Histological and Histochemical Investigations on Japanese Lizard

Okajimas Folia Anat. Jpn., 69(1): 25-34, May, 1992 Histological and Histochemical Investigations on Japanese Lizard Esophagus By Masatake IMAI and Taizo SHIBATA Department of Anatomy, Kanazawa Medical University. Uchinada-machi, Kahoku-gun, Ishikawa, 920-02, Japan Tokukazu IZUMI Department of Animal Reproduction, Ishikawa Agricultural College. Suematsu, Nonoichi-machi, Ishikawa-gun, Ishikawa, 921, Japan -Received for Publication, January 14, 1992- Key Words: Histology, Histochemistry, Esophagus, Bottle-shaped glands, Japanese lizard Summary: The authors previously investigated the bottle-shaped glands distributed in the lamina propria mucosae of the Japanese lizard and gecko. We made two sets of sections of the Japanese lizard at that time. The numerical values of the physical dimentions of the two individuals were as given table 1, showing that No. 2 was slightly smaller. Moreover we found very unusual tissue in the lower portion of the esophagus of No. 2. Therefore we excluded this individual from the preceding investigations. However, we made various observations, and the results of these investigations are as follows. 1. The lumen of the upper portion of the esophagus has no fold. However, the middle and lower portions formed very complicated folds. Therefore, the lumen was remarkably narrow. 2. The epithelium of the esophageal mucous membrane consisted of simple columnar cells and throughout each part, reacted strongly to PAS and moderately to AB (ph 2.5 and 0.5). It presented a dark blue (R18 ~ B13 of Blue-Purple-Red) color in response to PAS-AB (ph 2.5) and contained no pepsinogen granules. The esophageal upper portion of small individuals only exhibited the PAS reaction in this investigation. 3. A number of bottle-shaped glands were distributed in the lamina propria mucosae of the lower portion of the esophagus of each material. The glandular cells in the basal portion were most differentiated and contained a great number of pepsinogen granules. 4. The above-mentioned glands were extremely simple and glands of this type could not be found in textbooks and theses. Accordingly, we previously described them with the tentative name of shimple branched tubular glands, but subsequently found this to be erroneous. We assume that these glands are esophageal gastric glands. 5, Compound tubular glands are formed in the lamina propria mucosae of the human esophagus, but do not exist in the Japanese macaque, crab-eating monkey, horse, cow, swine, dog, cat, rabbit, mouse and rat. Dellmann-Brown also described the absence of such glands in the esophagus of the horse, swine, cow, goat, sheep, dog and cat. 6. We subsequently found compound tubular glands distributed in the lamina propria mucosae of the fowl, goose and wild duck esophagus. They similarly secreted pepsinogen granules. 7. We assume that these glands of the Japanese lizard and gecko have a phylogenic relation with the glands in the bird. The pepsinogen-granule-secreting cells in the snake do not extend into the lamina propria mucosae. 8. Accordingly, we consider that these cells in the snake are phylogenetically different to the glands in other reptiles. 9. We can not interpret the actual characteristics of the glands distributed in the lamina propria mucosae of the human esophagus. These glands are not found in other mammals. 10. We have never seen a more unusual tissue than that in the lower esophageal portion of material No. 2. it was composed of numerous large and small projections, all with slender cervical regions and long swollen ellipsoid bodies. We saw a few large projections with nuclei, but could not find any in the numerous remaining projections and it seems that no small nucleated projections developed. 11. The unusual tissue reacted strongly to PAS and AB (ph 2.5), moderately to AB (ph 0.5) and presented a dark blue color with PAS-AB (ph 2.5) stain. The cervix and the greater part of the body contiguous to the cervix reacted strongly to PAS, but the basal portion was negative and contained the nucleus. 12. The above-mentioned colored portions did not stain with hematoxylin-eosin stain. The authors performed histological and histochemical investigations on the Japanese lizard esophagus. This paper prevides a detailed description of the results. Materials and Methods Two Japanese lizards were used in this study. Numerical values of their physical dimensions are 25

