On behalf of the local organising committee I am pleased to invite you to join us for Intercongress Symposium of the 7th Asia and Oceania Society for Comparative Endocrinology (AOSCE). The 7th Intercongress Symposium of the AOSCE will be held at the National Taiwan Ocean University, Keelung, Taiwan from March 18 (Tuesday) to 23 March (Sunday), 2014.

Our AOSCE meetings provide fantastic opportunities not only to exchange and share research information, discoveries and advancements, but also provide us the chance to network with our colleagues and build new friendships.

This symposium with 127 papers in total covers all the areas of the endocrinology and physiology in the comparative aspects. The symposium will provide a great opportunity for endocrinologists to share their ideas/experiences and to discuss the various challenges that are being faced in this field. It is also a good opportunity to see how various collaborations can make significant contributions to our science field.

The 7th AOSCE Intercongress symposium being held in Taiwan will provide an excellent chance to not only participate in the congress but also to visit our NTOU campus and enjoy the cultures that is the essence of Taiwan. You will be able to tour the city of Keelung and Taipei as well as visit the historical town of Taiwan amongst other interesting things.

We appreciated the financial supports from the National Science Council (Ministry of the Science and Technology, Taiwan) and the special assistance from The Center of Excellence for the Oceans of National Taiwan Ocean University.

We look forward to seeing you here in Taiwan and anticipate a great scientific interaction and joyful stay.

Ching-Fong Chang, Ph. D. Chair of the organizer President & Professor, National Taiwan Ocean University 2, Pei-ning Rd., Keelung 20224, Taiwan [email protected]

1 Symposium Organization Local Organization (Taiwan) Ching-Fong Chang (Chair) Bon-chu Chung Pung-Pung Hwang Yung-Sen Huang Wen-Shiun Yueh Paulus Shyi-Gang Wang Chi-Ying Lee Meng-Chun Hu Hui-Min Su Leang-Shin Wu

International Organising Committee (Council Members) Kazuyoshi Tsutsu (Japan; President of the AOSCE) Bronwyn McAllan (Australia) Hao-Ran Lin (China) Zandong Li (China) Wei Ge (Hong Kong) Saumen Kumar Maitra (India) Shelley Bhattacharya (India) M. Michael Aruldhas (India) Umesh Rai (India) Bagher Amiri (Iran) Kaoru Kubokawa (Japan) Akiyoshi Takahashi (Japan) Kouhei Matsuda (Japan) Hyuk-Bang Kwon (Korea) Kyungjin Kim (Korea) Jae Young Seong (Korea) Ishwar Parhar (Malaysia) John Cockrem (New Zealand) Evelyn Grace de (Philippines) Jesus-Ayson (Philippines) Sukumal Chongthammakun (Thailand) Suchinda Malaivijitnond (Thailand) Ching-Fong Chang (Taiwan)

2 General Information Venue The seventh Intercongress Symposium of the Asia and Oceania Society for Comparative Endocrinology (AOSCE) will be held in the Conference Hall of PI and Administration building on the campus of National Taiwan Ocean University (NTOU), Keelung from March 18-23, 2014.

Transportation We will provide transport from the accommodation location in the downtown to the conference venue on the afternoon of March 18 (Tuesday), and on the following morning, and return transport in the evenings. If you get a city bus (bus line # 103 or 104) or a taxi from the downtown to University you can ask to be taken get off at the university gate of the NTOU.

Please be ready for the bus in the afternoon of March 18 and mornings of March 19-22 as follows:

March 18 (Tuesday) The front of Keelung City Hall and Evergreen Laurel Hotel 14:40 pm, 15:20 pm, 15:50 pm

March 19-22 (Wednesday-Saturday) The front of Keelung City Hall and Evergreen Laurel Hotel 08:25 am

Registration desk The registration desk will be opened throughout the conference in the Exhibition Room of the Administration Building, NTOU.

Conference location Opening ceremony, group photo and Opening Lecture on March 18 will be started from 16:20 pm at the Conference Hall of PI. The conference from March 19-22 will be 1F and 2F Auditorium of the Administration Building.

3 Scientific Programme Oral presentation Keynote lecture will be 45 minutes in length. Symposium lecture will be 30 minutes in length, with 5 minutes for questions. Oral presentation will be 20 minutes in length, with 3 minutes for questions and a 2 minutes gap between papers. Poster presentation The maximum poster size is 90 (width) x 120 cm (length). The posters will be ready on March 19 (Wednesday) and displayed (presented) on March 20 (17:25-18:10 pm) and March 21 (12:40-13:40 pm) in the Exhibition Hall. Poster presenters are asked to be at their posters during the poster sessions on March 20 and 21.

Excursions Free trips will be organized in the afternoons of March 21 (National Museum of Marine Science and Technology, Jiu-fen village) and 22 (Yeh-liu Geopark, Peace Island Geopark). An option tour (NTD 2100/USD 70) will be on March 23 (7:40 am - 22:00 pm) to the following locations: The One-Land of Retreat & Wellness, Chihu Mausoleum, National Chiang Kai-Shek Memorial Hall, Taipei 101, Hsin Tian Kong Temple. Bus is scheduled to leave in the morning of March 23 (7:40 am on Sunday) from the Keelung City Hall.

Council meeting The Council meeting of the AOSCE will be in the 2F meeting room of the Administration building during lunch period (12:10-13:30 pm) on March 21 (Friday). The lunches will be provided there.

The next generation lunch A special session (the next generation lunch) for young fellows will be organized in the 3rd Floor meeting room of Administration building during the lunch period (12:05-13:10 pm) on March 19 (Wednesday). Please bring your lunch box to the meeting room. All the young fellows (students, postdoctors and others) are welcomed.

Morning and afternoon teas and food Morning and afternoon teas, coffee, lunches and dinners are provided and will be served in the Exhibition Hall. Lunch box and dinner will be served. The lunch location could be in the Exhibition Hall or 2F Auditorium of the Administration building.

4 Climate The weather in Taipei/Keelung could be around 19 C sometimes with raining.

National Taiwan Ocean University (NTOU) NTOU was established in 1953 and grew to become the famous university focused on the marine science and technology. It has 6 colleges (College of Life Sciences, College of Ocean Science and Resource, College of Maritime Science and Management, College of Engineering, College of Electrical Engineering and Computer Science, College of Humanities and Social Sciences) with more than 8500 students, 400 faculty and 27 departments and Institutes (16 Bachelor of Sciences, 26 Master of Science and 17 Doctoral Program). The fields are related to ocean culture, marine education, marine law, marine affairs, fishery science, aquaculture, food science, marine biology, marine biotechnology, oceanography, navigation and management, ship-building, harbor and river engineering, ocean engineering, mechanical engineering, electrical engineering and others.

Keelung City Keelung city is just in the northest of Taipei (about 20 km) and it is the beginning of the “Northeast Coast National Park”. Keelung city is traditionally famous for the commercial harbor, fishery harbors, night market (Miaokou or temple mouth) with traditional and local food, famous forts, marine culture, sea coast, seafood and others. Nearby areas are also famous such as Yeh-liou Geopark, Peace Insland Geopark, Gold Ecological Park/Jiu-Fen village (gold- and coal-mining town in the past) and National Museum of Marine Science and Technology.

5 Institute of Marine General Department of 1. Conference Hall of PI Environmental purpose Environmental Biology Student Student Chemistry and building 1 and Fisheries Science 2. Exhibition hall dormitory dormitory Ecology Restaurant

3. Administration Building Department of Building for Marine NTOU Marine College of History Life Sciences Fountain Science Environmental

Building Museum Informatics Entrance

General Building of purpose humanities Department of Food Science Student building 3 1 dormitory Terrestrial Animal Restaurant Yen‐Ping Ocean hall building purpose General dormitory Recreation Experimental Student technology building Exhibition hall Library Center building 2

Swimming Department Bookstore pool Department of of Mechanical Administration Merchant Marine Ship and Mechatronic Building General Research Center

Department of Handling Engineering 3 Hai‐Kong Ivy Wang Simulator Post

6 Shipping and building Transportation Hall Labs office Management

Student Gymnasium Entrance Activity Center

Pei‐Ning Road

Entrance

Department Aquatic of Harbor College of Animal and River Electrical Engineering Center Engineering Engineering Department Department bulding 2 of Systems of Electrical Engineering Engineering & Naval Architecture

Taiwan University

Ocean National Hotel

Coast ‐ E Star 基隆港義一路 Rd Hotel st Evergreen 港西街 1

Yi Hotel

K 中正路 Harbor

Gangxi St. Xin 2nd Rd. 信二路 Train

Hall

Hotel Beidoo Keelung City Zhongzheng Rd. Keelung Station Keelung

Zhong 1st Rd.忠一路 Ren 2nd Rd.仁二路

孝二路 Hotel Huado Hotel

Rd. .義一路 Hotel Harbor HuaShin Keelung nd Rd View 2

st 1

Yi Xiao

Hotel HuaGuo Night Market Keelung Freeway

7 Programat Glance

March 18, 2014 Registration at NTOU Tuesday 13:00-18:00 Group Photo 16:20-16:25 Opening Ceremony and Opening Address 16:30-16:50 Keynote Lecture : a special session dedicated to the memory of Emeritus Pofessor Hideshi Kobayashil Campus tour 17:50-18:30 Dinner (NTOU, Keelung) 18:30-20:00 March 19, 2014 Registration at NTOU Wednesday 0830-17:00 Keynote Lecture, Symposium Lecture and Oral Presentation 09:00-18:15 Lunch, The next generation lunch (12:05-13:10) 12:05-13:10 Dinner (988 restaurant, Keelung) 18:30-20:00 March 20, 2014 Registration at NTOU Thursday 0830-17:00 Keynote Lecture, Symposium Lecture and Oral Presentation 09:00-17:25 Poster Presentation 17:25-18:10 Dinner (水園會館Le Jardin restaurant, Keelung) 18:30-20:00 March 21, 2014 Registration at NTOU Friday 08:30-14:00 Keynote Lecture, Symposium Lecture and Oral Presentation 09:00-14:25 Lunch, Council meeting (12:10-13:30), Poster Presentation (12:40-13:40) 12:05-13:40 Free Tour 1. 海科館National Museum of Marine Science and Technology , Keelung 2. 九份Jiufen, Taipei 14:30-18:00 Dinner (Evergreen Laurel Hotel, Keelung) and Performance 18:30-20:30 March 22, 2014 Registration Saturday 0830-12:00 Keynote Lecture, Symposium Lecture and Oral Presentation 09:00-12:15 Closing Ceremony and Closing Address 12:15-12:25 Lunch and Free Tour (和平島公園Peace Island Geopark , Keelung) 13:00-15:00 Free Tour (野柳地質公園Yehliu Geopark, Taipei) 16:00-18:00 Dinner (基隆廟口夜市Keelung Night Market, Keelung) 18:30-20:00 March 23, 2014 Optional Tour or Departure Sunday 08:00-20:00 March 24, 2014 Departure Monday

8 Program Schedule

DAY1 March 18, 2014 (Tuesday)

Time Program Venue Exhibition 1300-1630 Registration Room 1620-1625 Group Photo Opening Ceremony Chairman: Ching-Fong Chang (Taiwan) Conference 1630-1650 Opening Address: Kazuyoshi Tsutsui (Japan) Hall of PI

Chairman: Kyungjin Kim (Korea) Keynote Lecture-1 P.1 Yoshio Takei (Japan) Conference 1650-1740 What we learned from Dr. Kobayashi, the Founder of Comparative Hall of PI Endocrinology in Japan (Dedicated to the memory of Emeritus Professor Hideshi Kobayashi). 1750-1830 Campus tour (Fountain, NTOU History Museum, Ocean Center) 1830-2000 Dinner and Welcome reception (NTOU, Keelung)

9 DAY2 March 19, 2014 (Wednesday)

Time Program Venue Exhibition 0830-0900 Registration Room Chairman: Bronwyn McAllan (Australia) Keynote Lecture-2 P.2 1F 0900-0945 Bon-chu Chung (Taiwan) Auditorium Function and regulation of steroids in vertebrates. Symposia- Symposia- Molecular Endocrinology Neuroendocrinology Chairman: Olivier Kah (France) Chairman: Young-Joon Kim (Korea) Symposium Lecture-1 P.11 Symposium Lecture-2 P.12 Sylvie Dufour (France) Anderson On Lam Wong (China) Impact of whole genome duplications Novel pituitary actions of TAC3 gene 1F / 2F 0945-1015 on vertebrate neuroendocrine systems products in fish model. Auditorium with special focus on teleosts.

Symposium Lecture-3 P.13 Symposium Lecture-4 P.14 Wei Hu (China) Akiyoshi Takahashi (Japan) 1F / 2F 1015-1045 Transgenic Fish and its Reproductive Pleiotropic functions of Auditorium Containment Strategies. proopiomelancortin in fish.

Exhibition 1045-1105 Coffee Break Room Symposia- Symposia- Molecular Endocrinology Neuroendocrinology Chairman: Bronwyn McAllan Chairman: Takeshi Miura (Japan) (Australia) Oral Presentation-1 P.45 Oral Presentation-2 P.46 Hee-Dae Kim (Korea) Hui-Min Su (Taiwan) Epigenetic modulation of rhythmic Exposure to a maternal n-3 fatty acid- GnRH gene transcription by dynamic deficient diet during the brain 1F / 2F 1105-1125 histone modifications. development enhance the activity and Auditorium dysregulation of hypothalamic- pituitary-adrenal axis responses to stress in rat offspring later in life.

Oral Presentation-3 P.47 Oral Presentation-4 P.48 Sung Kook Chun (Korea) Sayaka Maehiro (Japan) Identification of small molecule Identification of a novel 1F / 2F 1125-1145 Cryptochrome inhibitor modulating transglutaminase gene that is Auditorium the molecular circadian clock. differentially expressed between the sexes in the medaka brain.

10 Oral Presentation-5 P.49 Oral Presentation-6 P.50 Solmi Cheon (Korea) Adimoolam Aruna (Taiwan) Glucocorticoid-mediated Period2 Involvement of corticotropin-releasing induction delays the phase of hormone and corticosteroid receptors 1F / 2F 1145-1205 circadian rhythm. in the brain-pituitary of tilapia during Auditorium cronic and acute salinity stress.

Lunch; 1205-1310 The next generation lunch (3F Meeting Room) organized by Guan-Chung Wu (Taiwan) Chairman: Sylvie Dufour (France) Keynote Lecture-3 P.3 1F 1310-1355 Ishwar Parhar (Malaysia): Auditorium Functional significance of kisspeptins in fish. Symposia- Symposia- Evolutionary and Developmental Neuroendocrinology Endocrinology Chairman: Ritsuko Ohtani-Kaneko Chairman: Jae Young Seong (Korea) (Japan) Symposium Lecture-5 P.15 Symposium Lecture-6 P.16 Olivier Kah (France) Satoshi Ogawa (Malaysia) 1F / 2F 1355-1425 Aromatase in the brain of fish: Role of tachykinins and tachykinin Auditorium expression, regulation and potential receptors in fish reproduction. functions. Symposium Lecture-7 P.17 Symposium Lecture-8 P.18 Guan-Chung Wu (Taiwan) Hae Chul Park (Korea) Tug-of-war of gonadal soma Regulation of oligodendrocyte 1F / 2F 1425-1455 transdifferentiation in the development by Hedgehog signaling Auditorium hermaphroditic fish. in the zebrafish CNS.

Chairman: Keerikkattil Paily Joy Chairman: Honoo Satake (Japan) (India) Oral Presentation-7 P.51 Oral Presentation-8 P.52 Jyoti Kamal Hazarika (India) Sugiyono (Japan) Histomorphological study on the Effect of cadmium on dopaminergic 1F / 2F 1455-1515 development of thymus of and serotonergic neurons and Auditorium Oryctolagus cuniculus . involvement of estrogen signaling pathway during early development of zebrafish. Oral Presentation-9 P.53 Oral Presentation-10 P.54 Chieh Jhen Chen (Taiwan) Shan-Ru Jeng (Taiwan) Characterization of Dmrt gene family Neuroendocrine gene expression 1F / 2F 1515-1535 in a scleractinian coral, Euhyllia levels at different ovarian Auditorium ancora . developmental stages in wild Japanese eel. Exhibition 1535-1555 Coffee Break Room

11 Symposia- Symposia- Endocrinology of Stem Cell Biology Neuropeptides and Receptors Chairman: Akiyoshi Takahashi Chairman: Bon-chu Chung (Taiwan) (Japan) Symposium Lecture-9 P.19 Symposium Lecture-10 P.20 Woong Sun (Korea) Jae Young Seong (Korea) 1F / 2F 1555-1625 Migration and differentiation of neural Evolutionary genomics for Auditorium progenitors following brain injury in identification of novel neuropeptides mice. and their receptors. Symposium Lecture-11 P.21 Symposium Lecture-12 P.22 Hong-Yo Kang (Taiwan) Billy KC Chow (China) Androgen receptor regulates stemness Transmembrane peptides as unique 1F / 2F 1625-1655 and differentiation of stem cells. tools to demonstrate the in vivo action Auditorium of GPCR hetero-complex of secretin and angiotensin. Chairman: Hans Uwe Dhams (Korea) Chairman: Hae Chul Park (Korea) Oral Presentation-11 P.55 Oral Presentation-12 P.56 Partha Roy (India) Jenn-Kan Lu (Taiwan) A novel pterostilbene derivative Molecular cloning and 1F / 2F 1655-1715 supresses prostate cancer cell growth characterization of feeding regulator- Auditorium irrespective of androgen receptor NPY and NPF genes and its status. applications in attractant development for finfish and crustacean. Oral Presentation-13 P.57 Oral Presentation-14 P.58 Tomoyuki Okutsu (Japan) Jong-IK Hwang (Korea) Development of a DNA microarray A novel glucagon-related peptide and its application to the elucidation (GCRP) and its receptor GCRPR 1F / 2F 1715-1735 of gene transcription dynamics in account for coevolution of their family Auditorium ovary fragments of Litopenaeus members in vertebrates. vannamei incubated with sinus gland extract. Oral Presentation-15 P.59 Oral Presentation-16 P.60 Yufeng Si (China) Ganesan Nagarajana (Taiwan) Genetic polymorphisms and DNA Effects of neuropeptide, methylation in exons CpG rich neurosteroidogenic enzymes and 1F / 2F 1735-1755 regions of PACAP gene and its effect estrogen receptors in the early brain of Auditorium on mRNA expression and growth protogynous fish: an in vivo approach. traits in Half smooth tongue sole (Cynoglossus semilaevis ). Oral Presentation-17 P.61 Oral Presentation-18 P.62 Ching-Hua Hu (Taiwan) Ratu Fatimah (Japan) Endocrine disruptor TCDD induces Suppression of aromatase by 1F / 2F 1755-1815 zebrafish CYP3A65 transcription via fadrozole and morpholino antisense Auditorium AHR2-dependent pathway. oligonucleotide alters activity of dopaminergic neuron in early development of zebrafish. 1830-2000 Dinner (988 restaurant, Keelung)

12 DAY3 March 20, 2014 (Thursday)

Time Program Venue Exhibition 0830-0900 Registration Room Chairman: Olivier Kah (France) Keynote Lecture-4 P.4 1F 0900-0945 Yoshitaka Nagahama (Japan) Auditorium Regulation of ovarian development and oocyte maturation in fish. Symposia- Symposia- Reproductive Endocrinology Brain and Behavioral Endocrinology Chairman: Ishwar Parhar (Malaysia) Chairman: Kouhei Matsuda (Japan) Symposium Lecture-13 P.23 Symposium Lecture-14 P.24 Kiyoshi Soyano (Japan) John Donad (Australia) Pheromonal control of final oocyte Appetite regulation in desert-adapted 1F / 2F 0945-1015 maturation and lunar-related spawning spinifex hopping mice during water Auditorium in grouper. deprivation.

Symposium Lecture-15 P.25 Symposium Lecture-16 P.26 Masatoshi Mita (Japan) M.C. Subhash Peter (India) Regulatory contribution of Gs- Serotonin and melatonin mitigate the proteins to 1-methyladenine stress response and promote the ease 1F / 2F 1015-1045 production in starfish ovarian follicle response in air-breathing fish. Auditorium cells stimulated by relaxin-like gonad- stimulating substance.

Exhibition 1045-1105 Coffee Break Room Symposia- Symposia- Reproductive Endocrinology Growth and Metabolism Chairman: Sylvie Dufour (France) Chairman: John Donad (Australia) Oral Presentation-19 P.63 Oral Presentation-20 P.64 Muhammad Ahya Rafiuddin Donlaporn Kittivanichkul (Thailand) (Indonesia) Changes in bone mineral density and Manipulation expression FSH content in pre-, peri- and post- 1F / 2F 1105-1125 (Follicle Stimulating Hormone) from menopausal cynomolgus monkeys. Auditorium striped catifsh (Pangasianodon hypopthalmus ) with hormonal manipulation during gonadal early development. Oral Presentation-21 P.65 Oral Presentation-22 P.66 Chiemi Miura (Japan) YeLin Jiang (China) Controlled feeding inhibits growth Integrated ecological impact of the 1F / 2F 1125-1145 reduction associated with spawning in equipment on the soft-shelled turtle Auditorium farmed yellowtail. breeding environment and growth and metabolism.

13 Oral Presentation-23 P.67 Oral Presentation-24 P.68 En-Lieng Lau (Taiwan) Hong-Yi Gong (Taiwan) Conserved sex-specific timing of Muscle-specific expression of giant 1F / 2F 1145-1205 meiotic initiation during sex grouper growth hormone in transgenic Auditorium differentiation in the protandrous zebrafish leads to muscle hypertrophy black porgy Acanthopagrus and hyperplasia. schlegelii . 1205-1300 Lunch Chairman: Keerikkattil Paily Joy (India) Keynote Lecture-6 P.6 1F 1300-1345 Kyungjin Kim (Korea) Auditorium BioClock Timing System: Impact of circadian clock molecule, Rev-erb alpha on mood regulation by dopamine. Symposia- Symposia- Brain and Behavioral Endocrinology Reproductive Endocrinology Growth and Metabolism Chairman: Wei Ge (China) Chairman: M.C. Subhash Peter (India) Symposium Lecture-17 P.27 Symposium Lecture-18 P.28 Takeshi Miura (Japan) Kouhei Matsuda (Japan) Control mechanisms of meiotic during Orexin A induces anxiety-like 1F / 2F 1345-1415 spermatogenesis in Japanese eel behavior in goldfish. Auditorium (Anguilla japonica ).

Symposium Lecture-19 P.29 Symposium Lecture-20 P.30 Deshou Wang (China) Takashi Yada (Japan) 1F / 2F 1415-1445 Roles of sex steroids on sex Ghrelin and food acquisition in Auditorium determination and maintenance in japanese eel. teleosts. Chairman: Jae Young Seong (Korea) Chairman: Chi-Ying Lee (Taiwan) Oral Presentation-25 P.69 Oral Presentation-26 P.70 Sherly Tomy (India) Yong Zhang (China) Identification of interacting partners Molecular cloning of four MRFs of gonad-inhibiting hormone of (MyoD、Myf5、MRF4、Myogenin) 1F / 2F 1445-1505 Penaeus monodon by yeast two- and their expression patterns during Auditorium hybrid analyses. fasting and refeeding in orange- spotted grouper, Epinephelus coioides . Oral Presentation-27 P.71 Oral Presentation-28 P.72 Wanshu Hong (China) Chika Nishitani (Japan) The role of melatonin in Restoration of lung degeneration by 1F / 2F 1505-1525 synchronizing semilunar spawning pueraria mirifica plant. Auditorium rhythm of the mudskipper Boleophthalmus pectinirostris .

Exhibition 1525-1545 Coffee break Room

14 Symposia- Symposia- Growth and Metabolism Reproductive Endocrinology Endocrinology Chairman: Deshou Wang (China) Chairman: Weiqun Lu (China) Symposium Lecture-21 P.31 Symposium Lecture-22 P.32 Wei Ge (China) Chi-Ying Lee (Taiwan) Kit system and its role in the paracrine Neuroendocrine response to pathogens 1F / 2F 1545-1615 regulation of follicle development in and its implication for the metabolism Auditorium zebrafish. of crustacean host.

Symposium Lecture-23 P.33 Symposium Lecture-24 P.34 Honoo Satake (Japan) Suchinda Malaivijitnond (Thailand) Neuropeptides and peptide hormones Anti-osteoporotic effects of 1F / 2F 1615-1645 of the ascidian, Ciona intestinals : phytoestrogens containing plant Auditorium Comparative views to novel follicle Pueraria mirifica : in vivo and in growth. vitro approaches. Chairman: Kiyoshi Soyano (Japan) Chairman: Jenn-Kan Lu (Taiwan) Oral Presentation-29 P.73 Oral Presentation-30 P.74 Hera Maheshwari (Indonesia) Deng Siping (China) Fecal testosterone profile and sperm Molecular cloning and dimorphic 1F / 2F 1645-1705 quality of dairy buffalo from north expression of Growth hormone (GH) Auditorium sumatera. in female and male spotted scat (Scatophagus argus ).

Oral Presentation-31 P.75 Oral Presentation-32 P.76 Priyajit Chatterjee (India) Wensheng Li (China) A super molecule reverts lipid Molecular characterization of 1F / 2F 1705-1725 induced insulin resistance. adiponectin receptors, its mRNA Auditorium expression, and subcellular location in the orange-spotted grouper (Epinephelus coioides ). Exhibition 1725-1810 Poster presentation and Coffee break Room 1830-2000 Dinner (水園會館Le Jardin restaurant, Keelung)

15 DAY4 March 21, 2014 (Friday)

Time Program Venue Exhibition 0830-0900 Registration Room Chairman: Ishwar Parhar (Malaysia) Keynote Lecture-7 P.7 1F 0900-0945 Saumen Kumar Maitra (India) Auditorium Melatonin in gut and pineal: A testimony of diversity in temporal pattern and its synchronizer. Symposia- Symposia- Environmental and adaptative Reproductive Endocrinology Endocrinology Chairman: Tomoko Soga (Malaysia) Chairman: Pung-Pung Huang(Taiwan) Symposium Lecture-25 P.35 Symposium Lecture-26 P.36 Keerikkattil Paily Joy (India) Hans Uwe Dhams (Korea) 1F / 2F 0945-1015 Catecholestrogens in final oocyte Does bioaccumulation and Auditorium maturation in the catfish biomagnification of EDC’s take place Heteropneustes fossilis : an overview. in marine fish? Symposium Lecture-27 P.37 Symposium Lecture-28 P.38 Akihiro Takemura (Japan) Weiqun Lu (China) Rhythms in fish reproduction : Neuro-endocrine control of the 1F / 2F 1015-1045 possible involvement of dopaminergic environmental adaptability in fish-a Auditorium activity in tidal-related spawings of a study of fish caudal neurosecretory tropical wrasse. system. Exhibition 1045-1105 Coffee Break Room Chairman: Ritsuko Ohtani-Kaneko Chairman: Wei Hu (China) (Japan) Oral Presentation-33 P.77 Oral Presentation-34 P.78 Xiaochun Liu (China) Taro Ikegami (Japan) Molecular cloning, characterization Is tide-related locmotion rhythm 1F / 2F 1105-1125 and expression profiles of Dax1 in controlled by external tidal stiumali or Auditorium protogynous orange-spotted grouper internal melatonin in the barred (Epinephelus coioides ). mudskipper? Oral Presentation-35 P.79 Oral Presentation-36 P.80 Wen-Shiun Yueh (Taiwan) Chunhua Zhu (China) The functions of estradiol-17β during Effects of Nonylphenol on 1F / 2F 1125-1145 oocyte maturation in multiple Vitellogenin Gene Expression and Auditorium spawning fish, yellowfin porgy. Gonad Development in Macrobrachium rosenbergii . Oral Presentation-37 P.81 Oral Presentation-38 P.82 Yung-Sen Huang (Taiwan) Noppadon Kitana (Thailand) Correlation between PTEN Herbicide utilization as an 1F / 2F 1145-1205 expression levels and ovarian environmental stressor on Auditorium development in the induced Japanese reproductive/endocrine functions of eel. sentinel species in agricultural areas.

16 Lunch (1205-1250), Poster presentation (1240-1340,Exhibition Room) 1205-1340 & Coucil meeting (1210-1330, 2F Meeting room)

Chairman: Hao-Ran Lin (China) Keynote Lecture-8 P.8 1F 1340-1425 Hao-Ran Lin (China) Auditorium Structure and functions of GnIH and GnIH receptor in teleost. Free Tour (海洋科技博物館National Museum of Marine Science and 1430-1800 Technology and 九份Jiufen, Keelung) 1830-2030 Dinner (Evergreen Laurel Hotel, Keelung) & Performance

17 DAY5 March 22, 2014 (Saturday)

Time Program Venue Exhibition 0830-0900 Registration Room Chairman: Billy KC Chow (China) Keynote Lecture-9 P.9 1F 0900-0945 Kazuyoshi Tsutsui (Japan) Auditorium Progress in comparative endocrinology by discovering novel neuropeptides and neurosteroids. Symposia- Symposia- Environmental and adaptative Invertebrate Endocrinology Endocrinology Chairman: Saumen Kumar Maitra Chairman: Yoshio Takei (Japan) (India) Symposium Lecture-29 P.39 Symposium Lecture-30 P.40 Young-Joon Kim (Korea) Pung-Pung Hwang (Taiwan) A sleep-stabilizing pathway in New insights into hormonal control of 1F / 2F 0945-1015 Drosophila composed of sex peptide body fluid ion homeostasis: zebrafish Auditorium receptor and its ligand, the as a model. myoinhibitory peptide. Symposium Lecture-31 P.41 Symposium Lecture-32 P.42 Shinya Shikina (Taiwan) Tomoko Soga (Malaysia) Yolk formation in a stony coral Impact of early life stress on anxiety 1F / 2F 1015-1045 Euphyllia ancora (Cnidaria, and reproduction. Auditorium Anthozoa): insight into the evolution of vitellogenesis in non-bilaterian animals. Exhibition 1045-1105 Coffee Break Room Chairman: Anderson On Lam Wong Chairman: Pung-Pung Huang (China) (Taiwan) Oral Presentation-39 P.83 Oral Presentation-40 P.84 Yung-Che Tseng (Taiwan) Minoru Uchiyama (Japan) Convergent evolutions of epithelial Osmoregulation in the crab-eating 1F / 2F 1105-1125 cell differentiation and frog, Fejervarya cancrivora . Auditorium neuroendocrine regulation in cephalopod and fish embryos. Oral Presentation-41 P.85 Oral Presentation-42 P.86 Hai Hui Ye (China) Mitsuyo Kishida (Japan) The presence and role of gnrh in the Nitrate (NO - ) and nitrite (NO - ) 1F / 2F 1125-1145 3 2 reproduction of the mud crab, Scylla perturb the activity of dopaminergic Auditorium paramamosain . neuron during early development of zebrafish.

18 Symposium Lecture-33 P.43 Symposium Lecture-34 P.44 Hosung Jung (Korea) Takashi Yoshimura (Japan) 1F / 2F 1145-1215 Xenopus as a model to study neuronal Evolution and origin of seasonal Auditorium axons. sensor in vertebrates.

