a concurrent expansion of theplasmalemma.

Although no internal structure is observed in the vesicles, there is a definiteincrease in filamentous material within the microvillus proper. This changeresulting from hormone application is reflected in the development of theterminal web region of the apical cell cytoplasm (cf. Plate 3, Figs. L & M).

DISCUSSION

Our data demonstrate a positive effect of cortisone, under the cultivationconditions chosen, upon the maturation of the striated border of the embryonicduodenal epithelium. The normal rate of differentiation of these cells in vivois approximated in vitro after hormonal administration. Untreated culturesfail to attain comparable stages of maturation either morphologically orphysiologically.

The onset of functional activity, as evidenced by the accumulation of alkalinephosphatase in the tissue, is stimulated by cortisone. As originally demonstratedby Moog & Nehari (1954), this in vitro effect indicates a direct influence of thehormone on the duodenal tissue. This response reaches maximal levels withlesser concentrations of hormone as tissue age increases, provided the tissue iscompetent to respond. In the present study, enzymatic activity is not visualizedin the 11-day tissue, but phosphatase activity is (1) induced in the 13- and 15-daycultures and (2) intensified in the 17- and 19-day tissues.

Maximal enzymatic activity is reached with a definite hormonal concentrationsuch that submaximal levels are elicited by concentrations above or below thisvalue. That is, the duodenal mucosa is increasingly responsive to cortisone upto a point where the induction of the phosphatase activity is optimal. This


intensification of enzyme activity is attributed both to an increase in the numberof reactive cells and to an increase in reactivity per cell.

Once the optimal hormonal concentration is exceeded, maximal enzymaticactivity is lost. Hence, cortisone has a repressive effect upon the enzymaticmachinery of the cell when given in excess. Other studies indicate that over-exposure to steroids is generally toxic to tissues (Karnofsky et al. 1951; Evans,1953; Grossfeld, 1959; Moscona & Karnofsky, 1960). Moreover, the degree oftoxicity shown in vitro varies with the cell strain and the specific corticosteroidutilized (Gillette & Buchsbaum, 1953; Holden et al. 1953; Grossfeld & Ragan,1954; Holden & Adams, 1957; Perlman et al. 1962).

Lasfargues & Di Fine (1951) have reported the diminution of alkalinephosphatase activity in 14-day tissue following administration of an adreno-cortical extract, 'eschatine', but they did not vary the hormone concentrationto reveal the extent of this effect. Moog & Nehari (1954) have attributed thesenegative results to the insensitivity of the tissue to hormone at that age as well asto cultivation conditions favoring the monolayering of cells. Our data, however,indicate that chick tissue is sensitive to corticoid influence as early as 13 days ofincubation. Consequently, Lasfargues & Di Fine's negative results might bestbe attributed to exposure to excess hormone levels, which the authors haveexpressed in 'dog units'.

In spite of the evidence for positive effects of cortisone on the duodenalmucosa, other in vitro studies have suggested little or no dependence of the tissueupon cortisone. Hancox and Hyslop (1953) report that 15-day tissue, whichexhibited positive phosphatase activity before cultivation, shows increasedactivity after 2-3 days in vitro without hormone. Hancox (1954) has reportedthat 12-day tissue, which is enzymatically negative at explantation, becomespositive after 4 days of cultivation. Moog & Nehari (1954) report that 16-daytissue responds similarly after 1-2 days in vitro. Such apparent indications ofsteroid independence can be explained by a latency in the appearance of theenzymatic activity. Adrenal function has been reported in the 12-day chickembryo (Dawson, 1953; Moog, 1959), so the tissue used in the above studiesmust have been exposed to the hormone. That this exposure, if sufficient, neednot be continuous is quite probable.

In addition to influencing enzymatic activity on the apical cell membrane,cortisone induces morphological changes in the striated border. As hormone isapplied to 19-day explants, the microvilli appear to elongate. This increase inlength continues during exposure to hormone levels which coincidently repressenzyme activity. Dissociation between the morphological and physiologicalaspects of differentiation of duodenal epithelia has been reported previously byKato (1959) and by Moog & Nehari (1954). These workers based their deductionon a change in enzymatic levels with no concurrent change in morphology, whileour data indicate structural changes with no corresponding increase inphosphatase activity.

