Gut 1993; 34:1057-1059 1057 Immunocytochemical localisation of parietal cells and G cells in the developing human stomach Gut: first published as 10.1136/gut.34.8.1057 on 1 August 1993. Downloaded from

E J Kelly, M Lagopoulos, J N Primrose

Abstract obtained from the University Departments of Previous studies on the distribution of parietal Pathology and Obstetrics and Gynaecology, cells and G cells in normal adult stomachs have St James's University Hospital, Leeds. The shown that in about 20% of specimens parietal specimens resulted from therapeutic abortions, cells extended to the pylorus. This study aimed miscarriages, and cot deaths. The ages of the to examine the distribution ofparietal cells and fetuses and babies are listed in Table I. The G cells in the body and antrum ofthe develop- maturity of fetuses is given in weeks ofgestation ing human stomach in relation to anatomical since the last menstrual period and was con- landmarks, using histological and immuno- firmed by antenatal ultrasound assessment and cytochemical methods. In all 15 fetal stomachs fetal foot length measurements. Infants who examined, parietal cells extended to the were studied after unexplained sudden infant pylorus and expressed intrinsic factor and death were all born at term and their ages are hydrogen-potassium-ATPase activity from given as weeks ofpostnatal life. week 13 of gestation. By contrast, in only one The specimens were immersed in buffered of the five infant stomachs did parietal cells formalin for a minimum of four days. The distal extend to the pylorus: this is identical to the pylorus and the anatomical antrum-corpus distribution in the adult. G cells developed in boundary (indicated by the point where the the antrum from 18 weeks' gestation and their nerve of Latarjet crossed the lesser curvature) distribution did not differ between the fetal and were marked with indian ink on both sides ofthe infant stomachs. These findings indicate that specimens. Two adjacent strips containing parietal cells disappear from the antrum of the mucosa from the corpus, antrum, and pylorus stomach in the third trimester of pregnancy, were then cut from the anterior and posterior but this process fails to occur in approximately aspects, resulting in four strips from each speci- 20% ofthe population. men. Sections, 8 [t thick, were stained using

(Gut 1993; 34: 1057-1059) haematoxylin and eosin and the peroxidase anti- http://gut.bmj.com/ peroxidase technique of Sternberger.4 Intrinsic factor and hydrogen-potassium The specialised cells of the stomach, including ATPase activity were mapped using murine Departments ofAnatomy the parietal cell (responsible for gastric acid and monoclonal antibodies (Dr Smolka, University and Surgery, University intrinsic factor secretion) and the G cell (gastrin of South Carolina, Charleston, South Carolina, of Leeds and St James's University Hospital, producing), have been the subject of consider- USA) and gastrin activity was mapped using a Leeds able study in both normal subjects and in those rabbit antiserum to human gastrin (Dako, High on September 28, 2021 by guest. Protected copyright. E J Kelly with peptic ulcer disease. In adults there seem Wycombe). The second antibodies were raised M Lagopoulos to be two anatomically distinct types ofstomach. in goat and swine respectively (Sigma, Poole) J N Primrose In the first type, parietal cells are found within and complemented with rabbit and mouse Correspondence to: Mr E J Kelly, Neonatal Unit, the corpus and G cells within the antrum, with peroxidase antiperoxidase complexes (Sigma). St James's University a transitional zone on the antrum-corpus The sections were stained with diamino Hospital, Beckett Street, 3,3 Leeds LS9 7TF. boundary containing both cell types (80% of the benzidine and counterstained with methyl Accepted for publication population). In the second, parietal cells extend green. 24 November 1992 to the pylorus, but G cells are also present in the A negative control, in which the primary antrum (20% of the population). This type of antibody was omitted, was performed for all stomach has been referred to as having an 'acid sections. The sections stained for intrinsic factor antrum'.I Both types of stomach are seen in and gastrin also had negative controls in which patients with duodenal ulcer disease23 and this their antisera were incubated with their respec- is relevant to the surgical treatment of the tive antigens (Sigma). The ages ofthefetuses and condition since patients with acid antra may All sections were viewed under a Leitz Dialux babiesfrom whom the be inadequately treated by highly selective 20 EB microscope using x 50 and x 100 objec- specimens were taken vagotomy. tives. The sections stained with haematoxylin Age No of Surprisingly, the origin of these two distinct and eosin and those stained for gastrin had the (wk) specimens types of stomach is unknown. In this study we point at which the parietal cells stopped distally Gestational age: have mapped the location of parietal cells and G and the point at which the G cells stopped 13 1 15 1 cells in the developing human stomach, using proximally marked on the surface of the slides. 18 2 specific immunocytochemistry, in order to As the stomachs varied greatly in size, data 19 4 are two was done the 20 3 determine whether there distinct popula- were normalised. This by using 21 2 tions in the fetus or whether a change in distribu- pylorus and the anatomical antrum-corpus 24 1 occurs find- 26 1 tion of parietal cells and G cells during boundary as reference points. Histological Postnatal age: fetal and postnatal life. ings were related to these anatomical landmarks. 2 1 The of both cell within the antrum 4 1 density types 10 1 of each specimen was determined by calculating 13 1 the number of each cell 21 1 Methods type per high power Fifteen fetal and five infant stomachs were field. Ten measurements were made indepen- 1058 Kelly, Lagopoulos, Primrose

