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Characterization of cholecystokinin and mucus-secreting goblet cell in longtooth grouper, Epinephelus bruneus

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Abstract
자바리(Epinephelus bruneus)는 식용어로 기호도가 높으며 현재 우리나라에서 양식대상어종으로 선정되어 양식기술 개발과 산업화에 적극적인 연구가 이루어지고 있다. 이 연구는 자바리의 최적화 양식기술 개발의 기초자료로 활용하기 위해 소화기관의 초기 발달특징, 빛 파장에 따른 성장 및 소화활성 특징, 소화호르몬 Cholecystokinin (CCK) 및 배상세포의 특징을 조사하였다.
자바리 소화기관의 초기발달 특징을 조사하기 위해 부화직후부터 부화 후 55일까지의 자어를 조직학적 및 분자생물학적 방법을 이용하여 실험을 수행하였다. 자바리는 부화직후 큰 난황을 가지고 있었으며, 부화 후 4일경 간과 각종 소화효소 분비에 관여하는 췌조직이 관찰되었다. 부화 후 5일경 장이 꼬이기 시작하여 소화관 길이가 증가하고 장 내강의 폭이 급격히 넓어지기 시작하며, 점액물질을 분비하여 소화활성을 높여주는 배상세포가 직장에서 관찰되었다. 부화 후 20일경 소화관 전장부와 중장부의 점막주름 상피층에서 배상세포가 관찰되었으며 부화일수가 경과할수록 배상세포의 수가 증가하는 특징이 관찰되었다. 부화 후 30일경 위에서 위산을 분비하는 위선이 관찰되었으며 소화면적을 넓혀주고 소화속도를 조절해주는 유문수와 유문괄약근이 관찰되었다. 각종 소화효소 분비촉진 및 장 연동운동에 관여하는 주요소화호르몬인 CCK의 경우 개구가 이루어지는 부화 후 2일경 발현되기 시작하였으며 처음으로 먹이생물을 섭이하는 부화 후 4일경 발현량이 급격히 증가하였다. 소화기관들의 기능적인 발달과 부화일수가 증가함에 따라 점차 CCK발현양상이 안정화되는 특징이 관찰되었다.
빛 파장에 따른 자바리의 성장 및 소화활성 특징을 조사하기 위해 자연광조건 (full spectrum), Green (520 nm), Red (590 nm), Blue (480 nm) 조건하에 12주간 사육 후 조직화학적 및 분자생물학적 방법을 이용하여 실험을 수행하였다. 성장률의 경우 Green파장에서 유의적으로 높게 나타났으며, 소화활성에 관여하는 CCK 및 배상세포의 활성도 Green파장에서 유의적으로 높게 관찰되었다.
자바리 고유의 CCK 및 배상세포의 특징을 조직화학적 및 분자생물학적 방법을 이용하여 조사하였다. 자바리 CCK cDNA길이는 535 bp 였으며, 다른 경골어류와의 상동성 조사결과 Red drum과 가장 높은 상동성 (95%)을 나타내었다. CCK 분비세포의 경우 소화관의 전장부와 유문수에 가장 많이 분포하였고 소수의 CCK 분비세포가 직장까지 분포하였다. 배상세포의 경우 식도를 제외한 소화관의 전장부와 유문수에서 가장 많이 분포하였으며 직장부로 갈수록 수가 감소하는 특징이 관찰되었다.
The longtooth grouper, Epinephelus bruneus is a commercially and recreationally valuable species and a promising candidate for marine aquaculture in Korea. Studies related to the ontogenetic development of the digestive system of marine fish larvae are an initial step in the identification and implementation of new feed alternatives for marine fish larvae. The purpose of this study was to describe the ontogenetic development of the digestive system of longtooth grouper larvae, and to characterize its cholecystokinin (CCK) and goblet cells in the hope of better understanding their organization and functionality in order to improve the rearing efficiency of longtooth grouper larvae. The larvae were kept in 35-metric-ton concrete tanks for 55 days. They were fed rotifer (Brachionus plicatilis) from 0 days after hatching (DAH) to 30 DAH. Artemia nauplii were given from 15 DAH to 55 DAH, and an artificial diet was used from 20 DAH onward.
At the time of hatching, the digestive system consisted of an undifferentiated straight tube laying over the yolk sac and an unopened mouth and anus. At 2 DAH, the digestive tract became wider and the anus opened. The epithelium of the intestine consisted of a single layer of columnar cells. CCK mRNA was first expressed at and was continually expressed until the end of the experiment. The mouth was open at 4 DAH. The liver and pancreas were observed, and the intestinal valve was formed by columnar epithelium in the posterior intestine. Mucosal folds were formed in the rectum, and goblet cells appeared in the buccopharyngeal cavity. At 5 DAH, the digestive tract started coiling and the yolk sac was almost depleted. At 10 DAH, the pancreas was completely diffused around the liver and intestine as observed in juveniles. At 20 DAH, goblet cells appeared in the anterior intestine. At 25 DAH, the stomach was morphologically differentiated and separated from the anterior intestine by the pyloric sphincter. At 30 DAH, the stomach was still changing in shape and increasing in size, and a number of gastric glands were observed. The pyloric ceca appeared as invaginations of the anterior intestine surrounding the stomach. At 35 DAH, canine-like teeth and taste buds were observed to protrude from both the oral valve and the posterior region of the buccopharyngeal cavity. The stomach size and the number of gastric glands increased continually.
Longtooth grouper larvae develop a well-differentiated digestive tract with a functional stomach at about 30 DAH. The weaning of longtooth grouper larvae to formulated feeds was started at 30 DAH at 25 °C once the functional stomach, pyloric sphincter, and pyloric ceca had formed.

