Cell Tiss. Res. 183, 33-50 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
Endocrine Cells in the Human Fetal Small Intestine* Pamela C. Moxey and Jerry S. Trier** Department of Medicine, Peter Bent Brigham Hospital, and Harvard Medical School, Boston, Massachusetts, USA
Summary. In this report we describe the time of appearance and ultrastructural features of enteroendocrine cells (EECs) in the h u m a n fetal small intestine (SB) between 9 and 22 weeks gestation. Thirteen distinctive EECs were identified in fetal SB. Two of these, not found in normal adult SB, appeared within the stratified epithelium of the proximal SB at 9-10 weeks. They were arbitrarily termed "primitive" and "precursor" cells. As in all fetal EECs, the pale cytoplasm o f the "primitive" cell contains a distinctive population o f secretory granules (SGs). Primitive cell SGs average 200-330 nm; some have dense cores with lucent halos while others are filled with a homogeneous dense or flocculent material. The SGs of the "precursor" cells are larger, averaging up to 1 gm in diameter and their contents vary in electron density. A third group of cells not described in normal adult SB was arbitrarily termed "transitional" cells. These have two populations of SGs; one resembles the SGs of the "precursor" cells, and the other resembles the SGs of some of the specific adult type EECs. Transitional EC, S, I and G cells are seen. In addition, mature appearing EC, S, G, I, L, D, and D 1 cells were identified by 12 weeks of gestation. The "primitive", "precursor", and "transitional" cells may represent sequential developmental precursors of adult type EECs. Key words: H u m a n fetal small intestine - Endocrine cells - Ultrastructure.
Introduction In adult h u m a n small intestine, at least 9 enteroendocrine cell types (EECs) with specific structural and/or immunohistochemical features have been described. Only limited information, however, is available regarding the time of appearance and Send offprint requests to: Dr. Pamela C. Moxey, Gastroenterology Division, Department of Medicine,
Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, MA 02115, USA * Supported by Research Grant AM-17537 from the National Institutes of Health, Besthesda, Maryland ** The authors would like to thank Ms. Linda Barstein for her excellent technical assistance
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u l t r a s t r u c t u r a l features o f E E C s in the fetal small intestine. The presence o f E E C s in h u m a n fetal small bowel (SB) by the 12th week is m e n t i o n e d in several u l t r a s t r u c t u r a l studies ( T o d d et al., 1970; B a r t m a n , 1972; V a r k o n y i et al., 1974), b u t these E E C s were n o t classified as to specific cell types. In a recent study (Osaka, 1975) a n a t t e m p t was m a d e to identify specific E E C s in h u m a n fetal SB based on u l t r a s t r u c t u r a l criteria. In this r e p o r t S, G - l i k e a n d D cells were illustrated, a n d i d e n t i f i c a t i o n o f EC, D 1, a n d L cells was claimed in the f o u r t h to fifth m o n t h s o f gestation. In o t h e r studies, i m m u n o f l u o r e s c e n c e techniques revealed cells i m m u n o r e a c t i v e to gastrin ( L a r s s o n et al., 1975; D u b o i s et al., 1976b), s o m a t o s t a t i n ( D u b o i s et al., 1976 a), a n d c h o l e c y s t o k i n i n ( D u b o i s et al., 1976 b) antisera in the SB o f 10 to 12 week h u m a n fetuses. In addition, cells with glucagon-like i m m u n o r e a c t i v i t y were localized in the h u m a n fetal SB b y 12 weeks o f gestation ( P o l a k et al., 1971), a n d cells exhibiting 5 - H T fluorescence were detected b y 13-14 weeks ( F a l c k et al., 1967). I n the p r e s e n t r e p o r t we describe the u l t r a s t r u c t u r a l features o f 13 m o r p h o l o g i c a l l y distinctive E E C s f o u n d in p r o x i m a l a n d distal SB o f 9 - 2 2 week h u m a n fetuses a n d a t t e m p t to classify these cells on the basis o f their u l t r a s t r u c t u r a l characteristics.
Materials and Methods Proximal and distal small intestinal tissue from 36 nonviable human fetuses 9 to 22 weeks old was obtained following therapeutic abortions by hysterotomy. Developmental age was determined by crown rump and/or crown-heel measurements (Patten, 1953). Tissue was placed in Liebowitz L-15 medium immediately after dissection and fixed within 30-90 min after surgical removal of the fetus. Samples from 28 fetuses were fixed for 1-2 h in chilled 1% osmium tetroxide in dichromate buffer containing 0.9 mM CaC12 (Dalton, 1955) and were then transferred to neutral 0.2 M phosphate buffered formalin for 30min. Tissue from 5 fetuses was fixed for I 2h in 2.5% cacodylate buffered glutaraldehyde containing 0.05 % CaCI 2. The tissue was then washed in cacodylate buffer for 1-24h and placed in cacodylate-buffered 1% osmium tetroxide for 1 h. Samples from the three remaining fetuses were fixed by both methods. Tissues were dehydrated and embedded in epoxy resin using standard methods. Sections 1 gm thick of epon embedded tissues were stained with Richardson's (Richardson et al., 1960) or a modified PAS procedure using Lillie's Schiff reagent (1951) and well-fixedareas were selected for thin sectioning. Thin sections were stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963), and examined with a Philips 300 electron microscope. Specific endocrine cell types were classified on the basis of the size, shape, electron density and average diameter of their secretory granules. To determine the average diameter of the granule population of each endocrine cell type, 10-40 randomly selected granule profiles per cell were measured. For each endocrine cell type, granules in 3 to 35 cells were measured.
Results The l u m e n o f the p r o x i m a l a n d distal SB o f the y o u n g e s t fetus (9 weeks) was lined by stratified epithelium. By 10 weeks, villi h a d a p p e a r e d in the p r o x i m a l intestine; m a n y o f these lined by simple c o l u m n a r epithelium. In the distal SB, villus f o r m a t i o n with r e p l a c e m e n t o f stratified epithelium by c o l u m n a r epithelium o c c u r r e d a p p r o x i m a t e l y one week later t h a n in the p r o x i m a l SB. T h i r t e e n E E C s were tentatively identified in the fetal SB by the 12th week. The g e s t a t i o n a l ages at which these various E C C s were first identified in the p r o x i m a l
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Fetal Intestinal Endocrine Cells Table 1, Age of appearance in weeks of gestation of human fetal intestinal endocrine cells Cell type
Proximal small intestine
Distal small intestine
Primitive Precursor Transitional EC Transitional S Transitional I Transitional G Mature-appearing S Mature-appearing D 1 Mature-appearing EC Mature-appearing I Mature-appearing L Mature-appearing D Mature-appearing G
9-10" 9-10a 10-11 10-11 10-11 10-11 9-10 9-10 10-I1 10-I1 13 10-11 11-12
11-12 15 13 11-12 10-11 11-12 10 11 11 12 10-11 ?b
a Stratified Epithelium b Possible D-like cells seen by 14-15 weeks
and distal SB of our material are summarized in Table 1. The morphological features of the secretory granules of each of these are summarized in Table 2. For comparison, Table 3 lists the location, secretory granule structure, and putative products of endocrine cells identified by others in adult h u m a n small intestine. Two distinct types of EECs not found in the normal adult SB could be identified along the basal lamina of the fetal stratified epithelium and the rudimentary villi of the proximal SB at 9-10 weeks. These cells were not seen in the stratified epithelium of the distal SB at this age. One of these cell types was arbitrarily termed"primitive" cell. This cell is characterized by its pale cytoplasm compared to adjacent epithelial cells, its large nucleus, the presence of free ribosomes, little formed endoplasmic reticulum (ER), numerous small mitochondria, and by secretory granules (SGs) whose fine structure varies. M a n y of the SGs have small, electron opaque cores surrounded by electron lucent regions and an incomplete membrane (Fig. 1 A, B). This discontinuity of the granule membrane m a y be a fixation artefact. A few similar granules are completely enclosed by an outer membrane. Other granules vary in electron density and often contain no or only very thin electron lucent halos (Fig. 1 B). The range of the mean diameter of these various granules in "primitive" EECs varies from 200 to 330 nm. These cells can be identified in the distal SB by 10-11 weeks gestation after villi have formed. The second distinct type of EEC identified within the stratified epithelium of the proximal SB at 9-10 weeks was arbitrarily termed a "precursor" cell. Such cells were not detected within the stratified epithelium of the distal SB at 9-10 weeks but could be identified, although infrequently, several weeks later in the simple columnar epithelium of villi. "Precursor" cells are characterized by large homogeneous cytoplasmic granules with an average diameter ranging from 530 to 1450 nm. These granules are consistently pale to moderately electron dense and are surrounded by a closely apposed membrane (Fig. 2A). As in all fetal EECs, the remainder of the pale cytoplasm contains free ribosomes, scattered glycogen