50 - G CELLS + Parietal cells G cells could not be detected in the fetuses of 13 + O G cells and 15 weeks' gestation. By contrast, specimens Gut: first published as 10.1136/gut.34.8.1057 on 1 August 1993. Downloaded from 40 - from 18 gestational weeks showed gastrin I o + activity (Fig 3), although at this stage cells were 30- + sparse. The number of G cells then increased 0 Z appreciably until 24 weeks of gestation (Fig 1). + + At all maturities G cells were limited, to the *t + 0 0 20- + @ + D anatomical antrum, and their distribution within + 0) o 0 the antrum did not differ between the fetal and 0 + 10 - 0 infant specimens (p=0 42). + 0 0 + Regression analysis of the density of G cells 8 0 + and parietal cells showed that there was a signifi- n) El LJl3 13 15 18 19 20 21 24 26 2 4 10 13 21 cant negative correlation between G cells and _- ~~~parietal cells, rho= -0-52(p=0-04). Gestational age (wk) Postnatal age (wk) None of the negative controls in which the Figure 1: The density (cells per high powerfield (HPF)) ofparietal cells and G cells in both primary antibody was omitted showed any stain- fetal and infant stomachs. ing after the application of 3,3 diamino benzi- dine. This was also true when gastrin and dently by two observers on each spiecimen and intrinsic factor were incubated together with the mean was calculated. their respective antibodies. Statistical analysis of the data wass performed using the Mann-Whitney U tesit for non- parametric data and Spearman's rank order Discussion correlation. This study shows that parietal cells, with their specialised functions of acid and intrinsic factor secretion, are present from 13 weeks of gesta- Results tion, and at this stage these cells occupy the whole stomach. Our results, therefore, agree PARIETAL CELLS with those of previous investigators who have Parietal cells were detected in all s,tomachs on noted that parietal cells are present in fetal slides stained with haematoxylin and eosin, from stomachs from 11 gestational weeks5 as differen- 13 weeks of gestation to 21 postnLatal weeks. tiated epithelial cells which represent adult

There was a difference in the disitribution of parietal cells in both structure and histochemical http://gut.bmj.com/ parietal cells between the fetal and infant speci- staining.6 By contrast, the G cells are not detect- mens. In all of the fetal specimens p)arietal cells able until 18 weeks of gestation and develop were noted to extend from the body, through the entirely within the anatomical antrum. Interest- antrum, to the pyloric region of the,,stomach. In ingly, this is the period in which parietal cells the five infant stomachs studied, howrever, exten- disappear from the same anatomical site in most sion of parietal cells into the antrunn was much' subjects. Previous studies in the adult have

more limited. In one case parietalI cells were shown that where the parietal cells retain their on September 28, 2021 by guest. Protected copyright. found to extend to the pylorus, in two they 'embryological' distribution, the G cells are extended through 20 and 22% of the antrum relatively sparse.7 It is interesting to speculate respectively, and in the remaining two infant that in the fetus the G cells may, by a means yet specimens no parietal cells were fc)und in the to be identified, lead to the disappearance of antrum. There was great diversity in the density parietal cells in their immediate vicinity. This is of parietal cells between specimens of different supported by our observation that the G cell and maturities (Fig 1). Intrinsic factor and hydrogen- parietal cell densities were negatively correlated. potassium ATPase activity were found in all Hydrogen-potassium ATPase, necessary for specimens, including those of 13 anLd 15 weeks' the secretion of hydrogen ions, is present from gestation. The location of this actiivity corres- the 13th gestational week in a functional form. ponded precisely with the location of parietal The timing of active hydrogen ion secretion, cells as determined by haematoxylin and eosin however, remains unknown. Gastric acid has staining (Fig 2). been shown during the first hour of life in both

