指導老師：褚俊傑 組員： 4A1H0002 林侑靜 4A1H0006 謝宜玲 4A1H0037 林芷彤

1. 生技英文論文報告Tissue and CellSerous goblet cells: The protein secreting cells in the oral cavity of a catfish, Rita rita (Hamilton, 1822) (Bagridae, Siluriformes) 指導老師：褚俊傑 組員：4A1H0002 林侑靜 4A1H0006 謝宜玲 4A1H0037 林芷彤

2. 出處：Tissue and Cell 46 (2014) 9– 14 標題： Serous goblet cells: The protein secretingcells in the oral cavity of a catfish, Rita rita (Hamilton, 1822) (Bagridae, Siluriformes) 作者： Madhu Yashpal, Ajay Kumar Mittal

3. Journal: Tissue And Cell Impact Factor:1.095

4. Introduction • Mucus, in the oral cavity of fish, is predominantly secreted by the mucous goblet cells. • Mucous goblet cells involved in diverse roles : 1.trapping of food particles 2.formation of food bolus 3.solubilization of food materials 4.Facilitation of mastication 5.initial digestion of starches and lipids 6.lubrication and protection of epithelial surfaces 7.cleansing the oral cavity 8.antimicrobial activity

5. Introduction • In teleost , the secretions elaborated by the cells, which are mainly characterized by the presence of distinct eosinophilic granules occupying large parts of the cytoplasm also contribute to the surface mucus. • These ‘granular’ cells, involved in proteinaceous rather than mucoid secretions, were first described and designated as “serous goblet cells” by Whitear .

6. Materials and methods 1. Fish samples 2. Histology and histochemistry 3. Examination and assessment of stained sections 4. Measurements

7. Fish samples • Samples Live specimens of R. rita (mean ± S.D., standard length, Ls,105 ± 6 mm; n = 10), irrespective of their sex were collected from river Ganges, Varanasi, Uttar Pradesh, India. • Maintained in a laboratory aquarium with a layer of sand at the bottom for 24–48 h at 25 ± 2◦C. • The fish were regularly fed with minced goat liver and sacrificed by exposure to ice cold water for an extended period of time (∼5 min) following Mittal and Whitear .

8. After the sacrifice, pieces of the roof and floor of the oral cavity were excised, rinsed in physiological saline and fixed in aqueous 1. Bouin’s fluid (18–22h) 2. Zenker’s fluid (24 h) 3. Helly’s fluid (24 h) for histological studies and in 10%Neutral Formalin (24 h) for protein histochemistry.

9. Histology and histochemistry • Intact sheets of the epithelium covering the roof and the floor of the oral cavity were separated by dissecting the fixed pieces of tissue under a Nikon stereoscopic SMZ-1B microscope (Nikon, Nippon Kogaku K.K., Tokyo, Japan) to remove the bones. • The tissues were dehydrated through an ascending series of ethanol concentrations, cleared in cedar wood oil and embedded in paraffin wax (58–60◦C; Merck Specialties Private Limited, Mumbai, India). • Sections were cut at a thickness of 6μm using a Leica semi-motorized rotary microtome.

10. The sections were mounted on ethanol-cleaned glass slides with-out any adhesive and were dried in an oven at 40◦C. • Sections were deparaffinized in xylene and were hydrated through a descending ethanol series. • For histological studies, the paraffin sections of aqueous Bouin’sfluid, Zenker’s fluid and Helly’s fluid fixed tissues were stained with 1. Ehrlich’s hematoxylin and eosin (H/E) 2. Papinicolaou’s stain 3. Verhoeff’selastin stain 4. Mallory’s triple stain 5. Altmann’s Acid Fuchsin 6. Regaud’s iron hematoxylin 7. Heidenhain’s iron hematoxylin

11. Sections of the 10% Neutral Formalin fixed tissues were subjected to a series of histochemical methods (Table 1)

12. Examination and assessment of stained sections • Tissues were examined using a Leitz ‘Laborlux S’ microscope equipped with a digital camera system for automatic microphotography and to record the results on a Vintron Pentium IV computer. • The histochemical methods used in this study were performed on tissue sections from several samples of the fish species and the results obtained from each method were consistently reproducible. • Evaluation of staining intensities was based on subjective estimates by all authors after examination of several tissue sections.

13. Measurements • Samples of 10 randomly selected sites in cross sections of each tissue were analyzed for the estimation. • Data thus obtained from ten specimens (n = 10 fish) irrespective of their sex were pooled and the results were expressed as mean ± S.D. • Statistical analyses of data were conducted using SPSS (ver.16.0) software. • All the data was subjected to one way analysis of variance (ANOVA) supplemented with Student–Neuman–Keuls (SNK) post hoc test.

14. Results • In R. rita, in addition to the mucous goblet cells and the club cells, the SGCs constitute the secretory components of the oral epithelium (Fig. 1a), which show striking differences in their density and dimensions (p < 0.05; Tables 2 and 3). • However, no significant differences in the area of the secretory cells were observed in the floor of the oral cavity.

15. The dorsal side of the velum showed statistical significance when the perimeter, diameter and major axis were analyzed, while other regions such as ventral side of the velum and tongue showed no significant difference compared to the lower jaw of the floor of the oral cavity (Table 3).