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Table 2. Endocrine cells in human fetal small intestine by 10-12 weeks gestation
Cell type
Secretory granules, characteristics
Primitive cell
Highly variable densities Small electron dense cores often present Average 200-330 nm
Precursor
1. Primitive cell granules may be present 2. Spherical Pale to moderately electron dense, homogenous Average 530-1450 nm
Transitional Enterochromaffin (EC) cell
1. Large precursor cell granules 2. Spherical to highly pleomorphic Uniformly electron dense Average 250-400 nm
Mature-appearing EC cell
Spherical to highly pleomorphic Uniformly electron dense Average 250-380 nm
Transitional S cell
1. Large precursor cell granules 2. Spherical or oval, may be irregular Moderate to highly electron dense Narrow halo may be present Average 150-210 nm
Mature-appearing S cell
Spherical or oval, may be irregular Primarily dense, but may be variable Narrow halo may be present Average 100-200 nm
Transitional G cell
1. Large precursor cell granules 2. Spherical Highly variable densities; empty or flocculent core common Average 220-400 nm
Mature-appearing G cell
Spherical Variable densities; empty or flocculent core common Average 280-450 nm
Transitional I cell
1. Large precursor cell granules 2. Spherical or oval, regular Uniformly electron dense Average 230-270 nm
Mature-appearing I cell
Spherical or oval, regular Moderate to highly electron dense, uniform Narrow halo may be present Average 220-280 nm
Mature-appearing EG or L cells
Spherical or oval Uniformly electron dense Average 320- 390 nm
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Fetal Intestinal Endocrine Cells Table 2 (continued) Cell type
Secretorygranules, characteristics
Mature-appearing D cell
Spherical Low to moderate electron density; uniform Closely applied membrane Average 250-300 nm
Mature-appearing D 1 cell
Spherical, regular Variable density; generallymoderate to highly electron dense Halo may be present Abundant microfilaments Average 140-190nm
particles, numerous small mitochondria, a Golgi complex, a few formed elements of smooth ER, rare profiles of rough ER, and aggregates of microfilaments. In several of these "precursor" cells, there is a second population of SGs morphologically similar to those found in the "primitive" cells (Fig. 2 B). Both "primitive" and "precursor" cells persisted during the second trimester but they appeared to decrease in frequency. Between the 10th and 12th weeks of gestation, four other distinctive cell types, collectively termed "transitional" cells appeared. These cells are characterized by two distinct populations of SGs. One, common to all "transitional" cells, is indistinguishable from the large, pale granules of the "precursor" cells (Figs. 3 A 6A). The second population is characteristic of a specific adult-type EEC, namely EC, S, I or G cells. Each "transitional" cell was classified by this second population of SGs. As indicated in Table 1, "transitional" I and G cells were never identified in the distal SB. In contrast "transitional" EC and S cells were present in proximal and distal SB by 13 weeks of gestation, though they were more frequent proximally. Mature adult-type EC, S, I and G cells were also present (Figs. 3 B-6 B). Transitional S, EC and mature-appearing S, EC and I cells were located in crypts and on villi whereas transitional I, G and mature-appearing G cells were identified only in crypts. After villus formation, both transitional and adult type EC cells were frequent. A representative fetal "transitional" EC and a mature-appearing fetal EC cell are illustrated in Figure 3. The "transitional" EC cell contains many typical adult-type, electron-opaque, pleomorphic SGs as well as several larger pale granules, averaging over 1 Ixm and resembling those in the fetal "precursor" cells. The pleomorphic SGs of fetal "transitional" and mature-appearing EC cells range between 250 and 400 nm in average diameter. Mature-appearing fetal EC cells were identified in the proximal SB throughout the 10th to 22nd week of gestation, and in the distal SB after the 1 lth week. A representative fetal "transitional" S cell and a mature-appearing fetal S cell are shown in Figure 4. In the "transitional" S cells, two populations of granules are apparent; one consists of the large, moderately electron dense granules characteristic of "precursor" cells and the second consists of small, round or oval electron opaque granules (Fig. 4A). In both the "transitional" S cell and the mature-
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Fig. 1. A "Primitive" cell bordering basal lamina of stratified epithelium of proximal SB of 9-10 week fetus. Note small mitochondria, supranuclear Golgi complex (G), abundant free ribosomes, and several characteristic SGs (arrows) with electron opaque cores and electron lucent halos. Some granule profiles surrounded by a continuous membrane (long arrows); others only partially surrounded (short arrows). • 8100. B Higher magnification of cytoplasm of "primitive" cell. Contents of SGs exhibit variable electron density. Some have electron opaque cores with relatively clear halos and incomplete membranes (long arrows); others are filled with electron opaque or flocculent material (short arrows), x 18,800
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Fig. 2. A "Precursor" cell from proximal SB of 9-10 week fetus. Cytoplasm characterized by large granules which average 530 nm in diameter (P). Other profiles (arrows) appear empty except for small amount of flocculent material, x 18,800. B "Precursor" cell from crypt of proximal SB of 9-10 week fetus. This cell has two distinct populations of secretory granules: one population is characteristic of "precursor" cells and consists of granules averaging 750 nm, second population resembles more heterogeneous granules typical of "primitive" ceils (arrows). x 14,000
a p p e a r i n g S cell, as in a d u l t S cells, the electron o p a q u e S G s r a n g e f r o m 100-210 n m in m e a n diameter. A few o f these dense granules have a thin electron lucent h a l o i n t e r p o s e d between the g r a n u l e core a n d the limiting m e m b r a n e . B o t h " t r a n sitional" S cells a n d a d u l t - t y p e S cells a p p e a r one week earlier in the p r o x i m a l SB t h a n in the distal SB (Table 1). The " t r a n s i t i o n a l " I cell a n d the m a t u r e - a p p e a r i n g I cell (Fig. 5) were identified in the p r o x i m a l SB b y 10-11 weeks o f gestation. T h e m a t u r e a p p e a r i n g I cells were
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Fig. 3. A "Transitional" EC cell from proximal SB of 10 11 week fetus. Large granules similar to those in "precursor" cells (P) are pale, and average over 1 lam in diameter. Adult-type EC cell granules (arrows) are pleomorphic and electron opaque, x 13,700. B Mature-appearing EC cell from distal SB of 10-11 week fetus. Electron dense granules are highly pleomorphic, and include round, discoid and ellipsoid shapes. This cell is morphologically indistinguishable from adult EC cells, x 11,000
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Fig. 4. A "Transitional" S cells from distal SB of 14-15 week fetus. "Precursor" type granules(P) vary in electron density and average 800 nm in diameter. Electron opaque granules (arrows) similar to those of mature S cells are also present; they average 200nm in diameter. • 14,300. B Mature-appearing S cell from crypt of distal SB of 14-15 weekfetus. Secretorygranules are electronopaque and average 180 nm in diameter. Note some microfilaments(arrow) among infranuclear granules, x 12,500
present distally by 11-12 weeks, whereas transitional I cells were not seen in distal SB at any age (Table 1). The "transitional" I cells contain large granules resembling those of the "precursor" cells and a second population of smaller granules similar to those in I cells of adult human intestine (Fig. 5 A). These smaller granules resemble those of S cells except that they are considerably larger, ranging in average diameter from 230 to 270 nm (Table 2). Similarly, in adult-type fetal I cells the round or oval uniformly electron opaque SGs range from 220 to 280 nm in mean diameter (Table 2 and Fig. 513). In a few granules, a narrow electron lucent halo is interposed between the granule core and its surrounding membrane. The fourth type of "transitional" cell in the fetal SB at 10-11 weeks is the "transitional" G cell. The large "precursor" type granules are uniform in electron density while the density of the smaller specific SGs varies considerably (Fig. 6A). The specific granules are spherical with a mean diameter ranging from 220 to 400 nm. Mature-appearing G-cells appeared in proximal SB at 11 to 12 weeks. As in adult G cells (Table 3) the contents of the fetal G cells SGs are heterogeneous and the mean diameter ranges from 280 to 450 nm in various cells (Table 2, Fig. 6 B). By the middle of the second trimester of gestation "transitional" G cells were not seen but mature-appearing G cells were still present.