26 M. Imai, T. Shibata and T. Izumi shown in table 1 below. The animals were killed under chloroform anesthesia. Samples were taken in as fresh a state as possible from the upper, middle and lower portions of the esophagus, fixed in buffered formalin, and cut into sections of about 6 la in thickness. Hematoxylineosin, Van-Gieson, PAS, AB (ph 2.5 and 0.5), PAS-AB (ph 2.5) stains, as well as Bowie's staining method for pepsinogen granule analysis were used. Observations The esophageal lumen of the upper portion did not form the fold, and therefore was extensive (Fig. 15). However, the middle and lower portions formed many folds, thus forming complicated lumens (Figs. 5, 9, 17, 23). The esophageal epithelium reacted strongly to PAS (Figs. 1, 6, 10, 16, 18, 25), weakly or moderately to AB (ph 2.5 and 0.5) (Figs. 2, 3, 7, 8, 11, 12, 19, 20, 26, 27) and presented a dark blue color with PAS-AB (ph 2.5) (Figs. 4, 10, 18). These epithelia did not contain pepsinogen granules (Fig. 22). A large number of bottle-shaped glands were distributed in the lamina propria mucosae of the lower esophageal region (Figs. 10, 11, 12, 13, 24, 25, 26). The glandula cells of these glands in the large individual contained a large number of pepsinogen granules (Fig. 14). The basal glandular cells of the glands showed variocolored staining with PAS stain. The histochemical reactions in these glands were the same as that of the esophageal epithelium (Figs. 10 13, 25, 26). These glands had no parietal cells and no glands existed in the esophageal tela submucosa. The authors found unusual tissue in the lower esophageal portion of the samll individual (No. 2). Numerous large and very small projections protruded from the funiculi. These reacted strongly to PAS, AB (ph 2.5), PAS-AB (ph 2.5) and moderately Tablc 1. to AB (ph 0.5). These funiculi did not stain with hematoxylin-eosin stain, and were empty canals or canaliculi. The projections were attached by a fine cervical portion to the thin walls of the canal or canaliculi. The cervical portion and the greater part of the body of the large projection was strongly PAS positive, and the basal portion of the body was negative. The nucleus was present in this portion (Fig. 33). However, the authors did not confirm the existence of the nucleus in the samll projections. Discussion The esophageal lumen in the upper portion of the Japanese lizard does not form any folds. Consequently, there is a wide lumen. However, the middle and lower portions form many folds. Therefore, the lumen in these regions is complicated. The esophageal mucous membrane in covered with a simple columnar epithelium and reacted strongly to PAS, weakly or moderately to AB (ph 2.5 and 0.5) and presented a dark blue color with PAS-AB (ph 2.5). The esophageal epithelium contained no pepsinogen granules. The rock snake did not form any kind of gland in the lamina propria mucosae, but the esophageal epithelium secretes pepsinogen granules5). A number of bottle-shaped glands of the Japanese lizard and gecko are distributed in the lamina propria mucosae of the lower portion of the esophagus. These glands are very simple and can not be found in textbooks and theses. Accordingly, we tentatively called them by the above-mentioned name). The same portion was most differentiated and contained a great number of pepsinogen granules. These glands had no parietal cells. Although, we consider that these glands are esophageal gastric glands, we mistakenly described them earliar as simple branched tubular glands4). The compound tubular glands are distributed in the lamina propria mucosae of the human3). They are generally called esophageal fundic (cardiac) glands and consist of two groups. The upper groups are distributed in the region of the cricoid cartilage and the V tracheal cartilage. Ishizawa7 reported that these glands are lacking in 30% 40% of adults, but exist in more than 90% of the fetuses. In other words, they have degenerative characteristics. The lower groups are distributed in the terminal portion of the esophagus and are often spread into the cardiac region of the stomach. The authors deny the existence of the cardiac glands based on the investigations of the same glands in the human, Japanese macaque, crab-eating monkey, swine, horse, cow, cat, dog, rabbit, motic

Histological and Histochemical Investigations on Japanese Lizard Esophagus 27 and rat,') and demonstrated that the glands distributed in the lamina propria mucosae in the human are the only ones to exist in mammals. These glands contain pepsinogen granules and parietal cells. The authors considered that these glands were undifferentiated gastric glands and proposed the terms of upper and lower esophageal gastric glands instead of upper and lower esophageal cardiac glands. In other words, the upper and lower esophageal gastric glands are more desirable terms than upper and lower esophageal cardiac (fundic) glands. Dellmann-Brown') also described the absence of the glands in the lamina propria mucosae of the esophagus in the horse, swine, goat, cow, sheep, dog and cat, but demonstrated their existence in the tela submucosa of the esophagus in the horse, swine, cow, goat, sheep, dog and cat. These glands also exist in humans3), and are termed esophageal glands. Accordingly, the human alone has these two kinds of glands in mammals, which is a significant finding. A large number of compound tubular glands are distributed in the lamina propria mucosae of the esophagus of birds2'8). They exist in large quantities in the upper and lower regions of the esophagus and are rarely observed in the middle portion. They contain many pepsinogen granules and parietal cells do not exist. However, we consider that they are esophageal gastric glands. Besides tubular superficial gastric glands in the mucous membrane of the stomach, there is an aggregation of large compound tubular glands in the tela submucosa. The authors consider that the superficial glands are undifferentiated and glands distributed in the tela submucosa are differentiated gastric glands judging from their pepsinogen granules and other features. A large number of simple bottle-shaped glands are distributed in the lamina propria mucosae of the lower esophageal region (Fig. A) of the Japanese lizard and gecko6). These glands do not exist in the upper and middle regions. They contain a great number of pepsinogen granules, but parietal cells do not exist. However, we consider that these glands are esophageal gastric glands. No glands exist in the tela submucosa. The rock snake does not form any glands in the lamina propria mucosae and tela submucosa of the esophagus. However, the pseudostratified ciliated epithelium in the upper esophageal mucous membrane contains pepsinogen granules5). The authors assume, according to the abovementioned facts, that the Japanese lizard and gecko have a deep phylogenic relation with birds. The authors have never seen a more unusual tissue in the lower esophageal region of specimen No. 2 (Figs. 23, 24, 28). It is composed of numerous large and small projections (Fig. 29), from the Fig. A. Schema of the bottle-shaped gland (The original drawing). 1: Esophageal epithelium. 2: Esophageal lumen.