16. 表２

17. 表３

18. (a) General structure of the oral epithelium constituted of the mucous goblet cells (arrows), the SGCs (barred arrows), the club cells (asterisks), the lymphocytes and the eosinophilic granular cells (arrowheads). (b) Same as (a) in higher magnification showing SGCs (barred arrows) and mucous goblet cells (arrows) in the epithelium.

19. (c) Mallory’s triple stain method, showing SGCs (stained deep red) and mucous goblet cells (stained light blue) in the outer layer of the epithelium covering upper jaw. (d) Papanicolaou’s stain method, displaying SGCs (stained deep orange red) and blue the mucous goblet cells (stained blue) in the outer layer of the oral epithelium. (e) Altman’s acid fucshin stain method, showing SGCs (stained red) and the mucous goblet cells (stained pink) in the outer layer of the oral epithelium.

20. (f) Verhoeff’s elastin staining method, showing SGCs (stained black) and the mucous goblet cells (arrows) in the epithelium. (g) Regaud’s iron hematoxylin staining method, showing SGCs (stained black) and the mucous goblet cells (stained brown). (h) Heidenhain’s iron hematoxylin staining method, showing SGCs (stained black) and the mucous goblet cells (arrows).

21. (i) Mercury-bromophenol blue method, demonstrating the protein nature of the SGCs stained deep blue with purple tinge. Reaction is feeble in the mucous goblet cells. (j) Acid solochrome Cyanine-R method, displaying positive reactions for basic proteins in the SGCs. (k) Same as (j) in higher magnification showing SGCs stained deep orange-red (acid solochrome cyanine-R).

22. (l) DMAB-nitrite method, indicating the presence of tryptophan and 3-indole derivatives in deep blue stained SGCs. (m) Performic acid Schiff’s Reaction method, showing the presence of cystine bound disulphide (—SS) groupsin pink stained SGCs.

23. Histochemistry • Strong blue with purple tinge reaction with the mercury-bromophenol blue (method 1; Fig. 1i) for general proteins and vivid orange red reaction with the acid solochrome cyanine-R for basic proteins (method 2; Fig. 1j and k)→ the granular content of SGCs are highlyproteinaceous in nature. • The granules stain intense blue with theDMAB-nitrite (method 3), which is abolished with prior iodination (method 4) → the presence of tryptophan and 3-indole derivativesin high concentrations (Fig. 1l).

24. Strong orange reaction with Mil-lon(method 5) that is abolished by prior iodination (method 6) → the presence of high amounts of tyrosine in these cells. • Moderate pink reaction with ninhydrin-schiff (method 7) that is abolished by prior deamination (method 8) →the presence of lysine (— NH2 ) groups in moderate concentrations. • A strong purplish red reaction with DDD (method 9), which is not obliterated with prior N-ethyl maleimideblock or iodine oxidation (methods 10 and 11) → the reaction is non-specific and is not due to the presence of cysteine bound sulphydryl(—SH) groups.

25. Positive reactions with performic acid-alcian blue (method 12) and performic acid-Schiff (method 14;Fig. 1m), which are abolished with prior thioglycollatereduction (method 13 and 15) →these cells contain cystine bound disulphide (—SS) groups. • The SGCs remain unstained with Sakaguchi reaction (method 16) → the absence of arginine.

26. Discussion • The SGCs, with strong eosinophilic granular contents, are primarily involved in the elaboration of proteinaceous secretions, form one of important constituents of the secretory elements in the epithelium at different regions of the oral cavity of R. rita. • Further, Petrie-Hanson and Peterman (2005) pointed out that these cells could be found in high numbers in fish gill and gut tissues.

27. The high proteinaceous content secreted by SGCs at the surface of the oral epithelium of R. rita may have defensive roles and acts synergistically with mucus glycoproteins to inhibit the adherence and proliferation of microorganisms. • In R. rita, the tryptophan-rich secretions of SGCs may assist in different physiological stages of digestion.

28. The present study also revealed that the proteinaceous secretions of SGCs are rich in various amino acids like tyrosine, lysine, cystine and may indicate the presence of enzymatic pre-cursors such as pepsinogen or digestive enzymes. • In addition to their role as substrates for protein synthesis, these amino acids may also play a vital role in neurotransmission, osmoregulation, stress response and antioxidative defense .

29. Since R. rita preferably dwells in muddy and dirty water it is conceivable that the proteinaceous secretion of SGCs provides a protective barrier to the epithelium against various pathogens and infections, abrasions during manipulation of food items in the oral cavity. • It is worth mentioning that the amphibians are evolved from freshwater fish and during the course of evolution, the SGCs in fishes may be considered as a significant representative of an ancient specialized line in the alimentary canal.

30. Conclusion • We conclude that the SGCs, in the oral cavity of R. rita, may possibly be involved in the secretion of a broad spectrum of proteins, similar to those found in mammalian saliva. • The secretions of SGCs in harmony with those of mucous goblet cells orchestrate various functions of mucus such as handling, maneuvering and driving of food items toward the esophagus, maintaining taste sensitivityand protection of the oral epithelium.

31. In addition, the SGCs may also be considered as the primary defensive cell of the oral epithelium of R. rita. • The results of the present study could function as the initial step to elucidate the precise roles of secretions of SGCs in the oral cavity of fish. • Further, such exploration of proteome of SGCs not only add knowledge to the oral physiology in fish.

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