1F 1215-1225 Closing Ceremony: Kazuyoshi Tsutsui (Japan) Auditorium 1300-1500 Lunch and Free Tour (和平島公園Peace Island Geopark , Keelung) 1600-1800 Free Tour (野柳地質公園Yehliu Geopark, Taipei) 1830-2000 Dinner (基隆廟口夜市Keelung Night Market, Keelung)

19 Program Schedule Poster list Symposia- Molecular Endocrinology1 Cho Rong Park (Korea) P.87 Park C.R.*, You D-.J., Kim D-.K., Moon M.J., Lee C., Oh S-.H., Ahn C., Seong J.Y., Hwang poster J-.I. 1-1 CXCL14 enhances proliferation and migration of cancer cells overexpressing the glycoproteins. Sumi Park (Korea) P.88 poster Park S.*, Seong J.Y., Hwang J. I. 1-2 A novel gonadotropin-releasing hormone-Ⅱ analog induces apoptosis prostate cancer cells. You Lee Son (Japan) P.89 poster Son Y.L.*, Ubuka T., Narihiro M., Fukuda Y., Hasunuma I., Yamamoto K., Belsham D.D., 1-3 Tsutsui K. Transcriptional activation of gonadotropin-inhibitory hormone by corticosterone. Kim Ha Da (Korea) P.90 poster Kim J.-H., Han K.-N. , Dahms H.-U.* 1-4 Molecular responses in the migrating Takifugu obscurus - gene regulation depends on tissue and reading time. Symposia- Environmental and adaptative Endocrinology 2 Khattapan Jantawongsri (Thailand) P.91 poster Jantawongsri K.*, Kitana J., Kitana N. 2-1 Stress and immune response of the rice frog Fejervarya limnocharis living in areas with different agricultural activity. Rangsima Pewphong (Thailand) P.92 poster Pewphong, R.*, Kitana, J., Kitana, N. 2-2 Incubating temperature affects development of endocrine cells in gonad of Malayemys macrocephala , a freshwater turtle from tropical asia. Jirarach Kitana (Thailand) P.93 Kitana, J.*, Meesawat S., Thammachoti P., Thitiphuree, T., Maneein, R., Chiewchanchai M., poster Pariyanonth P., Khonsue W., Kitana N. 2-3 Stress and health status based on hematological parameters of the rice field frog Hoplobatrachus rugulosus in captivity. Ying Jey Guh (Taiwan) P.94 poster Guh Y.J.*, Tseng Y.C., Yang C.Y., Hwang P.P. 2-4 Endothelin-1 regulates transepithelial H+ secretion in zebrafish. DaYaWa Shim (Korea) P.95 poster Shim D.M.-C., Dahms H.-U.*, Yang J., Wang L. 2-5 Endocrine effects of copper in aquatic animals – a review. Adimoolam Aruna (Taiwan) P.96 poster Aruna A.*, Nagarajan G., Chang C.F. 2-6 Differential expression of arginine vasotocin and isotocin mRNA to external salinity stress in black porgy (Acanthopagrus Schlegelii ) hypothalamus and pituitary.

20 Chia-Hao Lin (Taiwan) P.97 poster Lin C.H.*, Hu H.J., Hwang P.P. 2-7 The calciotropic hormone vitamin D regulates ion homeostasis in zebrafish (Danio rerio ). Symposia- Evolutionary and Developmental Endocrinology 3 Seongsik Yun (Korea) P.98 poster Yun S.*, Moon M.J., Park C.R., Cho E.B., Hwang J-.I., Seong J.Y. 3-1 Evolution and expansion of glp1 and glp1 receptor family. Hsiang-Hsu Pan (Taiwan) P.99 poster Pan H.H.*, Shikina S., Shao Z.F., Chang C.F. 3-2 Evolution and possible roles of heat shock protein 60 (Hsp60) in germline of metazoan animals. Symposia- Neuroendocrinology 4 & Neuropeptides and Receptors 5 Shogo Haraguchi (Japan) P.100 poster Haraguchi S.*, Hara S., Ubuka T., Tsutsui K. 4-1 Pineal allopregnanolone saves purkinje cells from apoptosis in the cerebellum. Ganesan Nagarajana (Taiwan) P.101 poster Nagarajan G.*,Aruna A., Chang C.F. 4-2 Potential role for neurosteroids in the early brain of orange-spotted grouper. Shan-Ru Jeng (Taiwan) P.102 poster Jeng S.R.*, Yueh W.S., Dufour S., Kah O., Chang C.F. 5-1 The expression and regulation of aromatase and estrogen receptors in Japanese eel. Symposia- Invertebrate Endocrinology 6 Shunsuke Moriyama (Japan) P.103 poster Moriyama S.*, Kawamura M., Kodera Y., Uchida K., Amano H. 6-1 Neuropeptide Y involves in the regulation of somatic growth of the Pacific abalone. Shinya Shikina (Taiwan) P.104 poster Shikina S.*, Shao Z.F., Chung Y.J., Chang C.F. 6-2 Expression of heat shock protein 60 (Hsp60) in the germline cells of a scleractinian coral Euphyllia ancora (Cnidaria, Anthozoa). Yi-Ling Chiu (Taiwan) P.105 poster Chiu Y.L.*, Shikina S., Chang C.F. 6-3 Exploration of the genes involving in sexual reproduction in a stony coral Euphyllia ancora . Symposia- Brain and Behavioral Endocrinology 7 Yasuto Narita (Japan) P.106 poster Narita Y.*, Okada N., Tsutiya A., Kaneko T., Ohtani-Kaneko R. 7-1 Dose 11KT increase GnRH3 neurons by enhancing neurogenesis in female mozambique? Yu Shinohara (Japan) P.107 poster Shinohara Y.* and Kobayashi M. 7-2 Sexual bipotentiality of the brain for olfactory processing and sexual behavior in goldfish. Chien-Ju Lin (Taiwan) P.108 poster Lin C.J.*, FanChiang Y.C., Dufour S., Chang C.F.* 7-3 Activation of brain steroidogenesis and neurogenesis at the time of gonadal sex differentiation in protandrous black porgy, Acanthopagrus schlegelii .

21 Symposia- Growth and Metabolism Endocrinology 8 Dong-Joo You (Korea) P.109 poster You D.J., Park C.R., Lee H.B., Seong J.Y., Hwang J.I.* 8-1 Suppression of NF-κB signaling by nme1 through direct interaction with IKKβ. Fumitaka kondo (Japan) P.110 poster Kondo F.*, Iwai T., Miura C., Miura T. 8-2 Analysis of cholecystokinin, peptide Y and Ghrelin in the appetite and digestion of pacific bluefin tuna (Thunnus orientalis ). Li-Jung Cheng (Taiwan) P.111 poster Cheng L.J.*, Liang C.C., Kong Z.L. 8-3 Effect of β-glucans on diabetic mouse regarding to natural killer cells. Symposia- Reproductive Endocrinology 9 Cheng-Han Chou (Taiwan) P.112 poster Chou C.H.*, Chiu H.C., Wu L.T., Chen C.H., Kang Y.R., Chang Y.S., Lee T.L., Huang W.T. 9-1 Gonadotropins affected the in vitro expression of neuropeptide and steroidogenesis in the gonad of tilapia. Rang Rang Dong (China) P.113 poster Dong R. *, Yang S., Fang L., Feng R., Sun Y., Zhang Y., Xie X., Wang D. 9-2 Effects of edcs on sex as demonstrated by feminization of artificially propagated southern catfish by feeding of Limnodilus spp . Hung-Ching Chiu (Taiwan) P.114 poster Chiu H.C.*, Chou C.H., Chen J.H., Chang Y.S., Lee T.L., Tang P.C., Huang W.T. 9-3 Neuropeptide affected protein expression in the gonad of tilapia in vitro . Katsuhisa Uchida (Japan) P.115 poster Uchida K.*, Hyodo S., Watanabe T., Kagawa H. 9-4 Morphological and molecular approaches on pituitary gland of the cloudy catshark, Scyliorhinus torazame . Linyan Zhou (China) P.116 poster Yu X., Wu L., Xie L., Yang S., Shi H., Wang D., Zhou L.* 9-5 Characterization of two duplicated star genes in a teleost, Nile tilapia (Oreochromis niloticus ). Lina Sun (China) P.117 poster Sun L., Jiang X., Xie Q., Zhou L., Nagahama Y., Wang D.* 9-6 Transdifferentiation of differentiated ovary into functional testis by long term treatment of aromatase inhibitor in Nile tilapia. Guang-Li Li (China) P.118 poster Li G.L*, Zhang M.Z., Deng S.P., Zhu C.H. 9-7 Molecular cloning and gene expression of Foxl2 in spotted scat (Scatophagus argus ). Zhang Yan (Japan) P.119 poster Zhang Y.*, Yamamoto Y., Sarida M., Hattori R.S., Strüssmann C.A. 9-8 Genetic sex determination in pejerrey: evidence from the testis-determining gene, AMHY. Shu Chun Cheng (Taiwan) P.120 poster Cheng S.C., Ting T.L., Kong Z.L.* 9-9 Effects of Basswood Culture Antrodia cinnamomea Ethanol Extracts on Reproductive Function in Streptozotocin - Nicotinamide Induced Diabetic Male Rats.

22 Masahiro Matsumoto (Japan) P.121 poster Matsumoto M.*, Uchida K., Watanabe T., Hyodo S., Nakamura M., Sato K., Kagawa H. 9-10 Egg shell formation in the oviducal gland of the cloudy catshark, Scyliorhinus torazame , elasmobranchs. Wen-Shiun Yueh (Taiwan) P.122 poster Yueh W.S.*, Jeng S.R., Haung P.H., Chiang S.D., Chang C.F. 9-11 The characters of final oocyte maturation in largescale mullet, Chelon macrolepis. Yung-Sen Huang (Taiwan) P.123 poster Zhu C.-W. , Lee Y.-H. , Chang C.-F., Huang Y.-S.* 9-12 Effects of the Cilostazol (a specific phosphodiesterase type Ⅲ inhibitor) on primary- secondary oocyte development in the induced Japanese eel. Chi Chen (Taiwan) P.124 poster Chen C.*,Wu G.C., Chang C.F. 9-13 The role of germ cell differentiation genes during testicular development and germ cell proliferation in black porgy, Acanthopagrusschlegelii . Jia-Wun Luo (Taiwan) P.125 poster Luo J.W.*,Wu G.C., Chang C.F. 9-14 The soma fate alter by the ectopic oocytes in the regenerated testis in black porgy, Acanthopagrusschlegelii . Ching-Hsuan Fan (Taiwan) P.126 poster Fan C.H.*, Wu G.C., Chang C.F. 9-15 The interaction between brain and gonad during gonadal differentiation in black porgy, Acanthopagrus schlegelii . Wei-Guan Tey (Taiwan) P.127 poster Tey W.G.*, Wu G.C., Chang C.F. 9-16 The functional change of germ and soma during sex reversal in Orange-spotted grouper. Symposia- Endocrinology of Stem Cell Biology 10 Ji Yeon Kim (Korea) P.128 poster Kim J.Y.*, Kim Y., Chung S., Jung H., Park S.H., Son G.H. 10-1 Identification of molecular signatures related to ischemic heart failure by gene expression analysis on human myocardial autopsy materials. Yunmi Kim (Korea) P.129 poster Kim Y.*, Kim J.Y., Kim J.W., Hwang J.J., Park S.H., Son G.H. 10-2 Cell death-associated 28S ribosomal RNA cleavage in postmortem tissues.

23 Abstract Contents Keynote Lecture KL-1 What we learned from Dr. Hideshi Kobayashi, the Founder of Comparative Endocrinology in Japan Takei Y. 1 KL-2 Function and regulation of steroids in vertebrates Chung B.C. 2 KL-3 Functional significance of kisspeptins in fish Parhar I.S. 3 KL-4 Regulation of ovarian development and oocyte maturation in fish Nagahama Y. 4 KL-5 Lipid induced insulin resistance is associated with immunity Cancelled 5 KL-6 BioClock Timing System: Impact of circadian clock molecule, Rev-erb alpha on mood regulation by dopamine Kim K. 6 KL-7 Melatonin in gut and pineal: A testimony of diversity in temporal pattern and its synchronizer Maitra S.K. 7 KL-8 Structure and Functions of Gonadotrophin-inhibitory hormone in teleost Zhang Y., Li S., Qi X., Liu X., Lin H.* 8 KL-9 Progress in comparative endocrinology by discovering novel neuropeptides and neurosteroids Tsutsui K. 9

Symposium Lecture SL-1 Impact of whole genome duplications on vertebrate neuroendocrine systems with special focus on teleosts Dufour S.*, Lafont A.G., Maugars G., Pasquier J., Rousseau K. 11 SL-2 Novel pituitary actions of TAC3 gene products in fish model Hu G., He M., Ko W.K.W., Wong A.O.L.* 12 SL-3 Transgenic Fish and its Reproductive Containment Strategies Hu W.* and Zhu Z.Y. 13

24 SL-4 Pleiotropic functions of proopiomelancortin in fish Takahashi A.*, Kobayashi Y., Mizusawa K. 14 SL-5 Aromatase, estrogens and brain development in teleost fishes Kah O.*, Cano-Nicolau J., Nasri H., Diotel N., Vaillant C., Gueguen M.M., Pellegrini E. 15 SL-6 Role of tachykinins and tachykinin receptors in fish reproduction Ogawa S. 16 SL-7 Tug-of-war of gonadal soma transdifferentiation in the hermaphroditic fish Wu G.C.*and Chang C.F. 17 SL-8 Regulation of oligodendrocyte development by hedgehog signaling in the zebrafish CNS Chung A.Y., Kim S., Kim E., Jeong I., Kim D., Park H.C.* 18 SL-9 Migration and differentiation of neural progenitors following brain injury in mice Sun W. 19 SL-10 Evolutionary genomics for identification of novel neuropeptides and their receptors Seong J.Y. 20 SL-11 Androgen receptor regulates stemness and differentiation of stem cells Kang H.Y. 21 SL-12 Transmembrane peptides as unique tools to demonstrate the in vivo action of a GPCR hetero-complex of secretin and angiotensin Lee L.T., Ng S.Y., Chu J.Y., Sekar R., Harikumar K.G., Miller L.J., Chow B.K.* 22 SL-13 Pheromonal control of final oocyte maturation and lunar-related spawning in grouper Soyano K.*, Izumida D. , Nakamura M. 23 SL-14 Appetite regulation in desert-adapted spinifex hopping mice during water deprivation Donald J.A.*, Hamid N.K.A., McLeod J.L., Takei Y 24 SL-15 Regulatory contribution of Gs-proteins to 1-methyladenine production in starfish ovarian follicle cells stimulated by relaxin-like gonad-stimulating substance Mita M.*, Haraguchi S., Takeshige Y., Watanabe M., Tsutsui K. 25

25 SL-16 Serotonin and melatonin mitigate the stress response and promote the ease response in air-breathing fish Peter M.C. 26 SL-17 Control mechanisms of meiotic initiation during spermatogenesis in Japanese eel (Anguilla japonica). Miura T.*, Higuchi M., Miura C. 27 SL-18 Orexin A induces anxiety-like behavior in goldfish Matsuda K.*, Shibata H., Nakamachi T. 28 SL-19 Role of sex steroids on sex determination and maintenance in teleosts Sun L., Tao W., Li M., Jang D., Zhou L., Wang D.* 29 SL-20 Ghrelin and food acquisition in japanese eel Yada T.*, Kaifu K., Kaiya H., Tsukamoto K. 30 SL-21 Kit System and Its Role in the Paracrine Regulation of Follicle Development in the Zebrafish Ge W.* and Yao K. 31 SL-22 Neuroendocrine response to pathogen and its implication in the metabolism of crustacean host Lin L.-J., Chang Y.-S., Huang W.-S., Tien Y.-C., Chen Y.-J., Lee C.-Y.* 32 SL-23 Neuropeptides and peptide hormones of the ascidian, Ciona intestinals: Comparative views to novel follicle growth. Satake H. 33 SL-24 Anti-osteoporotic effects of phytoestrogens containing plant Pueraria mirifica: in vitro and in vivo approaches Malaivijitnond S.*, Urasopon N., Tiyasatkulkovit W., Kittivanichkul D., Charoenphandhu N. 34 SL-25 Catecholestrogens in final oocyte maturation in the catfish Heteropneustes fossilis: an overview Joy K.P. 35 SL-26 Does bioaccumulation and biomagnification of EDC’s take place in marine fish? Dahms H.-U.* and Kim J.-H 36 SL-27 Rhythms in fish reproduction: possible involvement of dopaminergic activity in tidal-related spawnings of a tropical wrasse Takemura A.*, Takeuchi Y., Hur S.P. 37

26 SL--28 Neuro-endocrine control of the environmental adaptabilitty in fish – a study of fish caudal neurosecretoryr system Lu W. 38 SL--29 A sleep-stabilizingn pathway in Drosophila composed of sex peptide receptor and its ligand, the myoinhibitory peptide Kim Y.J. 39 SL--30 New insights into hormonal control of body fluid ion homeoostasis: zebrafish as a model Hwang P.P. 40 SL--31 Yolk formation in a stony coral Euphyllia ancora (Cnidaria, Anthozoa): insight into the evolution of vitellogenesis in non-bilaterian animals. Shikina S.*, Chen C.J., Chung Y.J., Shao Z.F., Liou J.Y., Tseng H.P., Lee Y.H., Chang C.F. 41 SL--32 Impacts of Early Life Stress on Anxiety and Reproduction Soga T.* and Parhar I.S. 42 SL--33 Xenopus as a model to studdy neuronal axons Jung H. 43 SL--34 Evolution and origin of seaasonal sensor in vertebrates Yoshimura T. 44

Oral Presentation OP-1 Epigenetic modulation of rhythmic GnRH gene transcriptiion by dynamic histone modifications Kim H.-D.* , Chung S., Son G.H., Kim K. 45 OP-2 Exposure to a maternal n-3 fatty acid-deficient diet during the brain development enhance the activity and dysregulation of hypothalamic-pituitary-adrenal axis responses to stress in rat offspring later in life Hsieh Y.J. and Su H.M.* 46 OP-3 Identification of small molecule Cryptochrome inhibitor modulating the molecular circadian clock Chun S.K.* , Chung S., Jang J, Son G.H., Suh Y.G., Kim K. 47 OP-4 Identification of a novvel transglutaminase gene that is differentially expressed between the sexes in the medaka brrain Maehiro S.*, Oka Y., Okubo K. 48

27 OP-5 Glucocorticoid-mediated Period2 induction delays the phase of circadian rhythm Cheon S. *, Park N., Kim K. 49 OP-6 Involvement of corticotropin-releasing hormone and corticosteroid receptors in the brain-pituitary of tilapia during cronic and acute salinity stress Aruna A.*, Nagarajan G., Chang C.F. 50 OP-7 Histomorphological study on the development of thymus of Oryctolagus cuniculus Hazarika J.K. 51 OP-8 Effects of cadmium on dopaminergic and serotonergic neurons and involvement of estrogen signaling pathway during early development of zebrafish Sugiyono* and Kishida M. 52 OP-9 Characterization of Dmrt gene family in a scleractinian coral, Euhyllia ancora Chen C.J. *, Shinya S., Chang C.F. 53 OP-10 Neuroendocrine gene expression levels at different ovarian developmental stages in wild Japanese eel Jeng S.R.*, Pen Y.T., Yueh W.S., Dufour S., Chang C.F. 54 OP-11 A novel pterostilbene derivative supresses prostate cancer cell growth irrespective of androgen receptor status Nikhil K, Sharan S, Roy P* 55 OP-12 Molecular cloning and characterization of feeding regulator-NPY and NPF genes and its applications in attractant development for finfish and crustacean Lu J.K.*,Song J.X., Chen T.Y. 56 OP-13 Development of a DNA microarray and its application to the elucidation of gene transcription dynamics in ovary fragments of Litopenaeus vannamei incubated with sinus gland extract Okutsu T.*, Kang B.J., Bae S-H., Wilder M.N. 57 OP-14 A novel glucagon-related peptide (GCRP) and its receptor GCRPR account for coevolution of their family members in vertebrates Park C.R., Moon M.J., Park S., Kim D.K., Cho E.B., Seong J.Y., Hwang J.I.* 58

28 OP-15 Genetic polymorphisms and DNA methylation in exons CpG rich regions of PACAP gene and its effect on mRNA expression and growth traits in Half smooth tongue sole (Cynoglossus semilaevis) Si Y. *, Wen H., He F. 59 OP-16 Effects of neuropeptide, neurosteroidogenic enzymes and estrogen receptors in the early brain of protogynous fish: an in vivo approach Nagarajan G.*, Aruna A., Chang C.F. 60 OP-17 Endocrine disruptor TCDD induces zebrafish CYP3A65 transcription via AHR2-dependent pathway Chang C.T., Chung H.Y., Su H.T., Tseng H.P., Tzou W.S., Hu C.H.* 61 OP-18 Suppression of aromatase by fadrozole and morpholino antisense oligonucleotide alters activity of dopaminergic neuron in early development of zebrafish Fatimah R.* and Kishida M. 62 OP-19 Manipulation expression FSH (Follicle Stimulating Hormone) from striped catifsh (Pangasianodon hypopthalmus) with hormonal manipulation during gonadal early development Rafiuddin, M.A*., Sudrajat, Agus Oman#, Widyastuti U., Suharsono, Alimuddin 63 OP-20 Changes in bone mineral density and content in pre-, peri- and post-menopausal cynomolgus monkeys Kittivanichkul D.* and Malaivijitnond S. 64 OP-21 Controlled feeding inhibits growth reduction associated with spawning in farmed yellowtail Miura C.*, Yoshihara Y. , Hayashi D. , Miura T. 65 OP-22 Integrated ecological impact of the equipment on the soft-shelled turtle breeding environment and growth and metabolism Jiang Y.l., Zhang J., Li X., Chen H.l., Hou G.J, Wang l. 66 OP-23 Conserved sex-specific timing of meiotic initiation during sex differentiation in the protandrous black porgy Acanthopagrus schlegelii. Lau E.L.*, Lee M.F., Chang C.F. 67 OP-24 Muscle-specific expression of giant grouper growth hormone in transgenic zebrafish leads to muscle hypertrophy and hyperplasia Gong H.Y. *, Chin H.Y., Tai T.Y., Tseng P.C., Huang S.C. 68

29 OP-25 Identification of interacting partners of gonad-inhibiting hormone of Penaeus monodon by yeast two-hybrid analyses. Tomy S.*, Saikrithi P. , James N. , Otta S.K., Balasubramanian C.P. , Subramoniam T., Ponniah A.G. 69 OP-26 Molecular cloning of four MRFs (MyoD、Myf5、MRF4、Myogenin) and their expression patterns during fasting and refeeding in orange-spotted grouper, Epinephelus coioides Zhang Y.*, Sang Q., Chen H., Zhang H., Liu X., Lin H. 70 OP-27 The role of melatonin in synchronizing semilunar spawning rhythm of the mudskipper Boleophthalmus pectinirostris Hong W.*, Hong L., Zhu W., Chen S. 71 OP-28 Restoration of lung degeneration by Pueraria mirifica plant Nishitani C.*, Limjunyawong N., Phumsatitpong C., Jaroenporn S., Nagaoka K., Watanabe G., Malaivijitnond S. 72 OP-29 Fecal testosterone profile and sperm quality of dairy buffalo from north sumatera Maheshwari H.*, Boediono A., Andriyanto, Handarini R., Nalley W.M., Yulnawati, Sjahfirdi L., Astuti P. 73 OP-30 Molecular cloning and dimorphic expression of growth hormone (GH) in female and male spotted scat (Scatophagus argus) Deng S.P., Wu Bo, Zhu C.H., Li G.L. 74 OP-31 A super molecule reverts lipid induced insulin resistance Priyajit C.*, Soma S.*, Sandip M.*, Soumik A., Maitrayee P., Sushmita B., Chaudhuri M.K., Subeer M., Samir B. 75 OP-32 Molecular characterization of adiponectin receptors, its mRNA expression, and subcellular location in the orange-spotted grouper (Epinephelus coioides) Qin C., Wang B., Sun C., Jia J., Li W.* 76 OP-33 Molecular cloning, characterization and expression profiles of Dax1 in protogynous orange-spotted grouper (Epinephelus coioides) Liu, X.*, Lin M., Li S., Chen H., Zhang Y., Lin H. 77 OP-34 Is tide-related locmotion rhythm controlled by external tidal stiumali or internal melatonin in the barred mudskipper? Ikegami T.*, Oyama Y., Takemura A. 78 OP-35 The functions of estradiol-17β during oocyte maturation in multiple spawning fish, yellowfin porgy Yueh W.S. *, Jeng S.R., Sun S.F., Kuo S.F. and Chang C.F. 79

30 OP-36 Effects of Nonylphenol on Vitellogenin Gene Expression and Gonad Development in Macrobrachium rosenbergii Zhu C.*, Li Y., Chen L., Chen S., Li G. 80 OP-37 Correlation between PTEN expression levels and ovarian development in the induced Japanese eel Huang Y.-S.1*, Gwo J.-C.2, Chen Y.-F.1, Chang C.-F. 81 OP-38 Herbicide utilization as an environmental stressor on reproductive/endocrine functions of sentinel species in agricultural areas Kitana N. *, Maneein, R., Thammachoti P., Thitiphuree, T., Kitana, J., Khonsue, W., Varanusupakul, P. 82 OP-39 Convergent evolutions of epithelial cell differentiation and neuroendocrine regulation in cephalopod and fish embryos Chen Y.C., Sung P.H., Lee J.R., Hu M.Y., Hwang P.P., Tseng Y.C. 83 OP-40 Osmoregulation in the crab-eating frog, Fejervarya cancrivora Uchiyama M. 84 OP-41 The presence and role of gnrh in the reproduction of the mud crab, Scylla paramamosain Feng B.Y., Bao C.C., Ye H.H.* 85 - - OP-42 Nitrate (NO3 ) and nitrite (NO2 ) perturb the activity of dopaminergic neuron during early development of zebrafish Jannat M. and Kishida M.* 86

Poster Presentation PP1-1 CXCL14 enhances proliferation and migration of cancer cells overexpressing the glycoproteins. Park C.R.*, You D-.J., Kim D-.K., Moon M.J., Lee C., Oh S-.H., Ahn C., Seong J.Y., Hwang J-.I. 87 PP1-2 A novel gonadotropin-releasing hormone-Ⅱ analog induces apoptosis prostate cancer cells. Park S.*, Seong J.Y., Hwang J. I. 88 PP1-3 Transcriptional activation of gonadotropin-inhibitory hormone by corticosterone. Son Y.L.*, Ubuka T., Narihiro M., Fukuda Y., Hasunuma I., Yamamoto K., Belsham D.D., Tsutsui K. 89

31 PP1-4 Molecular responses in the migrating Takifugu obscurus - gene regulation depends on tissue and reading time. Kim J.-H., Han K.-N. , Dahms H.-U.* 90 PP2-1 Stress and immune response of the rice frog Fejervarya limnocharis living in areas with different agricultural activity. Jantawongsri K.*, Kitana J., Kitana N. 91 PP2-2 Incubating temperature affects development of endocrine cells in gonad of Malayemys macrocephala, a freshwater turtle from tropical asia. Pewphong R.*, Kitana J., Kitana N. 92 PP2-3 Stress and health status based on hematological parameters of the rice field frog Hoplobatrachus rugulosus in captivity. Kitana, J.*, Meesawat S., Thammachoti P., Thitiphuree, T., Maneein, R., Chiewchanchai M., Pariyanonth P., Khonsue W., Kitana N. 93 PP2-4 Endothelin-1 regulates transepithelial H+ secretion in zebrafish. Guh Y.J.*, Tseng Y.C., Yang C.Y., Hwang P.P. 94 PP2-5 Endocrine effects of copper in aquatic animals – a review. Shim D.M.-C., Dahms H.-U.*, Yang J., Wang L. 95 PP2-6 Differential expression of arginine vasotocin and isotocin mRNA to external salinity stress in black porgy (Acanthopagrus Schlegelii) hypothalamus and pituitary. Aruna A.*, Nagarajan G., Chang C.F. 96 PP2-7 The calciotropic hormone vitamin D regulates ion homeostasis in zebrafish (Danio rerio). Lin C.H.*, Hu H.J., Hwang P.P. 97 PP3-1 Evolution and expansion of glp1 and glp1 receptor family. Yun S.*, Moon M.J., Park C.R., Cho E.B., Hwang J-.I., Seong J.Y. 98 PP3-2 Evolution and possible roles of heat shock protein 60 (Hsp60) in germline of metazoan animals. Pan H.H.*, Shikina S., Shao Z.F., Chang C.F. 99 PP4-1 Pineal allopregnanolone saves purkinje cells from apoptosis in the cerebellum. Haraguchi S.*, Hara S., Ubuka T., Tsutsui K. 100 PP4-2 Potential role for neurosteroids in the early brain of orange-spotted grouper. Nagarajan G.*,Aruna A., Chang C.F. 101

32 PP5-1 The expression and regulation of aromatase and estrogen receptors in Japanese eel. Jeng S.R.*, Yueh W.S., Dufour S., Kah O., Chang C.F. 102 PP6-1 Neuropeptide Y involves in the regulation of somatic growth of the Pacific abalone. Moriyama S.*, Kawamura M., Kodera Y., Uchida K., Amano H. 103 PP6-2 Expression of heat shock protein 60 (Hsp60) in the germline cells of a scleractinian coral Euphyllia ancora (Cnidaria, Anthozoa). Shikina S.*, Shao Z.F., Chung Y.J., Chang C.F. 104 PP6-3 Exploration of the genes involving in sexual reproduction in a stony coral Euphyllia ancora. Chiu Y.L.*, Shikina S., Chang C.F. 105 PP7-1 Dose 11KT increase GnRH3 neurons by enhancing neurogenesis in female mozambique? Narita Y.*, Okada N., Tsutiya A., Kaneko T., Ohtani-Kaneko R. 106 PP7-2 Sexual bipotentiality of the brain for olfactory processing and sexual behavior in goldfish. Shinohara Y.* and Kobayashi M. 107 PP7-3 Activation of brain steroidogenesis and neurogenesis at the time of gonadal sex differentiation in protandrous black porgy, Acanthopagrus schlegelii. Lin C.J.*, FanChiang Y.C., Dufour S., Chang C.F.* 108 PP8-1 Suppression of NF-κB signaling by nme1 through direct interaction with IKKβ. You D.J., Park C.R., Lee H.B., Seong J.Y., Hwang J.I.* 109 PP8-2 Analysis of cholecystokinin, peptide Y and Ghrelin in the appetite and digestion of pacific bluefin tuna (Thunnus orientalis). Kondo F., Iwai T., Miura C., Miura T. 110 PP8-3 Effect of β-glucans on diabetic mouse regarding to natural killer cells. Cheng L.J.*, Liang C.C., Kong Z.L. 111 PP9-1 Gonadotropins affected the in vitro expression of neuropeptide and steroidogenesis in the gonad of tilapia. Chou C.H.*, Chiu H.C., Wu L.T., Chen C.H., Kang Y.R., Chang Y.S., Lee T.L., Huang W.T. 112

33 PP9-2 Effects of edcs on sex as demonstrated by feminization of artificially propagated southern catfish by feeding of Limnodilus spp. Dong R. *, Yang S., Fang L., Feng R., Sun Y., Zhang Y., Xie X., Wang D. 113 PP9-3 Neuropeptide affected protein expression in the gonad of tilapia in vitro. Chiu H.C.*, Chou C.H., Chen J.H., Chang Y.S., Lee T.L., Tang P.C., Huang W.T. 114 PP9-4 Morphological and molecular approaches on pituitary gland of the cloudy catshark, Scyliorhinus torazame. Uchida K.*, Hyodo S., Watanabe T., Kagawa H. 115 PP9-5 Characterization of two duplicated star genes in a teleost, Nile tilapia (Oreochromis niloticus). Yu X., Wu L., Xie L., Yang S., Shi H., Wang D., Zhou L.* 116 PP9-6 Transdifferentiation of differentiated ovary into functional testis by long term treatment of aromatase inhibitor in Nile tilapia. Sun L., Jiang X., Xie Q., Zhou L., Nagahama Y., Wang D.* 117 PP9-7 Molecular cloning and gene expression of Foxl2 in spotted scat (Scatophagus argus). Li G.L*, Zhang M.Z., Deng S.P., Zhu C.H. 118 PP9-8 Genetic sex determination in pejerrey: evidence from the testis-determining gene, AMHY. Zhang Y.*, Yamamoto Y., Sarida M., Hattori R.S., Strüssmann C.A. 119 PP9-9 Effects of Basswood Culture Antrodia cinnamomea Ethanol Extracts on Reproductive Function in Streptozotocin - Nicotinamide Induced Diabetic Male Rats. Cheng S.C., Ting T.L., Kong Z.L.* 120 PP9-10 Egg shell formation in the oviducal gland of the cloudy catshark, Scyliorhinus torazame, elasmobranchs. Matsumoto M.*, Uchida K., Watanabe T., Hyodo S., Nakamura M., Sato K., Kagawa H. 121 PP9-11 The characters of final oocyte maturation in largescale mullet, Chelon macrolepis. Yueh W.S.*, Jeng S.R., Haung P.H., Chiang S.D., Chang C.F. 122

34 PP9-12 Effects of the Cilostazol (a specific phosphodiesterase type Ⅲ inhibitor) on primary-secondary oocyte development in the induced Japanese eel. Zhu C.-W. , Lee Y.-H. , Chang C.-F., Huang Y.-S.* 123 PP9-13 The role of germ cell differentiation genes during testicular development and germ cell proliferation in black porgy, Acanthopagrusschlegelii. Chen C.*,Wu G.C., Chang C.F. 124 PP9-14 The soma fate alter by the ectopic oocytes in the regenerated testis in black porgy, Acanthopagrusschlegelii. Luo J.W.*,Wu G.C., Chang C.F. 125 PP9-15 The interaction between brain and gonad during gonadal differentiation in black porgy, Acanthopagrus schlegelii. Fan C.H.*, Wu G.C., Chang C.F. 126 PP9-16 The functional change of germ and soma during sex reversal in Orange-spotted grouper. Tey W.G.*, Wu G.C., Chang C.F. 127 PP10-1 Identification of molecular signatures related to ischemic heart failure by gene expression analysis on human myocardial autopsy materials. Kim J.Y.*, Kim Y., Chung S., Jung H., Park S.H., Son G.H. 128 PP10-2 Cell death-associated 28S ribosomal RNA cleavage in postmortem tissues. Kim Y.*, Kim J.Y., Kim J.W., Hwang J.J., Park S.H., Son G.H. 129

Abstracts

Keynote Lecture‐1

What we learned from Dr. Hideshi Kobayashi, the Founder of Comparative Endocrinology in Japan

Takei Y. Laboratory of Physiology, Department of Marine Bioscience, Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Japan e-mail: [email protected]

At the end of 2012, the comparative endocrinologists in the world were struck by the sudden death of our great leader, Professor Hideshi Kobayashi, who is a real founder of comparative endocrinology in Japan. From his experience as secretary general of the 3rd International Symposium on Comparative Endocrinology held in Japan in 1962, he recognized the importance of comparative endocrinology and made tremendous efforts to organize a new society in Japan. His efforts were realized by inauguration of the Japan Society for Comparative Endocrinology in 1975 with him as the first president. He has also contributed greatly to the establishment of the Asia Oceania Society for Comparative Endocrinology (AOSCE), and one of his postdoctoral associates, Professor Susumu Ishii, served as the first president. As his last student of the University of Tokyo, the initial part of my talk will be devoted to introduce Dr. Kobayashi’s profound accomplishments in comparative endocrinology in the world. Then, I would like to introduce our works on the evolution of body fluid regulation in vertebrates, which have been influenced greatly by his tutelage. The body fluid regulation has been evolved greatly in vertebrates during the transition from aquatic to terrestrial life. In terrestrial tetrapods, the mechanisms have been developed for the retention of water and ions, particularly Na+ and Cl-. In aquatic fishes, on the other hand, regulation of water and ions are in the opposite direction; water is excreted and ions are retained when they are in fresh water but water is retained and ions are excreted when they are in seawater. By utilizing such differences among habitats, we can make an original story on the evolution of osmoregulatory hormone actions and can extract an essential action of the hormone. Through such experiences, I would emphasize how much comparative endocrinology is attractive. If the time allows, I would also introduce how comparative endocrinology can contribute to general endocrinology through discovery of new hormones. Takei et al., Gen. Comp. Endocrinol. 185: 107-108, 2013. Takei et al., Front. Neuroendocrinol. 28: 143-160, 2007.