Light characteristics are very specific in the aquatic environment. Fish vision and different light spectra perception are related to each species' natural habit. Light is one of the main environmental conditions and can be easily manipulated in artificial rearing settings. Cholecystokinin (CCK) and mucus-secreting goblet cells are the main regulators of digestion. In this study, we established whether the light spectrum (natural condition, full spectrum: green, 520 nm; red, 590 nm, and blue, 480 nm) influences growth performance and digestive activity related to CCK mRNA expression and mucus-secreting goblet cell activity in order to develop a good management protocol and optimal rearing system for the longtooth grouper. For each light spectrum, fish were reared 12 weeks under a flow-through system and fed commercial pellet diets once daily. At the end of the experiment, the final body weights differed among the fish reared under different light spectra. The highest growth performance value was observed in fish reared under the green light condition. On the other hand, the growth performances of fish in the natural and blue light conditions were drastically decreased in last 3 weeks of the experiment. CCK mRNA expression and mucus-secreting goblet cell activity were significantly higher in the fish under green light condition than in the fish under the natural, red, and blue light conditions. Rearing of the longtooth grouper under the green light condition had positive effects on fish growth performance and digestion.
We recommend that the appropriate light spectrum for the artificial culture of the longtooth grouper is the green light condition from the perspective of growth performance and the synergistic effects of CCK and mucus-secreting goblet cells. However, longer light treatment periods are needed in future investigations to clarify the effects of light spectrum on the longtooth grouper. Together with the findings of the present study, such studies would result in better understanding of the digestive physiology and contribute to the development of optimal rearing management for commercial production of the longtooth grouper.