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Fig. 5. A "Transitional" I cell from proximal SB of 10 11 week fetus. "'Precursor" type granules (P) average 750 nm in diameter. Sectioned I-like granules (arrows) are oval or round, average 270 nm in diameter and closely resemble those of adult I cells. • 16,100. B Mature-appearing I cell from distal SB of 20-21 week fetus. Cytoplasm is filled with abundant round electron dense SGs averaging 270 nm in diameter, mitochondria, arid occasional microfilaments. • 14,400 T h r e e a d d i t i o n a l a d u l t - t y p e E E C s w e r e t e n t a t i v e l y i d e n t i f i e d in the h u m a n fetal SB. T h e s e i n c l u d e the L, D 1 a n d D cells. U n l i k e m o s t fetal E E C s , the L cells w e r e first n o t e d d i s t a l l y at 10 to 11 w e e k s o f g e s t a t i o n b u t w e r e n o t f o u n d in the p r o x i m a l SB u n t i l t h e 13th w e e k ( T a b l e 1). L cells, as i l l u s t r a t e d in F i g u r e 7, r e s e m b l e m a t u r e a p p e a r i n g I cells (Fig. 5 B). B o t h c o n t a i n a p o p u l a t i o n o f r o u n d o r o v a l S G s o f
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Fig. 6. A Putative "transitional" G cell from proximal small intestine of 11-12 week fetus. "Precursor" type granules (P) average 900 nm in diameter. Smaller secretory granules which vary in electron density (arrows) are also present. • 23,900. B Mature-appearing G cell from proximal small intestine o f 10-11 week fetus. Secretory granules average 400 nm in diameter and their contents vary in electron density. Note similarity o f some granules (arrows) to those in Figure 6A. x 21,500
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Fig- 7. Mature-appearing L cell from proximal SB of 17 18 week fetus. Electron opaque granules average 390 nm in diameter, x 12,800 Fig.& PutativeD-cell fromproximalSBof9 10week fetus. Pale to moderately electron dense granules, averaging 280nm in diameter, are surrounded by a closely apposed membrane, x 15,700
relatively u n i f o r m electron density. However, the average d i a m e t e r o f the L cell granules is larger a n d ranges from 320 to 390 n m (Table 2). D cells a p p e a r e d in the p r o x i m a l SB between 10 a n d 12 weeks (Table 1, Fig. 8). A l t h o u g h some o f the D cells resemble G cells, the electron density o f D cell S G s is less h e t e r o g e n e o u s a n d the range o f the m e a n d i a m e t e r o f the D cell S G s varies less
Fetal Intestinal Endocrine Cells
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Fig. 9. Putative D 1 cell from distal SB of 15 week fetus. Cytoplasm contains a b u n d a n t granules averaging 170 n m in diameter, whose electron density varies. Cytoplasmic microfilaments (arrows) are interspersed a m o n g granules, x 19,000 Fig. 10. Cluster o f endocrine cells frequently found in crypts o f h u m a n fetal SB. In this particular group, located at crypt base of 13 week fetus, there are an EC cell, two I cells and an S cell. x 10,200
Table 3. S u m m a r y of features of h u m a n adult intestinal endocrine cells Cell type
Location
Secretory granule characteristics
Postulated products
Gastrin or G cell
Duodenum
Spherical Highly variable densities Halo m a y be present Average diameter range: 250-400 n m (Frexinos et al., 1973; Grube, 1976)
Gastrin
Enterochromaffin or EC cell
Entire length of small intestine
Highly pleomorphic Uniformly electron dense No halo Average diameter range: 300 6 0 0 n m (Kobayashi et al., 1970; Cavallero et al., 1972)
Motilin Substance P Serotonin
Secretin or S cell
Entire length of small intestine: Primarily d u o d e n u m and jejunum
Spherical, oval or irregular Moderate to highly electron dense Narrow halo may be present Average diameter range: 100 2 0 0 n m (Frexinos et al., 1973; Solcia et al., 1974)
Secretin
Intermediate or I cell
Primarily upper intestine Rarely in ileum
Spherical Uniformly electron dense Closely applied membrane; rarely a narrow halo Average diameter range: 240-280 n m (Frexinos et al., 1973; Polak et al., 1975)
Cholecystokinin
Large or L cell
Primarily lower Spherical j e j u n u m and Uniformly electron dense ileum N o halo Average diameter range: 300-400 n m (Labo et al., 1971; Osaka et al., 1973)
Enteroglucagon
Intestinal D cell
Primarily d u o d e n u m and jejunum
Spherical Low to moderately electron dense Closely applied membrane Average diameter range: 300-450 n m (Kobayashi et al., 1970; Cavallero et al., 1972)
Somatostatin
DI
Primarily d u o d e n u m and upper j e j u n u m
Spherical, regular Variable electron density Closely applied membrane; rarely narrow halo A b u n d a n t microfilaments Average diameter range: 140 180 n m (Cavallero et al., 1972; Solcia et al., 1974)
Gastric Inhibitory Polypeptide (GIP) or H u m a n Pancreatic Polypeptide (HPP)
K Cell
Entire length of small intestine: Primarily d u o d e n u m and upper jejunum
Spherical, ovoid Moderately electron dense, often with a double structure, i.e. darker core Halo m a y be present Average diameter range: 200-400 n m (Solcia et al., 1974; Solcia et al., 1975)
Gastric Inhibitory Polypeptide (GIP)
H cell
Primarily lower Spherical, may be angular j e j u n u m and Moderate electron density (Solcia et aI., 1974; ileum Solcia et al., 1975)
Vasoactive Intestinal Polypeptide (VIP)
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(Table 2; compare Figs. 6 B and 8). The D cell SGs are uniformly pale or moderately electron dense and are surrounded by a closely apposed membrane. D 1 cells are identified in the proximal small intestine at 9-10 weeks of gestation and by the 12th week are common in both the proximal and distal SB (Table 1). These putative D 1 cells contain abundant cytoplasmic microfilaments and the SGs have a mean diameter ranging from 140 to 190 nm (Fig. 9). The SGs resemble the S cell SGs in size (Table 2). The cells can be distinguished, however, by the electron density of the SG contents; D 1 SGs vary from pale to moderately electron dense while those of S cells are uniformly electron opaque (compare Figs. 4 and 9). L and D 1 cells are present on villi as well as within crypts. Accumulation of EECs composed of several cell types were frequently found in clusters at the base of the crypts. For example, the section shown in Figure 10 includes an EC, a putative S, and two I cells. In addition to the 13 types of fetal endocrine cells described above, there were additional cells with structural features suggestive of endocrine cells. These contained putative secretory granules but the morphology of the SGs was extremely variable and different from that of defined adult EECs thus precluding their classification. N o cells were identified which resembled the adult-type K or H cells (Table 3), and no EECs could be recognized within the lamina propria at any of the ages studied.