Keyword: history of comparative endocrinology, osmoregulation, environmental adaptation

1 Keynote Lecture‐2

Function and regulation of steroids in vertebrates

Chung B.C. Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan. email: [email protected]

Steroids are small lipophilic molecules derived from cholesterol. They are synthesized by a set of cytochromes P450 enzymes are present in all vertebrates. To understand the regulation and functions of steroids, we depleted Cyp11a1, which encodes cytochrome P450scc (cholesterol side-chain cleavage enzyme), in the first step of steroid biosynthetic pathway. Cyp11a1 knockout mice do not synthesize any steroid and die neonatally. Yet Cyp11a1 knockout fetuses survived because maternal steroid supplies. When the promoter of the Cyp11a1 gene was mutated, the production of CYP11A1 in the adrenal decreased by 15-fold, but these mice had normal life span. These mice were more prone to infection, and could not recover from inflammation easily. These studies reveal the importance of steroids in neonatal life and in stress response. To investigate the functions of steroids during embryogenesis, we resorted to oviparous zebrafish whose embryos develop outside maternal protection. When cyp11a1 was depleted by the injection of an antisense morpholino oligonucleotide, zebrafish embryos had shorter body axis because of a reduction of embryonic morphogenetic cell migration. This defect was alleviated when the immediate steroid product of CYP11A1, pregnenolone, was supplied. We found that pregnenolone controlled zebrafish embryonic cell migration and consequently morphogenesis by binding to a microtubule associated protein CLIP-170, thus stabilizing microtubules and promote cell migration. We found that pregnenolone changed the conformation to activate CLIP-170, thus promoting microtubule polymerization and increasing cell motility. This study reveals a previously unidentified role of steroids during early embryogenesis.

Key words: zebrafish, steroid, Cyp11a1

2 Keynote Lecture‐3

Functional significance of kisspeptins in fish

Parhar I.S. Brain Research Institute, School of Medicine and Health Sciences, Monash University Malaysia, PJ 46150, Malaysia. e-mail: [email protected]

Kisspeptin, encoded by the kiss1 gene, has the ability to activate its receptor, Kiss-R and known to play an essential role in vertebrate reproduction. We have identified two kisspeptin (kiss1 and kiss2) and two kisspeptin receptor types (Kiss-R1 and Kiss-R2) genes in the teleost species. In the zebrafish brain, kiss1 is expressed in the ventral habenula, while kiss2 is expressed in the hypothalamic nuclei. However, the functional significance and the existence of the two kisspeptin types in the same animal species remain unclear. Recently, we have identified the potential role of Kiss1 in serotonergic modulation as well as in alarm substance (AS)-evoked fear responses in the zebrafish. We further substantiated the role of habenular Kiss1 neurons using Kiss1 peptide conjugated with saporin (ribosome-inactivating protein), to selectively inactivate Kiss-R1 expressing neurons. Kiss1-SAP injection significantly altered the AS-evoked fear responses. These observations suggest that Kiss1 could be involved in social behaviours. On the other hand, the Kiss2 neural population in the hypothalamic nuclei has been viewed as a potent regulator of reproductive function in fish. We have previously identified Kiss-R (kissr2) in the Nile tilapia, and have shown its expression in gonadotropin-releasing hormone (GnRH) neurons. Kiss2 neurons are sensitive to thyroid hormone and to environmental temperature, factors that are important for the control of reproduction. These results suggest that the two kisspeptin systems may play independent roles in non-reproductive and reproductive functions.

Keyword: reproduction, GPR54, behaviour

3 Keynote Lecture‐4

Regulation of ovarian development and oocyte maturation in fish

Nagahama Y. South Ehime Fisheries Research Station, Ehime University, Matsuyama 790-8577, Japan e-mail: [email protected]

The two main roles of the ovary are the production of steroid hormones and the generation of mature oocytes. This lecture highlights recent advances in our understanding of regulation of ovarian development and oocyte maturation in fish. Emphasis will be on medaka (Oryzias latipes) systems, where many of these new findings have taken place. Loss- and gain-of-function evidence indicates that the formation of the ovary is initiated by estradiol-17 (E2) and estrogen receptor 2, together with Foxl2 and R-spondin 1. During oocyte growth, E2 stimulates vitellogenin (yolk precursor) synthesis in the liver. Recently, we showed that the depletion of E2 in adult breeding females of medaka and Nile tilapia resulted in a functional female-to-male sex reversal, indicating that gonochoristic fish maintain their sexual plasticity until adulthood and E2 plays a critical role in maintaining the female phenotype. After the oocyte is fully grown, it becomes ready for final oocyte maturation, which is regulated by three mediators, gonadotropin (LH), maturation-inducting hormone (MIH), and maturation-promoting factor (MPF). LH acts on the ovarian follicle layer to produce MIH (17,20-dihydroxy-4-pregnen-3-one, DHP). A large scale ovarian follicle transcriptome analysis utilizing a custom-designed medaka microarray resulted in the isolation of novel 17β-HSD12-like gene, which is dramatically up-regulated during maturational stage of medaka ovarian follicles. The recombinant 17-HSD12-like protein is able to efficiently convert 17-hydroxprogesterone (DHP precursor) to DHP. We conclude that 17β-HSD12-like protein is the major enzyme for the production of MIH in medaka. The DHP signal received on the oocyte surface is transduced into the oocyte cytoplasm for the formation and activation of MFP (cdc2 and cyclin B), the final mediator of oocyte maturation.

4 Keynote Lecture‐5

Cancelled Lipid induced insulin resistance is associated with immunity

Bhattacharya S. Visva-Bharati (A Central University), Santiniketan, India email: [email protected]

Adipocytes are the major cells in adipose tissue, there are other cells too, but these are the cells which could uptake lipid, store them and metabolize them. Adipocytes are endocrine cells, several hormones such as leptin, adiponectin etc are released from these cells and any defect in adipocytes would convert them to inflammatory cells, which secret pro-inflammatory cytokines therefore aggravate inflammation that causes insulin resistance. It is lipid which induces inflammatory status to adipocytes. We know that TLR4 or TLR2 receptors are on the membrane of macrophage cells which serve as the prime member of innate immunity system. Interestingly, TLR4 receptors in good numbers are also localized on the membrane of adipocytes which mediate inflammatory response. Question is how? This riddle has been fairly cleared by a current report from our laboratory. Previous findings implicated that fatty acid (FA) binds to TLR4 and triggers inflammatory signal. We showed that FA effect on adipocyte inflammation is not direct, but Fet-A binds to FA and present it to TLR4, then FA-Fet-A-TLR4 forms a ternary complex that activates NF-κB resulting overexpression of TNFα, IL-6, IL-1β genes, when these proinflammatory cytokines secreted from adipocytes they cause insulin resistance. Interestingly, lipid induced inflammation of adipose tissue attracts macrophage, they move towards adipose tissues, infiltrate into them and became resident there, termed as adipose tissue macrophage (ATM). ATMs are polarized from anti-inflammatory M2 subtype to proinflammatory M1 phenotype and vigorously release proinflammatory cytokines. Question is – Who drives them to adipose tissue and who converts them from M2 to M1 phenotype? All these remain unresolved. MCP1 or Monocyte chemoattractant protein 1, was thought to be acting as a chemoattractant, but recently found to be only partially potent. One can imagine the peak inflammatory status when both adipocytes and macrophages are cohabitants and releasing inflammatory cytokines which inflicts crucial metabolic disorders. Regulatory mechanism behind the macrophage migration and polarization in the niche of these endocrine cells remains yet unclear, primarily because of the lack of identification of signal(s) or factor(s), which appears to be originated from the adipose tissue ambience. We have recently discovered an endogenous signal which participates in this regulation; this will be discussed during my deliberation.

5 Keynote Lecture‐6

BioClock Timing System: Impact of circadian clock molecule, Rev-erb alpha on mood regulation by dopamine

Kim K. Department of Brain & Cognitive Science and Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea email: [email protected]

Circadian rhythm is involved in the regulation of physiology and behavior in mammals. The mammalian circadian timing system is organized in a hierarchy: The central circadian pacemaker residing in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus orchestrates numerous subsidiary local clocks in several regions of the brain and peripheral tissues. The molecular clock machinery has two interlocking feedback loops that drive the circadian oscillation in cell-autonomous and self-sustainable manner even at the single-cell level. It works through the transcription/translation and post-translational modifications that contribute to the fine regulation of molecular circadian clockwork. Following a brief overview of recent advance in chronobiology, I will discuss the novel functional link between the mood regulation by midbrain dopamine(DA) and circadian timing system through Rev-erb alpha. Genetic abrogation of Rev-erb alpha gene or pharmacological inhibition of Rev-erb alpha activity in the ventral midbrain induced mania-like behaviors in association of hyperdopaminergic state. Rev-erb alpha represses tyrosine hydroxylase(TH, a rate-limiting step of DA synthesis) gene transcription by competition with Nurr1(a crucial nuclear receptor for DA neuronal development and maintenance) and functions driving circadian expression of DA system. Furthermore Rev-erb alpha represses TH gene transcription by recruiting histone deacetylase 3(HDAC3) to the promoter region resulting in suppressive histone deacetylation.

Keyword: circadian rhythm, mood regulation, rev-evb, dopamine

6 Keynote Lecture‐7

Melatonin in gut and pineal: A testimony of diversity in temporal pattern and its synchronizer.

Maitra S.K. Department of Zoology, Visva-Bharati University, Santiniketan - 731 235, India. e-mail: [email protected]

Melatonin (5-methoxy-N-acetyltryptamine) is synthesized in both the pineal and gut in different vertebrates. In each tissue, arylalkylamine N-acetyltransferase (AANAT) is the key regulator of melatonin biosynthesis. While melatonin in the pineal is synthesized rhythmically with a daily peak at mid-night in synchronization with environmental light-dark cycle, temporal pattern and its synchronizer of gut melatonin in any species remained unknown. Accordingly, daily and seasonal profiles of melatonin and AANAT in the pineal and three broad regions of gut were studied under natural as well as diverse experimental conditions to identify their synchronizer in a surface dwelling carp Catla catla, for which the photo-neuro-regulatory mechanisms in pineal were known. In a daily cycle, concentrations of melatonin and the band intensity of a ~23kDa AANAT protein in each gut segment, irrespective of sampling months, exhibited a peak at midday, while such variables in the pineal showed maximum values during dark-phase. Experimental studies under altered long- (LP) or, short photoperiods (SP) or continuous illuminations (LL) or darkness (DD) revealed that none of the employed lighting conditions had any significant effects on daily profiles of melatonin and AANAT in gut. Conversely, rhythm features of pineal melatonin and AANAT were either modified under LP or SP or lost under LL and DD. Additional study on the effects of fasting, re-feeding, and time of food supply, notably, depicted feeding schedule as the synchronizer of daily profiles of gut melatoninergic system. Taken together, available data provide first evidence that melatonin is a chronobiotic molecule which in the pineal and gut exhibits different temporal pattern and employs different external cue as their synchronizer and, thereby, underline independent physiological functions.

Keyword: Carp, Gut, Melatonin, Pineal

7 Keynote Lecture‐8

Structure and Functions of Gonadotrophin-inhibitory hormone in teleost

Zhang Y., Li S., Qi X., Liu X., Lin H.* State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals, and the Guangdong Province Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China email: [email protected]

Gonadotropin inhibitory hormone (GnIH), via binding to GnIH receptor (GnIHR), plays a negative role on the avian and mammalian reproductive axis by inhibiting luteinizing hormone (LH) release. However, the biological significance of the GnIH/GnIHR system in other vertebrates is controversial. To demonstrate the presence of such a system in teleost, we have identified the orthologous gnih and gnihr genes in zebrafish and goldfish. The gnih precursor contains three putative LPXRFamide peptides. Three gnihrs genes (gnihr1, gnihr2 and gnihr3) were also identified and they are typical seven transmembrane G protein-coupled receptors. In situ hybridization results showed that gnihrs were localized to the hypothalamus and pituitary. Tissue expression studies revealed that gnih is mainly expressed in the brain, eye, testis, ovary and spleen, corroborating largely with the tissue expression patterns of the gnihrs. The biological action of the teleost GnIH on LH release was further investigated in goldfish in vivo. Intraperitoneal administration of the mature GnIH peptide could significantly reduce the basal serum LH level and sGnRH mRNA levels in goldfish. In vitro, GnIH showed no effect on GtH synthesis, but an inhibition of GnRH-stimulated LH and FSH synthesis. Implantation of goldfish GnIH peptides did not affect the serum estradiol level in female goldfish, but it did enhance the serum testosterone levels in males. Conversely, injecting goldfish GnIH peptides increased the expression of StAR and 3βHSD mRNA and decreased the expression of CYP19 mRNA significantly in the testis, but these genes remained unchanged in the ovary. In addition, goldfish GnIH peptides not only increased the expression of StAR, 3βHSD and decreased CYP19 mRNA but they also increased the expression of FSHR and LHR mRNA in testicular cells. However, they did not affect the expression of these genes in ovarian cells in vitro. GnIH may contribute to the sexual dimorphism of steroidogenesis in goldfish. These results provided that GnIH plays an important role in regulating of reproduction in teleost.

Keywords: gonadotrophin-inhibitory hormone (GnIH), GnIH receptor (GnIHR), GnRH, reproduction, inhibition.

8 Keynote Lecture‐9

Progress in comparative endocrinology by discovering novel neuropeptides and neurosteroids

Tsutsui K. Laboratory of Integrative Brain Sciences, Department of Biology and Center for Medical Life Science, Waseda University, Tokyo 162-8480, Japan e-mail: [email protected]

Identification of novel neurohormones, such as neuropeptides and neurosteroids, that regulate physiological processes is essential for the progress of comparative endocrinology. Our studies over the past two decades have significantly broadened the horizons of this field of research by identifying novel neuropeptides and neurosteroids in vertebrates that have opened new lines of scientific investigation in comparative endocrinology. Since the discovery of gonadotropin-releasing hormone (GnRH) in mammals at the beginning of 1970s, it was generally believed that GnRH is the only hypothalamic neuropeptide regulating gonadotropin release in vertebrates. In 2000, however, we discovered a novel hypothalamic neuropeptide that actively inhibits gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). It is now clear that GnIH is highly conserved across vertebrates, including humans, and serves a number of behavioral and physiological functions other than regulation of reproduction, providing enormous opportunity for investigators from a wide array of disciplines to study this neuropeptide. We have also established de novo synthesis and functions of neurosteroids in the brain of various vertebrates. Recently, we discovered 7-hydroxypregnenolone, a novel bioactive neurosteroid that acts as a key regulator for inducing locomotor behavior by means of the dopaminergic system. We further discovered that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol. The pineal gland actively secretes 7-hydroxypregnenolone and 3,5-tetrahydroprogesterone (allopregnanolone) that are involved in locomotor rhythms and neuronal survival, respectively. Subsequently, we have demonstrated their mode of actions and functional significances. This lecture summarizes the discovery of novel neuropeptides and neurosteroids and its contribution to the progress of comparative endocrinology.

Keyword: gonadotropin-inhibitory hormone (GnIH), 7-hydroxypregnenolone, 3,5-tetrahydroprogesterone, reproduction, locomotor behavior

10 Symposium Lecture‐1

Impact of whole genome duplications on vertebrate neuroendocrine systems with special focus on teleosts

Dufour S.1*, Lafont A.G.1, Maugars G.1, Pasquier J.1, Rousseau K.1 1Research Unit BOREA (Biology of Aquatic Organisms and Ecosystems), Museum National d’Histoire Naturelle, CNRS 7208, UPMC, IRD 207, UCBN, Paris, France. e-mail: [email protected]

Two rounds (1R and 2R) of whole genome duplications have occurred in early vertebrates, with an additional third round (3R) at the basis of the teleost lineage. These duplications have provided the genomic basis for morphological and physiological innovations in vertebrates. In particular, the teleost-specific 3R may have contributed to the remarkable diversification of this group, the largest amongst vertebrates. In addition to whole genome duplications, local events of gene duplications may have also increased the number of paralogs for some genes. Based on examples from the neuroendocrine gonadotropic axis, we are investigating the impact of genome/ gene duplications on the numbers, evolutionary histories and roles of neuroendocrine paralogs. Blast search, phylogeny and synteny analyses are performed in order to identify gene paralogs and retrace their origin and fate according to vertebrate lineages and species. To that aim we are taking advantage of the increasing number of available genomes including species of key-phylogenetic positions, such as a basal sarcopterygian, the caelacanth, a basal non-teleost actinopterygian, the spotted gar, or a basal teleost, the eel. Evolutionary scenarios reveal that the larger numbers of paralogs in teleosts as compared to mammals, do not always originate from the teleost-specific 3R but have various origins according to gene families. Paralog identification and evolutionary histories also allow to propose new and phylogenetically-supported gene nomenclatures. Following duplications, paralogs may have been conserved or lost, according to lineages and species. Conservation of multiple paralogs can be related to amplification or partition of pre-existing functions (subfunctionalization), or to emergence of new functions (neofunctionalization). In complement to phylogenetic studies, functional analyses including structure-activity relationships, tissue expression profiling and regulation, are performed on selected biological models, such as the eel, in order to infer evolutionary forces that may have contributed to the conservation of multiple paralogs.

Keywords: neuroendocrinology, reproduction, evolution, genome, teleosts

11 Symposium Lecture‐2

Novel pituitary actions of TAC3 gene products in fish model

Hu G., He M., Ko W.K.W., Wong A.O.L.* School of Biological Sciences, The University of Hong Kong, Hong Kong, China. e-mail: [email protected]

TAC3 is a member of tachykinins and its gene product neurokinin B (NKB) has recently emerged as a key regulator for luteinizing hormone (LH) via modulation of kisspeptin/GnRH system within the hypothalamus. In fish models, TAC3 not only encodes NKB but also an additional “tachykinin-like” peptide called NKB-related peptide (NKBRP) and the pituitary actions of these two TAC3 gene products are still unknown. Using grass carp as a model, the direct effects as well as the post-receptor signaling for TAC3 gene products were examined at the pituitary level. Grass carp TAC3 was cloned and confirmed to encode NKB and NKBRP. In carp pituitary cells, NKB and NKBRP did not affect LH release and gene expression but could up-regulate prolactin (PRL) and somatolactin  (SLα) secretion, protein production and transcript levels. The stimulation by the two TAC3 products on PRL and SLα release and mRNA levels were mediated by pituitary NK2 and NK3 receptors, respectively. Apparently, NKB- and NKBRP-induced SLα secretion and transcript expression were caused by subsequent activation of cAMP/PKA, PLC/IP3/PKC and Ca2+/CaM/CaMK-II cascades. The signaling mechanisms for the corresponding effects on PRL release and gene expression were also similar to that of SLα responses, except that PKC component was not involved. These findings indicate that TAC3 gene products did not play a role in LH regulation at the pituitary level in carp species but might serve as novel stimulators for PRL and SLα synthesis and secretion via overlapping post-receptor signaling mechanisms coupled to NK2 and NK3 receptors, respectively.

Keyword: NKB, NKBRP, NK2 & NK3 receptors, Somatolactin, Prolactin, Grass Carp

12 Symposium Lecture‐3

Transgenic Fish and its Reproductive Containment Strategies

Hu W.* and Zhu Z.Y. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072 e-mail: [email protected]

A fast-growing growth hormone transgenic homozygous common carp has been generated in our laboratory. One of the bottleneck issues for transgenic fish commercialized cultivation is the possible ecological risk posed by these fish. We developed several reproductive containment strategies to produce sterile transgenic fish to counteract the ecological risk of transgenic fish. Firstly, we produced a completely sterile GH transgenic triploid carp by conducting hybridization between diplontic common carp and allotetraploid Cyprinuscarpio carp. Secondly, we generated a sterile transgenic carp by inhibiting GnRH expression with an antisense transgene. Thirdly, we developed an on-off strategy to control fish reproduction. The fertility of transgenic fish is controllable by blocking dnd expression via GAL4/UAS inducible system in offspring. The On-off strategy to control transgenic fish reproduction makes it convenient to generate sterile offspring for its commercialization while the valuable traits are maintained in the fertile brood stock.

Keywords: Transgenic fish, Ecological risk, Reproductive containment

13 Symposium Lecture‐4

Pleiotropic functions of proopiomelancortin in fish

Takahashi A.*, Kobayashi Y., Mizusawa K. School of Marine Biosciences, Kitasato University, Sagamihara, Japan. e-mail: [email protected]

Adrenocorticotropic hormone (ACTH), -melanocyte-stimulating hormone (-MSH), and -endorphin (-END) are produced from proopiomelanocotin (POMC) by posttranslational processing in the pituitary gland. These peptides occasionally undergo some modifications. Receptors for ACTH and -MSH are melanocortin receptors (MCRs) and those for -END are opioid receptors. We found that ACTH and -END modulate the cortisol release from interrenal cells of goldfish by opposite functions. ACTH increased the cortisol release, whereas -END decreased basal cortisol release; moreover, -END inhibited ACTH activities. Using barfin flounder, we showed that in addition to ACTH, -MSH-related peptides (-MSH-rp) also stimulated cortisol release, although the potencies were lower than ACTH. These activities depended on the number of the acetyl groups at the N-terminal—the potencies of cortisol releasing activities were Di-Ac--MSH > Des-Ac--MSH >> -MSH. The different potencies would be caused by the formation of heteromers consisting of MC2R and MC5R. Additionally, -MSH-rp did not stimulate cortisol release in goldfish, in which only MC2R was found among the five MCR subtypes. The differential effects of -MSH-rp depending on the acetylation were also observed in flounder chromatophores. -MSH having one acetyl group exhibited the activities on xanthophores where MC5R was observed, whereas it showed negligible activities on melanophores where MC1R and MC5R were observed. The pleiotropic characters of POMC reside in the structure itself, because of the occurrence of several peptide hormones. Moreover, post-translational processing further diversifies the multiplicity. In addition, the presence of subtypes for MCR and opioid receptors and formation of heteromers contribute to the pleiotropism.

Keyword: adrenocorticotropic hormone, -melanocyte-stimulating hormone, -endorphin, cortisol release, pigment dispersion

14 Symposium Lecture‐5

Aromatase, estrogens and brain development in teleost fishes

Kah O. 1*, Cano-Nicolau J., Nasri H., Diotel N., Vaillant C., Gueguen M.M., Pellegrini E. 1Université de Rennes 1, Institut de Recherche Santé Environnement & Travail (IRSET), INSERM U1085, Campus de Beaulieu, 35042 Rennes cedex, France e-mail: [email protected]

In teleosts, aromatase B (cyp19a1b) is expressed in radial glial cells, a unique type of brain cells, which act as progenitors in developing and adult vertebrates. In addition, aromatase B expression is strongly up-regulated by androgens and estrogens, so that aromatase activity is highest in sexually mature fish, when sex steroid levels are elevated. These properties represent unique features of teleosts compared to other vertebrates and raise the question of their functional significance. Using zebrafish, we tested the hypothesis that estrogens affect adult neurogenesis and brain regeneration by modulating the neurogenic activity of radial glial cells. We found that in both male and female adult zebrafish, estrogenic treatments, inhibition of aromatase activity or blockade of nuclear estrogen receptors lead to significantly decreased cell proliferation in most regions examined. Migration of new-born cells was also significantly reduced. In zebrafish embryos and larvae, exposure to estradiol or ethynilestradiol also consistently leads to increased expression of cyp19a1b, as expected, but this is also accompanied by decreased expression of PCNA, further suggesting that estradiol inhibits cell proliferation in this model. From the results obtained so far, estrogens appear to inhibit cell proliferation of radial glial cells in embryos, juveniles, larvae and adult zebrafish. It also seems that estradiol does not favour cell proliferation after brain lesion, in contrast with the situation in birds and mammals. All these data further point to a peculiar function of aromatase and estrogens in the brain of fish. Because fish in general do not exhibit gonadal and somatic growth at the same time, our current hypothesis stipulates that inhibitory effects of estrogens or aromatizable androgens would reduce brain growth in maturing or mature fish, when energy demand for gonadal growth is highest.

Supported by INSERM, the ANR PROOF and the TC2N European project.

15 Symposium Lecture‐6

Role of tachykinins and tachykinin receptors in fish reproduction

Ogawa S. Brain Research Institute, School of Medicine and Health Sciences, Monash University Malaysia, PJ 46150, Malaysia. e-mail: [email protected]

Tachykinins are a family of neuropeptides, including substance P (SP), neurokinin A (NKA), and neurokinin B (NKB), that are encoded by the tac1 (SP and NKA) or tac3 (NKB) genes. Tachykinins are widely distributed in the central nervous system and have roles as neurotransmitters and/or neuromodulators. Recent studies in mammals have demonstrated the co-expression of NKB and kisspeptin and their co-modulatory roles over the control of reproduction. We have recently identified two kisspeptin-encoding genes, kiss1 and kiss2, in teleosts. However, such relationship between tachykinins and other reproductive neuroendocrine cells such as kisspeptin and gonadotropin-releasing hormone (GnRH) neurons has not been demonstrated in non-mammalian species. The zebrafish tac3a mRNA-containing cells were observed in the preoptic region, habenula, and hypothalamus, whereas the tac3b mRNA-containing cells were predominantly observed in the dorsal telencephalic area. Dual-fluorescent in situ hybridization showed no coexpression of tachykinins mRNA with kiss2 mRNA in hypothalamic nuclei or kiss1 mRNA in the habenula. In the tilapia, we found expression of NKB receptor genes (NK3A and NK3B) in GnRH neuron types. In addition, NK3A and NK3B were expressed in the hypothalamic region containing Kiss2 neurons. These results suggest the presence of reciprocal pathways among Kiss2, NKB and GnRH neurons in the regulation of reproduction in fish.

Keywords: neuroendocrine, kisspeptin, neurokinin B

16 Symposium Lecture‐7

Tug-of-war of gonadal soma transdifferentiation in the hermaphroditic fish.

Wu G.C.1,2*and Chang C.F.1,2 1Department of Aquaculture, 2Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung City, Taiwan. e-mail: [email protected]

Hermaphroditism has been reported from at least 500 species in the fish. No ancient ancestry and no single sex-determing mechanism are involved in the hermaphroditic fish. However, the origins and evolution of hermaphroditism in fish are far from understood. In the protandrous black porgy, we generated the abnormal testicular part of the ovotestis with estradiol-17b (E2) treatment, in which newly regenerated testis has ectopic oocytes. The ectopic oocytes in the regenerated testis do not undergo apoptosis; thus, a number of occytes are in the testis. Furthermore, the cells surrounding the oocytes are Dmrt1-positive cells (Sertoli cells) in the oocytes at an early stage. Then, as the Dmrt1 expression diminishes, Cyp19aia-positive cells (follicle cells) appear in the oocytes at a later stage. On the other hand, the number of oocytes in the regenerated testis is limited and the germ cells proliferation is arrested in the near region of ectopic oocytes. The candidate suppressor of self-renewing germ cells proliferation, Amh, was detected in the near region of oocytes in the regenerated testis. Thus, we demonstrated that oocytes are competent to create a microenvironment to protect against a testicular environment in black porgy. On the other hand, the number of ectopic oocytes in the testis is restricted through arrest the spermatogonia (or early germline cells) proliferation by Amh signlas. These results shed light on why the presence of more than one sex at a time existed during an evolutionary transition from gonochorism to hermaphroditism in fish.