Figure 2: Haematoxylin and eosin stained specimenfrom a 15 Figure 3: Immunocytochemical localisation ofgastrin in a weekfetus (original magnification xS00). A typicalparietal fetus ofgestational age 18 weeks (original magnification cell is indicated (arrow). x500). The G cells are indicated (arrow). Immunocytochemical localisation ofparietal cells andG cells in thedeveloping human stomach 1059

term and preterm infants of more than 32 weeks the antrum. Circulating gastrin is trophic to gestation.8 No data are available on acid secretion parietal cells,'4 but its paracrine effects are by more immature infants. unknown. It may be that the high local concen- Gut: first published as 10.1136/gut.34.8.1057 on 1 August 1993. Downloaded from In 1971, using a radioimmunoassay technique trations ofgastrin, or another factor produced by on homogenised tissue, Schwarz and Weber9 the G cell, is responsible for the loss of parietal suggested that intrinsic factor may be present cells. Alternatively, the development of G cells in fetal stomachs from at least 13 weeks of and the loss of parietal cells may be the result of gestation. We have shown, using immunohisto- other, as yet unidentified, factors. Why the chemistry, that intrinsic factor is present in process fails in 20% of the population is unclear parietal cells from 13 weeks of gestation. This but it has been noted that in patients with acid does not merely indicate that the framework for antra, G cells are qualitatively sparse.' Further intrinsic factor production is in place but that it is understanding of the processes and growth being produced actively by the parietal cell from factors involved in gastric epithelial develop- the end ofthe first trimester. ment in the fetus may allow the exact mecha- G cells have been found in the duodenum from nisms to be elucidated. 10 gestational weeks and in much lower numbers The authors wish to thank Dr Adam Smolka, Department of in the antrum from 19 gestational weeks.'" This Anatomy, University of South Carolina for the generous gift of agrees with our localisation of gastrin producing antibodies to intrinsic factor and H+/K+ ATPase. cells from 18 gestational weeks. The role of 1 Derbyshire SA, Lagopoulos M, Lee T, Primrose JN. peptide hormones produced by endocrine cells Distribution of the vagus nerve to the human pyloric antrum and possible surgical implications. Clin Anat 1990; in fetal development is incompletely understood 3:25-31. at present in both experimental animals and 2 Simms JM, Bird NC, Johnson AG. An unusual acid antrum. BrJ Surg 1985; 72: 12. humans. It seems probable, however, that since 3 Rogers MJ, Lagopoulos M, Primrose JN. Is the antrum factors such as gastrin are being produced by the clinically important after highly selective vagotomy for duodenal ulcer disease? BrJ7 Surg 1988; 75: 1249. developing stomach from about 18 gestational 4 Sternberger LA. Immunocytochemistry. 2nd ed. New York: weeks," they may have a role in the development J Wiley & Sons, 1979. 5 Salenius P. On the ontogenesis of the human gastric epithelial of the gastrointestinal tract. This is consistent cells. Acta Anat (Basel) 1962; 46: 1-76. with observations of Schulkes et al on fetal sheep 6 Norura Y. On the submicroscopic morphogenesis of parietal cells in the gastric gland of the human foetus. Z Anat in which gastrin has been detected in the gastric Entwicklungsgeschichte (Berlin) 1966; 125: 316-22. juices at a time when the intragastric acidity is 7 Naik KS, Lagopoulos M, Primrose JN. Distribution of antral G cells in relation to the parietal cells of the stomach and low. The authors argue that 'gastrin in fetal juice anatomical boundaries. Clin Anat 1990; 3: 17-24. could have a unique role in the in utero develop- 8 Euler AR, Bryne WJ, Meis PJ, Leake RD, Ament ME. Basal and pentagastrin stimulated acid secretion in newborn ment of the gastrointestinal tract'.'2 Indeed, if human infants. Pediatric Res 1979; 13: 36-7.

the developing human fetus provides some of its 9 Schwarz M, Weber J. Gastric intrinsic factor in the human http://gut.bmj.com/ foetus. ScandJ Gastroenterol 1971; 9 (suppl): 57-9. own nutrition,'3 digestive enzymes may need to 10 Larsson LI, Hakanson R, Sjoberg NO, Sundler F. be synthesised and secreted during interuterine Fluorescence histochemistry of the gastrin cell in fetal and adult man. Gastroenterology 1975; 68: 1152-9. life. 11 Hirsh-Marie H, Loisilier F, Touboul JP, Burtin P. From the small number of neonatal stomachs Immunochemical study and cellular localization of human pepsinogens during ontogenesis and in gastric cancer. Lab examined, it seems that the distribution of Invest 1976; 34: 623-32. parietal cells remains constant after birth, and so 12 Shulkes A, Chick P, Hardy KJ. Presence and stability of gastrin in the gastric juice offetal sheep. Clin Exp Phannacol it appears reasonable to assume that a definite Physiol 1984; 11: 45-52. on September 28, 2021 by guest. Protected copyright. change in distribution of parietal cells must 13 Friis-Hausen B. Body water metabolism in early infancy. Acta Paediatr Scand 1982; 296: 44-8. occur during the third trimester. This period 14 Johnston LR. The trophic action ofgastrointestinal hormones. coincides with the development of G cells within Gastroenterology 1976; 72: 278-87.