A Light and Electron Microscopic Study of Duodenal Epithelium of Chick Embryos Cultured in the Presence and Absence of Hydrocortisone

Vol. 53, No. 4 Printed in U.S.A.

GASTROENTEROLOGY

Copyright© 1967 by The Williams & Wilkins Co.

A LIGHT AND ELECTRON MICROSCOPIC STUDY OF DUODENAL EPITHELIUM OF CHICK EMBRYOS CULTURED IN THE PRESENCE AND ABSENCE OF HYDROCORTISONE W. 0. DoBBI Ns, III , M.D., J . C. HIJMANS, M .D., AND K . S. McCARTY, PH.D. Gastroenterology Research Laboratory, Veterans Administration Hospital, and Departments of 1l!Jedi6ne and Biochemisl1·y, Duke University Medical Cente1·, Durham, North Carolina

Numerous reports on t he morphology and biochemistry of duodena l villous epithelial cells in t he d eveloping chick embryo in vivo have appeared in recent years 1 • 2 • 3 (references 1 and 2 a re reviews of t his subject), and differentiation of embryonic chick duodenum h as been observed in organ cult ure. 2 • 4 - 11 Electron microscopic reports of t he in v ivo and in vitro develop ment of embryonic chick duodenum are confined only t o observations of microvilli of absorptive cells.12 • 13 This repor t is the first electron microscopic study of the differe ntiation of duodena l epithelium of 16clay- olcl chi ck embryos cultured in v itro for 4 clays in t he presen ce or absence of hydrocortisone. \Ve have been engaged in a program t o study ma turation a nd fun ctional differ entiation in t he duodenum of t he chick embryo by compa ring biochemical and morphologi cal observations, in vitro a nd in YiYo.H , 15 In t his report we have focused on some morphological aspects occurring in the 16- a nd 20-day embryo , contrastin g them to events in 16-clay-old tissu e cuiReceived April 13, 1967. Accepted May 26, 1967. Address requests for reprints to : W . 0. Dobbins, III, M.D., Veterans Administration Hospital, Durham, North Carolina 27705. Th is investigation was supported by Research Grants AM 95507, GM 12805-02, and AM 25-905-02 from the United States Public Health Service, by Grant P 363A from the American Cancer Society, and by grants from the Hanes Fund and Duke Endowment. Th e autho rs are particularly indebted to Mrs. Nell Peedin and to Mr. Emory L. Rollins for their expert technical assistance during this study. 557

turecl for 4 cl ays a nd thus attaining a chronological age of 20 clays. Since t he expla nted t issu es were removed from the intact anima l, a nd t herefo re from hormona l influ ences, t he explants were cultured in t h e presence or absence of hydrocortisone in the medium. Materials and Meth ods

Preparation of cultures . Fourteen-day embryonated Plymouth Rock-White Rock chicken eggs were obtained from the Farmer's exchange and were placed in a 37 C incubator until the day of the experiment. Sixteen-day chick embryo duodenums were cultured as described previously.1' Beak and toe measurements were used to verify embryonic age.1" Duodenal loops were removed and unfolded, and the pancreas was stripped off under a dissecting microscope. The gut was opened lengthwise and was sectioned transversely into 1- to 1.5-mm fragments. Duodenal tissue of each embryo was distributed serosa l side down on Millipore membranes (0.45 m,u) in 2 Falcon culture dishes, one of which contained 0.5 fJ.g of hydrocortisone per ml of culture medium (modified Eagle's medium supplemented with 10% calf serum, penicillin, and streptomycin). The other half of the duodenum served as control. Approximately 0.05 ml of medium was dropped on the surface of each explant. The cellulose humidification rings were moistened with 1.5 ml of sterile distilled water and t he culture vessels were incubated in a 5% CO,-air humidified chamber at 37 C. The medium was not changed during the 4 days of in cubation . Prepamtion fo r microscopy . Intact duodenums of 16-day and 20-day chick embryos and the duodenal fragments of 16-day embryos cultlu ed for 4 days in the presence or absence of hydrocortisone were prepared for microscopy as follows. For light mi croscopy, t he tissues were fixed