Keyword: ovotestis, Figla, Amh

17 Symposium Lecture‐8

Regulation of oligodendrocyte development by hedgehog signaling in the zebrafish CNS

Chung A.Y., Kim S., Kim E., Jeong I., Kim D., Park H.C.* Department of Biomedical Sciences, School of Medicine, Korea University, Ansan, Gyeonggido 425-707, Republic of Korea e-mail: [email protected]

A subset of ventral spinal cord precursors, known as pMN precursor cells, initially generate motor neurons and then oligodendrocyte progenitor cells (OPCs), which migrate and differentiate as myelinating oligodendrocytes in the developing neural tube. The switch between motor neuron and oligodendrocyte production by the pMN neural precursors is an important step in building a functional nervous system. Previously we have shown that morphogenetic gradient of Sonic hedgehog (Shh) induces olig2 expression in pMN precursor domains in the ventral spinal cord, and then Shh signaling is required for the generation of motor neuron and OPCs from the pMN precursors. However, it is not clear how Hh signaling instructs the complex sequence of motor neurons and OPC specification that occurs over time during development. The present study demonstrates that Indian Hedgehogb (Ihhb), previously known as Echidna Hedgehog, begins to be expressed in the floor plate cells of the ventral spinal cord at the time of OPC specification in zebrafish embryos. Ihhb loss-of-function analysis revealed that Ihhb function is required for OPC specification from pMN precursors by negatively regulating the proliferation of neural precursors. Finally, results showed that Sonic Hedgehog (Shh) could not replace Ihhb function in OPC specification, suggesting that Ihhb and Shh play separate roles in OPC specification. Altogether, data from the present study suggested a novel mechanism, mediated by Ihhb, for the sequential generation of motor neurons and oligodendrocytes from pMN precursors in the ventral spinal cord of zebrafish embryos.

Keyword: oligodendrocyte, Hedgehog signaling, pMN precursor, zebrafish

18 Symposium Lecture‐9

Migration and differentiation of neural progenitors following brain injury in mice

Sun W. Department of Anatomy, Korea University College of Medicine, Seoul, Korea e-mail: [email protected]

In adult mouse brain, new neurons are spontaneously produced in the restricted regions where neural stem cells (NSCs) maintain. In the subventricular zone, for example, neuroblasts are generated and these newly produced neuroblasts migrate toward the olfactory bulb through the rostral migratory stream. Upon injury, NSCs enhance their proliferation and often generate more neurons, thus promising the potential use of endogenous NSCs for the treatment of neuropathological conditions. This injury-induced neuroblast migration is controlled by multiple extracellular signals including SDF1, and these extracellular signals are transduced to evoke cellular events such as actin remodeling. We have found that ERM proteins are critical for the migration of neuroblasts. Recently, we also identified that novel neuroblastogenic region, subcallosal zone (SCZ), which contains independent pool of NSCs. Following brain injury, the production of SCZ-derived neuroblasts is increased and they migrate toward the injured brain region. However, most SCZ-derived cells in the injury penumbra failed to differentiate and were removed by apoptosis. Although overexpression of anti-apoptotic gene Bcl-xL in the SCZ partially promoted neuronal maturation, the majority of Bcl-xL overexpressing neuroblasts failed to mature and integrate into neuronal circuits. By supplement of neurotrophic factor BDNF, large proportion of Bcl-xL-rescued but atrophied neurons exhibited mature neuronal phenotypes, suggesting that NSCs may be utilized for brain repair with combination of anti-apoptotic and neurotrophic treatments.

Keyword: adult neurogenesis, subcallosal zone, brain injury, apoptosis, mouse

19 Symposium Lecture‐10

Evolutionary genomics for identification of novel neuropeptides and their receptors

Seong J.Y.1 1 Laboratory of G-Protein Coupled Receptors, Graduate School of Medicine, Korea University, Seoul 136-705, Republic of Korea e-mail: [email protected]

Neuropeptides and their G-protein-coupled receptors (GPCRs) have become diversified through evolutionary processes, such as gene/chromosome duplications and gene modification, generating families of related yet distinct peptides and receptors. Currently, the large accumulation of genome sequence information for various invertebrate and vertebrate species combined with recent advances in bioinformatic tools has allowed large-scale genome comparisons. Particularly, comparing entire genomes of evolutionarily distant taxa allows reconstructing the hypothetical chromosomes of vertebrate or chordate ancestor, facilitating the exploration of the origin and relationship of gene families of interest. Our lab has searched for the evolutionary lineages of neuropeptide and GPCR genes including novel neuropeptides and orphan GPCRs. Here, I will discuss discovery of novel neuropeptide genes and identification of their receptors using evolutionary genomics.

Keyword: Neuropeptide, GPCR, bioinformatics, evolution

20 Symposium Lecture‐11

Androgen receptor regulates stemness and differentiation of stem cells

Kang H.Y. 1,2 1Department of Graduate Institute of Clinical Medical Sciences, Chang Gung University, Kaohsiung, Taiwan. 2 Hormone Research Center, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan. e-mail: [email protected]

Androgens, principally testosterone and 5 alpha-dihydrotestosterone, play important roles in cellular genesis and differentiation, tissue development, and organogenesis. Their biological actions are mediated by a ligand-dependent nuclear transcription factor, the androgen receptor (AR). The AR is a member of the steroid hormone receptor family, which is found in a variety of stem/progenitor cells from sex differentiation during embryogenesis to the onset and the maintenance of reproductive capacity in sexual maturity and required for diverse physiological functions such as development, metabolism, and aging. Increasing evidence shows that AR in regulating stemness or differentiation of embryonic stem (ES) cells and tissue-specific adult stem cells. Here, we summary our recent studies on the roles of the androgen/AR signaling in various aspects of stem cell biology, including regulation of self-renewal in mesenchymal stem cells, proteotoxic stress in ES cells; reprogramming of adipogenesis to osteogenesis and regeneration of bone fracture repair. These studies provide insights into the therapeutic potential of targeting AR in stem cell therapy and in treating androgen-associated diseases.

Keyword: androgen receptor, stem cells, and differentiation

Reference: 1. Loss of androgen receptor promotes adipogenesis but suppresses osteogenesis in bone marrow stromal cells. Stem Cell Res. 2013 Sep;11(2):938-50. 2. Testosterone delivered with a scaffold is as effective as bone morphologic protein-2 in promoting the repair of critical-size segmental defect of femoral bone in mice. PLoS One. 2013 Aug;8(8):e70234. 3. Suppression of androgen receptor enhances the self-renewal of mesenchymal stem cells through elevated expression of EGFR. BBA-Mol Cell Res. 2013 May;1833(5):1222-34. 4. Androgen receptor inclusions acquire GRP78/BiP to ameliorate androgen-induced protein misfolding stress in embryonic stem cells. Cell Death Dis. 2013 Apr 25;4:e607.

21 Symposium Lecture‐12

Transmembrane peptides as unique tools to demonstrate the in vivo action of a GPCR hetero-complex of secretin and angiotensin

Lee L.T.1, Ng S.Y.1, Chu J.Y.1, Sekar R.1, Harikumar K.G.2, Miller L.J.2, Chow B.K.1* 1 School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong; 2 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona e-mail: [email protected]

Angiotensin and secretin share overlapping and interdependent osmoregulatory functions in the brain, and a possible mechanism is the formation of GPCR hetero-complex. In this report, we established that such a complex exists, and also that several transmembrane (TM) peptides of secretin receptor (SCTR) and angiotensin receptor (AT1aR) are able to inhibit receptor oligomerization and cAMP responses of SCTR. One of these peptide, ATM-1 affected only the formation and activity of the hetero-complex, was found to be active in vivo in mice by its injection into the lateral ventricle, thereby suppressing water-drinking behavior after hyperosmotic shock, similar to what has been observed in SCTR knockouts and in H-89-injected mice. Here, we show the in vivo action of SCTR/AT1aR receptor association in the central nervous system, in this case, in inducing water intake. These TM peptides are novel biochemical tools that can modulate GPCR functions by selective impact on homomers and/or heteromers. The potential of these TM peptides is tremendous, as we are only in the beginning of understanding the importance of receptor oligomerization in our body. The present study was supported by the Hong Kong Government RGC grant 770212M to LTO Lee, CRFHKU6/CRF/11G, HKU 764812M to BKC Chow, and the NIH Grant DK046577 to LJ Miller.

Keywords: GPCR oligomerization, secretin, angiotensin II, transmembrane peptide

22 Symposium Lecture‐13

Pheromonal control of final oocyte maturation and lunar-related spawning in grouper

Soyano K.1*, Izumida D. 1, Nakamura M.2 1Institute for East China Sea Research, Nagasaki University, Nagasaki, Japan. 2 Okinawa Churaumi Aquarium, Motobu, Okinawa, Japan. e-mail: [email protected]

Groupers are very important target species for fisheries industry and aquaculture in Asian, Oceanian, Caribbean, and Middle and Near East countries. Moreover, these species occupy an important position in the food chain of marine ecosystem. In addition, groupers have many interesting characteristics of gametogenesis and reproductive phenomena, which are rich in variation. We are conducting research by focusing on the lunar-related spawning and the pheromone-induced final oocyte maturation. Some species distributed in the sub-tropical and tropical regions, such as the honeycomb grouper Epinephelus merra and the white-streaked grouper Epinephelus ongus, have a gonadal development and spawn cycle linked to lunar phase. The honeycomb grouper and the white-streaked grouper spawn at just after the full moon and the last quarter moon, respectively. These groupers migrate to the suitable spawning ground from the habitat just prior to spawning and form fish spawning aggregation at the spawning ground. However, the fish has not yet completed the acquisition of maturational competence of oocyte. In our study, the final oocyte maturation in the female honeycomb grouper was induced in the fish exposed to rearing water of mature male during spawning season. The result suggests that the rearing water with mature male include a pheromonal substance that plays an important role on induction of final oocyte maturation in female. The pheromone stimulated testosterone synthesis and accelerated GTHs transcription in pituitary, while plasma levels of maturation-inducing hormones were not change. It appears that testosterone stimulated via the GTHs synthesis by pheromone has an important role on the induction of the morphological changes of oocyte in final oocyte maturation.

Keyword: grouper, pheromone, lunar-related spawning, final oocyte maturation, spawning migration

23 Symposium Lecture‐14

Appetite regulation in desert-adapted spinifex hopping mice during water deprivation

Donald J.A.1*, Hamid N.K.A.1, McLeod J.L.2, Takei Y.3 1School of Life and Environmental Sciences, Deakin University, Geelong, Australia, 3216. 2 School of Medicine, Deakin University, Geelong Australia, 3216. 3 Atmosphere and Ocean Research Institute, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, 277-8564 Chiba, Japan e-mail: [email protected]

Like many desert rodents, spinifex hopping mice, Notomys alexis, can maintain water balance without drinking. In contrast to the role of the kidney, little is known about the physiological mechanisms underpinning the metabolic production of water to offset obligatory water loss. We used water deprivation (WD) of hopping mice as a model to study food intake and its control in desert rodents. We found that hopping mice water-deprived for 28 days shows a cyclical food intake in which hypophagia for 5 days is followed by a sustained food intake that is greater than water-replete animals. In the initial cyclical phase of food intake (10-12 days of WD), the changes in appetite are driven by expected changes in plasma leptin and ghrelin and the expression of neuropeptide genes in the hypothalamus. However, the control of appetite in the sustained food intake phase (12-29 days of WD) shows some unusual characteristics. Plasma leptin decreases in parallel with fat loss but then increases again despite body fat stores not recovering, which is likely due to leptin expression in skeletal muscle and heart. WD had no effect on central leptin receptor mRNA and protein. Plasma ghrelin decreases during hypophagia but then increases significantly as food intake increases. However, it then decreased markedly despite a sustained food intake. Brain ghrelin mRNA expression was up-regulated during WD but the receptor mRNA was down-regulated. Brain NPY mRNA was up-regulated during WD. In conclusion, water-deprived hopping mice have a sustained food intake despite peripheral signals that should inhibit food intake. Therefore, it is likely that central control of appetite is probably critical in hopping mice for sustaining food intake to provide substrate to generate metabolic water when free water is unavailable.

Keywords: spinifex hopping mouse, water deprivation, leptin, ghrelin, neuropeptide Y

24 Symposium Lecture‐15

Regulatory contribution of Gs-proteins to 1-methyladenine production in starfish ovarian follicle cells stimulated by relaxin-like gonad-stimulating substance

Mita M.1*, Haraguchi S.1,2, Takeshige Y.1, Watanabe M.1, Tsutsui K.2 1Department of Biology, Faculty of Education, Tokyo Gakugei University, Tokyo 184-8501, Japan. 2Laboratory of Integrative Brain Sciences, Department of Biology and Center for Medical Life Science, Waseda University, Tokyo 162-8480, Japan e-mail: [email protected]

Gonad-stimulating substance (GSS) mediates oocyte maturation in starfish by acting on the ovarian follicle cells to produce the maturation-inducing hormone, 1-methyladenine (1-MeAde), which, in turn, induces the maturation of oocytes. The hormonal action of GSS on follicle cells involves its receptor, G-proteins and adenylyl cyclase. However, GSS failed to induce 1-MeAde and cAMP production in follicle cells during oogenesis. At the maturation stage, follicle cells acquired the potential to respond to GSS by producing 1-MeAde and cAMP. Adenylyl cyclase activity in follicle cells of fully grown stage ovaries was also stimulated by GSS in the presence of GTP. These activations depended on the size of oocytes in ovaries. The subunit of Gs-proteins was not detected immunologically in follicle cells in growing stage ovaries. Using a specific primer for G s, expression levels of G s in follicle cells were found to increase significantly as the size of oocytes in ovaries increased. These findings strongly suggest the potential of follicle cells to respond to GSS by producing 1-MeAde and cAMP is brought by de novo synthesis of G s-proteins. keyword: Gs-proteins, GSS, 1-MeAde, Adenylyl cyclase, Starfish

25 Symposium Lecture‐16

Serotonin and melatonin mitigate the stress response and promote the ease response in air-breathing fish

Peter M.C. Centre for Evolutionary and Integrative Biology, University of Kerala, Kariavattom, Thiruvananthapuram-695 581, Kerala, India e-mail: [email protected]

Fishes have evolved complex and multi-step neuroendocrine and physiologic mechanisms by which they maintain the physiologic homeostasis in challenging environment. The neuroendocrine circuitries can direct the physiologic plasticity of fish and thus accommodate the environmental oscillations. Serotonin (5-hydroxytryptamine) and melatonin (N-acetyl-5-methoxytryptamine) have been linked with the stress in fish. But the exact role of these hormones in stress and ease response have not yet understood in fish. We tested the in vivo and in situ effects of serotonin and melatonin on acid-base and ion transporter activities in an air-breathing fish. Ion-selective ATPase transporters that maintain ion gradients across plasma membrane and mitochondria of osmoregulatory epithelia were quantified in pre-stressed, stressed and post-stressed fish. Analyses of the response pattern of calcium, magnesium, sodium and proton pump activities to serotonin and melatonin clearly indicate protective roles of these neurohormones in these fish during stress condition. Treatment of serotonin or melatonin in post-stressed fish showed that these hormones suppress the magnitude of stress response while promoting recovery response. Overall, the data indicate that serotonin and melatonin can lower the stress response and promote the ease response in this fish (Supported by a DST Project grant).

Key words: serotonin, melatonin, stress response, ease response, fish

26 Symposium Lecture‐17

Control mechanisms of meiotic initiation during spermatogenesis in Japanese eel (Anguilla japonica).

Miura T.*, Higuchi M., Miura C. South Ehime Fisheries Research Center, Ehime University, Ainan, Japan e-mail: [email protected]

Meiosis is a unique and critical process in reproduction. Although the key molecular components of meiosis have been identified, the molecular mechanisms regulating the entry into this pathway remain unclear. A progestin in teleost fish, 17alpha, 20beta- dihydroxy- 4-pregnen- 3-one (DHP), is an essential component of the spermatogenesis pathway, particularly during the initiation of the first meiotic division. In the course of our investigations on the mechanisms underlying progestin-stimulated meiosis in spermatogenesis, we found that trypsin and taurine are up-regulated substances in testis through DHP treatment in Japanese eel. Trypsin is well known as a pancreatic enzyme that is typically secreted into the intestine to digest proteins, and taurine is an organic acid widely distributed in animal tissues. Using the Japanese eel model both in vivo and in vitro, we investigated the role of trypsin and taurine in meiotic process of spermatogenesis. As a result of this investigation, we clarified the following mechanisms of meiotic initiation in spermatogenesis. Trypsin and taurine production are induced by DHP stimulation in Sertoli cell. Trypsin stimulates the expression of taurine transporter, Slc6a6 on the membrane of spermatogonia. Consequently, induction of Slc6a6 expression results to uptake of taurine by spermatogonia. In spermatogonia, transported taurine supports the formation of active form of Spo11, which is involved in the formation of DNA double-strand breaks in meiotic prophase. And finally, the first meiotic division is initiated.

Key words: meiosis, progestin, trypsin, taurine, Spo11

27 Symposium Lecture‐18

Orexin A induces anxiety-like behavior in goldfish

Matsuda K.1,2*, Shibata H.1, Nakamachi T.1 1Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555, Japan 2Laboratory of Regulatory Biology, Graduate School of Innovative Life Science, University of Toyama, 3190-Gofuku, Toyama 930-8555, Japan e-mail: [email protected]

Orexin is a member of incretin family of peptides in vertebrates. Orexin acts as an orexigenic factor for the regulation of appetite and rhythmicity in rodents. In a goldfish model, intracerebroventricular (ICV) administration of orexin A affects not only food intake, but also locomotor activity. However, there is no information regarding the psychophysiological roles of orexin A in goldfish. Therefore, we investigated the effect of orexin A on psychomotor activity in this species. ICV administration of synthetic orexin A at 2 and 4 pmol/g body weight (BW) enhanced locomotor activity. Enhancement of locomotor activity by orexin A was attenuated by treatment with the orexin receptor antagonist, SB334867, at 10 pmol/g BW. Since intact goldfish prefer the black to the white background area, or the lower to the upper area of tank, we developed two types of preference test (light/dark and upper/lower preference tests) for measuring the time spent for fish in the white background area and the time spent to move from lower to upper area. ICV administration of orexin A at 4 pmol/g BW and the central-type benzodiazepine receptor inverse agonist FG-7142 (an anxiogenic agent) at 1-4 pmol/g BW both decreased the time spent in the white area, and increased the time spent to move from lower to upper area. The anxiogenic-like effects of orexin A were abolished by treatment with SB334867 at 10 pmol/g BW. These results indicate that orexin A can potently affect psychomotor activities in goldfish.

Keywords: goldfish; orexin A; ICV injection; psychomotor activity; anxiogenic-like action

28 Symposium Lecture‐19

Role of sex steroids on sex determination and maintenance in teleosts

Sun L., Tao W., Li M., Jang D., Zhou L., Wang D.* Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Science, Southwest University, Chongqing, 400715, China. e-mail：[email protected]

The Nile tilapia, a gonochoristic fish with XX/XY sex determination system, is one of the most important species in global aquaculture. It has been characterized as a good model for study of gonadal sex differentiation. Four pairs of gonadal transcriptomes from XX and XY fish at 5, 30, 90, and 180 days after hatching (dah) were sequenced using Illumina HiseqTM technology. The produced 28 Gb sequences were mapped to 21,334 genes. Almost all steroidogenic enzymes, including cyp19a1a, were up-regulated in XX gonads at 5 dah; but in XY gonads these enzymes, including cyp11b2, were significantly up-regulated at 90 dah, indicating that, at a time critical to sex determination, the XX fish produced estrogen and the XY fish did not produce androgens. The most pronounced expression of steroidogenic enzyme genes was observed at 30 and 90 dah for XX and XY gonads, corresponding to the initiation of germ cell meiosis in the female and male gonads, respectively. Both estrogen and androgen receptors were found to be expressed in XX gonads, but only estrogen receptors were expressed in XY gonads at 5 dah. This could explain why exogenous steroid treatment induced XX and XY sex reversal. In addition, females with differentiated ovary were masculinized by long term treatment with an aromatase inhibitor (Fadrozole). The reversed gonads developed into functional testes with fertile sperm. Furthermore, the Fadrozole induced sex reversal, designated as secondary sex reversal (SSR), was successfully rescued by supplement of exogenous E2. During SSR, the spermatogonia were transformed from oogonia; while Leydig and Sertoli cells probably came from the interstitial cells and granulosa cells, respectively. Decrease of serum E2 was detected earlier than increase of serum 11-KT indicating decrease of estrogen was the cause; while increase of androgen was the consequence of SSR. Transdifferentiation was driven by suppression of female pathway genes and activation of male pathway genes while the global gene expression profiles remained closer to the female control. Disruption of Dmrt1 in XY fish resulted in increased foxl2, cyp19a1a expression, serum E2 and 11-KT levels. On the contrary, deficiency of Foxl2 in XX fish exhibited varying degrees of oocyte degeneration and even complete sex reversal, significantly decreased cyp19a1a expression and serum E2 levels. Taken together, our results suggested that estrogen, but not androgen, plays important roles in sex determination, differentiation and maintenance of phenotypic sex in tilapia.

Keywords: Transcriptome analysis, sex steroids, sex reversal, transdifferentiation of germ cell and somatic cell, TALEN- and CRISPR/Cas9-mediated gene knockout, Nile tilapia

29 Symposium Lecture‐20

Ghrelin and food acquisition in japanese eel

Yada T.1*, Kaifu K.2, Kaiya H.3, Tsukamoto K.4 1National Research Institute of Aquaculture, Nikko, Japan. 2 Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Japan. 3 Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, Suita, Japan. 4 Graduate School of Bioresource Sciences, Nihon University, Fujisawa, Japan. e-mail: [email protected]

To investigate the involvement of ghrelin on habitat selection through the food acquisition, circulating concentrations and mRNA contents of ghrelin were measured in wild and cultured juveniles of Japanese eel. Glass eels caught at estuary showed no significant fluctuation of whole body contents of ghrelin mRNA during upstream migratory periods from December to March. Experimental starvation of cultured elvers for 4 weeks resulted in a retardation of growth, although there was no significant influence on ghrelin mRNA contents. On the other hand, in cultured yellow eels, 4-weeks starvation increased stomach mRNA contents and plasma concentrations of ghrelin. After refeeding, the both parameters decreased to the levels observed in the control fish with continuous feeding, suggesting a stimulatory role of ghrelin in feeding regulation in eel. In the field study, again, wild yellow eels captured in freshwater habitats showed significantly higher levels of stomach mRNA contents and plasma concentrations of ghrelin than the fish obtained from brackish water habitats (estuary and gulf areas). Stomach fullness index of eels captured in brackish water was significantly greater than those captured in freshwater. Moreover, from the age determination with annual rings on otolith, growth rates of wild eels were higher in brackish water than in fresh water. Possible interactions across ghrelin, feeding, and growth will be discussed in relation to the habitat selection through the food acquisition in fresh or brackish waters.

Keyword: eel, feeding, ghrelin, growth, habitat

30 Symposium Lecture‐21

Kit System and Its Role in the Paracrine Regulation of Follicle Development in the Zebrafish

Ge W.*, 1, 2 and Yao K. 1 1 School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong; 2 Faculty of Health Sciences, University of Macau, Taipa, Macau e-mail: [email protected]; [email protected]

In the zebrafish, the Kit system consists of two ligands (Kitlga and Kitlgb) and two receptors (Kita and Kitb). Interestingly, Kitlga and Kitb are localized in the somatic follicle cells, but Kitlgb and Kita are expressed in the oocyte. Using recombinant zebrafish Kitlga and Kitlgb, we demonstrated that Kitlga preferentially activated Kita whereas Kitlgb specifically activated Kitb. In support of this, Kitlgb triggered a stronger and longer MAPK phosphorylation in follicle cells than Kitlga, whereas Kitlga but not Kitlgb activated MAPK in the denuded oocytes, in agreement with the distribution of Kita and Kitb in the follicle and their specificity for Kitlga and Kitlgb. A functional study showed that Kitlga and Kitlgb both suppressed spontaneous oocyte maturation. Our results provided strong evidence for a Kit-mediated bi-directional communication system in the zebrafish ovarian follicle. The expression of Kitlga in the follicle cells suggests that this ligand could be a target of the Kit system that is subject to endocrine and paracrine controls. To test this, we did a series of experiments on the regulation of Kitlga by hCG (endocrine hormone), IGF-I (endocrine hormone and paracrine factor), and PACAP (paracrine factor) in vitro. Our results showed that IGF-I played dual roles in regulating Kitlga expression. It stimulated Kitlga expression via PI3K/Akt pathway but suppressed its expression through MAPK. Similarly, both hCG and PACAP suppressed IGF-I-stimulated Kitlga expression in cultured follicle cells, probably via cAMP that signaled through PKA and Epac. Interestingly, these two pathways also exhibited opposing effects on the expression of Kitlga, with cAMP-PKA pathway being stimulatory and cAMP-Epac inhibitory. These results suggest that the Kit system is likely subject to regulation by multiple endocrine and paracrine factors, but its response to each factor may vary at different stages depending on the relative strength of the downstream signaling pathways.

Keyword: Kit and Kit ligand, ovary, zebrafish

31 Symposium Lecture‐22

Neuroendocrine response to pathogen and its implication in the metabolism of crustacean host

Lin L.-J.1, Chang Y.-S.2, Huang W.-S.3, Tien Y.-C.1, Chen Y.-J.1, Lee C.-Y.1* 1 Department of Biology, National Changhua University of Education, Changhua 50058, Taiwan. 2 Department of Molecular Biotechnology, Da-Yeh University, Changhua 51591, Taiwan. 3 Department of Biology, National Museum of Natural Science, 1, Kuan-Chien Rd., Taichung, 404, Taiwan e-mail: [email protected]

We are interested in studying the effect of pathogens on the expression and release of metabolic hormones of decapod hosts and the consequences on metabolism. It is discovered that crustacean hyperglycemic hormone (CHH), a polypeptide hormone originally identified in an eyestalk neuroendocrine complex, is also expressed in circulating hemocytes and hemocyte-producing tissue (the hematopoietic tissue). After host infection by the white spot syndrome virus (WSSV), release of CHH into hemolymph was acutely enhanced at an early stage of viral replication cycle, while hemolymph glucose levels remained constant. CHH gene expression in circulating hemocytes and hematopoietic tissue, but not in the eyestalks, was significantly increased after WSSV infection. Knocking down CHH gene expression by CHH double-stranded RNA significantly altered the number of circulating hemocytes. Considered the effects of CHH on metabolism, it is likely that the virus-induced CHH expression and release are involved in regulating glucose uptake and its utilization by tissues. Metabolic significance of the neuroendocrine response to viral challenge is currently under investigation.

Keyword: crustacean hyperglycemic hormone, pathogen, metabolism

32 Symposium Lecture‐23

Neuropeptides and peptide hormones of the ascidian, Ciona intestinals: Comparative views to novel follicle growth.

Satake H. 1 1Bioorganic Research Institute, Suntory Foundation for Life Sciences, Osaka, Japan. e-mail: [email protected].

The critical phylogenetic position of the ascidian, Ciona intestinalis, as a protochordate suggested its potential appliciability as a model organism in diverse fields of comparative biology involving neuropeptides and hormones. However, only a few ascidian peptides were structurally and functionally characterized from ascidians. In our recent study, a combination of mass spectrometric analyses and database-searching detected 35 peptides. These peptides are categorized into three types. i) orthologs of vertebrate peptides including tachykinin, calcitonin, galanin, RF-amide peptides, and neurotensin-like peptides. ii) vasopressin and GnRH-related peptide possessing ascidian-specific molecular forms and/or functions. iii) novel family peptides such as LF peptides and YFL/V peptides. These results showed that ascidians, unlike other invertebrates, possess a variety of homologs and/or prototypes of vertebrate neuropeptides and peptide hormones, and that several ascidian peptides were diverged in ascidian-specific evolutionary lineages. We also proved the presence of unique regulatory mechanisms of GnRH receptor functions via heterodimerization. Furthermore, we have substantiated a novel protease-associated follicle growth pathway regulated by tachykinins using C. intestinalis, which is the first functional characterization of tachykinins in ovaries of any organisms. In addition, our recent studies have been veryfing that this novel tachyninergic protease-directed follicle growth mechanism is essentially conserved in mammalian early growth development. These findings provide new insight into the molecular and functional evolution and diversity of peptidergic systems in chordates.

Keyword: ascidian, Ciona intestinalis, neuropeptide, peptide hormone, follicle development

33 Symposium Lecture‐24

Anti-osteoporotic effects of phytoestrogens containing plant Pueraria mirifica: in vitro and in vivo approaches

Malaivijitnond S.1*, Urasopon N.1, Tiyasatkulkovit W.1,2, Kittivanichkul D.1, Charoenphandhu N.2 1National Primate Research Center of Thailand and Department of Biology Faculty of Science, Chulalongkorn University, Bangkok, 10330 Thailand 2Faculty of Agriculture, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand 3Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. e-mail: [email protected]

Pueraria mirifica (PM) is an endemic Thai plant which contains at least 17 phytoestrogens in the part of tuberous roots. Its estrogenic activity has been clearly verified on reproductive organs in laboratory animals (rodents and monkeys) and humans. Thus, it has been later tested and elicited the positive effects on bone cells. PM extract could simulate the expression of the genes associated with osteoblast bone formation, i.e., ALP and type I collagen and the gene associated with osteoclastogenic inhibition (bone anti-resorption), i.e., OPG/RANKL ratio in rat osteoblast-like UMR106 cells, primary rat osteoblast cells and primary baboon osteoblast cells. Further, the preventive effects of PM on bone loss induced by gonadectomy in SD female and male rats were assessed. Bone loss in trabecular and cortical bones of the various sites of axial bone (fourth lumbar vertebral body) and long bones (tibia and femur) after gonadectomy was dose-dependently prevented by 10-1000 mg/kg BW/day of PM extract after 90-day treatment. The anti-osteoporotic effect of PM extract was comparable to that of 17beta-estradiol, both in vitro and in vivo studies. The next steps of the study are to evaluate the mechanism of action of PM extract on rat and monkey osteoclast cells and to assess its anti-osteoporotic effect in cynomolgus macaques (Macaca fascicularis). If the positive effects have been evident, this will corroborate the high potential of the PM herb to be developed as anti-osteoporotic drug for human use in the near future.

Keywords: bone mass, monkey, rat, osteoblast, osteoclast

34 Symposium Lecture‐25

Catecholestrogens in final oocyte maturation in the catfish Heteropneustes fossilis: an overview

Joy K.P. Department of Zoology, Centre of Advanced Study, Banaras Hindu University, Varanasi-221005, India. e-mail: [email protected]

Catecholestrogens (CEs) are metabolites of estrogens, formed through stepwise conversions involving hydroxylation, methylation and conjugation for excretion largely through the bile. However, their distribution in tissues other than the liver and in estrogen synthesis/catabolic sites, especially in estrogen target organs, led to speculations on their possible physiological significance. CEs have been shown to participate in a number of reproductive functions such as gonadotropin release, ovarian steroidogenesis, prostaglandin secretion, egg transport, uterine weight increase, parturition and ovarian angiogenesis in mammals. However, such studies are few in non mammals. The principal CEs are 2-hydroxy- and 4-hydroxy estrogens, and 2- and 4- methoxy estrogens. In the catfish ovary, the CEs and estrogen-2- hydroxylase (EH) showed seasonal variations and periovulatory changes, inverse with E2 suggesting metabolic conversions. Furthermore, 2-OHE2 inhibited P450 aromatase activity. The EH activity was stimulated by hCG and IGF-1 and inhibited by steroids, progesterone (P4) being the most potent one. The hydroxyestrogens stimulated germinal vesicle break down (GVBD), 2-hydroxyestrogens being more effective than 4-hydroxyestrogens. 2-methoxyestrogen was a poor stimulator of GVBD but promoted mortality of the follicles. 2-OHE2 inhibited testosterone and E2, and stimulated progestins, P4,

17-OHP4 and 17, 20β-dihydroxy-4-pregnen-3-one (maturation-inducing steroid, MIS). These effects seem to be mediated through adrenergic or E2 receptor or both. Prostaglandin (PGF2α and PGE2) levels were stimulated by 2-OHE2, the effect being mediated through the above mentioned receptor mechanisms. At the signal transduction level, the involvement of cAMP-protein kinase A, MAP kinase and protein kinase C pathways was indicated depending on targets. It is suggested that CEs may provide the requisite trigger (steroidogenic shift) for the resumption of meiotic maturation of the follicles.