Discussion
In this study, we describe morphologically distinctive "primitive", "precursor", 4 different "transitional", and 7 different adult-type EECs in the human fetal small intestine by the 12th week of gestation. Only the 7 adult-type EECs have been described in the normal adult intestinal mucosa. The other 6 fetal EECs are distinctive and do not have adult correlates. It seems likely that two of these, the "primitive" and "precursor" cells, are immature endocrine cells that appear transiently during intestinal maturation. This is supported by their early appearance in the stratified epithelium at 9-10 weeks, their ultrastructural features which suggest immaturity, their apparent decrease in number with increasing age, and their absence in normal adult SB. There is additional evidence that the "primitive" cell may represent an immature or undifferentiated endocrine cell. Cells morphologically resembling "primitive" cells have been illustrated in regenerating rabbit pancreas and carcinoma of the pancreas in hamsters (Boquist and Falkmer, 1969), and in an endocrine tumor of the human pancreas (Greider and Elliot, 1964). Morphologically similar cells have also been illustrated in the pancreas of lower vertebrates, specifically the teleosts (Nakamura and Motoyoshi, 1971 ; Falkmer and Patent, 1972). The "precursor" cells with their extremely large secretory granules are unique among EECs. Endocrine cells with comparable secretory granule morphology have not been reported in other gastrointestinal tissues. A potential relationship of the "precursor" cells to other fetal endocrine cells is suggested by the occurrence of certain EECs with 2 distinct populations of granules. These include some of the
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"precursor" cells themselves which contain granules resembling those found in "primitive" cells in addition to their extremely large granules. Another group of cells with two distinct populations of SGs is the"transitional" cells. In"transitional" cells one population of granules is morphologically indistinguishable from the large granules noted in "precursor" cells. The second population varies from cell to cell and may resemble the specific SGs found in adult EC, S, I, or G cells. We therefore suggest that the "transitional" cells may be developmental precursors of specific adult EECs. The only other situation in which cells resembling "transitional" cells have been described is in the gastric mucosa from a patient with pernicious anemia (Forssman, 1976), a condition which has been associated with endocrine cell hyperplasia (Rubin, 1973). In summary, we suggest that the "primitive", "precursor", and "transitional" cells may represent sequential developmental precursors of adult type EECs in fetal small intestine. The apparent differentiation of fetal endocrine cells within the intestinal mucosa and the absence of identifiable endocrine cells within the lamina propria at any age studied do not provide support for the APUD concept of a neural crest origin of intestinal endocrine cells (Pearse, 1973). We are unable, however, to exclude the possibility that precursors of endocrine cells derived from the neural crest migrated into the mucosa prior to the 9th week of gestation. The appearance of diverse EECs occurred largely in concert with villus formation at 9-10 weeks (Table 1). During the same period there appeared to be an increase in the total number of EECs per area of mucosa. This impression is supported by quantitative studies (Singh, 1963) in which it was noted that the number of EECs per unit length of mucosa increased progressively between the 8th to 16th weeks of gestation in both proximal and distal SB and then gradually decreased at later gestational ages. In the rat, the frequency of EECs within the intestinal epithelium is greatest at birth, and decreases postnatally (Josephson and Altmann, 1973). Using specific antisera and immunofluorescent techniques it has been shown that G cells (Larsson et al., 1975; Dubois et al., 1976 b), D cells (Dubois et al., 1976 a), I cells (Dubois et al., 1976 b), and L cells (Polak et al., 1971) are present in fetal small intestine by 12 weeks. These findings indicate that at least some fetal EECs synthesize and store polypeptides immunologically similar to adult hormones. The early appearance and apparent differentiation of fetal EECs within the fetal SB by the 12th week suggests that these cells may have functional roles during development. For example, perhaps gastrin produced by fetal G-cells has atrophic role in human intestinal development similar to gastrin's trophic effect on adult gastrointestinal tissues of a variety of species (Johnson, 1976). Similarly, CCK of intestinal I cells may have atrophic role in the human fetus as it does in the adult rat pancreas (Mainz et al., 1973). Another potential function of the EECs in utero might be regulation of gastrointestinal motility. Active motility in utero is suggested by studies in which circulation of amniotic fluid through the gastrointestinal tract has been shown late in pregnancy (Pritchard, 1965). In the adult gastrointestinal tract gastrin, CCK, secretin, serotonin, and motilin have all been shown to affect motility (Brown, 1974; Walsh and Grossman, 1975; Rayford et al., 1976), and the cell types which produce these hormones have been tentatively identified in human fetal SB.
Fetal Intestinal Endocrine Cells
49
References Bartman, J.: Ultrastructure of human foetal small intestinal mucosa in early gestation. J. Path. 106, viiiix (1972) Boquist, L., Falkmer, S.: The significance of agranular and ciliated islet cells. In: The structure and metabolism of the pancreatic islets (S. Falkmer, B. Hellman and I.B. Taljedal, eds.), pp. 25-35. Oxford-New York: Pergamon Press 1969 Brown, J.C.: Candidate hormones of the gut: Motilin. Gastroenterology 67, 734-735 (1974) Cavallero, C., Vassallo, G., Capella, C., Solcia, E.: Ultrastructure of intestinal endocrine cells in man. In: Advances in gastrointestinal endoscopy. Proc. 2nd World Congress of Gastroint. Endosc. (G. Marcozzi and M. Crespi, eds.), pp. 449-455. Rome and Copenhagen: Piccin Medical Books 1972 Dalton, A.J.: A chrome-osmium fixative for electron microscopy. Anat. Rec. 121, 281 (1955) Dubois, P.M., Paulin, C., Dubois, M.P.: Gastrointestinal somatostatin cells in the human fetus. Cell Tiss. Res. 166, 179-184 (1976a) Dubois, P.M., Paulin, C., Chayvialle, J.A.: Identification of gastrin-secreting cells and cholecystokininsecreting cells in the gastrointestinal tract of the human fetus and adult man. Cell Tiss. Res. 175, 351356 (1976 b) Falck, G., H~kanson, R., Owman, C.H., Sjrberg, N.O.: Monoamine storing cells of the enterochromaffin type in gastrointestinal tract of human fetus. Acta physiol, scand. 71, 403-404 (1967) Falkmer, S., Patent, G.J.: Comparative and embryological aspects of the pancreatic islets. In: Handbook of physiology, Vol. 1, Sect. 7 (Steiner, D.G., Freinkel, N., eds.), pp. 1-23. Baltimore, Md.: Williams and Williams 1972 Forssman, W.G.: The ultrastructure of the endocrine cells in the normal and pathological gastrointestinal mucosa. In: Chromaffin, enterochromaffin and related cells (R.E. Coupland, T. Fujita, eds.), pp. 227-241. Amsterdam: Elsevier Scientific Publishing Company 1976 Frexinos, J., Balas, D., Ribet, A. : Les cellules endocrines de la musque duodrnale chez l'homme, l~tude ultrastructurale. Biol. Gastroenterol. 6, 105-117 (1973) Greider, M.H., Elliot, D.W.: Electron microscopy of human pancreatic tumors of islet cell origin. Amer. J. Path. 44, 663-678 (1964) Grube, D.: The endocrine cells of the gastrointestinal epithelium and the metabolism of biogenic amines in the gastrointestinal tract. Progress in Histochem. Cytochem. 8, 1-128 (1976) Johnson, L.R.: The trophic action of gastrointestinal hormones. Gastroenterology 70, 278-288 (1976) Josephson, R.L., Altmann, G.G.: Distribution of diazo-positive (argentaffin) cells in the small intestine of rats of various ages. Amer. J. Anat. 136, 15-22 (1973) Kobayashi, S., Fujita, T., Sasagawa, T.: The endocrine cells of human duodenal mucosa. An electron microscopy study. Arch. Histol. Jap. 31, 477-494 (1970) Labo, C., Gasbarrini, G., Miglio, F.: Aspects ultrastructuraux de la muqueuse duodrno-intestinale humaine. I. l~tude de la muqueuse duod+nointestinale humaine normale. Ann. Gastroent+rol. Hrpatol. 7, 293-312 (1971) Larsson, L.I., H~kanson, R., Sj6berg~ N.O., Sundler, F.: Fluorescence histochemistry of the gastrin cell in fetal and adult man. Gastroenterology 68, 1152-1159 (1975) Lillie, R.D.: Simplification of the manufacture of Schiff reagent for use in histochemical procedures. Stain Technol. 26, 163-165 (1951) Mainz, D.L., Black, O., Webster, P.D. : Hormonal control of pancreatic growth. J. clin. Invest. 52, 23002304 (1973) Nakamura, M., Motoyoshi, Y.: Ultrastructural studies on the islets of Langerhans of the carp. Z. Anat. Entwickl.-Gesch. 134, 61-72 (1971) Osaka, M.: Fine structure of the basal-granulated cells in human fetal duodenum. Arch. Histol. Jap. 38, 307-320 (1975) Osaka, M., Sasagawa, T., Fujita, T.: Endocrine cells in human jejunum and ileum: An electron microscopy study of biopsy material. Arch. Histol. Jap. 35, 235 248 (1973) Patten, B.M.: Human embryology. 2nd ed. New York-London: Blakiston Company Inc. 1953 Pearse, A.G.E.: Cell migration and the alimentary system: Endocrine contribution of the neural crest to the gut and its derivatives. Digestion 8, 372-385 (1973) Polak, J., Coulling, I., Bloom, S., Pearse, A.G.E.: Immunofluorescent localization of secretin and enteroglucagon in human intestinal mucosa. Scand. J. Gastroent. 6, 739-744 (1971) Polak, J.M., Pearse, A.G.E., Bloom, S.R., Buchanan, A.MJ., Rayford, P.L., Thompson, J.C.: Identification of cholecystokinin-secreting cells. Lancet 1975 II, 1016-1025