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in Bouin's solution , embedded in paraffin, and serially sectioned at 4 p.,. The duodenums from 6 to 12 embryos were prepared in each group. The intact duodenums were cut in cross section and the duodenal fragments were sectioned parallel to t he villi in order to avoid artifacts of tangentiall y cut villi. Only those slides containing \Yell or!ented villi were used for the

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study. Sections were stained with hematoxylin and eosin or with periodic acicl-Schiff reaction (PAS) follmYecl by hematoxylin counterstaining. Every third section of serially cut tissue was used to count cells in mitosis.'" Mitotic cells wore counted in 100 villi. Onl y those villi which were well oriented ::mel which \Yere sectioned :-dong th e entire length \\·ere examined. o~mium-

Fros. 1 to 4. Photomicrogra phs of Bouin-fixed, paraffin-embedded sections of embryonic ehick duodenum. Sections 11·ere stain ed wi th PAS reac tion and hemato xylin. FJG. 1 (abo ve ). Villi of norm al16-cla? ebiek cluoclenmn. A faint brush border is apparent (X 1000). Fro. 2 (b elou·). Distal po rtions of Yilli of norm al 20-day chicle Surface epi thelia! cells possess a prominent brush border. Note nu cleated ery t hro cytes (arrow) wi thin th e vascular chann els ( X 1000).

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fixed, Epon-cmbedded tissue was cut at 1.0 to 1.5 fL using a glass knife and stained by Richardson's method." For electron microscopy, tissues from 5 embryos in each group were placed in ice-cold 3\ls% osmium tetroxide buffered with 0.05 M sodium cacodyla te or with 0.1 M sodium bica rbon ate.'" F ive minutes later the specimens were removed, cut into 1-mm slices, ::mel returned to the fixing solution for 1 to 2 hr. After fixation, the specimens "·ere dehydrated a nd embedded in epoxy resin by the method of Luft." Thin sections were collected on carbon-coated copper grids and stained with aqueous uranyl magnesium acetate'0 and lead citrntc."' Pho tog raphs 1rere taken IYith an R CA 3F elect ron microscope at original magnification of 1,300 to 16,400 t imes.

Results

Light Nficroscopic Ob serva tions Sixteen-clay embryos. The duodenal mucosa had short and predominantly leafshaped villi. Surface epithelial cells were low columnar in shape althou gh the cells at villous tips tended to be cuboidal in shape. A faint brush border was demonstrated with hematoxylin and eosin staining and was slightly accentuated by PAS stainin g (fig. I) . Nuclei were quite large in relation to the cytoplasm and con tain ed prominent nucleoli. Lipid droplets, characterized by their strong affinity for methyl ene blue-azure II, were Eeen at the bases of approximately half of the cells (fig. 5) . Gob let cell s were rarely id entified. Surface epithelial mitotic cells were evenly distributed th roughout the length of the v illi. Th e mitotic counts are shown in tabl e 1. Th e lam ina propria was usua lly inconspicuous, although some villi had a cellular lamina propria with earl y vascularization. Twenty-clay embryos . The v illi were lon ger than in the duodenum of the 16-day embryos, were predomin antly fingersha ped, and contained a well-vascularized cellular lamina propria. The epithelial cells were columnar (figs. 2 and 6) . The brush border was more prominent in the hematoxylin and eosin-stain ed sections and was heavily stain ed following the PAS reaction. Nuclei were small er in relation to the cytoplasm but still contain ed prominent nucleoli. Surface epithelial cell mitoses