Keyword: catfish ovary, catecholestrogens, meiotic maturation, steroidogenic shift

35 Symposium Lecture‐26

Does bioaccumulation and biomagnification of EDC’s take place in marine fish?

Dahms H.-U.1* and Kim J.-H.2 1 Department of Life Science, College of Natural Science, Sangmyung University, Seoul 110-743, Korea. 2 Department of Fisheries and Oceans, Centre for Aquaculture and Environmental Research, 4160 Marine Drive, West Vancouver, BC, V7V 1N6, Canada e-mail: [email protected]

Pollutants are known to accumulate and to move through trophic levels within ecosystems. Bioaccumulation occurs within a trophic level and represents the concentration increase of a substance in certain tissues of organisms due to absorption from food and the environment. Biomagnification (or bioamplification) is the increase in concentration of a substance that occurs along food chains. Biomagnification commonly results from chemical persistence, food chain energetics, or rate of internal degradation and excretion. For enhanced biomagnification the pollutant must be long-lived, mobile, soluble in fats, and biologically active. There is concern about these phenomena because even small environmental concentrations can find their way into organisms at high concentrations. Among xenobiotics are endocrine disrupting chemicals (EDCs) capable of adversely affecting the function of endocrine systems, leading to changes in growth, development, and reproduction of exposed animals. Although the occurrence and implications of steroid estrogens in the environment has received some attention, there is only limited evidence for bioaccumulation in wild fish. Some laboratory studies show environmental estrogens bioconcentrated in the bile of fish prior to excretion leaving high concentrations in this specific medium. Similarly, there is no consistent indication of biomagnification of xenobiotics and EDCs observed along the food web. For example, PCDD/Fs and PCBs behave quite differently along aquatic food chains: PCDD/Fs concentrations were lower in higher tropic-level organisms with fish presenting a distinct PCDD/Fs compared to congener profiles.

Keywords: Bioaccumulation, biomagnification, EDCs, estrogenic chemicals.

36 Symposium Lecture‐27

Rhythms in fish reproduction: possible involvement of dopaminergic activity in tidal-related spawnings of a tropical wrasse

Takemura A.1*, Takeuchi Y.1, Hur S.P.1,2 1Department of Chemistry, Biology and Marine Science, University of the Ryukyus, Okinawa, Japan, 2Marine Environmental Institute, Jeju National University, Jeju, Korea [email protected]

Most tropical wrasses show a daily pattern of spawning with gamete release typically near daytime high tide. It is hypothesized that environmental cues in relation to daily and tidal cycles are perceived by fish and transduced as internal stimuli. To gain insight into these issues, involvement of monoamines in mediating endogenous day–night and tidal rhythms in the threespot wrasse, Halichoeres trimaculatus, were examined. Levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC, a metabolite of DA) in the brain of this species were measured with high-performance liquid chromatography and electrochemical detection. DOPAC and the metabolic rate of DA activity (DOPAC/DA) increased during the day and decrease during the night for fish held under a natural photoperiod. Intraperitoneal injection of melatonin resulted in a significant reduction in DOPAC/DA. DOPAC/DA was lower in fish held at 3 m compared to 0 m depth, suggesting that hydrostatic pressure influences DA metabolic rate. In vitro culture of ovaries from pressurized fish in the presence of human chorionic gonadotropin resulted in an increase in 17 ,20 -dihydroxy-4-pregnen-3-one in the medium. These results indicate that light and hydrostatic pressure control dopaminergic activity in the brain of threespot wrasse. Wrasses bury themselves in the sand at the bottom of the ocean at night and arrest their movement. Therefore, darkness and hydrostatic pressure under stable conditions in sands may be used to synchronize its spawning activity.

Keyword: brain, dopamine, hydrostatic pressure, sex steroids, tide, wrasse

37 Symposium Lecture‐28

Neuro-endocrine control of the environmental adaptability in fish – a study of fish caudal neurosecretory system

Lu W. College of Fisheries and Life Science, Shanghai Ocean University e-mail: [email protected]

Neuroendocrine systems play an important role in the adaptive physiology of animals. Stress response system is the key regulatory system to ensure that the maximum adaptive capacity of organism is matched to their physiological requirements in order to survive in changing environment. The caudal neurosecretory system (CNSS) is a part of stress response system, a neuroendocrine structure unique to fish. Large peptide-synthesizing neurons, Dahlgren cells, located in the terminal segments of the spinal cord, project to a neurohaemal organ, the urophysis, from which neuropeptides are released into the circulation. CNSS is the major site of expression and secretion of CRF, UI and UII. These peptides are differentially expressed with co-localisation of up to two in a single cell. Dahlgren cells display a range of electrical firing patterns, including characteristic bursting activity, which is dependent on L-type Ca2+ and Ca-activated K+ channels. Electrophysiological and mRNA expression studies have examined changes in response to altered physiological demands. Bursting activity is more robust and more Dahlgren cells are recruited in seawater compared to freshwater adapted fish and this is mirrored by a reduction in mRNA expression for L-type Ca2+ and Ca-activated K+ channels. Acute seawater/freshwater transfer experiments support a role for CNSS in adaptation to changing environmental salinity. We hypothesise that the Dahlgren cell population is reprogrammed during salinity adaptation, and this is seen as changes in gene expression profile and electrical activity. The CNSS shows striking parallels with the hypothalamic neurohypophysial system, providing a highly accessible system for studies of neuroendocrine mechanisms.

Key word: Stress, Endocrinology, CNSS

38 Symposium Lecture‐29

A sleep-stabilizing pathway in Drosophila composed of sex peptide receptor and its ligand, the myoinhibitory peptide

Kim Y.J. School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, South Korea. e-mail: [email protected]

Sleep occurs in accordance with other biological processes including reproduction. Here, we demonstrate that the sex peptide receptor (SPR), known essentially for its role in female reproduction, is also important in stabilizing sleep in both males and females. Mutants lacking either the SPR or its central ligand, myoinhibitory peptide (MIP), fall asleep with normal frequencies but have difficulty in maintaining sleep. Our analyses have mapped the SPR sleep function to pigment dispersing factor (pdf) neurons, an arousal center. MIP downregulates intracellular cAMP levels in pdf neurons through SPR. In response to sleep loss, MIP is secreted from a few specific brain neurons innervating pdf neurons. These results indicate that MIP-SPR signaling stabilize sleep by feeding a slow-acting inhibitory input into the arousal center. Because sex peptide, the seminal fluid-borne ligand of SPR, circulates in hemolymph of the mated female, SPR may integrate sleep-pressure and reproductive-state coding signals, and modulate sleep.

39 Symposium Lecture‐30

New insights into hormonal control of body fluid ion homeostasis: zebrafish as a model

Hwang P.P. Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan e-mail: [email protected]

Animal body fluid homeostasis is tightly controlled by hormones. Many hormones have been proposed to control the ionic and acid-base regulation mechanisms in fish; however, most of those proposed pathways still lack convincing evidences to support. Zerbafish, an emerging model with advantages of forward/reverse genetic manipulation and molecular physiological approaches, has been recently used to study regulatory physiology. In zebrafish, different types of ionocytes were identified to carry out ion transport functions through respective sets of ion transporters, and the molecular mechanisms of ionocytes proliferation and differentiation were also dissected. These provide excellent platform to precisely study the ion transport pathways and ionocytes that a hormone targets. Our loss- and gain-of-function studies demonstrated that isotocin stimulates the proliferation of epidermal stem cells and differentiation of ionocyte progenitors by regulating the P63 and Foxi3a transcription factors, consequently enhancing the functional activities of ionocytes. Similarly, the cortisol/glucocorticoid receptor signaling stimulates ionocytes differentiation through the mediation of Foxi3a. Stanniocalcin, on the other hand, suppress the ion transport functions by down-regulating the ionocytes differentiation through Foxi3a. Our recent experiments further investigated the involvement of endothelin signaling in the activation (without translational regulation) of the H-ATPase mediated acid secretion function in a specific group of acid-secreting ionocytes upon acute acidification in environment. As such, these hormones control zebrafish ionic and acid-base homeostasis through regulations of the activities/functions of target ion transporters or ionocytes at the level(s) of transcription, translation or post-translation, and/or different developmental stages of the cells.

Keyword: isotocin, cortisol, stanniocalcin, endothelin, ion regulation

40 Symposium Lecture‐31

Yolk formation in a stony coral Euphyllia ancora (Cnidaria, Anthozoa): insight into the evolution of vitellogenesis in non-bilaterian animals.

Shikina S.1*, Chen C.J.2,3, Chung Y.J.2, Shao Z.F.2, Liou J.Y.2, Tseng H.P.2, Lee Y.H.4, Chang C.F.1,2 1Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan. 2Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan. 3Department of Oceanography, National Taiwan University, Taipei, Taiwan. 4Tungkang Biotechnology Research Center, Fisheries Research Institute, Tungkang, Taiwan. e-mail: [email protected]

Vitellogenin (Vg) is a major yolk protein precursor in numerous oviparous animals. Numerous studies in bilateral oviparous animals have shown that Vg sequences are conserved across taxa and that Vgs are synthesized by somatic-cell lineages, transported to and accumulated in oocytes, and eventually used for supporting embryogenesis. In non-bilateral animals (Polifera, Cnidaria, and Ctenophora), which are regarded as evolutionarily primitive, although Vg cDNA has been identified in two coral species from Cnidaria, relatively little is known about the characteristics of yolk formation in their bodies. To address this issue, we identified and characterized two cDNA encoding yolk proteins, Vg and egg protein (Ep), in the stony coral Euphyllia ancora. RT-PCR analysis revealed that expression levels of both Vg and Ep increased in the female colonies as coral approached the spawning season. In addition, high levels of both Vg and Ep transcripts were detected in the putative ovarian tissue, as determined by tissue distribution analysis. Further analyses using mRNA in situ hybridization and immunohistochemistry determined that, within the putative ovarian tissue, these yolk proteins are synthesized in the mesenterial somatic cells but not in oocytes themselves. Furthermore, Vg proteins that accumulated in eggs were most likely consumed during the coral embryonic development, as assessed by immunoblotting. The characteristics of Vg that we identified in corals were somewhat similar to those of Vg in bilaterian oviparous animals, raising the hypothesis that such characteristics were likely present in the oogenesis of some common ancestor prior to divergence of the cnidarian and bilaterian lineages.

Keyword: coral, vitellogenin, egg protein, Euphyllia ancora, oogenesis

41 Symposium Lecture‐32

Impacts of Early Life Stress on Anxiety and Reproduction

Soga T. 1* and Parhar I.S. 1 1Brain Research Institute, School of Medicine and Health Sciences Monash University, Sunway, Malaysia e-mail: [email protected]

Early-life stress lastingly influences brain development and the underlying expression of emotional control such as anxiety and reproductive activity during adolescence and adulthood. Exposure to neonatal or early postnatal animals to synthetic glucocorticoid (Dexamethasone; DEX) impacts on the hypothalamo-pituitary adrenal (HPA) and hypothalamo-pituitary gonadal axis. This results in high anxiety-like behavior, delayed puberty and disrupts estrus cyclicity which persist into adulthood. We have recently shown in neonatal DEX treated mice slow growth and high anxiety using open field test. Furthermore, we have found that down-regulation of hypothalamic gonadotropin-releasing hormone (GnRH)-kisspeptin signaling identified as an essential factors for pubertal onset and gonadotropins secretion, with delays puberty and causes irregular estrus cycles in neonatal DEX treated female mice. In addition, we have shown neonatal DEX induces expression of hypothalamic gonadotropin-inhibitory hormone (GnIH), negative regulator for preoptic GnRH neurons, and expression of GnIH receptors in the preoptic area (POA) of mice. Considerable progress has been made concerning the mechanisms underlying DEX induced reproductive dysfunction by enhancement of GnIH signaling in the POA where GnRH neurons are mainly located. These results suggest that early-life stress induced anxiety is linked to the deregulation of HPA and down regulation of kisspeptin and up-regulation of GnIH signaling may contribute to the developmental cues that modulate the GnRH neuronal activity critical for reproduction, pubertal onset and normal estrus cyclicity.

Keyword: stress, reproduction, GnRH, Kisspeptin, GnIH

42 Symposium Lecture‐33

Xenopus as a model to study neuronal axons

Jung H. Yonsei University e-mail: [email protected]; [email protected]

Proper neural function depends on the precision and stability of neural connections, which are made by axons. The distal tip of a growing axon receives numerous external signals along the way to its target, to which it must promptly respond in order to make appropriate navigational decisions. An increasing body of evidence indicates that the distal axon can respond to extrinsic cues without the help of its cell body. This subcellular signaling autonomy requires local protein synthesis, and recent evidence shows that axonal protein synthesis also plays a key role in mature axons, where it is required for their long-term survival. I will introduce an emerging link between local protein synthesis and axon’s ability to interact with the environment and the advantages of Xenopus to study the biology of the axon

43 Symposium Lecture‐34

Evolution and origin of seasonal sensor in vertebrates

Yoshimura T.1-3 1 Institute of Transformative Bio-Molecules and 2Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, and 3Division of Seasonal Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan e-mail: [email protected]

Animals living outside the tropics use changes in day length to adapt to seasonal changes in environment, but the molecular and endocrine mechanisms underlying seasonal time measurement are not fully understood. The Japanese quail is a robust model for the study of these mechanisms because of its rapid and dramatic response to changes in photoperiod. We have demonstrated that local thyroid hormone catabolism within the mediobasal hypothalamus (MBH) by thyroid hormone-activating enzyme (type 2 deiodinase: DIO2) regulates photoperiodism. Functional genomics analysis in quail demonstrated that long day stimulus induces thyrotropin (thyroid stimulating hormone: TSH) production in the pars tuberalis (PT) of the pituitary gland, which triggers DIO2 expression in the ependymal cells of the MBH. In mammals, nocturnal melatonin secretion provides an endocrine signal of the photoperiod to the PT that contains melatonin receptors in high density. We have also demonstrated the involvement of TSH signalling pathway in mammals by using the TSH receptor null mice. Although fish also exhibit robust seasonal responses, they do not possess an anatomically distinct PT. We found expression of TSH, DIO2, and rhodopsin family genes in the saccus vasculosus (SV), suggesting the existence of a photoperiodic signalling pathway from light input to neuroendocrine output. Although physiological function of SV has been a mystery for several centuries, functional analysis suggested that the SV acts as a seasonal sensor in fish. I would like to review the current understanding of the mechanism underlying photoperiodism in vertebrates.

Keyword: photoperiodism, thyrotropin, pars tuberalis, photoreceptor

44 Oral Presentation‐1

Epigenetic modulation of rhythmic GnRH gene transcription by dynamic histone modifications

Kim H.-D.1*, Chung S.2, Son G.H. 3, Kim K.1 1Department of Brain & Cognitive Sciences and Department of Biological Sciences, Seoul National University, Seoul 151-742, KOREA. 2Department of Anatomy; 3Department of Legal Medicine, Korea University College of Medicine, Seoul 136-705, KOREA. e-mail: [email protected]

Gonadotropin-releasing hormone (GnRH) is a pivotal hypothalamic neurohormone governing reproduction and sexual development. The pulsatile release of GnRH into the portal vessel is essential for pituitary gonadotrope function. To determine whether the GnRH gene expression exhibits rhythmic oscillations at the level of gene transcription, GnRH primary transcript levels were measured in immortalized GnRH-expressing GT1 cells after a serum treatment for synchronization. Primary transcript levels of GT1 cells exhibited rhythmic oscillation with an hour interval, and this rhythm was not abolished by inhibition of translation. However the oscillation was profoundly disrupted by the changes in histone acetylation and methylation states. Kinetic chromatin immunoprecipitation assay revealed that histone acetylation and methylation also showed ultradian kinetics in the GnRH genomic regions. Furthermore, histone methylations at the transcription start site of GnRH gene showed anti-phasic cycles between transcriptional active (H3K4-Me3) and repressive (H3K9-Me2) histone markers. These results implicate that dynamic chromatin modifications may confer the ultradian oscillation of GnRH transcription.

Keyword : Gonadotropin-releasing hormone (GnRH), Transcriptional regulation, Epigenetic regulation, Ultradian rhythm, Histone modification

45 Oral Presentation‐2

Exposure to a maternal n-3 fatty acid-deficient diet during the brain development enhance the activity and dysregulation of hypothalamic-pituitary-adrenal axis responses to stress in rat offspring later in life

Hsieh Y.J. and Su H.M.* Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan e-mail: [email protected], [email protected]

Brain docosahexaenoic acid (DHA, 22:6n-3) accumulates rapidly during brain development and is essential for normal neurological function. The aim of this study was to examine whether DHA deficiency during brain development leads to dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress later in life. Rats were exposed to an n-3 fatty acid-deficient or n-3 fatty acid-adequate diet from embryo to weaning at 3-weeks-old via maternal intake throughout the pregnancy and lactation, and then were changed to chow diet till sacrificed at 10-week-old. We found that the maternal rats fed n-3 fatty acid-adequate diet showed significant higher licking/grooming and arch-back nursing behavior than the maternal rats fed n-3 fatty acid-deficient diet. Exposure to the maternal n-3 fatty acid-deficient diet during the brain development resulted, at weaning, in a significant decrease in hypothalamic and hippocampal DHA levels and a reduced male offspring body weight. DHA deficiency during the brain development significantly increased and prolonged restraint stress-induced changes in colonic body temperature and serum corticosterone levels, caused a significant increase in sensitivity to dexamethasone negative feedback, and enhanced depressive-like behavior in the forced-swimming test and anxiety-like behavior in the plus-maze test in later life. These results suggest that DHA deficiency during brain development leads to excessive HPA responses and blunted HPA negative regulation to stress and elevated behavioral indices of depression and anxiety in adulthood.

Keyword: docosahexaenoic acid, HPA axis, corticosterone, stress, depression

46 Oral Presentation‐3

Identification of small molecule Cryptochrome inhibitor modulating the molecular circadian clock

Chun S.K.1*, Chung S.2, Jang J.4, Son G.H.4, Suh Y.G.4, Kim K.1 1Department of Brain & Cognitive Sciences and Department of Biological Sciences, Seoul National University, Seoul 151-742, KOREA. 2Department of Anatomy; 3Department of Legal Medicine, Korea University College of Medicine, Seoul 136-705, KOREA.4College of Pharmacy, Seoul National University, Seoul 151-742, KOREA e-mail: [email protected]

Circadian rhythm, a biological oscillation with a period of about 24 hours, is maintained by genetically determined a time-keeping system. Disruption of normal circadian rhythm linked to the onset of various human diseases, including metabolic and mood disorders. In the present study, we aimed to identify novel small molecule modulators for the molecular circadian clockwork. To find this small molecule, we designed a unique two-step cell-based screening strategy based on E-box-mediated transcriptional activity and screened more than 1000 compounds. We identified a compound (KS15) as a small molecule inhibitor for both CRY1 and 2 (CRY1/2) which are key negative components of the mammalian circadian clockwork. This compound directly interacted with CRY1/2 and enhanced E-box-mediated transcription in a CRY1/2-dependent manner. Moreover, KS15 attenuated the circadian oscillation of Per2-Luc and Bmal1-dsLuc activities in cultured fibroblasts, indicating the KS15 can functionally inhibit the action of CRY1/2. In summary, the present study provides a novel chemical inhibitor of CRY1/2 function, thereby activating CLOCK/BMAL1-evoked E-box-mediated transcription. Further optimizations and subsequent functional studies of this compound may lead to development of efficient therapeutics of several disorders with circadian nature.

Keyword : Cryptochrome, Circadian rhythm, Small molecule, Cell-based screening

47 Oral Presentation‐4

Identification of a novel transglutaminase gene that is differentially expressed between the sexes in the medaka brain

Maehiro S.1*, Oka Y.1, Okubo K.2 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan. 2Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan. e-mail: [email protected]

Vertebrates exhibit differences between sexes in a variety of behavioral and physiological traits. Many of these differences presumably reflect underlying sex differences in the brain. To dissect the molecular and cellular basis of sexual differentiation of the teleost brain, which maintains marked sexual plasticity throughout the life, we searched for genes differentially expressed between the sexes in the brain of medaka Oryzias latipes. Here we identified one gene with highly female-biased expression, f13a1a (encoding coagulation factor a, which belongs to the transglutaminase family). This gene showed approximately 15-fold higher expression in females than males at the whole brain level. Female-specific expression of f13a1a was observed in several nuclei in the preoptic area, including the parvocellular portion of the magnocellular preoptic nucleus (PMp) and the magnocellular portions of the magnocellular preoptic nucleus (PMm), which have been implicated in controlling pituitary hormone secretion and sexual behavior, respectively. We then examined the effects of sex steroid hormones on f13a1a expression by using females subjected to ovariectomy followed by sex steroid hormone treatment. Expression of f13a1a in the PMp and PMm was significantly decreased by ovariectomy and was fully restored by subsequent treatment with estrogen, suggesting that the female-specific expression of f13a1a in these nuclei arises from the stimulatory action of estrogen secreted by the ovary. The sexually dimorphic expression of f13a1a may contribute to sex differences in some behavioral and/or physiological traits.

Keywords: sex differences, medaka, brain, sex steroid hormone

48 Oral Presentation‐5

Glucocorticoid-mediated Period2 induction delays the phase of circadian rhythm

Cheon S. 1,3,4*, Park N.2,3 , Kim K.1,2,3,4 1Interdisciplinary Program in Neuroscience, Seoul National University, Seoul, Korea. 2Department of Biological Sciences, Seoul National University, Seoul, Korea. 3Brain Research Center for the 21st Century Frontier R&D Program in Neuroscience, Seoul National University, Seoul, Korea. 4Department of Brain & Cognitive Sciences, Seoul National University, Seoul, Korea. e-mail: [email protected]

Circadian rhythm regulates overall physiology and behavior with about 24-hr period. The master clock is resided in the suprachiasmatic nucleus (SCN) of the hypothalamus, but peripheral tissues also have molecular circadian clock machineries. Therefore, to maintain the robust circadian rhythm, the synchronization between the SCN and peripheral clocks is important. Glucocorticoid (GC) is one of the synchronizing signals that can induce the expression of circadian clock genes, such as Period2 (Per2). In the present study, we found that GC prominently induced Per2 expression and delayed the circadian phase. To understand how GC induced the prominent Per2 expression, compared to other synchronizing signals, we investigated molecular mechanisms of GC-mediated Per2 induction. The sequence analysis of Per2 promoter revealed that the overlapping GRE and E-box is responsible for GC-mediated Per2 induction. Interestingly, the GRE in Per2 promoter required Bmal1 for GC responsiveness, whereas other GRE-containing promoters, such as Period1 and mouse mammary tumor virus, still responded to the synthetic GC in the absence of Bmal1. Bmal1-dependent GC responsiveness might be due to Bmal1-dependent binding of the GC receptor to Per2 GRE. To know the functional importance of GC-mediated Per2 induction, we performed the rescue experiment using the adenoviruses, which express the fusion protein of PER2 and LUCIFERASE under the control of Per2 promoter. When Per2 could not respond to the synthetic GC because of the mutation of GRE or E-box, the phase of circadian oscillation failed to be delayed when compared to that of the wild type. Therefore, the present study reveals that GC-mediated Per2 induction by the novel regulatory mechanism is important for delaying the circadian rhythm.

Keyword: glucocorticoid, circadian rhythm, Period2, Bmal1

49 Oral Presentation‐6

Involvement of corticotropin-releasing hormone and corticosteroid receptors in the brain-pituitary of tilapia during cronic and acute salinity stress

Aruna A.1*, Nagarajan G.1, Chang C.F.1,2 Department of Aquaculture and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung 20224, Taiwan email: [email protected]

The mRNA expression of corticotropin-releasing hormone (CRH) and the hormone receptors CRH-receptor/CRH-R, glucocorticoid receptor 1/2 (GR1/2) and mineralocorticoid receptor (MR) were studied in the brain and pituitary of tilapia (Oreochromis mossambibus) during chronic and acute salinity stress by quantitative real-time (Q-PCR) analysis. The results indicated that the transcripts of CRH and CRH-R were increased in the forebrain, whereas elevated hypothalamic CRH suppressed the CRH-R mRNA in the pituitary in seawater (SW) fish in long term salinity stress. In addition, we performed in situ hybridisation analysis to localize and differentiate the CRH, CRH-R, GR1, GR2 and MR transcripts in the brain of freshwater (FW)- and SW-acute acclimated tilapia during salinity stress. In virtually all transcripts, the hybridization signal was significantly abundant in the SW-acute acclimated tilapia brain, especially in the dorsal ventral cephalon (Vd), dorsal nucleus preopticus pars magnocellularis (NPOmc) and dorsal nucleus preopticus pars parvocellularis (NPOpc). Furtermore, we analyzed the effects of salinity stress on the expression and localization of CRH in nucleus lateralis tuberis (NLT) and nucleus preopticus (NPO) of hypothalamus, GR2, MR, CRH-R, pro-opiomelanocorticotropin (POMC) transcripts in pituitary during FW and SW acclimation at acute stress. The results indicated that the CRH transcripts increased in NLT of the hypothalamus and rostral pars distalis (RPD) of the pituitary at SW fish. On the other hand, the CRH-R, GR2 and MR transcripts were more widely expressed in RPD and pars intermedia (PI) of pituitary at SW than FW fish. The data highlighted that the SW acclimation elevated these transcripts in pituitary may be related to the activation of the hypothalamic pituitary interrenal (HPI)-axis. Furthermore, the more abundant expression of CRH-R, GR2 and MR transcripts in RPD after SW transfer implied that the anterior pituitary was highly involved in addition to the posterior pituitary during SW acclimation. In addition, dual in situ hybridization results revealed that the transcripts of CRH-R, GR2 and MR with POMC were highly co-localized in corticotrophs of the anterior pituitary. The co-localization of POMC with CRH-R and corticosteroid receptors suggests that glucocorticoids may directly regulate the HPI-axis function not only at the hypothalamic level but also at the pituitary level.

Key words: corticosteroid receptors, POMC, CRH-R

50 Oral Presentation‐7

Histomorphological study on the development of thymus of Oryctolagus cuniculus

Hazarika J.K. Department of Zoology Darrang College, Tezpur, Affiliated to Gauhati University Assam, India Email: [email protected]

The thymus plays a pivotal role in the development, sustenance and integrity of the immune system. A thorough understanding of this important gland is therefore very essential. Oryctolagus cuniculus commonly known as rabbits are extensively used in different biomedical researches. No systematic study has been done on the postnatal development of the thymus of rabbit. In this study thirty nine numbers of apparently healthy (irrespective of sex) postnatal rabbits of different age group were utilized. The thymus of rabbit was invested by thin connective tissue fibres and cells from which projected the connective tissue septa that partially divided the gland into lobules. Hassall’s (thymic) corpuscles were observed in the medulla of each lobule. Myoid cells were found in the medulla of thymic lobules. The micrometrical values of cortex of thymus showed a decreasing trend with the increase in the age of the animal. Medullary tissue and Hassall’s corpuscles of the thymus of rabbit increased as the age of the animal advanced.

Key Words: thymus, rabbit, histomorphological, cortex, medulla.

51 Oral Presentation‐8

Effects of cadmium on dopaminergic and serotonergic neurons and involvement of estrogen signaling pathway during early development of zebrafish

Sugiyono* and Kishida M. Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan. e-mail: [email protected]

Cadmium is a heavy metal toxicant and known to bind to estrogen receptor. Although early life exposure of cadmium is associated with increased risk of behavioral alteration, its mechanism still needs to be elucidated. Previously we have shown that estrogen regulates the activity of dopaminergic and serotonergic neurons to control motor activity. Therefore, in this study we investigated the effects of cadmium on dopaminergic and serotonergic neurons in zebrafish embryos and larvae. We found that cadmium exposure decreased the response to tactile stimulation at 48-96 hpf (0.1-10 M), whereas spontaneous swimming activity was induced at 96 hpf (0.1 M). Exposure to L-dopa (dopamine agonist) reversed the decrease in response to tactile stimulation caused by cadmium at 48 hpf, and exposure to fluphenazine (dopamine receptor blocker) abolished the swimming activity caused by cadmium at 96 hpf. However, cadmium exposure did not change the expression of tyrosine hydroxylase. Further investigation showed that serotonin synthesis was decreased by cadmium at 48 hpf. Effects of cadmium on response to tactile stimulation and swimming activity were reversed by addition of quipazine (serotonin agonist) and methysergide (serotonin receptor blocker), respectively. On the other hand, ICI (estrogen receptor blocker) did not reverse the effect of cadmium on the response to tactile stimulation, indicating the effect of cadmium was not through estrogen receptor. However, interestingly, ICI blocked the induction of swimming activity, while estrogen exposure did not induce spontaneous swimming. These results suggest that the effect of cadmium on motor behavior is mediated through both dopamine and serotonin signaling pathways. In addition, cadmium may act on spontaneous swimming through estrogen signaling pathway.

Keyword: Cadmium, zebrafish, behavior, dopamine, serotonin.

52 Oral Presentation‐9

Characterization of Dmrt gene family in a scleractinian coral, Euhyllia ancora

Chen C.J.1,2*, Shinya S.2, Chang C.F.2 1 Institute of Oceanography, National Taiwan University, Taipei, Taiwan 2 Department of Aquaculture, National Oceanic Taiwan University, Keelung, Taiwan e-mail: [email protected]

The Dmrt (doublesex and mab3-related transcription factor) family, a name originated from a combination of two sexual-related genes, doublesex (dsx) in Drosophila melanogaster and male abnormal (mab-3) in Caenorhabditis elegans. The identification of Dmrt family is based on a DM domain with a zinc-finger like DNA-binding motif containing cysteine-rich residues. It is well known that Dmrt gene family plays important roles in sexual determination, sexual differentiation and neuron development. In this research, 7 different types of Dmrt genes (EaDmrtA, EaDmrtB, EaDmrtC, EaDmrtE, EaDmrtF, EaDmrtG, EaDmrtH) were found in Euphyllia ancora. Those 7 EaDmrt genes are divergent from the bilateral Dmrt gene family and were clustered into 7 distinct lineages based on phylogenetic analysis. All EaDmrt genes could be detected in both sexuality of their tentacles, putative gonads, mesentarial filaments by semi-quantitative PCR analysis. However, the relative expression pattern of two Dmrt genes (EaDmrtC and EaDmrtE) showed significantly variations in spatial distribution and also sexuality. Particularly, the majority of DmrtE was expressed in female gonads (One-way anova, p<0.05). In this research, we found the first female specific Dmrt gene in invertebrate, and this female specific EaDmrtE may play a crucial role in oogenesis.