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P.C. Moxey and J.S. Trier
Pritchard, J.A.: Deglutition by normal and anencephalic fetuses. Obstet. Gynecol. 25, 289-297 (1965) Rayford, P.L., Miller, T.A., Thompson, J.C.: Secretin, cholecystokinin and newer GI hormones. New Engl. J. Med. 294, 1093-1100 (1976) Reynolds, E.S. : The use of lead nitrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Richardson, K.C., Jarrett, L.K., Fincke, E.H.: Embedding epoxy resins for ultrathin sectioning for electron microscopy. Stain Technol. 35, 313-323 (1960) Rubin, W.: A fine structural characterization of the proliferated endocrine cells in atrophic gastric mucosa. Amer. J. Path. 70, 109 117 (1973) Singh, I.: The prenatal development of enterochromaffin cells in the human gastro-intestinal tract. J. Anat. (Lond.) 9"7, 377-387 (1963) Solcia, E., Capella, C., Buffa, R., Bettini, R., Vassallo, G. : Istochimica ed ultrastruttura delle cellule endocrine dell'apparato digerente. Riv. Istoch. Norm. Pat. 18, 63-94 (1974) Solcia, E., Polak, J. M., Buffa, R., Capella, C., Pearse, A.G.E.: Endocrine cells of the intestinal mucosa. In: Gastrointestinal hormones (J.C. Thompson, ed.), pp. 155-168. Austin: University of Texas Press 1975 Todd, M.E., Dunn, J.S., Bagshaw, V.: The fine structure of human fetal tissues. J. Anat. (Lond.) 106, 188189 (1970) V/trkonyi, T., Gergely, G., Vat6, V. : The ultrastructure of the small intestinal mucosa in the developing human fetus. Scand. J. Gastroent. 6, 495 500 (1974) Walsh, J., Grossman, M.I.: Gastrin, Part I. New Engl. J. Med. 292, 1324-1332 (1976) Watson, M.L.: Staining of tissue sections for electron microscopy with heavy metals. J. biophys. biochem. Cytol. 4, 475-478 (1958)
Accepted April 8, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
Endocrine Cells in the Human Fetal Small Intestine* Pamela C. Moxey and Jerry S. Trier** Department of Medicine, Peter Bent Brigham Hospital, and Harvard Medical School, Boston, Massachusetts, USA
Summary. In this report we describe the time of appearance and ultrastructural features of enteroendocrine cells (EECs) in the h u m a n fetal small intestine (SB) between 9 and 22 weeks gestation. Thirteen distinctive EECs were identified in fetal SB. Two of these, not found in normal adult SB, appeared within the stratified epithelium of the proximal SB at 9-10 weeks. They were arbitrarily termed "primitive" and "precursor" cells. As in all fetal EECs, the pale cytoplasm o f the "primitive" cell contains a distinctive population o f secretory granules (SGs). Primitive cell SGs average 200-330 nm; some have dense cores with lucent halos while others are filled with a homogeneous dense or flocculent material. The SGs of the "precursor" cells are larger, averaging up to 1 gm in diameter and their contents vary in electron density. A third group of cells not described in normal adult SB was arbitrarily termed "transitional" cells. These have two populations of SGs; one resembles the SGs of the "precursor" cells, and the other resembles the SGs of some of the specific adult type EECs. Transitional EC, S, I and G cells are seen. In addition, mature appearing EC, S, G, I, L, D, and D 1 cells were identified by 12 weeks of gestation. The "primitive", "precursor", and "transitional" cells may represent sequential developmental precursors of adult type EECs. Key words: H u m a n fetal small intestine - Endocrine cells - Ultrastructure.
Introduction In adult h u m a n small intestine, at least 9 enteroendocrine cell types (EECs) with specific structural and/or immunohistochemical features have been described. Only limited information, however, is available regarding the time of appearance and Send offprint requests to: Dr. Pamela C. Moxey, Gastroenterology Division, Department of Medicine,
Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, MA 02115, USA * Supported by Research Grant AM-17537 from the National Institutes of Health, Besthesda, Maryland ** The authors would like to thank Ms. Linda Barstein for her excellent technical assistance
34
P.C. Moxey and J.S. Trier
u l t r a s t r u c t u r a l features o f E E C s in the fetal small intestine. The presence o f E E C s in h u m a n fetal small bowel (SB) by the 12th week is m e n t i o n e d in several u l t r a s t r u c t u r a l studies ( T o d d et al., 1970; B a r t m a n , 1972; V a r k o n y i et al., 1974), b u t these E E C s were n o t classified as to specific cell types. In a recent study (Osaka, 1975) a n a t t e m p t was m a d e to identify specific E E C s in h u m a n fetal SB based on u l t r a s t r u c t u r a l criteria. In this r e p o r t S, G - l i k e a n d D cells were illustrated, a n d i d e n t i f i c a t i o n o f EC, D 1, a n d L cells was claimed in the f o u r t h to fifth m o n t h s o f gestation. In o t h e r studies, i m m u n o f l u o r e s c e n c e techniques revealed cells i m m u n o r e a c t i v e to gastrin ( L a r s s o n et al., 1975; D u b o i s et al., 1976b), s o m a t o s t a t i n ( D u b o i s et al., 1976 a), a n d c h o l e c y s t o k i n i n ( D u b o i s et al., 1976 b) antisera in the SB o f 10 to 12 week h u m a n fetuses. In addition, cells with glucagon-like i m m u n o r e a c t i v i t y were localized in the h u m a n fetal SB b y 12 weeks o f gestation ( P o l a k et al., 1971), a n d cells exhibiting 5 - H T fluorescence were detected b y 13-14 weeks ( F a l c k et al., 1967). I n the p r e s e n t r e p o r t we describe the u l t r a s t r u c t u r a l features o f 13 m o r p h o l o g i c a l l y distinctive E E C s f o u n d in p r o x i m a l a n d distal SB o f 9 - 2 2 week h u m a n fetuses a n d a t t e m p t to classify these cells on the basis o f their u l t r a s t r u c t u r a l characteristics.
Materials and Methods Proximal and distal small intestinal tissue from 36 nonviable human fetuses 9 to 22 weeks old was obtained following therapeutic abortions by hysterotomy. Developmental age was determined by crown rump and/or crown-heel measurements (Patten, 1953). Tissue was placed in Liebowitz L-15 medium immediately after dissection and fixed within 30-90 min after surgical removal of the fetus. Samples from 28 fetuses were fixed for 1-2 h in chilled 1% osmium tetroxide in dichromate buffer containing 0.9 mM CaC12 (Dalton, 1955) and were then transferred to neutral 0.2 M phosphate buffered formalin for 30min. Tissue from 5 fetuses was fixed for I 2h in 2.5% cacodylate buffered glutaraldehyde containing 0.05 % CaCI 2. The tissue was then washed in cacodylate buffer for 1-24h and placed in cacodylate-buffered 1% osmium tetroxide for 1 h. Samples from the three remaining fetuses were fixed by both methods. Tissues were dehydrated and embedded in epoxy resin using standard methods. Sections 1 gm thick of epon embedded tissues were stained with Richardson's (Richardson et al., 1960) or a modified PAS procedure using Lillie's Schiff reagent (1951) and well-fixedareas were selected for thin sectioning. Thin sections were stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963), and examined with a Philips 300 electron microscope. Specific endocrine cell types were classified on the basis of the size, shape, electron density and average diameter of their secretory granules. To determine the average diameter of the granule population of each endocrine cell type, 10-40 randomly selected granule profiles per cell were measured. For each endocrine cell type, granules in 3 to 35 cells were measured.