T A BLE

Chick

1. Number of mitoses per 100 vill1; 16D - --

Counts per 100 villi

55 58 66 63 63

Ave rage

61

16D +4D+ t6D +

m-

20D

--- - - - --23 69 49 115 26 10 12 60 30 24 56 40 79 28 33 34.6

22.4

75.8

(table I) were more frequent than in the 16-day -olcl embryo and were confined to the lower half of the villi as also shown by Overton and Shoup. 12 Occasionally, lipid droplets were seen at the base of the mucosal cells (fig. 6). Goblet cells were freq uently iden tifiecl in the surface epithelium. 01·gan cultures. Duodenum of 16-day embryos cul t ured for 4 cl ays wi thout hydrocortisone (16D + 4D -) . The villi at the center of the expl ant were sli ghtly more elongated than those of a 16-c\ay chi ck (figs. 3 and 7) , bu t were shorter than villi of the 20-day chick. At the edges of the explant the villi tended to be flattened. The lamina propria appeared similar to that of the 16-day embryo . Epithelial cells resembled those of the 20-clay embryo. The brush border was more prominent than that of the 16-clay chick but slightly less prominent than that of the 20-day chicle Occasionally, dense in clusion bodies were seen in the cells. Some of th ese inclusion bodi es were within large vacuol es (fig. 3). Lipid droplets were uncommon (fig. 7). Mitotic figures were confined to the lower ha lf of the v illi and were less frequent than in the 16-day embryo (table 1). Goblet cells were as numerous as in the 20-clay chick. Above some of the villi were sma ll cells with dense, eosinophilic cytopl asm and pyknotic nuclei (presumab ly degen erating cells). Duoden um of 16-clay embryos cultured for 4 cl ays with hydrocortisone (16D + 4D +) . The appearance of the villi, epitheli a l cells, and brush borders of these explants (figs. 4 and 8) was indistingui shble from those explants cultured without the hormon e, except that more cells with inclusion bodies were present (fig. 8). Cells

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Fro. 3 (above). Villi of 16-day chick duodenum cul tured in absence of hydro co rtisone. P resum ably degenerating cells are present above t he villi. Occasional inclusion bodies (arrow) a re presen t wi thin epithelial cells (X 1000 ) . FIG. 4 (below) . Villi of 16-day chick duodenum cultured in presence of hydroco rtisone. The villi are f!U ite similar to villi of 16-day chick duod enum cultured in absence of hydrocortiso ne. Inclusion bodies (arrow ) a re present (X 1000).

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in mitosis were encountered more fre quently t han in cultures without the hormone (table 1). Goblet cells appeared t o be more frequent t han in the 20-day embryo (fig. 8). Electron NI icroscopic Observations

Most observations were made of epit helial cells of the villous tip since no obvious differences could be detected between these and t he crypt cells. Sixteen-day embryos . Microvilli were sparse and proj ected in random directions from the free surface of the epithelial cells (fig. 9). Because of this random projection, many were cut in cross section, t hus giving the appearance of vesicles lying above the plasma membrane. Serial sectioning showed that these apparent vesicles were processes of transversely cut microvilli. Occasional fin e fil aments, the surface coat, radiated from the outer dense leaflets of the microvillous plasma membrane (fig. 10). Intracellular filaments from cores of the microvilli projected into a t hin, poorly defined terminal web (figs. 10 and 11 ) . Large vacuoles often ·were present in the terminal web area. The lateral plasma membranes were generally straight but had occasional interdigitations. The membranes of adjoining cells were closely apposed apically with prominent tight junct ions at the luminal surface (figs. 9 and 11 ). D esmosomes were observed at irregular intervals along the lateral surfaces. Widened intercellular spaces were often present at the basal half of the cells at villous tips. The lateral membranes of the crypt cells were closely apposed throughout their length. There was a t hin basal lamina below t he membranes at t he base of the cells. The basally placed nuclei were large and contained inconspicuous nuclear chromatin concentrated on the inner aspect of t he nuclear envelope. The chromatin was interrupted by areas of rarification adjacent to nuclear pores. Nucleoli were prominent. The ou ter membrane of the nuclear envelope often had attached ribonucleoprotein particles and was frequently continuous with cytoplasmic elements of smooth and granular endoplasmic retic-