12


The height of the striated border normally increases during maturation.Microvilli elongate in vivo 6-fold between days 16 and 22, with the greatestchange occurring at day 21. This increase in surface area of the micro villi parallelsan increase in alkaline phosphatase activity along the apical cell surface (Overton& Shoup, 1964).

Exposure to cortisone in vitro stimulates an increase in apical plasmalemmalsurface on the duodenal epithelium. This increase is complicated by vesiculationof the tips of the microvilli. Although the microvillous membrane is expanded,there is no significant increase in height of microvilli compared to 19-day controls.

Present attempts to explain vesiculation of microvilli are at best speculative.Other work with corticosteroids, however, might be linked with the resultsreported here. Fell & Thomas (1961) have demonstrated that hydrocortisoneretards the resorptive effects of Vitamin A on skeletal tissue in vitro. The vitaminis thought to disrupt membrane integrity and to increase membrane permeability(Lucy, 1964). These effects are exerted on both the plasmalemma (Dingle &Lucy, 1962; Dingle et al. 1962; Glauert et al. 1963) and intracellular membranes(Dingle, 1961; Fitton Jackson & Fell, 1963; Lucy et al. 1963). Hydrocortisoneis thought to counteract the influence of Vitamin A by stabilizing the membranesystems of the cell.

Cortisone, likewise, might increase the integrity or stability of the apical cellmembrane and in so doing might inhibit increases in the surface area of thisportion of the cell membrane. If so, the hormone would be stimulating precociousmembrane-bound phosphatase activity, yet concomitantly restricting membraneexpansion which is linked developmentally to and which varies directly withenzymatic activity.

Vesiculation of the cell membrane, under the influence of cortisone, mightrepresent the cell's attempt to maintain equilibrium between the physiologicaland morphological phases of differentiation. The small portions of the membraneand cytoplasm released as vesicles would no longer be identified as part of thecell. Consequently, when the duodenal epithelium is exposed to concentrationsof cortisone greater than that yielding maximal enzyme activity, no dispropor-tionate increase in apical surface area results.

SUMMARY

1. The effects of cortisone on the striated border of the embryonic chickduodenal epithelium during in vitro maintenance are described.

2. The application of cortisone induces phosphatase activity in 13- and 15-daytissue and intensifies this activity in 17- and 19-day explants.

3. The phosphatase activity obtained varies in quantity depending on the ageof the tissues and the concentration of hormone. The sensitivity of the tissue tohormone increases with age; that is, a lower cortisone concentration is needed toelicit a maximal enzymatic response in older tissues. As hormone concentration


is increased for all explants from 13 to 19 days, phosphatase activity increases toa maximum and thereafter decreases.

4. Cortisone induces a morphological change in the striated border of 19-daytissue. The apical ends of microvilli are pinched off to form numerous spheroidvesicles. A possible explanation for this phenomenon is discussed.

RESUME

La maturation in vitro du duodenum embryonnaire traite par la cortisoneII. La bordure stride

1. Les effets de la cortisone sur la bordure striee de l'epithelium duodenal del'embryon de Poulet cultive in vitro sont decrits.

2. L'application de cortisone induit l'activite phosphatasique dans le tissude 13etde 15 jours et intensifies cette activite dans les explants de 17etde 19jours.

3. L'activite phosphatasique obtenue varie quantitativement selon l'age dutissu et la concentration en hormone. La sensibilite du tissu a l'hormone s'accroitavec l'age; c'est-a-dire que plus les tissus sont ages plus la concentration decortisone necessaire pour produire la reponse enzymatique maximum est basse.Lorsqu'on augmente la concentration d'hormone pour tous les explants de 13 a19 jours, l'activite phosphatasique augmente jusqu'a un maximum puis decroit.

4. La cortisone induit un changement morphologique de la bordure strieedans le tissu provenant d'embryons de 19 jours. Les terminaisons apicales demicrovillosites se fragmentent pour former de nombreuses vesicules plus oumoins spheriques. Une explication possible de ce phenomene est discutee.

ACKNOWLEDGEMENT

This work was supported by U.S.P.H.S. Grant RG-6729.

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(Manuscript received 28fh April, 1965)

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