Results The l u m e n o f the p r o x i m a l a n d distal SB o f the y o u n g e s t fetus (9 weeks) was lined by stratified epithelium. By 10 weeks, villi h a d a p p e a r e d in the p r o x i m a l intestine; m a n y o f these lined by simple c o l u m n a r epithelium. In the distal SB, villus f o r m a t i o n with r e p l a c e m e n t o f stratified epithelium by c o l u m n a r epithelium o c c u r r e d a p p r o x i m a t e l y one week later t h a n in the p r o x i m a l SB. T h i r t e e n E E C s were tentatively identified in the fetal SB by the 12th week. The g e s t a t i o n a l ages at which these various E C C s were first identified in the p r o x i m a l
35
Fetal Intestinal Endocrine Cells Table 1, Age of appearance in weeks of gestation of human fetal intestinal endocrine cells Cell type
Proximal small intestine
Distal small intestine
Primitive Precursor Transitional EC Transitional S Transitional I Transitional G Mature-appearing S Mature-appearing D 1 Mature-appearing EC Mature-appearing I Mature-appearing L Mature-appearing D Mature-appearing G
9-10" 9-10a 10-11 10-11 10-11 10-11 9-10 9-10 10-I1 10-I1 13 10-11 11-12
11-12 15 13 11-12 10-11 11-12 10 11 11 12 10-11 ?b
a Stratified Epithelium b Possible D-like cells seen by 14-15 weeks
and distal SB of our material are summarized in Table 1. The morphological features of the secretory granules of each of these are summarized in Table 2. For comparison, Table 3 lists the location, secretory granule structure, and putative products of endocrine cells identified by others in adult h u m a n small intestine. Two distinct types of EECs not found in the normal adult SB could be identified along the basal lamina of the fetal stratified epithelium and the rudimentary villi of the proximal SB at 9-10 weeks. These cells were not seen in the stratified epithelium of the distal SB at this age. One of these cell types was arbitrarily termed"primitive" cell. This cell is characterized by its pale cytoplasm compared to adjacent epithelial cells, its large nucleus, the presence of free ribosomes, little formed endoplasmic reticulum (ER), numerous small mitochondria, and by secretory granules (SGs) whose fine structure varies. M a n y of the SGs have small, electron opaque cores surrounded by electron lucent regions and an incomplete membrane (Fig. 1 A, B). This discontinuity of the granule membrane m a y be a fixation artefact. A few similar granules are completely enclosed by an outer membrane. Other granules vary in electron density and often contain no or only very thin electron lucent halos (Fig. 1 B). The range of the mean diameter of these various granules in "primitive" EECs varies from 200 to 330 nm. These cells can be identified in the distal SB by 10-11 weeks gestation after villi have formed. The second distinct type of EEC identified within the stratified epithelium of the proximal SB at 9-10 weeks was arbitrarily termed a "precursor" cell. Such cells were not detected within the stratified epithelium of the distal SB at 9-10 weeks but could be identified, although infrequently, several weeks later in the simple columnar epithelium of villi. "Precursor" cells are characterized by large homogeneous cytoplasmic granules with an average diameter ranging from 530 to 1450 nm. These granules are consistently pale to moderately electron dense and are surrounded by a closely apposed membrane (Fig. 2A). As in all fetal EECs, the remainder of the pale cytoplasm contains free ribosomes, scattered glycogen
36
P.C. Moxey and J.S. Trier
Table 2. Endocrine cells in human fetal small intestine by 10-12 weeks gestation
Cell type
Secretory granules, characteristics
Primitive cell
Highly variable densities Small electron dense cores often present Average 200-330 nm
Precursor
1. Primitive cell granules may be present 2. Spherical Pale to moderately electron dense, homogenous Average 530-1450 nm
Transitional Enterochromaffin (EC) cell
1. Large precursor cell granules 2. Spherical to highly pleomorphic Uniformly electron dense Average 250-400 nm
Mature-appearing EC cell
Spherical to highly pleomorphic Uniformly electron dense Average 250-380 nm
Transitional S cell
1. Large precursor cell granules 2. Spherical or oval, may be irregular Moderate to highly electron dense Narrow halo may be present Average 150-210 nm
Mature-appearing S cell
Spherical or oval, may be irregular Primarily dense, but may be variable Narrow halo may be present Average 100-200 nm
Transitional G cell
1. Large precursor cell granules 2. Spherical Highly variable densities; empty or flocculent core common Average 220-400 nm
Mature-appearing G cell
Spherical Variable densities; empty or flocculent core common Average 280-450 nm
Transitional I cell
1. Large precursor cell granules 2. Spherical or oval, regular Uniformly electron dense Average 230-270 nm
Mature-appearing I cell
Spherical or oval, regular Moderate to highly electron dense, uniform Narrow halo may be present Average 220-280 nm
Mature-appearing EG or L cells
Spherical or oval Uniformly electron dense Average 320- 390 nm
37
Fetal Intestinal Endocrine Cells Table 2 (continued) Cell type
Secretorygranules, characteristics
Mature-appearing D cell
Spherical Low to moderate electron density; uniform Closely applied membrane Average 250-300 nm
Mature-appearing D 1 cell
Spherical, regular Variable density; generallymoderate to highly electron dense Halo may be present Abundant microfilaments Average 140-190nm
particles, numerous small mitochondria, a Golgi complex, a few formed elements of smooth ER, rare profiles of rough ER, and aggregates of microfilaments. In several of these "precursor" cells, there is a second population of SGs morphologically similar to those found in the "primitive" cells (Fig. 2 B). Both "primitive" and "precursor" cells persisted during the second trimester but they appeared to decrease in frequency. Between the 10th and 12th weeks of gestation, four other distinctive cell types, collectively termed "transitional" cells appeared. These cells are characterized by two distinct populations of SGs. One, common to all "transitional" cells, is indistinguishable from the large, pale granules of the "precursor" cells (Figs. 3 A 6A). The second population is characteristic of a specific adult-type EEC, namely EC, S, I or G cells. Each "transitional" cell was classified by this second population of SGs. As indicated in Table 1, "transitional" I and G cells were never identified in the distal SB. In contrast "transitional" EC and S cells were present in proximal and distal SB by 13 weeks of gestation, though they were more frequent proximally. Mature adult-type EC, S, I and G cells were also present (Figs. 3 B-6 B). Transitional S, EC and mature-appearing S, EC and I cells were located in crypts and on villi whereas transitional I, G and mature-appearing G cells were identified only in crypts. After villus formation, both transitional and adult type EC cells were frequent. A representative fetal "transitional" EC and a mature-appearing fetal EC cell are illustrated in Figure 3. The "transitional" EC cell contains many typical adult-type, electron-opaque, pleomorphic SGs as well as several larger pale granules, averaging over 1 Ixm and resembling those in the fetal "precursor" cells. The pleomorphic SGs of fetal "transitional" and mature-appearing EC cells range between 250 and 400 nm in average diameter. Mature-appearing fetal EC cells were identified in the proximal SB throughout the 10th to 22nd week of gestation, and in the distal SB after the 1 lth week. A representative fetal "transitional" S cell and a mature-appearing fetal S cell are shown in Figure 4. In the "transitional" S cells, two populations of granules are apparent; one consists of the large, moderately electron dense granules characteristic of "precursor" cells and the second consists of small, round or oval electron opaque granules (Fig. 4A). In both the "transitional" S cell and the mature-
38
P.C. Moxey and J.S. Trier
Fig. 1. A "Primitive" cell bordering basal lamina of stratified epithelium of proximal SB of 9-10 week fetus. Note small mitochondria, supranuclear Golgi complex (G), abundant free ribosomes, and several characteristic SGs (arrows) with electron opaque cores and electron lucent halos. Some granule profiles surrounded by a continuous membrane (long arrows); others only partially surrounded (short arrows). • 8100. B Higher magnification of cytoplasm of "primitive" cell. Contents of SGs exhibit variable electron density. Some have electron opaque cores with relatively clear halos and incomplete membranes (long arrows); others are filled with electron opaque or flocculent material (short arrows), x 18,800
Fetal Intestinal Endocrine Cells
39
Fig. 2. A "Precursor" cell from proximal SB of 9-10 week fetus. Cytoplasm characterized by large granules which average 530 nm in diameter (P). Other profiles (arrows) appear empty except for small amount of flocculent material, x 18,800. B "Precursor" cell from crypt of proximal SB of 9-10 week fetus. This cell has two distinct populations of secretory granules: one population is characteristic of "precursor" cells and consists of granules averaging 750 nm, second population resembles more heterogeneous granules typical of "primitive" ceils (arrows). x 14,000