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nlum. Centrioles frequently were present in the apical third of the cells. A small Golgi complex in the paranuclear or supranuclear region consisted of parallel arrays of flat cisternae, numerous small vesicles, and occasional vacuoles (figs. 9, 13, and 14). Mitochondria, varying in size, were randomly distributed t hroughout t he cytoplasm. The cristae were transversely oriented and dense intramitochondrial granules were present. Occasionally, a huge mitochondrion was noted. M oderate numbers of free cytoplasmic ribonucleoprotein particles were present. Smooth and granular elements of endoplasmic reticulum were present in small amounts (figs. 9 to 14). Intracellular fibrils, mostly confined to t he periphery of the cells, were closely associated with the lateral cell membrane and especially with desmosomes (figs. 9 and 12 ) . Granules with a diameter of about 250 A, presumably glycogen granules, ·were scattered about the cytoplasmic matrix. Prominent accumulations of glycogen were sometimes seen (fig. 12). Large heterogeneous and smaller homogeneous dense bodies were present in most cells (figs. 13 and 14). These bodies were enclosed within prominent membranes in which trilaminar unit structure was easily demonstrated. The matrix within the heterogeneous dense bodies contained myelin figures, vesicles with equally prominent membranes, and a homogeneous substance that may represent lipid droplets. Some of t he heterogeneous dense bodies were multivesicular and others contained structures with a crystalline array. These dense bodies probably represented lysosomes as demonstrated histochemically by Behnke22 in fetal rat duodenum. The presence of cells in mit osis, of goblet cells, and of large lipid droplets at the base of many cells was confirmed by electron microscopy. Twenty -day embryos . Some of t he differences observed in the 20-day embryos (fig. 15) when compared to 16-day embryos were as follows. The microvilli ·were well developed, elongated, and projected in a perpendicular direction from the free surface of the cells. The surface coat was prominent and easily identified. Interdigi-

• •

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tations between lateral plasma membran es ·were more frequent and the intercellular space at the basal half of the cells was generally widened. Nuclei occupied less of the total cell space. Some mitochondria were elongated and some were bizarre in shape. No giant mitochondria were seen. There were more elements of smooth and granular endoplasmic reticulum with a slight predominance of the granular reticulum. Ribosomes were more concentrated throughout the cytoplasmic matrix . There were fewer glycogen granules. The heterogeneous dense bodies were larger and more frequent and their internal structure was more elaborate. Occasionally, membranebounded granules containing a slightly dense homogeneous material were seen in the apical portion of t he ce ll. They probably represented "secretory granules." 2 :' One argentaffin cell was identified by its characteristic dense granules. 23 Organ cultures. Duodenum of 16-clay embryos cu ltured for 4 days without hydrocortisone (fig. 16) . The epithelial cells were well preserYecl, and the cell junction s and basal lamin a remained intact. The microYilli and the surface coat were comparable to that of the 20-clay old embryo, indicating that considerable clenlopment had occurred in Yitro. The following differences in the structl!i'es of the mucosal cells of these explants as compared with the surface cells of a 20day old embryo were noted. The mitochondria tend ed to be larger. Some mitochondria were quite elongated whi le others

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were rounded with the cristae oriented in a spokelike manner. The dense granules within these mitochondria appeared to be increased in concentration but not in size. :Many small dense bodies and occasional huge heterogeneous dense bodies were present. The latter may represent autophagic vacuoles since they contained identifiable cell organelles su ch as mitochondria, nuclei, and myelin figures (fig. 16). These autophagic vacuoles corresponded to the inclusion bodies seen by light microscopy. Some of these autophagic vacuoles did not have a well defined membrane, but they were separated from the surrounding cytop lasmic matrix by apparent clefts within the tissue. Lipid droplets and glycogen accumu lations were less frequent . The remainder of the cell structures were comparable to those of a 20-day embryo. An occasional goblet cell and argentaffin cell were present. Duodenum of 16-day embryos cultured for 4 clays with hydrocortisone (fig. 17) . The epithelial ce lls were simil ar to those seen in the duodenal explants cultured without hydrocortisone. There were no ap preciable differences in the microvilli, but the surface coat was more prominent (figs. 17 and 18). The mitochondria were similarly enlarged and had an elaborate structure. Intrace llular fibrils and the basal lamina were sligh tly more prominent. There were, however, some strikin g differences in the structures of some cells. Many cells had huge autophagic vacuoles. Multivesicu lar bodies and heterogeneous dense