28 M. Imai, T. Shibata and T. Izumi funiculus-like tissue (Fig. 29) with slender cervices. These parts and the greater part of the ellipsoid bodies adjacent to the cervices reacted strongly to PAS and the terminal portions of the bodies were PAS negative. We found a few large projections which contained nuclei (Fig. 33). These projections and funiculus-like tissue reacted strongly to PAS (Fig. 29) and AB (ph 2.5) (Fig. 30), moderately to AB (ph 0.5) (Fig. 31) and presented a dark blue color with PAS-AB (ph 2.5) stain (Fig. 32). The funiculus-like tissue did not stain with hematoxylineosin stain (Figs. 35, 36). In other words, they are not funiculi, but are canals and canaliculi, filled with the above-mentioned reacted substances. Probably the projections adhere to thin wall of the canals and canaliculi. We are continuing with observations to clarify the actual characteristics of the unusual tissue with smaller animals than specimen No. 2. Histology. Lea & Febiger, Philadelphia, 1976 (Japanese edition, 165. Gakusosha. Translators: Okano, M. Kano, Y. Daigo, M. and Makita, T.) 2) Hodges, R. D.: The Histology of the Fowl, 46. Academic Press. London, New York, San Francisco, 1974. 3) Imai, M., Shibata, T., Moriguchi, K. and Yamamoto, M.: Do the cardiac glands exist? 3. Humans. Okjimas Folia Anat. Jpn. 65: 51-70, 1988. 4) Imai, M., Shihata. T., Moriguchi, K., Yamamoto, M. and Hayama, H.: Proventricular glands in the fowl. Okajimas Folia Anat. Jpn. 68: 155-160, 1991. 5) Imai, M., Shibata, T. and Moriguchi, K.: Pepsinogen granules in the esophageal epithelium of the rock snake. Okajimas Folia Anat. Jpn. 68: 231-234, 1991. 6) Imai, M., Shibata, T., Moriguchi, K. and Hayama, H.: Glands distributed in the lamina propria mucosae of the esophagus in the gecko and Japanese lizard. Okajimas Folia Anat. Jpn. 68: 289-294, 1991. 7) Ishizawa, M.: Manual of histology, vol. 2, 228-230, 246. Nihon Isho Press. Tokyo-Kyoto, 1951 (in Japanese). 8) Shibata, T., Imai, M., Moriguchi, K., Takada, Y. and Hayama, H.: Actual characteristics of the glands distributed in the lamina propria mucosae of the fowl esophagus. Okajimas Folia Anat. Jpn. 68: 41-46, 1991. References 1) Dellmann, H. D. -Brown, E. M.: Textbook of Veterinary

Histological and Histochemical Investigations on Japanese Lizard Esophagus 2C I Explanation of Figures (All pictures are of Japanese lizard esophagus. Figs. 1-14 are of material No. 1 and Figs. 15-36 are of material No. 2) I Fig. 1. Simple columnar epithelium in the esophageal upper portion reacted strongly to PAS stain. x 303 Fig. 2. and 3. The epithelium in the esophageal upper portion reacted weakly or moderately to AB (ph 2.5: Fig. 2. ph 0.5: Fig. 3) stain. x 303 Fig. 4. The epithelium in the esophageal upper portion. PAS-AB (ph 2.5) stain presented a dark blue color. x 303 Fig. 5. The mucous membrane in the esophageal middle portion form folds. Van Gicson stain. x 75 Fig. 6. The epithelium in the esophageal middle portion reacted strongly to PAS stain. x 303