Key words: Dmrt gene family, gonochoric E. ancora, diversity, female specific, oogenesis,

53 Oral Presentation‐10

Neuroendocrine gene expression levels at different ovarian developmental stages in wild Japanese eel

Jeng S.R.1*, Pen Y.T.1, Yueh W.S.1, Dufour S.2 , Chang C.F.3,4 1 Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung, Taiwan 2 Muséum National d'Histoire Naturelle, Research Unit BOREA, Biology of Aquatic Organisms and Ecosystems, CNRS 7208 Paris, France 3 Department of Aquaculture, 4 Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan e-mail: [email protected]

The aim of the present study was to decipher the molecular aspects of various endocrine parameters of BPG axis at different ovarian developmental stages in wild yellow and silver female Japanese eels. In this study, the GSI of the wild female eels were in the range of

0.18-2.3 %, corresponding to yellow, pre-silver and silver stages. Both serum E2 and T concentrations significantly increased with ovarian development indicating a significant activation of steroidogenesis during silvering. The significant increase in pituitary FSH-β and LH-β transcripts suggests that activation of pituitary gonadotropin expression is likely responsible for the significant ovarian development observed during silvering. The mGnRH and the three GnRHRs transcripts did not change in the brain and pituitary suggest that the GnRH system is not significantly activated during silvering. Our results on KissR suggest that the stimulatory kisspeptin system is not yet activated in silver eels. The TH transcripts in the brain did not change in wild female Japanese eels during silvering. A significant decrease in D2BR expression in the forebrain and pituitary was observed, with no changes in D2AR. The decrease in the pituitary expression of D2BR during silvering would allow a reduced inhibitory effect of DA. We may raise the hypothesis that this regulation of D2BR gene expression is involved in the slight activation of the pituitary gonadotropin and gonadal activity that occur at silvering.

Keywords: Kisspeptin, GnRH, dopamine, dopamine D2 receptor, eel

54 Oral Presentation‐11

A novel pterostilbene derivative supresses prostate cancer cell growth irrespective of androgen receptor status

Nikhil K., Sharan S., Roy P.* Molecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, Uttarakhand, India email: [email protected]

Chemotherapy and anti-hormonal therapy are the most common treatments for non-organ-confined prostate cancer (PCa). However, the effectiveness of these therapies is limited, thus necessitating the development of alternative approaches. The present study focused on analyzing the role of pterostilbene (PTER)-isothiocyanate (ITC) conjugate - a novel class of hybrid compound synthesized by appending an ITC moiety on PTER backbone - in regulating the functions of androgen receptor (AR), thereby causing apoptosis of PCa cells. The conjugate molecule caused 50% growth inhibition (IC50) at 40 ± 1.12 and 45 ± 1.50 µM in AR positive (LNCaP) and negative (PC-3) cells, respectively. Reduced proliferation of PC-3 as well as LNCaP cells by conjugate correlated with accumulation of cells in G2/M phase and induction of caspase dependent apoptosis. Both PI3K/Akt and MAPK/ERK pathways played an important and differential role on conjugate-induced apoptosis of these PCa cells. While AKT inhibitors greatly sensitized PC-3 cells to conjugate-induced apoptosis, on the contrary, apoptosis was accelerated by ERK inhibition in case of LNCaP cells both culminating in cleaved caspase 9 protein expression. Moreover, anti-androgenic activity of the conjugate was mediated by decreased expression of AR and its co-activators (SRC-1, GRIP-1), thus interfering in their interactions with AR. All these data suggests that conjugate-induced inhibition of cell proliferation and induction of apoptosis are partly mediated by the downregulation of AR, Akt, and ERK signaling. These observations provide a rationale for devising novel therapeutic approaches for treating PCa by using conjugate alone or in combination with other therapeutics.

Keywords: Pterostilbene-isothiocyanate conjugate, Prostate cancer, Apoptosis, Androgen receptor, Dihydrotestosterone

55 Oral Presentation‐12

Molecular cloning and characterization of feeding regulator-NPY and NPF genes and its applications in attractant development for finfish and crustacean

Lu J.K.*, Song J.X., Chen T.Y. Department of Aquaculture, National Taiwan Ocean University, keelung, Taiwan 20224 e-mail: [email protected]

The hypothalamus has long been recognized as the focal brain region regulating food intake. The hypothalamus integrates peripheral signals relaying information about nutritional status and body stores and mediates appropriate changes in the expression of orexigenic (appetite-stimulating) and anorexigenic (appetite-repressing) factors. There are distinct neuronal circuits within the hypothalamus which produce the neuropeptides controlling food intake. In order to better understand the hormonal regulation of feeding in finfish, we have cloned the cDNAs encoding the appetite-related hormones NPY and NPY receptor from orange-spot grouper (Epinephelus coioides). The result indicated NPY is mainly expressed in the hypothalamus. We examined peri-prandial changes in mRNA expression of NPY and NPY receptor during a daily feeding period. We also had determined NPY changes in expression in a state of negative energy balance induced by food deprivation. The neuropeptide F (NPF) are proposed by some to be the homologs of vertebrate neuropeptide Y and NPF could mainly regulate food intake, metabolism, reproduction, and stress reaction in invertebrate. We have identified and characterized NPF-encoding transcripts from the penaeid shrimp (Litopenaeus vannamei) and Chinese mitten crab (Eriocheir sinensis). The results indicated that NPF play a role in the regulation of appetite and feeding behavior in crustacean. We have also demonstrated that the endogenous NPY and NPF genes expression level can be used as indicators in screening attractant candidate as functional additives for finfish and crustacean aquafeeds.

Keywords: feeding regulator, neuropeptide, NPY, NPF, attractant

56 Oral Presentation‐13

Development of a DNA microarray and its application to the elucidation of gene transcription dynamics in ovary fragments of Litopenaeus vannamei incubated with sinus gland extract

Okutsu T.1*, Kang B.J.1, Bae S-H.1, Wilder M.N.1 1Fisheries Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Japan. e-mail: [email protected]

Shrimp are an economically important target of aquaculture world-wide. In many commercial species, however, it remains difficult to rear broodstock, especially in females, under artificial environments because much remains unknown concerning reproductive mechanisms. In order to obtain a better understanding of the molecular mechanisms of shrimp reproduction, we employed the whiteleg shrimp, Litopenaeus vannamei, and second-generation sequencing to obtain full transcriptomic information on gene expression in the ovary and in several somatic tissues in adult females. Approximately 1.86 million clones were sequenced and assembled, and 72,434 contigs and 41,025 singlets were obtained. Based on these sequences, 146,565 probes were designed and subsequently used for the development of a DNA microarray. Microarray analysis was then performed for the purpose of comparing gene expression levels between freshly excised ovaries and ovary fragments that were incubated in vitro with/without sinus gland extracts (SGE) for 8 hours at 28 °C. In ovaries that were incubated for 8h without SGE, 19 genes were found that were expressed at levels of one-fourth those in ovaries that were unincubated. Furthermore, in ovaries that were incubated for 8h with SGE, the same 19 genes were found to be expressed at levels 4-fold higher than those in unincubated ovaries (e.g., troponin C isoform 1, sulfate transporter). On the other hand, following 8h of incubation without SGE, there were 27 genes that were expressed in ovaries at levels 4-fold higher than those in unincubated ovaries. Upon incubation for 8h with SGE, the same 27 genes were found to be expressed at levels of one-fourth those in unincubated ovary (e.g., vitellogenin, plasmid mobilization protein). Based on the above, a DNA microarray in L. vannamei was successfully established and microarray analysis revealed the differential expression of genes under the effects of SGE. We expect that further analysis using this DNA microarray will help achieve a better understanding of molecular reproductive mechanisms in L. vannamei.

Key words: Shrimp, microarray, ovary

57 Oral Presentation‐14

A novel glucagon-related peptide (GCRP) and its receptor GCRPR account for coevolution of their family members in vertebrates

Park C.R., Moon M.J., Park S., Kim D.K., Cho E.B., Seong J.Y., Hwang J.I.* Laboratory of G-protein Coupled Receptors, Graduate School of Medicine Korea University, Seoul, Republic of Korea e-mail: [email protected]

The glucagon (GCG) family consists of GCG, glucagon-like peptide 1 (GLP1), and GLP2, which are derived from a common GCG precursor, and the glucose-dependent insulinotropic polypeptide (GIP). These peptides interact with their cognate receptors which belong to the secretin-like G protein-coupled receptor family. We used bioinformatics to identify genes encoding a novel GCG-related peptide (GCRP) and its cognate receptor, GCRPR. The GCRP and GCRPR genes were found in representative tetrapod taxa such as anole lizard, chicken, and Xenopus, and in teleosts. However, they were not present in mammals and zebrafish. Phylogenetic and genome synteny analyses showed that GCRP emerged through two rounds of whole genome duplication (2R) during early vertebrate evolution. GCRPR appears to have arisen by local tandem gene duplications from a common ancestor of GCRPR, GCGR, and GLP2R after 2R. Biochemical ligand-receptor interaction analyses revealed that GCRP had the highest affinity for GCRPR compared with other GCGR family members. Stimulation of chicken, Xenopus, and medaka GCRPRs activated Gs-mediated signaling. In contrast to chicken and Xenopus GCRPRs, medaka GCRPR also induced Gq/11-mediated signaling. Chimeric peptides and receptors showed that the K16M17K18 and G16Q17A18 motifs in GCRP and GLP1, respectively, may at least in part contribute to specific recognition of their cognate receptors through interaction with the receptor core domain. In conclusion, we present novel data demonstrating that GCRP and GCRPR evolved through gene/genome duplications followed by specific modifications that conferred selective recognition to this ligand-receptor pair.

Keyword: glucagon-related peptide, Bioinformatics, glucagon, evolution, G-protein

58 Oral Presentation‐15

Genetic polymorphisms and DNA methylation in exons CpG rich regions of PACAP gene and its effect on mRNA expression and growth traits in Half smooth tongue sole (Cynoglossus semilaevis)

Si Y. 1 *, Wen H. 1, He F. 1 Fisheries College, Ocean University of China, Qingdao, PR China e-mail: [email protected]

The pituitary adenylate cyclase-activating polypeptide (PACAP) is a new type of hypophysiotropic hormone and plays an important role in regulating the synthesis and secretion of growth hormone(GH) and gonadotropin(GtH). The research on the relationship between single nucleotide polymorphism (SNP) in PACAP gene and different growth traits would contribute to explain its function during the process of growth. Moreover, epigenetic modifications, especially DNA methylation at the CpG site of the SNPs plays an important role in regulating gene expression. The results showed that one SNP in PACAP gene of male half smooth tongue sole(Cynoglossus semilaevis) was identified. The mutation (c.C151G) was located in CpG rich region of PACAP gene. According to multiple comparison analysis, this SNP was significantly associated with body weight, body length, liver weight, net weight and Hepatosomatic index（HSI）(P<0.05). Furthermore, the transition from C to G added a new methylation site of the PACAP gene. Intriguingly, this SNP was significantly related to the expression and the methylation level of PACAP gene, individuals with GG genotype had higher growth traits and gene expression level than those with CG and GG genotypes. Moreover, the methylation content of GG genotype was lower than CG and GG genotypes. This implied that the mutation and methylation status of PACAP gene could influence growth traits and this locus could be regarded as a candidate genetic or epigenetic marker for half smooth tongue sole molecular breeding.

Keyword: PACAP, SNP, DNA methylation, Growth, Molecular markers

59 Oral Presentation‐16

Effects of neuropeptide, neurosteroidogenic enzymes and estrogen receptors in the early brain of protogynous fish: an in vivo approach

Nagarajan G.1,2*, Aruna A.2 , Chang C.F.1,2 1Center of Excellence for the Oceans, 2Department of Aquaculture, National Taiwan Ocean University, Keelung 20224, Taiwan E-mail: [email protected]

The orange-spotted grouper (Epinephelus coioides) brain exhibits a unique feature of early development during gonadal sex differentiation. In recent year, we are focusing the functional development of early brain in emphasis to neuroestogen synthesis. The neuropeptide arginine-vasotocin (AVT) having versatile physiological functions in non-mammalian vertebrates. However, the functional association between AVT, neurosteroidogenic enzymes and estrogen receptors (ers) in relation to estrogen synthesis in the early brain of teleoets remains elusive. Therefore, we studied the developmental expression patterns of AVT and their V1 type receptor (AVT-RV1) at different developmental ages (90 to 150-dah; day after hatching) in relation to neurosteroidogenesis and estrogen signalling. AVT and AVT-RV1 mRNAs were significantly expressed during development with higher abundance at 110-dah in the forebrain and hypothalamus. Further, AVT mRNA was localized in three magnocellular neuronal populations of the preoptic areas (POA) such as parvocellular (PMpc), magnocellular (PMmc) and gigantocellular (PMgc) preoptic neurons. Intriguingly, the AVT transcripts in those neurons were more abundant in 120-dah. Next, dual fluorescence in situ hybridization analysis show that AVT gene was highly coexpressed with steroidogenic enzyme genes and ers in the PMpc, PMmc and PMgc neurons, indicating potential for functional associations. Cyp19a1b immunorecative (ir) positive fibres were also found in close proximity of AVT expressing neurons. Moreover, in vivo data of AVT-injected groupers showed a significant increase in both cell and mRNA levels of steroidogenic enzymes and estrogen receptors. Further, exogenous estradiol (E2) strongly up regulated AVT mRNAs in the grouper brain. Taken together, the present study demonstrated that modulation of neuropeptide may mediate some of the possible expressions of neurosteroids and reproductive related functions during the early brain development. keywords: brain, steroidogenic enzymes, estrogen receptors, AVT, gonadal sex differentiation

60 Oral Presentation‐17

Endocrine disruptor TCDD induces zebrafish CYP3A65 transcription via AHR2-dependent pathway

Chang C.T.1, Chung H.Y.1, Su H.T.1, Tseng H.P.1, Tzou W.S.1,2, Hu C.H.1,2* 1Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan 2Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan e-mail: [email protected]

CYP3A proteins are the most abundant CYPs in the liver and intestines and play a pivotal role in drug metabolism. In mammals, CYP3As are induced by various xenobiotics through processes mediated by PXR. Previously, we have identified CYP3A65 in zebrafish, a CYP3A ortholog that is constitutively expressed in gastrointestinal tissues and can be upregulated by dexamethasone and rifampicin treatment. In addition, the CYP3A65 gene could also be induced by tetrachloro-dibenzo-p-dioxin treatment. However, the underlying mechanism of TCDD-mediated CYP3A65 transcription is unclear. Here we generated two transgenic zebrafish, Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP), which contain 2.1 and 5.4 kb 5’ flanking sequences, respectively, of the CYP3A65 gene upstream of the EGFP. Both transgenic lines exhibit an EGFP expression pattern in larval gastrointestinal tissues that is similar to that of the endogenous CYP3A65 gene. Moreover, EGFP expression can be significantly induced by TCDD exposure during the larval stage. In addition to TCDD, EGFP expression can be stimulated by kynurenine, a putative AHR ligand produced from tryptophan metabolism. The AHRE elements in the upstream regulatory region of the CYP3A65 gene are indispensible for basal and TCDD-induced transcription. In addition, the AHR2 DNA and ligand-binding domains are required to mediate effective CYP3A65 transcription. In addition to the zebrafish CYP3A65 promoter, the AHRE sequences are also present in many teleost CYP3 promoters but not in those of mammalian CYP3 genes, suggesting that AHR/AHR2-mediated transcription is likely a common regulatory mechanism for teleost CYP3 genes. It may also reflect the different environments that terrestrial and aquatic organisms encounter.

61 Oral Presentation‐18

Suppression of aromatase by fadrozole and morpholino antisense oligonucleotide alters activity of dopaminergic neuron in early development of zebrafish

Fatimah R.* and Kishida M. Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan. e-mail: [email protected]

Estrogen, synthesized from the conversion of testosterone by aromatase, has been considered as a general neuroregulatory factor affecting neuronal growth and development in the brain. Unlike mammals, teleost fish has two different isoforms, ovarian (cyp19a) and brain (cyp19b) aromatase, which are expressed predominantly in the corresponding tissues. It has been known that teleost fish exhibit high level of brain aromatase activity, which is 100 to 1000 fold greater compared to the mammals. Indeed, the level of brain aromatase and estrogen receptor mRNAs increase rapidly after 12 hours post fertilization (hpf) in zebrafish embryos implying a possible increase in estrogen production in brain. To investigate the role of estrogen produced in brain during early development, the effects of inhibition of aromatase on dopaminergic neuron were analyzed. Fertilized eggs were exposed to fadrozole, an aromatase inhibior, and the expression of tyrosine hydroxylase (TH), a rate-limiting enzyme for dopamine production, and the motor activities such as the response to tactile stimulation at 72 hpf and swimming distance at 6 dpf (day post fertilization) were measured. Fadrozole exposure decreased the expression of TH and the motor activities in a dose dependent manner.

The decrease caused by fadrozole was reversed by addition of E2. Then we further verified the role of estrogen in brain by microinjection of morpholino antisense oligonucleotide (MO) targeted to cyp19b to the fertilized eggs. MO injection decreased the expression of TH and motor activities, which was reversed by incubation with E2. Taken together, the results demonstrate that it is likely brain-formed estrogen produced by the action of cyp19b regulates activities of dopaminergic neurons to modulate the motor activity in early development of zebrafish.

Keyword: brain aromatase, dopaminergic neuron, zebrafish

62 Oral Presentation‐19

Manipulation expression FSH (Follicle Stimulating Hormone) from striped catifsh (Pangasianodon hypopthalmus) with hormonal manipulation during gonadal early development

Rafiuddin M.A.1*, Sudrajat, Agus Oman1#, Widyastuti U.2, Suharsono2, Alimuddin1 1.Laboratory of Reproduction and Genetics of Aquatic, Departement Aquaculture, Bogor Agriculture University, Indonesia. 2.Laboratory of BIORIN (Biotechnology Research Indonesia and the Netherland), Research center for Bioresources and Biotechnology, Bogor Agriculture University, Indonesia. # corresponding author : [email protected]

Aquaculture activities are one of the activity can develop in Indonesia. The activitity in aquaculture include hatchery, rearing, and growth out in captivity exhibit. Increasing to develop aquaculture production have dependence in broodstock management ih hatchery. Almost all fish reared in captivity exhibit some form of reproductive dysfunction. In females, there is often failure to undergo final oocyte maturation, ovulation and spawning; while in males milt production may be reduced and of low quality. The research is proposed for measured the physiology respond and expression FSH β subunit in molecular aspect on gonadal development in female of striped catfish. The increasing of expression would be using by hormonal and chemical meterial. The hormone which use in this research is PMSG and chemical material is antidopamine. For molecular aspect using isolation total RNA, synthesis of cDNA, and for the physiology aspect using by histology as descriptive analysis. The result of this research showed there is effect of inducing hormonal and chemical material in fish in moleculer and physiology aspect. The increasing of expression in FSH β subunit caused by induce the combination PMSG and AD. Other hand, the histology proof that the induce PMSG and AD can be development of oocyte in fish. The incresing expression of striped catfish after induced by PMSG and AD so that the consistency seed production can be guaranted.

63 Oral Presentation‐20

Changes in bone mineral density and content in pre-, peri- and post-menopausal cynomolgus monkeys

Kittivanichkul D.* and Malaivijitnond S. National Primate Research Center of Thailand and Department of Biology Faculty of Science, Chualalongkorn University, Bangkok, 10330 Thailand e-mail: [email protected]

It has been well-known that the bone structure and reproductive hormone profiles in cynomolgus monkeys (Macaca fascicularis) are similar to those of humans and they become animal model of the choice for anti-osteoporosis drug development regarding the US-FDA guideline. However, the basic knowledge on estrogen deficiency and bone loss during transition of the climacteric stage in these monkeys has not been elucidated. Pre-menopausal, regular menstrual cycle (n=30), peri-menopausal, irregular menstrual cycle (n=12) and post-menopausal monkeys with >1 years of complete cessation of menstruation (n=32) were measured bone mineral density (BMD) and bone mineral content (BMC) at the distal radius and proximal tibia, both metaphysis and diaphysis, by pQCT machine. Blood sera were collected and measured for bone formation and resorption markers and estrogen levels. Serum estrogen levels were significantly lower in post-menopausal monkeys than those of peri- and pre-menopausal monkeys and were correlated with the reduction of the BMD and BMC at radius and tibia. Comparing between the use of BMC and BMD as a criterion for the justification of bone loss, changes in BMC seemed to be more promising. Changes of both BMD and BMC at radius are more pronounced than those of tibia and suggested to be used as a marker of bone loss in aged monkeys. Interestingly, bone loss was particularly observed at cortical site in both radius and tibia of post-menopausal monkey, indicating a high risk of bone fracture. The bone formation and resorption markers which are on the measurement process and will present in the symposium will help to shed light on the mechanism of actions of estrogen deficiency on changes of the bone detected.

Keyword: Bone mass, cynomolgus monkeys, estrogen deficiency, menopause, pQCT

64 Oral Presentation‐21

Controlled feeding inhibits growth reduction associated with spawning in farmed yellowtail

Miura C.*, Yoshihara Y. , Hayashi D. , Miura T. Research Group for Reproductive Physiology, South Ehime Fisheries Research Center, Ehime University, 1289-1 Funakoshi, Ainan 798-4292, Japan e-mail: [email protected]

Growth initiates sexual maturation in fish but the onset of maturity also leads to a marked loss in body weight. To avoid the growth loss associated with spawning, yellowtail (Seriola quinqueradiata) farmers have attempted to restrict the diets of their farmed fish in the period just before the breeding season. Our study of restricted diets in farm-cultured yellowtails indicated that the growth of a diet-restricted group gradually increases through the breeding season compared with the controls. In the spawning season, the diet-restricted group had smaller gonads than the controls in both sexes. The gonad somatic index rapidly increased in the control group compared with the restricted group. These findings confirm that a restriction of the diet suppresses gonad development during the breeding season. The sex steroid hormone, 17 lpha, 20beta-diOHprog, was found to be associated with this dietary control of gametogenesis as it showed decreased levels in the restricted groups compared with the controls. To further investigate the functions of 17 lpha, 20beta -diOHprog on fish growth, this hormone was administered intraperitoneally to juvenile yellowtails. Implanted fish showed a remarkable increase in body weight compared with the control group and the lipase activities of the dorsal and ventral muscles of 17 lpha, 20beta -diOHprog-injected fish were also higher than those in the control group. We conclude from our data that the growth inhibition caused by sexual maturation in farmed fish may be avoided by inhibiting gametogenesis through dietary regulation.

Key words: growth, gonad, feeding control, aquaculture

65 Oral Presentation‐22

Integrated ecological impact of the equipment on the soft-shelled turtle breeding environment and growth and metabolism

Jiang Y.l.1,3, Zhang J.1, Li X.2,3, Chen H.l.2,3, Hou G.J.1,3, Wang l.4* 1Fishery Institute, Anhui Academy of Agricultural Sciences, 40, Nongke Nan Road, ( 230031) HeFei, China. e-mail: [email protected] 2 Anhui Province XiJia agricultural development limited company, Bengbu, China. 3 Anhui province turtle breeding and aquaculture health engineering technology research center,Bengbu,China. 4 Anhui biological engineering school,HeFei,China.

Young Mitten choose this test for the study, the average size of 65g, 4 notch test pools are 2 * 2.5 * 0.7m3, test pool settings manually hidden objects, mesh basking platform, configure micro water equipment, recycled water organisms purification systems, nano- pore aeration systems, light warming systems, climate control systems and other ecological equipment, controls no more than four ecological pool equipment. Test and control temperatures are 30-31 degrees, feeding the feed are independent configurations health- turtle feed, from September 20, 2012 - October 20, feeding time is 30 days, every 15 days at random from each pool of 50 samples measured body weight and food consumption of turtles; for pH, ammonia, nitrite, dissolved oxygen, COD five indicators were detected, and heterotrophic bacteria, Asia digestive bacteria, digestive bacteria culture testing. The results show that the equipment is provided with a test group of ecological Youbie bred breeding ponds average weight , the absolute growth rate , the instantaneous growth rate , respectively, 100.28g, 1.068g / d, 3.538% / d, t test , test control groups differ significantly (P <0.01), respectively, higher than the control pond 27.5% 28.4% 10.6% ; test pool nurtured Youbie shiny bright , clean surface , blood red, and the large size of normal activity Youbie high proportion showed good quality ; experimental group dissolved oxygen in water 8-9mg / L, pH7.5-8.0, ammonia, nitrite , COD highest values were 0.19mg / L, 0.01 mg / L, 4.53 mg / L, significantly better than the control group ; significantly increased compared with the control test pool pool protein efficiency , feed conversion rate 23.85% . The test set greenhouse turtles breeding ponds provide theoretical ecology equipment parameters.

Fund: National Spark Program (2012GA710053); National Bureau projects (G20123400046); Anhui Science and Technology research project (12010302063,1301 c063012); Anhui International Cooperation Project (11030603030); Anhui turtle breeding class engineering and health culture technology Research Center, Anhui Branch [2010] No. 123; hospital project priorities and nurturing emerging discipline (13A0502) First Author: Chiang Lin (1963 -), male, Anhui Shucheng people, Bachelor of Science, Research Associate / turtle class Anhui Engineering Technology Research Center, chief expert, the main research directions: aquatic animal breeding and ecological E-mail: jiangyelin @ qq.com * Corresponding author, Wang Ling (1965 -), female, Anhui Hefei, E-mail: [email protected].

66 Oral Presentation‐23

Conserved sex-specific timing of meiotic initiation during sex differentiation in the protandrous black porgy Acanthopagrus schlegelii.

Lau E.L.1*, Lee M.F.2, Chang C.F.3,4 1Eastern Marine Biology Research Center, Fisheries Research Institute, Taitung 961, Taiwan 2 Department of Aquaculture, National Penghu University of Science and Technology, Penghu 880, Taiwan 3Center of Excellence for the Oceans 4Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan e-mail: [email protected]

Meiosis is an essential mechanism of gametogenesis for all sexually reproducing species. In vertebrates, one conserved aspect of sex differentiation is that female embryonic germ cells enter meiosis earlier than male germ cells. In some lower vertebrates, female germ cells proliferate prior to entering meiosis, whereas male cells remain in mitotic arrest. Protandrous black porgy fish, Acanthopagrus schlegelii, have a dramatic life cycle involving a characteristic sex change. Black porgy are functional males for their 1st and 2nd spawning seasons, but approximately half of the fish transform into females during their 3rd year. We cloned the black porgy homologs of dosage suppressor of mck1 homolog (dmc1), and synaptonemal complex protein 3 (sycp3), and examined their expression profiles as well as those of cytochrome P450 family 26 genes (cyp26: cyp26a and cyp26b), retinaldehyde dehydrogenases (raldh: raldh2 and raldh3), retinoic acid receptors (rars: rarα, rarβ, rarγand rarγb), retinoid X receptors (rxrs: rxrα, rxrβ and rxrγ) and deleted azoospermia-like (dazl) during gonadal sex differentiation by RT-PCR, qRT-PCR and immunohistochemistry. Our results show that during gonadal development, germ cells located in ovarian tissue proceed into meiosis earlier than germ cells in testicular tissue. Furthermore, treatment with estradiol-17β (E2), which induced cyp26 expression, blocked dazl and raldh expression and reduced the expression of rars, rxrs, dmc1 and sycp3. This unique model therefore suggests that the temporal differences in meiosis initiation between females and males are conserved during gonadal sex differentiation in hermaphroditic vertebrates.

Keywords: meiosis, sycp3, dmc1, cyp26, estradiol

67 Oral Presentation‐24

Muscle-specific expression of giant grouper growth hormone in transgenic zebrafish leads to muscle hypertrophy and hyperplasia

Gong H.Y.1,2*, Chin H.Y.1, Tai T.Y.1, Tseng P.C.1, Huang S.C.1 1Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan. 2Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan. e-mail: [email protected]

In this study, two growth hormone (GH) transgenic zebrafish lines with weak and strong expression of giant grouper (Epinephelus lanceolatus) GH driven by zebrafish muscle-specific promoter/enhancer were established to investigate the paracrine and autocrine effects of GH in teleost. The 3-month GH transgenic zebrafish showed growth enhancement in body weight up to 92% and in total body length up to 31% as compared to control fish. Hypertrophy was observed in the muscle myofibers with an increase of 39% and 76% in the myofiber area of 3-month weak and strong GH transgenic zebrafish, respectively. The adult transgenic zebrafish also exhibited stratified hyperplasia at the discrete germinal layer under the skin. The increase in Pax7 positive muscle satellite cells at 24 hpf GH transgenic zebrafish embryos and up-regulation of Pax7 in 2.5-month transgenic zebrafish were also observed. Quantitative RT-PCR analysis revealed that giant grouper GH significantly increased expression of GHR, IGF1, MRFs (MyoD, Myogenin, Myf5, Mrf4), and MEF2D in the muscle of 2.5-month transgenic zebrafish via autocrine action to promote the growth of transgenic zebrafish. The paracrine effect of giant grouper GH in the 2.5-month transgenic zebrafish was showed by the up-regulation of IGF1, IGF2b and GHR in the liver. It was reported that the transcription factor MEF2D acted downstream of IGF1 and cooperated with MRFs to regulate skeletal myogenesis. Furthermore, we discovered that a (CA)n polymorphic microsatellite maker in the 3’-UTR of giant grouper MEF2D gene was strongly associated with body weight and can be applied for marker assisted selection of giant grouper.

Keyword: giant grouper, growth hormone, muscle-specific, transgenic zebrafish, MEF2D

68 Oral Presentation‐25

Identification of interacting partners of gonad-inhibiting hormone of Penaeus monodon by yeast two-hybrid analyses.

Tomy S.*1, Saikrithi P. 1, James N. 1, Otta S. K. 1, Balasubramanian C. P. 1, Subramoniam, T. 2, Ponniah A.G. 1 1 Genetics and Biotechnology Division, Central Institute of Brackishwater Aquaculture, # 75, Santhome High Road, RA Puram, Chennai -600028, Tamilnadu, INDIA 2 Centre for Climate Change Studies, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Road, Chennai - 600 119, Tamilnadu, INDIA email: [email protected]

Reproductive dysfunction of captive penaeid shrimp population is the major stumbling block in shrimp aquaculture. Gonad-inhibiting hormone (GIH), a neuropeptide secreted from the X-organ sinus gland complex in eyestalk inhibits vitellogenesis. The molecular mechanism involved in GIH-mediated signaling cascade is poorly understood. Knowledge of specific protein interactions of GIH will help to gain better understanding of the biological process. Potential interactors of GIH was identified using GAL4 based yeast two-hybrid assay in Penaeus monodon. GIH was cloned from the eyestalks, restriction digested and finally cloned into the MCS of the plasmid pGBKT7 (DNA-binding domain vector) to form a recombinant plasmid pGBKT7-GIH (bait) and was confirmed by sequencing. The bait was verified negative for autoactivation. An ovarian cDNA library was constructed and fused to the transcriptional activation domain vector (pGADT7) and was transformed to competent Y187 yeast strain. Yeast two-hybrid screening assay was performed by mating of Y2Hgold containing the bait with Y187 that containing ovary cDNA library. Four positive interactions were obtained indicating probable interacting partners for GIH through which it exerts its regulatory role in vitellogenesis. Works are being carried out to characterize them.

Keywords: P. monodon, penaeid shrimp, reproduction,Gonad inhibiting hormone, yeast two hybrid assay

69 Oral Presentation‐26

Molecular cloning of four MRFs (MyoD、Myf5、MRF4、Myogenin) and their expression patterns during fasting and refeeding in orange-spotted grouper, Epinephelus coioides

Zhang Y.*, Sang Q., Chen H., Zhang H., Liu X., Lin H. School of Life Sciences, Sun Yat-sen University, Guangzhou, China e-mail: [email protected]

Myogenic regulatory factors (MRFs），including MyoD, Myf5, MRF4 and myogenin, play important roles in the proliferation and differentiation of vertebrate skeletal muscle. In order to explore the expression patterns of MRFs during fasting and refeeding，four genes of MRFs were cloned from the white muscle in orange-spotted grouper, E. coioides. Sequence analysis showed that the four MRFs had the conserved domain namely bHLH with about 60 amino acids. Phylogenetic tree analysis implied the four types of MRFs might evolve from two duplication of a single ancestral gene. MyoD/Myf5 and Myogenin/MRF4 clustered closer respectively. RT-PCR data demonstrated the four MRFs were all highly expressed in white muscle and red muscle and weakly expressed in eye and skin. After fasting for three weeks, the body weight was significantly decreased. Refeeding for one week, relative somatic growth rate in refed orange-spotted grouper was higher than that of the control, indicating a process of compensatory growth. Data from quantitative RT-PCR revealed that the mRNA level of MRF4 was highly increased in the first fasting week and all the expressions of four MRFs were significantly up-regulated in the third fasting week. Refeeding for one week allowed the expressions of Myf5, MRF4 and Myogenin return to normal levels. However, the level of MyoD was still higher compared to the control group. We speculated that MyoD might be a molecular factor that contributed to the compensatory growth.