FIGS. 5 to 8. Photomicrographs of o;,mi um-fixed, Epon-ernbedded sec tions of embryonic chick duod enum. S2dions were stained by Richardson 's method. Frc. 5 (above lefL) . Villus of normal 16-day chick duod enum showing, in addition to features seen in figme 3, lipid droplets (a rrow) at the base of most smface epithel ial cells (X 1000). FrG. 6 (abo ve right). Villus tip of normal 20-day duod enum showing fewer li pid droplets at the base of surface epith elial cells than seen in the 16-day chick. Goblet cells are present in the adjacent villus (arro ws ) (X 1000). FIG. 7 (belo w left). Villus of 16-day chick duodenum cultured in absence of hydrocortisone. There are occasional inclusion bodies (arrow) and absence of basal li pid droplets (X 1000). FIG. 8 (below right) . Villus of 16-day chick duodenum cultured in presence of hydrocortisone . Inclusion bodies (arrow), occasional basal li pid droplets, and, in this vi llus, rathe r frequ ent goblet cells (g) are present (X 1000).

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FIG. 9

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bodies were more frequent and were oft('n enlarged. Small dense bodies were increased in number (fig. 17). Profiles of the endoplasmic reticulum often were dilated. The space within the nuclear envelope was distended and the outer envelope was more often seen to be continuous with dilated profiles of the endoplasmic reticulum. Other cell components were indist inguishable from cell components of those tissues cultured without hydrocort isone. It must be emphasized that, except for those cells with t he above-described differences, many cells were entirely similar t o tho:::e seen m tissue cultured without the hormone. Discussion

Light morphological and histochemica l development of the epithelium of duodenal villi of chick embryos has been investigated by Moog1 • 2· 8 • v. 24 -27 and by Hinni and Watterson .3 Moog has shown that t he intestinal tract of t he chick embryo undergoes rapid development during t he 4clay period from 16 to 20 clays. Epithelial cells at 17 clays were small and cuboidal, and had large basophilic nuclei. At 20 clays the cells ·were taller and columnarshaped with smaller nuclei and more basophilic cytoplasm. During the period of rapid differentiation, t he cells accumulated and then lost glycogen 27 ; the striated border appeared and assumed its definitive form; and t he t otal nitrogen content of the mucosa doubled.1 • 2 At the same time, the low phosphatase level characteristic of undifferentiated tissue shifted to the high phosphatase level of functioning epithelium.24 J\!Ioog has furth er shown that exogenous cortisone acetate induced precocious phosphatase accumulation and t hat this was accompanied by precocious differentia-

565

tion of t he epithelial cells of the duodenum.24 Growth retardation and weight loss, usua lly resulting from corticoid administration,28· 29 did not occur when corticoids were injected at 16 to 18 days. Instead, body weight and yolk sac weight of embryos injected at those stages were slightly higher at 18 and 19 days t han those of untreated controls.24 In organ culture, phosphatase also accumulated in duodenal fragments and cortisone acetate accelerated the process. 2· 7· s. 9, 1 3 Moog found that events in vitro did not parallel t hose occurring in the intact intestines, but the various processes comprising differentiation could be somewhat dissociated. In particular, duodenal epithelial cells often failed to achieve the columnar shape seen in in vivo controls despite phosphatase accumulation at t he microvillous border. In organ cultures in the presence of cortisone, H ayes 7 found that the villi in duodenal explants matured at approximately the in vivo rate for 48 hr, but did not do so in the absence of cortisone. Further, Moog found that villi from 16-clay duodenal explants shorten and disappear if cultivated longer than 2 days, but, in the presence of hydrocortisone, lon g villi persisted to the end of t he experiment, which vvas 5 clays.9 In our light microscopic observations, we have confirmed the rapid development of the duodenal mucosa in vivo from the 16th to t he 20t h day as noted by others. 1 - 12 The short, predominant ly leaf-shaped villi with inconspicuous lamina propria of the 16-day-old embryo changed in 4 days to t he long, finger-shaped villi containing a well-vascularized lamina propria. The brush border became more prominent and stained heavily with the PAS reaction.