a p p e a r i n g S cell, as in a d u l t S cells, the electron o p a q u e S G s r a n g e f r o m 100-210 n m in m e a n diameter. A few o f these dense granules have a thin electron lucent h a l o i n t e r p o s e d between the g r a n u l e core a n d the limiting m e m b r a n e . B o t h " t r a n sitional" S cells a n d a d u l t - t y p e S cells a p p e a r one week earlier in the p r o x i m a l SB t h a n in the distal SB (Table 1). The " t r a n s i t i o n a l " I cell a n d the m a t u r e - a p p e a r i n g I cell (Fig. 5) were identified in the p r o x i m a l SB b y 10-11 weeks o f gestation. T h e m a t u r e a p p e a r i n g I cells were
40
P.C. Moxey and J.S. Trier
Fig. 3. A "Transitional" EC cell from proximal SB of 10 11 week fetus. Large granules similar to those in "precursor" cells (P) are pale, and average over 1 lam in diameter. Adult-type EC cell granules (arrows) are pleomorphic and electron opaque, x 13,700. B Mature-appearing EC cell from distal SB of 10-11 week fetus. Electron dense granules are highly pleomorphic, and include round, discoid and ellipsoid shapes. This cell is morphologically indistinguishable from adult EC cells, x 11,000
Fetal Intestinal Endocrine Cells
41
Fig. 4. A "Transitional" S cells from distal SB of 14-15 week fetus. "Precursor" type granules(P) vary in electron density and average 800 nm in diameter. Electron opaque granules (arrows) similar to those of mature S cells are also present; they average 200nm in diameter. • 14,300. B Mature-appearing S cell from crypt of distal SB of 14-15 weekfetus. Secretorygranules are electronopaque and average 180 nm in diameter. Note some microfilaments(arrow) among infranuclear granules, x 12,500
present distally by 11-12 weeks, whereas transitional I cells were not seen in distal SB at any age (Table 1). The "transitional" I cells contain large granules resembling those of the "precursor" cells and a second population of smaller granules similar to those in I cells of adult human intestine (Fig. 5 A). These smaller granules resemble those of S cells except that they are considerably larger, ranging in average diameter from 230 to 270 nm (Table 2). Similarly, in adult-type fetal I cells the round or oval uniformly electron opaque SGs range from 220 to 280 nm in mean diameter (Table 2 and Fig. 513). In a few granules, a narrow electron lucent halo is interposed between the granule core and its surrounding membrane. The fourth type of "transitional" cell in the fetal SB at 10-11 weeks is the "transitional" G cell. The large "precursor" type granules are uniform in electron density while the density of the smaller specific SGs varies considerably (Fig. 6A). The specific granules are spherical with a mean diameter ranging from 220 to 400 nm. Mature-appearing G-cells appeared in proximal SB at 11 to 12 weeks. As in adult G cells (Table 3) the contents of the fetal G cells SGs are heterogeneous and the mean diameter ranges from 280 to 450 nm in various cells (Table 2, Fig. 6 B). By the middle of the second trimester of gestation "transitional" G cells were not seen but mature-appearing G cells were still present.
42
P.C. Moxey and J.S. Trier
Fig. 5. A "Transitional" I cell from proximal SB of 10 11 week fetus. "'Precursor" type granules (P) average 750 nm in diameter. Sectioned I-like granules (arrows) are oval or round, average 270 nm in diameter and closely resemble those of adult I cells. • 16,100. B Mature-appearing I cell from distal SB of 20-21 week fetus. Cytoplasm is filled with abundant round electron dense SGs averaging 270 nm in diameter, mitochondria, arid occasional microfilaments. • 14,400 T h r e e a d d i t i o n a l a d u l t - t y p e E E C s w e r e t e n t a t i v e l y i d e n t i f i e d in the h u m a n fetal SB. T h e s e i n c l u d e the L, D 1 a n d D cells. U n l i k e m o s t fetal E E C s , the L cells w e r e first n o t e d d i s t a l l y at 10 to 11 w e e k s o f g e s t a t i o n b u t w e r e n o t f o u n d in the p r o x i m a l SB u n t i l t h e 13th w e e k ( T a b l e 1). L cells, as i l l u s t r a t e d in F i g u r e 7, r e s e m b l e m a t u r e a p p e a r i n g I cells (Fig. 5 B). B o t h c o n t a i n a p o p u l a t i o n o f r o u n d o r o v a l S G s o f
Fetal Intestinal Endocrine Cells
43
Fig. 6. A Putative "transitional" G cell from proximal small intestine of 11-12 week fetus. "Precursor" type granules (P) average 900 nm in diameter. Smaller secretory granules which vary in electron density (arrows) are also present. • 23,900. B Mature-appearing G cell from proximal small intestine o f 10-11 week fetus. Secretory granules average 400 nm in diameter and their contents vary in electron density. Note similarity o f some granules (arrows) to those in Figure 6A. x 21,500
44
P.C. Moxey and J.S. Trier
Fig- 7. Mature-appearing L cell from proximal SB of 17 18 week fetus. Electron opaque granules average 390 nm in diameter, x 12,800 Fig.& PutativeD-cell fromproximalSBof9 10week fetus. Pale to moderately electron dense granules, averaging 280nm in diameter, are surrounded by a closely apposed membrane, x 15,700
relatively u n i f o r m electron density. However, the average d i a m e t e r o f the L cell granules is larger a n d ranges from 320 to 390 n m (Table 2). D cells a p p e a r e d in the p r o x i m a l SB between 10 a n d 12 weeks (Table 1, Fig. 8). A l t h o u g h some o f the D cells resemble G cells, the electron density o f D cell S G s is less h e t e r o g e n e o u s a n d the range o f the m e a n d i a m e t e r o f the D cell S G s varies less
Fetal Intestinal Endocrine Cells
45
Fig. 9. Putative D 1 cell from distal SB of 15 week fetus. Cytoplasm contains a b u n d a n t granules averaging 170 n m in diameter, whose electron density varies. Cytoplasmic microfilaments (arrows) are interspersed a m o n g granules, x 19,000 Fig. 10. Cluster o f endocrine cells frequently found in crypts o f h u m a n fetal SB. In this particular group, located at crypt base of 13 week fetus, there are an EC cell, two I cells and an S cell. x 10,200
Table 3. S u m m a r y of features of h u m a n adult intestinal endocrine cells Cell type
Location
Secretory granule characteristics
Postulated products
Gastrin or G cell
Duodenum
Spherical Highly variable densities Halo m a y be present Average diameter range: 250-400 n m (Frexinos et al., 1973; Grube, 1976)
Gastrin
Enterochromaffin or EC cell
Entire length of small intestine
Highly pleomorphic Uniformly electron dense No halo Average diameter range: 300 6 0 0 n m (Kobayashi et al., 1970; Cavallero et al., 1972)
Motilin Substance P Serotonin
Secretin or S cell
Entire length of small intestine: Primarily d u o d e n u m and jejunum
Spherical, oval or irregular Moderate to highly electron dense Narrow halo may be present Average diameter range: 100 2 0 0 n m (Frexinos et al., 1973; Solcia et al., 1974)
Secretin
Intermediate or I cell
Primarily upper intestine Rarely in ileum
Spherical Uniformly electron dense Closely applied membrane; rarely a narrow halo Average diameter range: 240-280 n m (Frexinos et al., 1973; Polak et al., 1975)
Cholecystokinin
Large or L cell
Primarily lower Spherical j e j u n u m and Uniformly electron dense ileum N o halo Average diameter range: 300-400 n m (Labo et al., 1971; Osaka et al., 1973)
Enteroglucagon
Intestinal D cell
Primarily d u o d e n u m and jejunum
Spherical Low to moderately electron dense Closely applied membrane Average diameter range: 300-450 n m (Kobayashi et al., 1970; Cavallero et al., 1972)
Somatostatin
DI
Primarily d u o d e n u m and upper j e j u n u m
Spherical, regular Variable electron density Closely applied membrane; rarely narrow halo A b u n d a n t microfilaments Average diameter range: 140 180 n m (Cavallero et al., 1972; Solcia et al., 1974)
Gastric Inhibitory Polypeptide (GIP) or H u m a n Pancreatic Polypeptide (HPP)
K Cell
Entire length of small intestine: Primarily d u o d e n u m and upper jejunum
Spherical, ovoid Moderately electron dense, often with a double structure, i.e. darker core Halo m a y be present Average diameter range: 200-400 n m (Solcia et al., 1974; Solcia et al., 1975)
Gastric Inhibitory Polypeptide (GIP)
H cell
Primarily lower Spherical, may be angular j e j u n u m and Moderate electron density (Solcia et aI., 1974; ileum Solcia et al., 1975)
Vasoactive Intestinal Polypeptide (VIP)
Fetal Intestinal Endocrine Cells
47
(Table 2; compare Figs. 6 B and 8). The D cell SGs are uniformly pale or moderately electron dense and are surrounded by a closely apposed membrane. D 1 cells are identified in the proximal small intestine at 9-10 weeks of gestation and by the 12th week are common in both the proximal and distal SB (Table 1). These putative D 1 cells contain abundant cytoplasmic microfilaments and the SGs have a mean diameter ranging from 140 to 190 nm (Fig. 9). The SGs resemble the S cell SGs in size (Table 2). The cells can be distinguished, however, by the electron density of the SG contents; D 1 SGs vary from pale to moderately electron dense while those of S cells are uniformly electron opaque (compare Figs. 4 and 9). L and D 1 cells are present on villi as well as within crypts. Accumulation of EECs composed of several cell types were frequently found in clusters at the base of the crypts. For example, the section shown in Figure 10 includes an EC, a putative S, and two I cells. In addition to the 13 types of fetal endocrine cells described above, there were additional cells with structural features suggestive of endocrine cells. These contained putative secretory granules but the morphology of the SGs was extremely variable and different from that of defined adult EECs thus precluding their classification. N o cells were identified which resembled the adult-type K or H cells (Table 3), and no EECs could be recognized within the lamina propria at any of the ages studied.