Cholecystokinin (CCK) and mucus-secreting goblet cells play a key role in the digestive function of vertebrates including fish. CCK accelerates the release of pancreatic enzymes, contracts the gallbladder, and regulates intestinal peristalsis. The mucus secretion of goblet cells in vertebrates including fish plays important roles in the absorption of easily digestible substances and prevents the mucous membrane from being damaged by physical or chemical substances. The amino acid sequence of CCK is well conserved; the fish homology has only single substitution throughout vertebrates. Recently, the CCK gene has also been identified in several fish, and 2 major clusters of teleost CCK were reported after phylogenetic analysis. As a part of a molecular biological study on the longtooth grouper digestive tract, we have isolated CCK cDNA from the brain and the digestive tract using PCR. The cDNA of CCK was 535 bp in length. Phylogenetic analysis revealed that the longtooth grouper CCK subunit was a fish CCK2. Longtooth grouper CCK mRNA expression was detected in the brain (the telencephalon, optic tectum, medulla and hypothalamus, cerebellum, and pituitary) and the digestive tract except for the stomach, suggesting that longtooth grouper CCK could act as a central neuropeptide and as a neurotransmitter in the digestive tract. CCK-producing cells in the digestive tract of the longtooth grouper were scattered throughout the digestive tract. The highest frequency of CCK-producing cells was observed in the anterior intestine portion and pyloric ceca, with a very small number of cells distributed as far as the rectum. Mucus-secreting goblet cells in the digestive tract of the longtooth grouper were found to differ remarkably in their regional distributions and relative frequencies. High frequencies of mucus-secreting goblet cells were found in the digestive tract of the longtooth grouper, mainly in the anterior intestine portion and pyloric ceca, but not the esophagus; the frequency decreased slightly toward the rectum.
Our result suggests that food digested by gastric acid in the stomach moves on and is then delayed in the anterior (including the pyloric ceca) and mid intestine portion, thereby ensuring effective stimulation of the CCK-producing cells. In addition, the distribution pattern of the CCK-producing cells closely resembled that of mucus-secreting goblet cells. In the longtooth grouper, CCK and mucus-secreting goblet cells seem to be well adapted to promoting optimal control of the digestive process.
Author(s)
허상우
Issued Date
2011
Awarded Date
2011. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000005590
Alternative Author(s)
Hur, Sang-Woo
Affiliation
제주대학교
Department
대학원 해양생명과학과
Advisor
이영돈
Table Of Contents
List of figures ⅰ
List of tables ⅳ

Chapter I. Ontogenetic development of the digestive system of the longtooth grouper 1
Abstract 2
1. Introduction 4
2. Materials and Methods 6
2.1. Larvae and rearing condition 6
2.2. Fish sampling and growth measurement 8
2.3. Histological observation 8
2.4. Sampling for the ontogenetic expression of CCK 9
3. Results 11
3.1. Growth 11
3.2. General development of the digestive system 13
3.3. Ontogenetic expression of CCK 20
4. Discussion 23

Chapter II. Effects of light spectrum on growth performance, goblet cell activation, and CCK expression in the longtooth grouper 31
Abstract 32
1. Introduction 34
2. Materials and Methods 36
2.1. Specimens and rearing condition 36
2.2. Measurements and sampling 39
2.3. Histochemical analysis 39
2.4. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for CCK gene expression 40
2.5. Statistical analysis 41
3. Results 42
3.1. Growth 42
3.2. Effects of different light spectra on mucus-secreting goblet cells 44
3.3. Effects of different light spectra on CCK gene expression 51
4. Discussion 55

Chapter III. Identification and characterization of CCK, CCK-A receptor, and mucus-secreting goblet cells in the longtooth grouper 60
Abstract 61
1. Introduction 63
2. Materials and Methods 65
2.1. Specimens 65
2.2. Molecular cloning of CCK and CCK-A receptor 65
2.2.1. RNA extraction and cDNA synthesis 65
2.2.2. Polymerase chain reaction (PCR) 66
2.2.3. Sequence analysis 67
2.3. Tissue distribution and expression of CCK and CCK-A receptor 70
2.3.1. RT-PCR for tissue distribution and expression analysis 70
2.3.2. Real-time quantitative RT-PCR for CCK 71
2.4. Characteristics of CCK-producing cells and mucus-secreting goblet cells 73
2.4.1. Immunohistochemistry 73
2.4.2. Histochemistry 74
2.5. Statistical analysis 74
3. Results 75
3.1. Cloning of the longtooth grouper CCK and CCK-A receptor 75
3.1.1. CCK and CCK-A receptor 75
3.2. Tissue distribution of the longtooth grouper CCK and CCK-A receptor 80
3.3. Characteristics of CCK-producing cells and mucus-secreting goblet cells 83
3.3.1. CCK-producing cells 83
3.3.2. Mucus-secreting goblet cells 84
4. Discussion 88
국문요약 93
References 95
Degree
Doctor
Publisher
제주대학교 대학원
Citation
허상우. (2011). Characterization of cholecystokinin and mucus-secreting goblet cell in longtooth grouper, Epinephelus bruneus
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