FIG . 9. An electron micrograph of duodenal surface epithelial cells in 16-day chick embryo . Mi crovilli (Jl!!V) a re spars2 and proj ect random ly . The terminal web contains vesicles and vacuoles (V) . The cell junction (J) consists of a tight junction apically with an interm ediate junction below and desmoso mes scattered a long the late ral surfaces. The nucleus (N) is large and contains prominent nucleoli (Nu ). Mitochondria (M) vary in size and contain very few small dense granules. Riboso mes are sparsely scattered throughout the cytoplasmic matrix. The endoplasmic reticulum is sparse as is the Golgi co mplex (G) . The cells lie above a t hin basal la mina (arrows). D ense bodies ( d ) and multivesicular bodies (m v b) are fr equent ( X 15,250).

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FIGs. 10 to 14

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567

Electron microscopy showed striking de- (large, heterogeneous, dense bodies convelopment of the microvi lli which changed taining identifiable cell organelles) present from sparse, randomly directed proj ections in the cultured tissue. These alterations, to well developed, elongated, and orderly particularly the appea rance of autophagic arranged microvilli. The surface coat be- vacuoles, suggested that some cellular decame prominent and easily identified. The generation was t aking place in organ culsmooth and granular endoplasmic reticu- tme despite the evidence of continued delum became more prominent. Ribosomes velopment. were more concentrated throughout the \Vhen cortisone was added to the organ cytoplasmic matrix. Occasional argentaffin cultures, differentiation of t he villi, epicells made their appearance, and goblet cells thelial cells, and mi crovilli occurred to a similar degree as in 4-day cultures without increased in number. In organ culture, the duodenum of the cortisone. \Ve did not confirm the observa16-day embryo in general retain ed its tion t hat villous maturation beyond 48 hr viability and integrity during a 4-day in vitro requires the presence of hydroperiod. Villi elon gated only sli ghtly; the cortisone.'· 0 Goblet cells, epithelial inclulamin a propri a remained un changed; and sion bodies, and epitheli al cell mitoses vvere mitoses dec reased in numb er. However, more prominent in the presence of hydromaturation of epithelial cells and micro- cortison e. In electron microscopic studies, villi seemed to occm at a similar rate in t he subcellul ar structures in most epithelial viYo and in Yitro. The epithelia l cells cells of the exp lants cultured with hydrochan ged fro m low columnar or cuboidal t o cortison e did not differ from those maincolumn ar sha pe, and the mi croYilli ma - tain ed without hydrocortisone. Bo th often tured into t he \Ye ll deve loped brush border were similar to th e 20-day-old embryo in characteristic of t he 20-day -old embryo. vivo. The surface coat, however, was better Differentiat on of the Go lgi a pparatus, deve lop ed in the hydro cortisone-treated endoplasmi c reticu lum, ribosomes, and nu- cultmes. This finding correlates well with clei appea red to be similar in vitro and in the demonstration that alka line phospha viYO. Gob let cell and argentaffin cell di f- tase and intestinal disaccharidases are ferentiation was confirmed in vitro." Uti li- largely confined to the microvillous surface zation of glycogen and lipid droplets ap - coat immed iately adjacent to the outer peared simila r in vivo and in vitro . leafle t of the uni t membrane,31 - 33 and that HoweYer, in vitro cul tured tissue differed these enzymes are induced by hydrocortiin se,·era l major aspects from the 20-day - ~ on e in vivo an d in vitro. 12 • H, 1 5 old in viYo organ. First, the mit ochondri a \Ve did not find t hat cort isone caused a we re often larger an d pret:ented a greater pinching off of t he apical ends of the variety of shapes t han in the in vivo t issue, microvilli to form spheroid vesicles as rea finding a lso noted in embryonic chicken port ed by Hay es.13 Serial sectioning demliver cultured in vitro for 3 days.ao Second, onstrated that th e apparent vesicles above there were many autophagic vacuoles the mi crovilli were processes of t angenFIGS. 10 to 14. E lectron mi crographs of po rti ons of epithelial cells of 16-day embryoni c chi ck du odenum. FIG. 10 (ab ove left). Illustrates fin e filam ents radi ating from outer dense leaflet of mi cro,·illous plasma mr mbrnn e. Note longitud inally arranged fi ne fi lam ents within core of t he micro,·illus e n t he left (X 45,000). FIG. 11 (above right). Cross section of tight junctional co mplex (l) . Note fi laments (arrow) \\'ithin co re of mi crovilli. Cen triole (G) ( X 45,000). FIG. 12 (b elow left) . Glycogen accumul ation (Gl) with adj acen t fi la ments (/). Endoplasm ic reticulum (ER) ( X 45,000). FrG. 13 (middle right) . H ete rogeneo us dense bodies adjacen t to Golgi complex ( X 31,000 ). FrG. 14 (b ollom Tight). Small dense bodies adj acen t to Golgi co mplex ( X 31,000).