Keywords: Myogenic regulatory factors, fasting and refeeding, expression pattern, Epinephelus coioides

70 Oral Presentation‐27

The role of melatonin in synchronizing semilunar spawning rhythm of the mudskipper Boleophthalmus pectinirostris

Hong W.*, Hong L., Zhu W., Chen S. College of Ocean and Earth Sciences, Xiamen University, Xiamen 361005, P. R. China e-mail:[email protected]

The mudskipper Boleophthalmus pectinirostris, a burrow-dwelling fish inhabiting intertidal mudflats, is a restricted semilunar-synchronized spawner. To understand the mechanisms regulating this semilunar spawning rhythm, we determined the serum melatonin levels, investigated the in vivo and in vitro effects of melatonin on sexual hormonal production and cloned all melatonin receptor subtypes (mtnr1a1.4, mtnr1a1.7, mtnr1b and mtnr1c). The results showed that the levels of serum melatonin exhibited two cycles, each cycle with a peak within one month; the first peak was observed 2 days after the last lunar quarter, and the second ones, at the first lunar quarter. When females were intraperitoneally injected with melatonin, the levels of serum 17α,20β-DHP were observed significantly higher at dosage of 50ng/g body weight. Melatonin at concentration of 100pg/mL significantly induced the production of 17α,20β-DHP from the ovarian tissues in vitro. Expression of three melatonin receptor subtypes (excluding the mtnr1c) was found in the ovaries. In contrast, the expression of all receptor subtypes was found in the diencephalon and the pituitary. In the fully-grown follicles, only mtnr1a1.7 mRNA was detected in both the isolated follicle layers and denuded oocytes. The transcript levels of both mtnr1a1.4 in the diencephalon and mtnr1a1.7 in the ovary displayed two cycles within one lunar month, and peaked around the first and last lunar quarters. Taken together, these results provide the first evidence that melatonin and its receptors are involved in the synchronization of the semilunar spawning rhythm in female mudskipper by acting through the HPG axis and/or directly on ovarian tissues to stimulate the production of DHP.

Keywords: Boleophthalmus pectinirostris, Melatonin, semilunar spawning rhythm, 17α,20β- DHP

71 Oral Presentation‐28

Restoration of lung degeneration by Pueraria mirifica plant

Nishitani C. 1,2*, Limjunyawong N.1, Phumsatitpong C.1, Jaroenporn S.1, Nagaoka K.2, Watanabe G.2, Malaivijitnond S.1 1Faculty of Science, Chulalongkorn University, Bangkok, Thailand 2Laboratory of Veterinary Physiology, Faculty of Agriculture, Tokyo University and Agriculture and Technology, Tokyo, Japan

Emphysema, a disease with alveolar degeneration, can cause an expiratory airflow limitation and ultimately lead to death. Although estrogen has been reported to play a role on alveolar regeneration, the undesirable side effects regarding estrogen use are also concerned. Pueraria mirifica (PM), a phytoestrogen-rich herb, is become our focal alternative treatment. We determined if PM can restore the alveolar degeneration in estrogen deficient mice. We found that after 21 days of ovariectomy in mice the mean linear intercept (MLI) of alveoli was significantly increased (38.1±0.5 µm) in comparison with the shame control (33.1±0.6 µm) (p<0.0001). Thus, the mice were ovariectomized and kept longer for 28 days and fed with 10, 100 mg/kgBW/day of PM or 10 µg/kgBW/day of 17β-estradiol (E2) for 21 days. The

MLIs significantly decreased after PM and E2 treatment dose-dependently. Thus, PM seems to be a potential candidate for emphysema treatment. However, since emphysema is always happened together with lung cancer in sole patient. To assess the safety of PM use, the proliferative effect of PM on lung cancer cell, A549, were determined in vitro. A549 cells were exposed to PM at concentrations of 0-10 ug/ml for 24 hours. Viability and proliferation of cells were assessed. PM at only a concentration of 10 µg/ml stimulated A549 cell viability and proliferation. Taken together, low doses of PM should be suggested for emphysema treatment and the emphysema patient with lung cancer disease should use PM with careful.

Keyword: estradiol, alveoli, ovariectomy, Pueraria mirifica, A549

72 Oral Presentation‐29

Fecal testosterone profile and sperm quality of dairy buffalo from north sumatera

Maheshwari H.1*, Boediono A.1, Andriyanto1, Handarini R.2, Nalley W.M. 3, Yulnawati4, Sjahfirdi L.5 , Astuti P.6 1Department of Anatomy, Physiology and Pharmacology, Faculty of Veterinary Medicine Bogor Agricultural University, Jl. Agatis Darmaga 16154, Bogor, Indonesia, 2Faculty of Agricultural, Djuanda University, 3Faculty of Animal Science, Nusa Cendana Univesity, 4RC Biotechnology, Indonesia Institute of Science, 5Faculty of Mathematic and Natural Sciences University of Indonesia, 6Faculty of Veterinary Medicine, Gadjah Mada University. e-mail: [email protected]; [email protected]

Murrah buffalo is one of the dairy buffaloes, that is limited distributed in the province of North Sumatra, Indonesia as well as several other areas in Sumatra with a population that is not too high. This buffalo has great potential to be developed intensively as a dairy animal other than cows and goats to support milk production in this country. The application of Artificial Insemination (AI) for this animals might also be performed to minimized inbreeding so that reproductive physiology as the basis is very important to be elucidated. The present study was designed to observe testicular function of male dairy buffalo through analysis of fecal testosterone and analysis of the sperm quality. Five adult male dairy buffaloes were used for this study. Fecal samples were collected every day at 0700-0800am and at 1600-1700pm for seven consecutive days and stored at -200C until assayed. Prior to hormone analysis, samples were lyophylized, pulverized and extracted in 80% of methanol. Testosterone concentration was analyzed by using radioimmunoassay (RIA). Ejaculate sperm was collected once at the first day of fecal sample collectetion. The results showed fluctuations in daily testosterone profile of all males with the level of testosterone in the morning seemed to be higher than that of afternoon level. From the result could also be seen that there were slight differences in individual concentration and profile, but this profile still represented normal testicular endocrine function of those males. However, the sperm quality was different amongst those males, and only sperm with motility of 60% above was processed for frozen semen production.

Keywords: dairy buffalo, fecal testosterone, testicular function, sperm quality, North Sumatera

73 Oral Presentation‐30

Molecular cloning and dimorphic expression of growth hormone (GH) in female and male spotted scat (Scatophagus argus)

Deng S.P.1,2, Wu Bo1,2, Zhu C.H.1,2, Li G.L.1,2, * 1 Fisheries College, Guangdong Ocean University, Zhanjiang, China;2 Key Laboratory of Aquaculture in South China Sea for Aquatic Economic Animal of Guangdong Higher Education Institutes, Guangdong Ocean University, Zhanjiang, China *Corresponding author: Li G.L. e-mail: [email protected]

Spotted scat (Scatophagus argus) exhibits a typical sexual growth dimorphism in which the females grow faster than the males. Growth hormone (GH) is best known as an anterior pituitary hormone fundamental in regulating growth. And it may be associated with the mechanisms of sexual growth dimorphism in teleost. To clarify the roles in sexual growth dimorphism in spotted scat, the gh cDNA was isolated from the pituitary by using homologous cloning and RACE method. The cDNA contains a 66 bp 5′-UTR, a 606 bp open reading frame (ORF) encoding 202 amino acids with a 17 amino acid signal peptide followed by a 185 amino-acid mature polypeptide and a 241 bp 3′-UTR. Phylogenetic analysis of the spotted scat and other vertebrate’s GH show that the Perciformes species are identical with the traditional taxonomy on the level of suborder. And there is no divergent GH gene paralog could be identified in both spotted scat and other Perciformes species. Alignment of GH amino acid residues suggested the sequence identity between the spotted scat and other Perciformes species was very high. The qRT-RT PCR analysis demonstrated that the female spotted scat gh mRNA levels was approximately 4.61 and 6.4 times higher than that of the male at one and half a years and above two years old, respectively. And in the male fish, the gh mRNA levels were higher at blow one year old when the fish were immature than the fish at one and half a years and above two years old when the fish were mature. These results imply that the sexual growth dimorphism may be ascribable to the gh levels in pituitary in spotted scat.

Key words: Scatophagus argus, sexual growth dimorphism, Growth hormone, pituitary

74 Oral Presentation‐31

A super molecule reverts lipid induced insulin resistance

Priyajit C.*1, Soma S.*1, Sandip M.*1, Soumik A.1, Maitrayee P.1, Sushmita B.2, Chaudhuri M.K.3, Subeer M.4 , Samir B. 1 Department of Zoology, Visva-Bharati (A Central University), Santiniketan1, RCBT, Gurgaon2, Tezpur University, Assam3, Division of Cellular Endocrinology, National Institute of Immunology4, India email: [email protected]

Insulin resistance is the primary cause of type 2 diabetes. Although it is known to be affected by excess lipid, underlying mechanism remains unclear. We have recently demonstrated that Fetuin-A (Fet-A, α2-Heremans Schmid Glycoprotein- AHSG) disrupts adipocyte function by inducing inflammation of this vital cell, the only cell in the body capable of storing lipid. In inflamed adipocyte significant decrease of leptin, adiponectin and PPARγ occurred which are regulatory molecules for lipid metabolism. Interestingly, lipid also induces FetA gene expression in liver. The question at this point would be how FetA converts normal adipocytes to inflamed adipocytes. In addition we observed FetA first binds to fatty acids and then present it to TLR4 in the extracellular domain of adipocytes which recognizes FetA as an endogenous ligand. This transforms adipocyte to an inflammatory cell that secretes inflammatory cytokines. In our presentation we will describe the intervention of FetA induced adipocyte inflammation which secretes inflammatory cytokines causing insulin resistance. We have developed a novel compound termed as super molecule to prevent insulin resistance (SPIR). It is interesting to note that SPIR binds to insulin receptor with equal avidity as insulin receptor as insulin and transduce the signals for glucose uptake through Glut 4 activation. SPIR, surprisingly, also acts at the level of lipid induced insulin resistance through the inhibition of FetA. It augments adipogenesis by mesenchymal stem cells. These new clone of adipocytes have significant role for reducing extracellular lipid load through the upregulation of leptin, adiponectin and PPARγ. These results suggest SPIR could be an effective molecule in dealing the lipid induced loss of insulin sensitivity and insulin resistance.

Keyword: Adipocyte, lipid, Insulin resistance, Fetuin A, SPIR

75 Oral Presentation‐32

Molecular characterization of adiponectin receptors, its mRNA expression, and subcellular location in the orange-spotted grouper (Epinephelus coioides)

Qin C., Wang B., Sun C., Jia J., Li W.* State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, Sun Yat-Sen University, No. 135, Xingang Xi Road, Guangzhou, P. R. China. e-mail: [email protected] (Wensheng Li)

Adiponectin is an abundantly secreted adipokine from adipose tissue in mammals, which plays important roles in the regulation of glucose and lipid metabolism. The biological function of adiponectin is mediated by at least two receptors (AdipoR1 and AdipoR2). Although both of them were identified in mammals, there are few researches about adiponectin and its receptors in teleosts. In this study, two types of adiponectin receptors have been isolated and characterized in the orange-spotted grouper (Epinephelus coioides). The cDNAs of grouper AdipoR1 and AdipoR2 are 1444 bp and 2034 bp in length, encoding proteins of 376 amino acids and 375 amino acids, respectively. Multiple alignment results showed that there was a variable region at the N-terminal of AdipoR1/R2, which has never been reported. Both AdipoR1 and AdipoR2 were found to be widely expressed in various tissues of grouper. Compared to AdipoR2, AdipoR1 expressed at higher levels in the nervous system and pituitary gland, but at lower levels in some peripheral tissues, including heart, liver, adipose tissue, stomach, intestine and especially gonad. Fasting and refeeding experiments showed that the mRNA expressions of AdipoR1/R2 were up-regulated by fasting in the muscle and adipose tissue of grouper, and restored rapidly to normal levels after refeeding. However, the mRNA expressions of AdipoR1/R2 in the hypothalamus and liver of grouper were insensitive to fasting. By indirect immunofluorescence, we demonstrated that grouper AdipoR1/R2 were integral membrane proteins; the C-terminals were extracellular, while the N-terminals were intracellular.

Key words: Adiponectin receptors; tissue distribution; fasting; subcellular location

76 Oral Presentation‐33

Molecular cloning, characterization and expression profiles of Dax1 in protogynous orange-spotted grouper (Epinephelus coioides)

Liu, X.*, Lin M., Li S., Chen H., Zhang Y., Lin H. School of Life Sciences, Sun Yat-sen University, Guangzhou, China e-mail: [email protected]

Dax1 is a dosage-sensitive gene isolated from the DSS loci of Xp21 in human. Its overexpression will lead to sex reversal in XY individuals, while its mutation will cause a disease called AHC. Evidences have shown that Dax1 plays an important role in the sex determination and sex differentiation in vertebrates. Its product DAX1 can function as a transcriptional regulator by interacting with several factors, such as P450arom, StAR, P450scc, 3βHSD, ER, AR, PR and TRβ. In the present study, a 1412 bp gDax1 cDNA was isolated from a protogynous hermaphroditic fish, orange-spotted grouper (Epinephelus coioides). Phylogenetic tree showed that gDAX1 separated from tetrapod DAX1 and clustered together with that of other fish species, and analysis of amino acid sequences revealed that the C-terminus of gDAX1 exhibits the most conservation throughout evolution. Tissue expression of gDax1 showed a broad distribution pattern with a higher level in the tissues of the brain-pituitary-ovary axis. gDax1 mRNA levels in the gonadal tissue maintained at fairly low levels during the stages of formation of ovarian lumen and proliferation of gonia，then significantly increase at primary-growth stage of oocyte and reached the maximum value at the matured fish with the vitellogenic stage of oocyte, while dropped significantly as the oocyte degenerated. During MT-induced sex change from previtellogenic stage of female to functional male, gDax1 transcripts in the gonadal tissues were significantly down-regulated at late-bisexual stage and male-stage of fish. Taken together, the results of the present study strongly indicated the involvement of gDax1 in the regulation of ovary development and maturation, as well as sex reversal in orange-spotted grouper.

Keyword: Dax1, Epinephelus coioides, expression profile, ovarian development, sex reversal

77 Oral Presentation‐34

Is tide-related locmotion rhythm controlled by external tidal stiumali or internal melatonin in the barred mudskipper?

Ikegami T.*, Oyama Y., Takemura A. Department of Chemistry, Biology, and Marine Science, Faculty of Science, University of the Ryukyus, Senbaru 1, Nishihara, Okinawa, 903-0213, Japan e-mail: [email protected]

Organisms inhabiting the intertidal zone are affected by tidal and day-night cycles, and likely show integrated activities in relation to both cycles. Although it is speculated that the faced cycles are utilized for entrainment of rhythmic activities in locomotion, migration, and/or feeding, it remains unknown how rhythmic activities are controlled externally and/or internally. The barred mudskipper, Periophthalmus argentilineatus, is an intertidal amphibian fish in tropical and subtropical regions. This species is active on the surface of mudflats during low tide. According to tidal movement, it migrates from the mudflat to the land, and then is inactive during high tide. The aim of this study was to investigate the eco-physiological mechanism of tidal-related activity in this species under laboratory conditions. Locomotion was monitored in an aquarium under conditions of light and darkness (LD12:12), constant darkness (DD), reversed LD (DL12:12), and constant light (LL). Locomotion was also monitored in a tide-generating aquarium under LL conditions. There was no rhythmicity in locomotion in the aquarium with a constant water depth under any conditions. In contrast, locomotion showed tide-related rhythm in the tide-generating aquarium: it was significantly higher during low tides than high tides. When melatonin release from the cultured pineal organ was examined under LD, DD, and LL, day-low and nigh-high variation with a peak during the dark period was observed under LD, but not under DD and LL. Melatonin injection failed to alter locomotion activity. It is suggested that the locomotion of this species is externally controlled by tidal stimuli, but not internally by melatonin.

Keywords: Mudskipper; Tidal rhythm; Locomotion; Melatonin; Injection

78 Oral Presentation‐35

The functions of estradiol-17β during oocyte maturation in multiple spawning fish, yellowfin porgy

Yueh W.S. 1*, Jeng S.R.1, Sun S.F.1, Kuo S.F.1 , Chang C.F.2,3 1 Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung, Taiwan 2 Department of Aquaculture, 3 Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan e-mail: [email protected]

Yellowfin porgy, Acanthopagrus latus, is a protandrous hermaphrodite with group-synchronous oocyte development ovaries for multiple spawning. High concentrations of plasma estradiol-17β (E2) were detected in yellowfin porgy during spawning season; however, the functions of E2 in this stage are still unclear. In present study, we are measure plasma E2 levels; colon ers (erα, erβ1, erβ2, gper) from gonad and liver, measure the expression by QPCR in different diameter of oocyte before and during spawning season to find the function of E2 during spawning stage epically at final oocyte maturation (FOM) stage. The results shown that plasma E2 levels and cyp19a transcripts were increased in the FOM stage. Erα, erβ1, erβ2 expressions were increased significantly (p<0.05) during spawning season. Gper expressions were increased in spawning season and have significantly high levels in FOM. The expressions of erα, erβ1, gper were increased significantly in oocyte at vitellogenesis stage by LHRH-A treatments. In male fish with previtellogenic stage ovarium, plasma vitellogenin levels and HSI were increased by E2 treatments; GSI were increased only by HCG with E2 treatments; the expression of erα were increased both by LHRH-A and HCG treatments. The results indicated that erα were associated with vitellogenin synthesis in liver, ovarian development and yolk accumulation; Gper might be regulating reentry into the meiotic cell cycle in oocyte. E2 might be control multiple spawning by ers in different growth stage of oocyte in ovary.

Keywords: estrogen receptor, oocyte maturation, multiple spawning, yellowfin porgy

79 Oral Presentation‐36

Effects of Nonylphenol on Vitellogenin Gene Expression and Gonad Development in Macrobrachium rosenbergii

Zhu C.*, Li Y., Chen L., Chen S., Li G. Fisheries College, Guangdong Ocean University, Zhanjiang, 524025, China e-mail: [email protected]

The effects of different doses nonylphenol (NP) (0.1mg/L, 0.2mg/L, 0.4mg/L) on vitellogenin gene expression and gonad development of Macrobrachium rosenbergii were researched with the molecular biology、histology and microtechnology. The results showed that vitellogenin (VTG) expression was observably increase in hepatopancreas and ovarian of female M. rosenbergii at exposure in NP, The effect of low dose group were stronger than medium and high dose groups. Expression of VTG gene of hepatopancreas and testis in male M.rosenbergii was also promoted at exposure in NP, of which dose effect relationships existed in hepatopancreas, induction effect were more obviously with the doses increased, but significantly higher expression of VTG in the testis was only observably in the low dose groups. The development of testis and ovary in M.rosenbergii was inhibited at exposure in NP, inhibition of testicular and ovarian development gradually increased with the exposed time extending.

80 Oral Presentation‐37

Correlation between PTEN expression levels and ovarian development in the induced Japanese eel

Huang Y.-S.1*, Gwo J.-C.2, Chen Y.-F.1 , Chang C.-F.2 1Depart. Life Sci., National University of Kaoshiung, Kaoshiung, Taiwan. 2 Depart, Aquaculture, National Taiwan Ocean University, Keelung, Taiwan. e-mail: [email protected]

PTEN (phosphatase and tensin homolog on chromosome 10) has well-known roles as a negative regulator of the PI3K/AKT pathway and seems more prominent in a swift biological process (e.g. folliculogenesis and angiogenesis). We studied the roles of PTEN on folliucogenesis in the Japanese eel, Anguilla japonica, to investigate PTEN functions from the base of Evolution. Merits to use the Japanese eel to address PTEN roles on folliculargenesis are that follicular development is blocked in fresh water milieu, but advance of this process can be induced. Two forms of PTEN have been cloned in the Japanese eel confirmed by immunological methods and molecular techniques. Cellular distributions of these two PTEN were different, PTEN-short was concentrated in perinuclear area, PTEN-long was more ubiquitous. The different expression profiles were confirmed in primary culture of gas glands cells treated by various factors (PPAR agonists, IGF-1, bFGF) in vitro. PTEN-long form plays a more direct role on signaling transductions in vitro, PTEN-short seems more close to development in vivo. Furthermore, PTEN plays an important role in the early follicular development, the ovarian development in the induced eels corresponded to the decrease of ovarian PTEN expression, vice versa. Testosterone (T) has showed to ameliorate the early ovarian development in the eel, we demonstrated that the benefits of T on eel early ovarian development can be attributed to its PTEN-inhibitor role. Our data suggested the function of PTEN on ovarian development is conserved during the Evolution. Two forms of PTEN indeed play different roles in the primitive vertebrate. The relation or correlation between the mammalian counterparts is needed to be elucidated in the future; this might provide more insights in the PTEN functions.

Keyword: Anguilla japonica, development, ovary, PTEN, steroid

81 Oral Presentation‐38

Herbicide utilization as an environmental stressor on reproductive/endocrine functions of sentinel species in agricultural areas

Kitana N.1,2 *, Maneein R.1, Thammachoti P.1, Thitiphuree T.1, Kitana J.1,2, Khonsue W.1,2, Varanusupakul P.3 1Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. 2Center of Excellence in Biodiversity, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. 3Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand e-mail: [email protected]

Application of herbicides in agricultural area can lead to environmental contamination and adverse effects on non-target organisms. In this research, paddy field in northern part of Thailand, an area with intensive use of atrazine, glyphosate and paraquat, was selected as a study site. Screening for herbicides (atrazine, glyphosate and paraquat) in this area showed contamination of atrazine in water at the field with intensive herbicide utilization. Representative of vertebrate (rice frog, Fejervarya limnocharis) and invertebrate (rice field crab, Esanthelphusa nani) living in paddy field were thus selected as sentinel species to test for influence of herbicides on reproductive/endocrine functions of non-target organisms. Frogs and crabs were collected from a field with intensive herbicide utilization and a reference field with minimal herbicide utilization. Herbicide residue analysis showed that detectable levels of these herbicides were found in animals from both sites with a significantly higher level of atrazine in the contaminated site animals. In frogs, morphometric and gravimetric studies showed that frogs from the contaminated site had a significantly lower condition factor (indicative of health), and a significantly higher ovarian weight compared to those of the reference site, possibly due to stimulating effect of herbicide on ovarian growth. In crabs, relative body weight of male crabs in the contaminated site was significantly lower, and their sexually dimorphic traits (abdominal area and major claw size) were significantly smaller compared to those of the reference site, potentially due to demasculization effect of herbicide. Overall, site-related differences in health status and gravimetric-morphometric parameters indicate that herbicide utilization in agricultural area could act as environmental stressor on reproductive/endocrine functions of these non-target organisms.

Keyword: condition factor, GSI, rice field crab, rice frog, sexually dimorphic trait

82 Oral Presentation‐39

Convergent evolutions of epithelial cell differentiation and neuroendocrine regulation in cephalopod and fish embryos

Chen Y.C.1, Sung P.H.1, Lee J.R.2, Hu M.Y.2, Hwang P.P.2 , Tseng Y.C.1* 1 Department of Life Science, National Taiwan Normal University, Taipei City, Taiwan. 2 Institute of Cellular and Organismic Biology, Academia Sinica, Taipei City, Taiwan e-mail: [email protected]

Recent studies have already proved that cephalopods are powerful acid-base regulators as most teleosts, which tightly regulate their pH homeostasis; however the differentiation and endocrine controlled of acid-base regulatory machinery are virtually unexplored in this mollusks. In the present study, cephalopod Sepia pharaonis was utilized as a modal to study the epithelial cell’ differentiation and neuroendocrine regulation regarding embryos ionic homeostasis. High-CO2 incubation was used to monitor those relative physiological adaptation in S. pharaonis. The epidermal ionocytes’ differentiation marker, ΔNp63, is detected on the epidermis and yolk sac of S. pharaonis embryos. ΔNp63 mRNA expression was further affected by acid stress in parallel with that in teleosts. In addition, foxi3, the upstream factor related to ionocytes’ differentiation was also observed and cloned in S. Pharaonis. Moreover, an oxytocin receptor homolog, cephalotocin receptor 2 (CTR2), was up-regulated by 16-35 % under hypercapnic stress. Evident expression of CTR2 was observed in stage-28 embryos as well as the expressions of Na+/K+-ATPase (NKA) in functional gills at the same developmental stage. The present work suggests that in convergence to teleosts, cephalopod embryos have evolved sophisticated evolution pathway about epithelium differentiation and neuroendocrine regulation to cope with acid-base disturbances during their oviparous development.

Keyword: cephalopods, cephalotocin receptor, Np63, differentiation

83 Oral Presentation‐40

Osmoregulation in the crab-eating frog, Fejervarya cancrivora

Uchiyama M. Dept. of Biol., Univ. of Toyama, Toyama, Japan. e-mail: [email protected]

The euryhaline crab-eating frog inhabits mangrove swamps in Southeast Asia. Following acclimation to hyperosmotic seawater, body weight decreased, while plasma Na+, Cl−, and urea concentrations, hematocrit value (Ht)), and osmolality significantly increased. Plasma Ang II, Aldo, and AVT were maintained within relatively narrow concentration ranges in the control frogs; however, plasma Ang II and AVT increased in seawater-acclimated frogs. There was a significant correlation between plasma osmolality or Ht and AVT as well as Ang II but not Aldo. Plasma Ang II was significantly correlated with Aldo. During seawater acclimation, there were significant increases in mRNA expressions of the hepatic urea cycle enzyme, Arginase I (ARG I), renal urea transporter (UT), and water channel. Thus, osmoregulatory mechanism probably functions to increase urea synthesis and prevents excessive losses of urea and water in seawater. The renin–angiotensin system may play pivotal roles in body fluid regulation under volemic and osmotic stress. Although most larval anurans are ammoniotelic until the completion of metamorphosis, tadpoles of F. cancrivora synthesized urea and increased expression of urea-cycle enzyme (CPS I and ARG I) and UT mRNAs in diluted sea water environments. Thus, tadpoles of F. cancrivora have developed the ability to use urea as an osmolyte during the late development of larval phase (TK stages IV–X).

Keyword: saltwater tolerance, angiotensin II, arginine vasotocin, urea transporter, water channel

84 Oral Presentation‐41

The presence and role of gnrh in the reproduction of the mud crab, Scylla paramamosain

Feng B.Y., Bao C.C., Ye H.H. * College of Ocean and Earth Sciences, Xiamen University, Xiamen 361005, China e-mail: [email protected]

The objectives of this study were to investigate the presence of immunoreactive GnRH (irGnRH) in the mud crab, Scylla paramamosain, and evaluate its biological activity in the reproduction. Mammalian (m) and octopus (oct) irGnRH were detected in the brain and thoracic ganglion by immunohistochemistry. When mGnRH or octGnRH were added to neurosecretory cells in vitro, the calcium current increased with the whole-cell clamp technique. Transcriptomics analysis showed that GnRH pathway genes, such as GnRHR, PKC, JNK, EGFR were found in the brain of S. paramamosain. The expression of vitellogenin mRNA in ovary increased after the addition of mGnRH or octGnRH in vitro. Taken together, the results suggest that irGnRH play an important role in reproduction of the mud crab.

Key words: GnRH, reproduction, mud crab

85 Oral Presentation‐42

- - Nitrate (NO3 ) and nitrite (NO2 ) perturb the activity of dopaminergic neuron during early development of zebrafish

Jannat M. and Kishida M.* Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan email: [email protected]

With a view to consider the increasing concern over nitrogen pollution in the aquatic environment, we investigated effects of nitrate and nitrite on the activity of dopaminergic neuron in zebrafish embryos and larvae. Fertilized eggs were exposed to nitrate-N (NO3-N)

(0.1-100 mg/L) and nitrite-N (NO2-N) (0.1-100 mg/L) up to 6 dpf. NO3-N exposure reduced the tyrosine hydroxylase (TH) expression in dopaminergic neurons at 48 hpf and the swimming distance of the larvae at 6 dpf, whereas the response to tactile stimulation at 48 and

72 hpf did not change. On the other hand, NO2-N reduced the TH expression, response to tactile stimulation and swimming distance. When the embryos were exposed to NO3-N or

NO2-N together with an estrogen receptor blocker (ICI 182780), the decreases in TH expression and motor behavior caused by NO3-N or NO2-N alone were reversed, suggesting nitrate and nitrite exert their effects through estrogen receptor. When diphenyleneiodonium (DPI) was used to inhibit oxidation/reduction reactions in the cell, DPI blocked the effect of - - both NO3 and NO2 indicating that both of them are active anion to act on dopaminergic neuron. To detect NO production, DAF-FM-DA staining was performed at 5 dpf. The intensity of DAF-FM-DA staining increased in a dose dependent manner in NO2-N exposed larvae, whereas NO3-N exposure had no obvious effect. The present study demonstrates that - - both NO3 and NO2 can act as neurotoxicants to perturb the activity of dopaminergic neuron in early development of zebrafish. Moreover, the ability of nitrate and nitrite to affect through estrogen receptor suggests they act as endocrine disruptors.

Keyword: nitrate, nitrite, dopaminergic neuron, zebrafish

86 Poster Presentation 1‐1

CXCL14 enhances proliferation and migration of cancer cells overexpressing the glycoproteins

Park C.R.1*, You D-.J. 1, Kim D-.K. 1, Moon M.J. 1, Lee C. 2, Oh S-.H. 3, Ahn C. 4, Seong J.Y. 1, Hwang J-.I. 1 1 Graduate School of Medicine; Korea University, 73 Inchon-ro, Seongbuk-gu, Seoul 136-705, Republic of Korea 2 Life Sciences Division, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791 Republic of Korea 3 College of Pharmacy, Gachon University, Incheon, 406-840, Republic of Korea 4 Transplantation Research Institute, Cancer Research Institute, Seoul National University, Yongun-dong, Jongno-gu, Seoul 110-799, Republic of Korea e-mail: [email protected]

CXCL14 is a chemokine family member that is involved in various cellular responses. Although constitutive CXCL14 expression in normal epithelial cells may help protect against infection by activating immune systems, its expression in cancer cells has raised controversy regarding the its possible role in tumorigenesis. However, the underlying mechanisms for this disparity remain unknown. In the present study, we found that CXCL14 binds to various cell types. Interestingly, binding was prevented by heparan sulfate and N-acetyl neuraminic acid. Next, we examined the effect of CXCL14 binding in NCI-H460 and NCI-H23. CXCL14 enhanced proliferation and migration in NCI-H460 cells. A reporter gene assay with various transcription factor response elements revealed that only NF-κB signaling was activated by CXCL14 in NCI-H460 cells, which was blocked by BAPTA-AM, TCPA, brefeldin A. Exogenous expression of some glycoproteins such as syndecan-4, podoplanin, CD43 enhanced CXCL14 binding and NF-κB activity. Collectively, these results demonstrate that CXCL14 binding to glycoproteins harboring heparan sulfate proteoglycans and sialic acids leads proliferation and migration of some cancer cells.