30 M. Imai, T. Shibata and T. Izumi II II Figs. 7. and 8. The epithelium in the esophageal middle portion reacted weakly or moderately to AB (ph 2.5: Fig. 7, ph 0.5: Fig. 8) stain. x 303 Fig. 9. A large number of the bottle-shaped glands in the folds of the lower esophageal portion. Van Gieson stain. x 75 Fig. 10. The bottle-shaped glands (1, 2) in the lower esphageal portion. PAS stain. x 303 3: Duct of the glands. Fig. 11. and 12. The bottle-shaped glands (G) reacted moderately to AB (ph 2.5: Fig. 11, ph 0.5: Fig. 12) stain x 303

Histological and Histochcmical Investigations on Japanese Lizard Esophagus 31 III HI Fig. 13. The bottle-shaped glands (G) presented a dark blue color with PAS-AB (ph 2.5) stain. x 151 Fig. 14. A large number of pepsinogen granules in the bottle-shaped glands. Bowie's staining method for pepsinogen granule analysis. x 750 Fig. 15. The upper part of the esophagus. Folds do not form and esophageal lumen is extended. Van Gieson stain. x 75 Fig. 16. The esophageal epithelium of the upper portion is simple columnar and react strongly to PAS stain. x 150 Fig. 17. The esophageal lumen of the middle portion. A large number of folds of mucous membrane have formed. Van Gieson stain. x 75 Fig. 18. The epithelium in the middle portion of the esophagus reacted strongl) to PAS stain. x 150

32 M. Imai, T. Shibata and T. Izumi IV IV Fig. 19. The same epithelium as that in Fig. 18 reacted moderately to AB (ph 2.5) stain. x 150 Fig. 20. The same epithelium as that in Figs. 18 and 19 reacted weakly to AB (ph 0.5) stain. x 150 Fig. 21. The same epithelium as that in Figs. 18-20 presented a dark blue color with PAS-AB (ph 0.5) stain. x 150 Fig. 22. The epithelium in the esophageal mucous membrane did not contain pepsinogen granules. Bowie's staining method for pepsinogen granule analysis. x 750 Fig. 23. The bottle-shaped glands and unusual projects. This tissue appeared in the lower portion of the esophagus. PAS-AB (ph 2.5) stain. x 46 B: Bottle-shaped glands. E: Esophageal lumen. F: Funiculus. 1-4: Unusual projects. Fig. 24. 1 4: unusual tissue and numerous characteristic large and small projections can be observed. 5: Folds of the esophageal mucous membrane and the same bottle-shaped glands can be seen. This tissue appeared in the lower portion of the esophagus of No. 2. PAS stain. x 46

Histological and Histochemical Investigations on Japanese Lizard Esophagus 33 V V Fig. 25. Bottle-shaped glands (G) and epithelium in the lower esophageal portion reacted strongly to PAS stain. x 303 Fig. 26. Bottle-shaped glands (G) and epithelium in the lower esophageal portion reacted moderately to AB (ph. 2.5) stain. x 303 Fig. 27. Eipthelium in the lower esophageal portion reacted weakly to AB (ph 0.5) stain. x 303 Fig. 28. Unusual tissue. See the explanation of Figs. 23 and 24. PAS-AB (ph 2.5) stain. x 150 Fig. 29. Funiculus and projections in the unusual tissue reacted strongly to PAS stain. x 303 Fig. 30. The same structures in Fig. 29 reacted moderately to AB (ph 2.5) stain. x 15(1

34 M. Imai, T. Shibata and T. lzumi VI VI Fig. 31. The same structures as in Fig. 29 reacted weakly to AB (ph 0.5) stain. x 150 Fig. 32. The same structures as in Fig. 29 presented a dark blue color with PAS-AB (ph 2.5) stain. x 303 Fig. 33. Four projections have nuclei ( ). PAS stain. x 303 Fig. 34. Funiculus-like portion in Fig. 33 reacted strongly to PAS. PAS stain. x 303 Fig. 35. PAS, AB (ph 2.5 and 0.5) and PAS-AB (ph 2.5) positive funiculus-like portion did not stain with hematoxylin-eosin stain. Namely, the portion is the canal and material (A) in the canal is probably a hematoxylin-eosin stainable substance. Hematoxylineosin stain. x 303 Fig. 36. Positively reacted funiculi in the unusual tissue to PAS, AB (ph 2.5 and 0.5), PAS-AB (ph 2.5) did not stain with Hematoxylineosin stain, namely funiculi are nothing but canals filled with the above-mentioned colored substance. Hematoxylin-eosin stain. x 76 C: Canal : Unusual projects. S: Some substance in canals stained with Hematoxylin-eosin stain.