Fra . 15. E lectron micrograph of duodenal epithelial cells of 20-day chick embryo. Note well developed microvilli with fa in t surface coat. Ribosomes and endop lasmic reticulum are more prominent than in the 16-day chicle Nuclei (N) are relatively smaller. Nucleolus (NV), Golgi (G) lamellae, and vesicles contain small dense bodies (arrow) and large, heterogeneous dense bodies (D) are close by. Glycogen (Gl) and lipid droplets ( L) are presen t in small deposits. A th in basal lamina can be seen in the left lo wer corner (arrow ) (X 15,250) . 568

Fw. 16. E lectron micrograph of sligh tly tangentially sectioned duodenal epithelial cells of 16D + 4D- chick embryo. Note well preserved appearance of the cells. Some mitochondria (M ) are quite large and the Golgi com plex (G) and endoplasmic reticulum (ER) are prominent. Autophagic vacuoles (A) possibly in various stages of formation are present. Multivesicul ar body (m v b). Glycogen (G l). Basal lamina (arrows ) ( X 10,600).

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F I G.

17

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FIG . 18. Electron micrograph showing prominent surface coat of micro villi of 16D chi ck emb ryo . Compare to 20-day chi ck emb ryo shown in figme 15 (X 15,'250) .

tially cut microvilli, the basal ends of 1rhich "·ere not in the plane of the original section. Prominent dilation of the endoplasmic reticu lum was seen only in cortisonetreated tissues. Autophagic vacuoles, multiYesicular bodies, and small dense bodies (virgin lysosomes? 3 4 ) were much more fre quent in tissu e cultured in the presence of hydrocortisone. Such evid enc es of cellular autoph agy by lysosomes have been fo und to occur und er normal circumstances such as during intestinal differenti ation in the rat,22 as well as under pathological concli t ion s .~ 5-~8 B ecause of the accompanying prominent dilation of the endopl asmic reticulum and t he alterations in the mitochondri a, t he autophagic vacuoles in the cortisone-t reated cu ltures proba bly represent a cellula r response to injury. 36 Although it has been suggested that corticoids have a protective effect against cell injury, possibly a result of their ability to stabi lize lysosom al membranes, 35 · 3 7 there was greater evid ence of cell dam age in hydrocortisone-cu ltured tissue under our conditions. The increased number of dense mitochondrial granul es in our explants may be clue to an altered metabolic state of the

+ 4D•

ce ll. 39 The peculi ar, spokelike arrangement of the mitochondrial cristae and the formation of large, bizarre-shaped mitochondria may occur in a variety of situ at ions (as, for example, during development, or after changes in nutrition and endocrine state), but often are considered to be a manifestation of cell injury. 36 Since these changes were more pronounced in cells of hydrocortisone-treated cultures, and because excessive cortison e is known to cause liver mitochondria to increase in vo lume, 40 it would appear that hydrocortisone was in part responsible for some of themitochonclri al cha nges observed. The mechanism of action of hydrocortisone at the cellular leve l is not clearY· 42 In cell cultures high doses of corticosteroids severely da maged or inhibited the proliferation of cells, apparently through a reduction of respiration combined with a rise in gly colysis, while low doses increased respiration and cell proli feration. 42 In the cu ltured chick duodenum , low doses of hydro~ortisone appeared to increase cell proliferati on as judged by mitotic index of treated versus un treated cultures, but there appeared to be a greater degree of cellular degenerati on in the treated cu lt ures as judged by the number of extruded pyknotic