Discussion
In this study, we describe morphologically distinctive "primitive", "precursor", 4 different "transitional", and 7 different adult-type EECs in the human fetal small intestine by the 12th week of gestation. Only the 7 adult-type EECs have been described in the normal adult intestinal mucosa. The other 6 fetal EECs are distinctive and do not have adult correlates. It seems likely that two of these, the "primitive" and "precursor" cells, are immature endocrine cells that appear transiently during intestinal maturation. This is supported by their early appearance in the stratified epithelium at 9-10 weeks, their ultrastructural features which suggest immaturity, their apparent decrease in number with increasing age, and their absence in normal adult SB. There is additional evidence that the "primitive" cell may represent an immature or undifferentiated endocrine cell. Cells morphologically resembling "primitive" cells have been illustrated in regenerating rabbit pancreas and carcinoma of the pancreas in hamsters (Boquist and Falkmer, 1969), and in an endocrine tumor of the human pancreas (Greider and Elliot, 1964). Morphologically similar cells have also been illustrated in the pancreas of lower vertebrates, specifically the teleosts (Nakamura and Motoyoshi, 1971 ; Falkmer and Patent, 1972). The "precursor" cells with their extremely large secretory granules are unique among EECs. Endocrine cells with comparable secretory granule morphology have not been reported in other gastrointestinal tissues. A potential relationship of the "precursor" cells to other fetal endocrine cells is suggested by the occurrence of certain EECs with 2 distinct populations of granules. These include some of the
48
P.C. Moxeyand J.S. Trier
"precursor" cells themselves which contain granules resembling those found in "primitive" cells in addition to their extremely large granules. Another group of cells with two distinct populations of SGs is the"transitional" cells. In"transitional" cells one population of granules is morphologically indistinguishable from the large granules noted in "precursor" cells. The second population varies from cell to cell and may resemble the specific SGs found in adult EC, S, I, or G cells. We therefore suggest that the "transitional" cells may be developmental precursors of specific adult EECs. The only other situation in which cells resembling "transitional" cells have been described is in the gastric mucosa from a patient with pernicious anemia (Forssman, 1976), a condition which has been associated with endocrine cell hyperplasia (Rubin, 1973). In summary, we suggest that the "primitive", "precursor", and "transitional" cells may represent sequential developmental precursors of adult type EECs in fetal small intestine. The apparent differentiation of fetal endocrine cells within the intestinal mucosa and the absence of identifiable endocrine cells within the lamina propria at any age studied do not provide support for the APUD concept of a neural crest origin of intestinal endocrine cells (Pearse, 1973). We are unable, however, to exclude the possibility that precursors of endocrine cells derived from the neural crest migrated into the mucosa prior to the 9th week of gestation. The appearance of diverse EECs occurred largely in concert with villus formation at 9-10 weeks (Table 1). During the same period there appeared to be an increase in the total number of EECs per area of mucosa. This impression is supported by quantitative studies (Singh, 1963) in which it was noted that the number of EECs per unit length of mucosa increased progressively between the 8th to 16th weeks of gestation in both proximal and distal SB and then gradually decreased at later gestational ages. In the rat, the frequency of EECs within the intestinal epithelium is greatest at birth, and decreases postnatally (Josephson and Altmann, 1973). Using specific antisera and immunofluorescent techniques it has been shown that G cells (Larsson et al., 1975; Dubois et al., 1976 b), D cells (Dubois et al., 1976 a), I cells (Dubois et al., 1976 b), and L cells (Polak et al., 1971) are present in fetal small intestine by 12 weeks. These findings indicate that at least some fetal EECs synthesize and store polypeptides immunologically similar to adult hormones. The early appearance and apparent differentiation of fetal EECs within the fetal SB by the 12th week suggests that these cells may have functional roles during development. For example, perhaps gastrin produced by fetal G-cells has atrophic role in human intestinal development similar to gastrin's trophic effect on adult gastrointestinal tissues of a variety of species (Johnson, 1976). Similarly, CCK of intestinal I cells may have atrophic role in the human fetus as it does in the adult rat pancreas (Mainz et al., 1973). Another potential function of the EECs in utero might be regulation of gastrointestinal motility. Active motility in utero is suggested by studies in which circulation of amniotic fluid through the gastrointestinal tract has been shown late in pregnancy (Pritchard, 1965). In the adult gastrointestinal tract gastrin, CCK, secretin, serotonin, and motilin have all been shown to affect motility (Brown, 1974; Walsh and Grossman, 1975; Rayford et al., 1976), and the cell types which produce these hormones have been tentatively identified in human fetal SB.
Fetal Intestinal Endocrine Cells
49
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Pritchard, J.A.: Deglutition by normal and anencephalic fetuses. Obstet. Gynecol. 25, 289-297 (1965) Rayford, P.L., Miller, T.A., Thompson, J.C.: Secretin, cholecystokinin and newer GI hormones. New Engl. J. Med. 294, 1093-1100 (1976) Reynolds, E.S. : The use of lead nitrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Richardson, K.C., Jarrett, L.K., Fincke, E.H.: Embedding epoxy resins for ultrathin sectioning for electron microscopy. Stain Technol. 35, 313-323 (1960) Rubin, W.: A fine structural characterization of the proliferated endocrine cells in atrophic gastric mucosa. Amer. J. Path. 70, 109 117 (1973) Singh, I.: The prenatal development of enterochromaffin cells in the human gastro-intestinal tract. J. Anat. (Lond.) 9"7, 377-387 (1963) Solcia, E., Capella, C., Buffa, R., Bettini, R., Vassallo, G. : Istochimica ed ultrastruttura delle cellule endocrine dell'apparato digerente. Riv. Istoch. Norm. Pat. 18, 63-94 (1974) Solcia, E., Polak, J. M., Buffa, R., Capella, C., Pearse, A.G.E.: Endocrine cells of the intestinal mucosa. In: Gastrointestinal hormones (J.C. Thompson, ed.), pp. 155-168. Austin: University of Texas Press 1975 Todd, M.E., Dunn, J.S., Bagshaw, V.: The fine structure of human fetal tissues. J. Anat. (Lond.) 106, 188189 (1970) V/trkonyi, T., Gergely, G., Vat6, V. : The ultrastructure of the small intestinal mucosa in the developing human fetus. Scand. J. Gastroent. 6, 495 500 (1974) Walsh, J., Grossman, M.I.: Gastrin, Part I. New Engl. J. Med. 292, 1324-1332 (1976) Watson, M.L.: Staining of tissue sections for electron microscopy with heavy metals. J. biophys. biochem. Cytol. 4, 475-478 (1958)
Accepted April 8, 1977