FrG. 17. E lectron micrograph of duodenal epithelial cells of 16D + 4D+ chi ck embryo. Microvilli a re well developed and the surface coat is quite prominent. Mitochondria vary considerably in size and iu so me the mi tochondrial membran es are disru pted ( small arrow) . Endoplasmic reticulum (ER) is dilated an d often continuous with the outer mem brane of the nuclea r envelope. Note the autophagic ,·acuoles (A) and the frequent dense bodies (cl) . A Goblet cell (GC) is present, as well as an epithelial cell in mitosis (Mi) . Th e basal lamin a is indi cated by th e large arrow ( X 7,500).

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cells and organelle changes described above. The greater degr ee of cellular degeneration seen in t he treated cultures could be related to increased cell extrusion from the villous tips, t he physiological mechanism of epithelia l shedding of adult in testinal epithelium.3· 43 However, these degenerative changes were evenly distributed t hroughout the villous length and, furthermore, cell extrusion zones were not observed. Extrusion zones at apices of villi do no t occur normally unt il 22 days of incubation.3 H ydrocortisone does induce enzyme activity in t his culture system a nd the induction is selective in that invertase activity is increased while lactic dehydrogenase activity is not increased. 14 • 15 Hydrocortisone is known t o in crease t he activit ies of a seri es of liver enzymes,4 4 - 46 and it is thought t hat new r ibonucleic acid synthesis is required to support this enzyme forma tion because actinomy cin completely inhibits hydrocortisone indu ction.45 • 46 vVe have shown an incr ease in the specific activity of ribonucleic acid both in vivo and in vitro when t issues from 15- to 16-day chick embryos were incubated with hydrocortisone.14 Inter esting changes in cell division occurred during organ culture of t he duodenum for 4 days . T ab le 1 shows that t he average number of cells in mitosis per 100 villi in the duodenum of a 16-day -old chick embryo was 61. As m aturity progressed, t he number increased to 76 in the 20-day-old chick embryo . I n duodenal explan ts maintained for 4 days in the absence of hydro cortisone, t he average number of ce lls in division was 22 per 100 villi . This number increased to 35 when the tissue was ma intained in medium with hydrocortisone. \Vhen compared to a 16-day-old embryo, there was an obvious decrease in mi totic activity in a ll cul tures, probably because of a sma ller number of cells entering mitosis. Fina lly, we suggest t hat this in vitro system may well serve as an idea l method for t esting the effects of a variety of substances upon embryonic intestina l epithelimn. For instance, the effects of neomycin,

gluten, a nd glia din might be tested on t his system. Preliminary studies concernmg t he effects of t hese substances are a lready in progress in our laboratory. Summary \~T e have confirmed, usin g light m icroscopy, the rapid development of embryoni c chick duodenal mucosa in vivo from the 16th to t he 20th day. Using electron microscopy, we have described for t he first time these stages of differentiation. In organ cu lture the duodenum of t he 16-day embryo in general retain ed its via bility durin g a 4-day culture period. Maturation of epit helial cells occurred at a similar r ate in vivo and in vitro. However, the presence of mito chondria l enlargement and aut ophagic vacuoles in t he cultured tissue suggested t hat some cellular degener ation was taking place. Differentiation of epithelial cells occurred to a similar degree when hydrocortisone was added t o t he organ cu ltures. The surface coat was better developed in the hydrocort isone-treated cultures, a finding t hat correlates with t he indu ction of invertase by hydro cortisone in embryonic duodenum. H ydrocortisonetreated t issue showed prominent dilation of endoplasmic reticulum and moderate autophagy, suggesting that the addition of hydrocortisone to the organ culture was in part injurious to the culture. This organ culture system m ay well be ut ilized for testing the effects of a variety of substances upon embryonic intestinal epithelium.

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