Keyword: CXCL14, glycoprotein, NCI-H460 cells

87 Poster Presentation 1‐2

A novel gonadotropin-releasing hormone-Ⅱ analog induces apoptosis prostate cancer cells

Park S.1*, Seong J.Y.1 , Hwang J. I. 1 1 Laboratory of GPCR and Signal Transduction, Medical Science Department, School of Medicine, Korea University, Seoul, Republic of Korea e-mail: [email protected]

Gonadotropin-releasing hormone-I (GnRH-I) and its analogs have been used for a treatment of hormone dependent disease, particularly for androgen-dependent prostate cancer patients. However, because of the androgen-independency of the cancer in advanced stages, researchers are required new medical treatments. In previous reports, we demonstrated the GnRH-II antagonist Trp-1 inhibited proliferation and stimulated the autophagic death of various prostate cancer cells, including androgen-independent cells. Here, we investigated the effect of SN09-2 on the growth of PC3 prostate cancer cells. SN09-2, a novel GnRH-II derivative, induced the cell death of prostate cancer cells but had no effect on cells derived from other tissues. Compared with Trp-1, SN09-2 remarkably reduced prostate cancer cell growth, even at low concentrations. SN09-2-induced PC3 cell growth inhibition was related with decreased membrane potential in mitochondria where the analog was accumulated, and increased mitochondrial and cytosolic reactive oxygen species. SN09-2 induced lactate dehydrogenase release into the media and annexin V-staining on the PC3 cell surface, indicating that the analog stimulated prostate cancer cell death by activating apoptotic signaling pathways. Furthermore, cytochrome c release from mitochondria to the cytosol and caspase-3 activation were significantly increased in a dose- and time-dependent manner. SN09-2 also inhibited the growth of PC3 cells xenotransplantaed into nude mice. These results demonstrate that SN09-2 directly affects mitochondrial dysfunction, leading to apoptosis of prostate cancer cells as well as growth inhibition.

Keyword: GnRH-II analog, apoptosis, prostate cancer

88 Poster Presentation 1‐3

Transcriptional activation of gonadotropin-inhibitory hormone by corticosterone

Son Y.L.1*, Ubuka T.1, Narihiro M.1, Fukuda Y.1, Hasunuma I.1, Yamamoto K.1, Belsham D.D.2, Tsutsui K.1 1Department of Biology, Waseda University, Tokyo, Japan. 2Department of Physiology, University of Toronto, Toronto, Canada. e-mail: [email protected]

The inhibitory effect of stress on reproductive function is potentially mediated by high concentrations of circulating glucocorticoids (GC) acting via the GC receptor (GR). Recently, it has been shown that the expression of gonadotropin-inhibitory hormone (GnIH) is induced by stress, indicating that GnIH may play a role in stress-induced reproductive dysfunction. However, it is not yet known whether GC-bound GR is directly involved in GnIH transcription. In this study, we demonstrated the localization of GR mRNA in GnIH neurons in quail, suggesting that GC can directly regulate GnIH transcription. We next showed that 24 h treatment with corticosterone (CORT, the major GC in vertebrates) increases GnIH mRNA expression in the quail diencephalon. We further investigated the mechanism of activation of GnIH transcription by CORT using a GnIH-expressing neuronal cell line, rHypoE-23, derived from rat hypothalamus. We found the expression of GR mRNA in rHypoE-23 cells and increased GnIH mRNA expression by 24 h CORT treatment. We finally demonstrated that CORT directly regulates the promoter activity of rat GnIH gene, and identified that a glucocorticoid response element (GRE) at −1530 bp of rat GnIH 5’ flanking region is a functional GRE responsible for the CORT-induced GnIH transcription by recruiting GR. These results provide a molecular basis for transcriptional activation of GnIH under stress by demonstrating that CORT directly induces GnIH transcription by recruitment of GR to its promoter.

Keyword: stress, gonadotropin-inhibitory hormone, glucocorticoid, glucocorticoid receptor, corticosterone

89 Poster Presentation 1‐4

Molecular responses in the migrating Takifugu obscurus - gene regulation depends on tissue and reading time

Kim J.-H.1, Han K.-N. 2, Dahms H.-U.3* 1 Department of Fisheries and Oceans, Centre for Aquaculture and Environmental Research, 4160 Marine Drive, West Vancouver, BC, V7V 1N6, Canada. 2 Department of Ocean Sciences, Inha University, Incheon, South Korea. 3 Department of Life Science, College of Natural Science, Sangmyung University, Seoul 110-743, Korea

Spatiotemporal gene expression is the activation of genes within specific tissues of an organism, depending on specific developmental time windows and environmental changes such as stressor exposure. In order to find reliably consistent biomarkers for xenobiotics such as for EDCs, the tissue and time-dependent expression of mRNA was studied by real-time PCR in the migrating teleost T. obscurus exposed to two representative EDCs (bisphenol A and 4-tert-octylphenol). HSP70, HSP90, MR and FKBP52 mRNA expression level was much higher than GR and PEPCK expression. GR mRNA expression level was significantly upregulated after 48 h in both EDC treated fish groups compared to the control. Both groups also showed down-regulation patterns after 48 h. The initial expression of PEPCK in both groups was down-regulated after 24 h. In the bisphenol A treated group, the expression of FKBP52 showed substantially high up-regulation from 6 h to 48 h after exposure, and was then strikingly downregulated at 72 h. Our results provide insights into the EDCs metabolizing system of T. obscurus and offers baseline information for further research related to this commercially important aquaculture fish species.

Keywords: Pufferfish, biomarker, fold-change, EDCs, glucocorticoid receptor, gene expression, Takifugu obscurus.

90 Poster Presentation 2‐1

Stress and immune response of the rice frog Fejervarya limnocharis living in areas with different agricultural activity

Jantawongsri K.*, Kitana J., Kitana N. Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand e-mail: [email protected]

Agrochemicals have been intensively used in many agricultural areas especially in Nan Province, northern part of Thailand. More than 90% of agrochemicals used in this area are herbicides, particularly atrazine, paraquat and glyphosate. Previous reports indicated that herbicide contamination and adverse health effects were found on the rice frog Fejervarya limnocharis living in paddy fields at Nan Province. Since contamination of herbicides may influence disease emergence by acting directly or indirectly upon the immune system of amphibian, it is interesting to investigate potential effects of herbicide utilization on stress and immune response of frog living in agricultural areas. F. limnocharis, a sentinel species for herbicide contamination, were collected every three months in 2013 from two study sites including 1) an agricultural area with intensive herbicide usage and 2) a reference agricultural area with no herbicide usage for almost 10 years. After euthanasia, frogs were evaluated for stress as determined from plasma corticosterone levels and non-specific immune function as determined from differential leucocyte count and ratio of neutrophil: lymphocyte (N: L ratio). In addition, a separate group of frogs was subjected to a single immunization with TiterMax®Gold adjuvant and evaluation for stress and non-specific immune function afterward. The results showed that there were site-related differences in plasma corticosterone levels, percentage of the white blood cell and N: L in the rice frogs living in agricultural areas. These results could give sight into effects of stress induced by environmental contamination as a result of herbicide utilization on immune function in amphibians.

Keyword: amphibian, corticosterone, differential leucocyte count, immunization, herbicide

91 Poster Presentation 2‐2

Incubating temperature affects development of endocrine cells in gonad of Malayemys macrocephala, a freshwater turtle from tropical asia

Pewphong R.1*, Kitana J.1,2, Kitana N.1,2 1Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand 2Center of Excellence in Biodiversity, Faculty of Science, Chulalongkorn University, Bangkok, Thailand e-mail: [email protected]

Temperature is an important environmental factor that has profound effect on reproduction and development of freshwater turtles. Although most of the information on their reproductive/developmental biology has been investigated in temperate species, certain reports on tropical species suggested the similar sensitivity to temperature change. Prior studies on Malayemys macrocephala, a freshwater turtle found in lowland areas of Southeast Asia, showed that incubating temperatures could influence somatic and gonadal development, and suggested that sex determination in this tropical species is temperature-dependent. Current research aimed to examine effect of incubating temperature on development of endocrine cells in the gonad of M. macrocephala. Turtle eggs were collected from rice fields in central part of Thailand during 2011-2012. Eggs were randomly divided and incubated in microprocessor-controlled incubators at three temperatures (26°C, 29°C and 32°C). Upon hatching, gonad of turtle was examined histologically for structural organization, germ cell development and presence of endocrine cells in the gonad. Current results confirmed prior observations that sex ratio of hatchlings shows a temperature-dependent pattern. Incubating eggs at 26°C, 29°C and 32°C resulted in male-biased, 1 male: 1 female, and female-biased sex ratios, respectively. Development of endocrine cells in testis was classified according to the presence/absence as well as the active/inactive state of Leydig cell and Sertoli cell in testicular cords, whilst similar evaluation on theca cell and granulosa cell was used in the ovary. Association between incubating temperature and the stage of endocrine cell development in gonad was found. These results suggested that temperature can affect gonadal development of the tropical freshwater turtle, potentially via roles of differential endocrine function as a result of incubating temperature.

Keyword: granulosa cell, Leydig cell, Sertoli cell, temperature-dependent sex determination, theca cell

92 Poster Presentation 2‐3

Stress and health status based on hematological parameters of the rice field frog Hoplobatrachus rugulosus in captivity

Kitana J.1,2 *, Meesawat S.1, Thammachoti P.1, Thitiphuree T.1, Maneein R.1, Chiewchanchai M.2, Pariyanonth P.1, 2, Khonsue W.1,2, Kitana N.1,2 1Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand 2Center of Excellence in Biodiversity, Faculty of Science, Chulalongkorn University, Bangkok, Thailand e-mail: [email protected]

Frog is well known as an alternative protein source for people in many countries. With increasing demand, farmed frog become a stable alternative to wild caught animal. However, like other farmed animals, the captive frog has to cope with stress induce lesion and diseases and, in the worst case, mass mortality. Since health status of frog in conventional farm is usually based on visual examination, we thus aimed to develop preemptive health evaluation program for frog culture. In this study, a standard frog culture facility of the Huai Hongkhrai Royal Development Study Center, Chiangmai Province, Thailand was used as a study site. Blood of healthy adult and sub-adult frogs was sampled during wet season (October 2013) and subjected to hormonal and hematological evaluations. Stress was evaluated from plasma level of corticosterone (ELISA) and health status was evaluated from hematocrit (microtube centrifugation), and blood cell morphology, erythrocyte count and differential leukocyte count (Romanowsky's stain of blood smear). Our results showed that these parameters were age specific with no significant sex-related difference. Correlation between corticosterone and hematological parameters were further assessed. In addition, normal reference ranges for these parameters were set up and validated with separate groups of healthy and unhealthy frogs. It was found that although hematocrit is a direct proportion of erythrocyte count and simple to find, its correlation with stress hormone and sensitivity to differentiate between healthy and unhealthy frogs were not definite. On the contrary, blood cell morphology and leukocyte count, especially a neutrophil:lymphocyte ratio, seems to be better indicatives of health. Combined hormonal and hematological evaluations of this kind could be further developed and used in health assessment program of frog farming in this region.

Keyword: corticosterone, erythrocyte, frog farming, hematocrit, leukocyte

93 Poster Presentation 2‐4

Endothelin-1 regulates transepithelial H+ secretion in zebrafish

Guh Y.J.1,2*, Tseng Y.C.3, Yang C.Y.2,4, Hwang P.P.2 1. Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan. 2. Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan. 3. Department of Life Science, National Taiwan Normal University, Taipei, Taiwan. 4. Institute of Fisheries Science, National Taiwan University e-mail: [email protected]

Endothelin-1 (EDN1) is an important regulator of H+ secretion in mammalian kidney. However, little is known about the role of EDN1 in acid-base regulation of non-mammalian vertebrates. The aim of this study is to examine the effects of EDN1 and its receptors on the transepithelial H+ secretion in zebrafish. Expressions of EDN1 and one of its receptors, EDNRAa, were stimulated in zebrafish acclimated to acidic (pH4) water. By applying a scanning ion-selective electrode technique on non-invasive measurement of H+ secretion, EDN1 overexpression was found to enhance the proton secretion at 3-dpf embryonic skin. EDNRAa loss-of-function significantly decreased EDN1- and acid-induced H+ secretion. The inhibiton of EDN1-ehanced H+ secretion by vacuolar H+-ATPase inhibitor (bafilomycin A1) suggested that EDN1 exerts its action by regulating the H+-ATPase-mediated H+ secretion. EDN1 does not appear to affect H+ secretion through either altering the abundance of H+-ATPase or affecting the cell differentiation of H+-ATPase-rich (HR) ionocytes, because the reduction in secretion upon ednraa knockdown was not accompanied by decreased expression of H+-ATPase or reduced HR cell density. This suggests that EDN1 may achieve its action by regulating post-translational process(es) and may exert an acute effect soon after acid challenge. Expression of EDN1 was increased in 2h after acidic water treatment, implying EDN1 is involved in short term regulation of acid secretion. These findings demonstrated for the first time that EDN1 is involved in regulating acid-base homeostasis in fish.

Keywords: Endothelin-1, H+ secretion, zebrafish

94 Poster Presentation 2‐5

Endocrine effects of copper in aquatic animals – a review

Shim D.M.-C.1, Dahms H.-U.2*, Yang J.3, Wang L.3 1 Department of Integrated Ecological & Environmental Engineering, Graduate School of Sangmyung University, Seoul 110-743, Korea. 2 Department of Life Science, College of Natural Science, Sangmyung University 7 Hongij-dong, Jongno-gu, Seoul 110-743, Korea. 3 Laboratory of the Bio-effect and Molecular Mechanism of Classical Environmental Pollutants, School of Life Science, Shanxi University, 92 Wucheng Road, Taiyuan 030006, Shanxi Province, China.

Organisms build up homeostatic mechanisms and regulate free Cu concentrations strictly, since copper is an essential element. When Cu concentrations reach toxic levels, DNA damage, oxidative stress, and a disrupted osmotic ion-homeostasis may occur. Previous research revealed that Cu is involved in chloride and sodium transport systems in the gills of freshwater animals. A reduction in branchial sodium uptake occurs by inhibiting the Na+/K+- ATPase by nonspecific binding to thiol groups on the subunits of the transporter, and by binding to the Mg2+ binding site. Besides, Cu induces a competitive inhibition at the apical Na+-channel. Increased sodium loss leads to changes in permeability. This is caused by a displacement of calcium by copper at the cellular tight junctions shown for freshwater fish. These changes result in a net loss of Na+, an increase in blood viscosity and blood pressure, a compensatory tachycardia and cardiac failure when exposed to acute toxic Cu concentrations controlling ion osmoregulation. Hormones such as cortisol, prolactin (PRL), growth hormone and thyroid hormones, which are regulating ion osmoregulation are affecting and being affected by heavy metal toxicants like Cu. PRL functions in balancing water and electrolytes. In addition it is equally active in a wide range of metabolic and biological functions, such as metabolism, growth, development, reproduction, behavior and immunoregulation. PRL's ability to increase plasma ion concentrations and decrease the permeability of osmoregulatory surfaces to water, plays an important role in adapting to this situation. Thyroid hormones are involved in development and metabolism. Most thyroid hormone effects are mediated at the genomic level via binding to nuclear thyroid hormone receptors (TRs) which are provided by the two forms TRα and TRβ.

Key words: Copper, endocrine regulation, homoeostasis, environmental pollution.

95 Poster Presentation 2‐6

Differential expression of arginine vasotocin and isotocin mrna to external salinity stress in black porgy (Acanthopagrus Schlegelii) hypothalamus and pituitary

Aruna A.1*, Nagarajan G.1, Chang C.F.1,2 1Department of Aquaculture and 2Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung 20224, Taiwan e-mail: [email protected]

The nanopeptide arginine vasotocin (AVT) and isotocin (IT) and their receptors, including AVT-receptor and (AVT-R) and IT-receptor (IT-R) are considered to be involved in the osmotic stress to maintain the homeostasis in teleosts. In the present study, we quantified and localized the AVT, AVT-R, IT and IT-R transcripts in seawater (SW) and freshwater (FW) acclimated black porgy, (Acanthopagrus schlegelii) hypothalamus and pituitary at day 1, day 7 and day 30. The results showed that the increased transcript of AVT in pars magnocellularis (PMmc) and pars parvocellularis (PMpc), and IT-R in PMmc and PMpc in the hypothalamus at day 1 implied that the AVT also have the ability to stimulate IT-R during FW transfer. And, the increased expression of AVT and IT transcripts at day 1 and day 7, respectively highlighted that those transcripts were differentially sensitive to the external salinity as well as different exposure time. Furthermore, the increased expression of IT-R in forebrain and hypothalamus indicated that IT-R may auto regulated by itself during osmotic stress for successful acclimation. Finally, the in situ hybridization of AVT, IT and IT-R transcript revealed that the PMpc play an essential role during osmotic stress. During FW transfer, the integrated function of the brain and pituitary greatly involved to maintain the osmotic homeostasis in the black porgy. key words: AVT, AVT-R, IT, IT-R and black porgy

96 Poster Presentation 2‐7

The calciotropic hormone vitamin D regulates ion homeostasis in zebrafish (Danio rerio)

Lin C.H.*, Hu H.J., Hwang P.P. Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan, R.O.C. e-mail: [email protected]

Vitamin D is well known in calcium regulation in fish. In previous study, the vitamin D serum level of salmon was different between seawater and freshwater adaptation. This result implied vitamin D may also involve in sodium and chloride (major ions in water) regulation in fish. However, so far this issue is still unclear. In the present study, zebrafish was used as an animal model to explore the effect of vitamin D in ion regulation in fish. Whole-body chloride, calcium, and sodium contents, mRNA expressions of ion transporters and the number of epithelial ionocytes were increased in zebrafish embryos with vitamin D treatment. To further explore the regulatory mechanism of vitamin D in ion regulation, foxi3a and foxi3b (ionocyte differentiation marker) mRNA expressions and the density of P63 (an epidermal stem cell marker)-positive cells of zebrafish embryos with vitamin D treatment were analyzed. The foxi3a and foxi3b mRNA expressions were upregulated, but density of P63-positive cells was not regulated in zebrafish embryos with vitamin D treatment. Taken together, vitamin D could upregulate mRNA expressions of ion transporters and densities of ionocytes by stimulating foxi3a and foxi3b expression, consequently enhancing the ion uptake in zebrafish.

Keyword: vitamin D, ion regulation, ionocyte, zebrafish

97 Poster Presentation 3‐1

Evolution and expansion of glp1 and glp1 receptor family

Yun S.1*, Moon M.J.1, Park C.R.1, Cho E.B.1, Hwang J-.I.1, Seong J.Y.1 1Graduate School of Medicine, Korea University, Seoul, Republic of Korea e-mail: [email protected]

Glucagon like peptide 1 (GLP1) is an intestinal incretin regulating glucose homeostasis through stimulation of insulin secretion from pancreatic beta cells and inhibiting appetite by acting on the brain. Thus, it is a promising therapeutic agent for the treatment of type-2 diabetes mellitus and obesity. Studies using synteny and reconstructed ancestral chromosomes suggest that families for GLP1 and its receptor GLP1R have emerged from secretin-like GPCR and their peptide ligand genes through two rounds (2R) of whole genome duplication and local gene duplications before and after 2R. Exon duplications have also contributed to expansion of the peptide family members. Specific changes in the amino acid sequence following exon/gene/genome duplications have established distinct yet related peptide and receptor families. These specific changes also confer selective interactions between GLP1 and GLP1R. This information may provide critical insight for the development of potent therapeutic agents targeting GLP1R.

Keyword: Evolution, Exon, GLP1, GLP1R, G protein-coupled Receptor, Genome, Gene, Duplication

98 Poster Presentation 3‐2

Evolution and possible roles of heat shock protein 60 (Hsp60) in germline of metazoan animals

Pan H.H.1＊, Shikina S.2, Shao Z.F.3, Chang C.F.2,3 1.National Taiwan Ocean University Institute of Bioscience and Biotechnology 2.National Taiwan Ocean University Center of Excellence for the Oceans 3.Department of Aquaculture, National Taiwan Ocean University e-mail：[email protected]

Heat shock protein 60 (Hsp60) is one of the most evolutionarily conserved molecules across taxa throughout prokaryotic bacteria to mammals. Studies in mammal so far discovered that Hsp60 is highly expressed in reproductive organ including germline cells in both sexes, suggesting that Hsp60 may play important function in the mammalian reproduction. Thus far, however, few researches have been reported about the presence of Hsp60 in the reproductive organs of non-mammalian animals including invertebrates. In the present study, we hypothesized that Hsp60 expression in germline is evolutionarily conserved throughout metazoans. To verify this hypothesis, we produced a highly specific antibody against conserved regions of Hsp60 throughout metazoans and performed immunohistochemical analysis on various metazoans reproductive organs. It was confirmed that Hsp60 were present at a high level in the germline cells of cnidarians (corals, anemone), mollusks (oysters, abalone), arthropod (shrimp), and vertebrates (mouse, frog, fish). These results suggested that Hsp60 might possess an evolutionarily conserved fundamental role in metazoan germline.

Key words：heat shock protein 60 (hsp60), germ cell

99 Poster Presentation 4‐1

Pineal allopregnanolone saves purkinje cells from apoptosis in the cerebellum

Haraguchi S.1*, Hara S.1, Ubuka T.1, Tsutsui K.1 1Department of Biology, Waseda University, Tokyo, Japan. e-mail: [email protected]

Until recently, it was generally believed that neurosteroids are produced in neurons and glial cells in the central and peripheral nervous systems. Here, we show that allopregnanolone (ALLO), a neurosteroid, is exceedingly produced in the pineal gland, an endocrine organ located close to the brain, compared with the brain, and pineal ALLO acts on the Purkinje cell, a principal cerebellar neuron, to prevent apoptosis in the juvenile quail. We first demonstrated that the pineal gland is a major organ of neurosteroidogenesis. A series of experiments using molecular and biochemical techniques have further demonstrated that the pineal gland produces a variety of neurosteroids de novo from cholesterol in the juvenile quail. Importantly, ALLO was far more actively produced in the pineal gland than the brain. Pinealectomy (Px) decreased ALLO concentration in the cerebellum and induced apoptosis of Purkinje cells, whereas administration of ALLO to Px quail chicks prevented apoptosis of Purkinje cells. We further found that Px significantly increased the number of Purkinje cells that expressed active caspase-3, a key protease in apoptotic pathway, and daily injection of ALLO to Px quail chicks decreased the number of Purkinje cells expressing active caspase-3. These results indicate that the neuroprotective effect of pineal ALLO is associated with the decrease in caspase-3 activity during the early stage of neuronal development. We thus provide new evidence that the pineal gland is an important neurosteroidogenic organ and pineal ALLO may be involved in Purkinje cell survival during development. This is a new function of the pineal gland for the formation of neuronal circuits in the developing cerebellum.

Keyword: apoptosis, neurosteroids, allopregnanolone, pineal gland, Purkinje cell

100 Poster Presentation 4‐2

Potential role for neurosteroids in the early brain of orange-spotted grouper

Nagarajan G.1,2*, Aruna A.2 , Chang C.F.1,2 1Center of Excellence for the Oceans, 2Department of Aquaculture, National Taiwan Ocean University, Keelung 20224, Taiwan e-mail: [email protected]

The regulatory role of neurosteroids in the early brain during gonadal sex differentiation is unclear. The aim of this study was to investigate the expression and cellular localization of key steroidogenic enzymes in the early brain of the protogynous orange-spotted grouper Epinephelus coioides and the temporal expressions has been correlated with gonadal sex differentiation. In this study, we showed that peak of temporal expressions of neurosteroidogenesis and estrogen receptivity occurs in the early brain during gonadal sex differentiation. Further, in situ hybridization (ISH) analysis showed that cyp11a1, hsd3b1 and cyp17a1 transcripts were widely expressed in several discrete brain regions, especially the intense expression in the forebrain, with an overall similar expression pattern. High density in the cyp19a1b/Cyp19a1b expression was detected in radial glial cells. Thus, the expression of grouper cyp19a1b/Cyp19a1b is restricted to radial glial cells, suggesting estrogens can modulate their activity. In addition, we observed abundant immunoreactive (ir) PCNA in the irCyp19a1b positive radial glial cells of the grouper fish, suggested that radial cells are progenitor cells in the early orange-spotted grouper brain. Next, by combining Cyp19a1b immunohistochemistry (IHC) with florescence ISH (FISH) of cyp11a1, hsd3b1 and cyp17a1, we showed that sub-cellular localization of cyp11a1, hsd3b1 and cyp17a1 transcripts, in partial, appeared to be in Cyp19a1b radial glial cell soma. Moreover, exogenous estradiol (E2) increased the expression of cyp17a1 and cyp19a1b/Cyp19a1b in the brain of grouper. Taken together, a significant peak expression of key genes and the intense expressions of cyp19a1b/Cyp19a1b in radial glial cells suggested a role in neurogenesis by the auto-regulation of locally synthesized E2 in the brain of the orange-spotted grouper during gonadal sex differentiation.

Keywords: brain, steroidogenic enzymes, cyp19a1b, estradiol, brain development, gonadal sex differentiation

101 Poster Presentation 5‐1

The expression and regulation of aromatase and estrogen receptors in Japanese eel

Jeng S.R.1*, Yueh W.S. 1, Dufour S.2, Kah O.3, Chang C.F.4,5 1 Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung, Taiwan 2 Muséum National d'Histoire Naturelle, Research Unit BOREA, Biology of Aquatic Organisms and Ecosystems, CNRS 7208 Paris, France 3 Research Institute in Health, Environment and Occupation, INSERM U1085, Université de Rennes 1, Campus de Beaulieu, Rennes, France 4 Department of Aquaculture, 5 Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan e-mail: [email protected]

Eel species possess a single aromatase gene, CYP19a1, which is expressed in both gonad and brain. Female eels treated with catfish pituitary homogenate (CPH) to induce sexual maturation showed an increase in the expression of CYP 19a1 and aromatase enzymatic activity in the brain and in the ovaries. Treatments with sex steroids (estradiol-17β, E2 or testosterone, T) revealed that the increase in CYP19a1 expression in the brain may result from

E2-specific induction. In contrast, the increase in CYP19a1 expression in the ovaries of CPH-treated eels is a result of steroid-independent control. The CYP19a1 is expressed only in radial glial cells of the brain and in pituitary cells. The aromatase-expressing radial glial cells exhibit proliferative activity in both the brain and the pituitary. These data indicate that CYP19a1of Japanese eel exhibits characteristics similar to those reported for the brain specific CYP19a1b gene in teleosts having duplicated CYP19a1 genes. In CPH-treated eels, the expression of ESR-α was significantly increased in the brain. No change was observed in

ESR-β in any organ. Steroid treatments induced an upregulation by E2 of ESR-α, but not ESR-β expression, in the brain, pituitary and ovaries. Our results also showed that aromatase-positive cells were not ESR-α-positive cells in the brain of Japanese eel.

Keywords: aromatase, estrogen receptor, eel

102 Poster Presentation 6‐1

Neuropeptide Y involves in the regulation of somatic growth of the Pacific abalone

Moriyama S.1*, Kawamura M.1, Kodera Y.2, Uchida K.3, Amano H.1 1School of Marine Biosciences, Kitasato University, Sagamihara, Japan. 2School of Sciences, Kitasato University, Sagamihara, Japan.3Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan. e-mail: [email protected]

Neuropeptide Y (NPY) is known to plays key role in the regulation of food uptake, energy balance and reproductive behavior in vertebrate and invertebrate. Pacific abalone Haliotis discus hannai, is one of the most important aquaculture species in the Tohoku and Hokkaido areas of Japan. In an attempt to understand growth regulation in the abalone, abalone NPY homolog (AbNPY) was identified by cDNA cloning and peptide determination from cerebral ganglion cDNA. Two abNPY cDNAs, named abNPY-1 and 2, were determined from the abalone cerebral ganglion. abNPY-1 cDNA was identical with abNPY-2 in the signal peptide, mature peptide and C-terminal extension peptide, but lacked 26 consecutive nucleotides located in the 3’noncording region of abNPY-2. Two abNPYs, which consist of 26 and 39 amino acids (AA) with a C-terminal RPRF amide peptides, were determined from acid-acetone extract of the cerebral ganglion by reverse-phase HPLC and LC-MS/MS. abNPY shows high homology with C-terminal region of other molluscan NPYs. AbNPY immunoreactive cell bodies, which reacted with antibody raised against AbNPY fragment, were detected in the peripheral region of the cerebral ganglion of abalone, and were observed to send fibers into the neuropill. Higher AbNPY mRNA levels were observed in the cerebral ganglion of larger-sized abalone than in those of smaller abalone. Intramuscular injections of juvenile abalone with the synthetic AbNPY of 26 AA at a dose of 2.5 µg/g body mass at 7-day intervals resulted in an increase in body mass. These results suggest that NPY homolog is present in the cerebral ganglion and this peptide is involved in the regulation of somatic growth of the Pacific abalone.

Keyword: Abalone, Growth, Neuropeptide Y

103 Poster Presentation 6‐2

Expression of heat shock protein 60 (Hsp60) in the germline cells of a scleractinian coral Euphyllia ancora (Cnidaria, Anthozoa)

Shikina S.1*, Shao Z.F.2, Chung Y.J.2, Chang C.F.1,2 1. Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan 2. Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan e-mail: [email protected]

Heat shock protein 60 (Hsp60), a member of the mitochondrial chaperonin family, is highly conserved cellular stress proteins present in every organism from bacteria to man. In mammalians, HSP60 have been shown to be highly expressed in early stages of germline cells in both sexes, suggesting that HSP60 might play an important role during gametogenesis. In invertebrate, however, little research thus far has been conducted on the expression of Hsp60 in germline cells. To accumulate the knowledge of Hsp60 in reproduction, in the present study, we investigated the expression of Hsp60 in germline cells in the testis and ovary of a scleractinian coral Euphyllia ancora. The full-length of E. ancora hsp60 cDNA was elucidated, and a specific antibody against the Hsp60 was successfully generated. Immunohistochemical analysis of male corals revealed that Hsp60 was strongly expressed in spermatogonia and early spermatocytes, but not in the postmeiotic spermatids or spermatozoa. On the other hand, immunohistochemical analysis of female corals revealed that Hsp60 were strongly expressed in oogonia and early oocytes, and weakly expressed in later stage oocytes that exceeded 200 mm in diameter. Immunoreactivity were also detected in the somatic cells in the tentacles and mesenterial filament in both sexes. Furthermore, western blotting analysis of unfertilized mature eggs proved the presence of Hps60, suggesting that Hsp60 may be a maternally inherited to the next generation. These results demonstrated that Hsp60 is expressed in germline cells in the corals, suggesting that it might play a fundamental role in gametogenesis in corals.

Key words: heat shock protein 60 (hsp60), coral, Euphyllia ancora, germ cell

104 Poster Presentation 6‐3

Exploration of the genes involving in sexual reproduction in a stony coral Euphyllia ancora

Chiu Y.L.*1, Shikina S.2, Chang C.F.1,2 1.Department of Aquaculture, National Taiwan Ocean University 2.Center of Excellence for the Oceans, National Taiwan Ocean University E-mail：[email protected]

Coral reefs, which are among the most biologically diverse ecosystem on the planet, continually decline worldwide due to the influences of climate changes and human impacts. Aquaculture of corals is expected as an excellent methodology to propagate target coral species and conserve their genetic resources. Better understanding of molecular and endocrine mechanisms underlying sexual reproduction would allow us to induce the germ cell development, maturation, and spawning of corals under artificial rearing condition, however, are scarce. To increase our understanding of the molecular mechanisms underlying coral sexual reproduction, we utilized the technique of suppression subtractive hybridization (SSH) to identify genes that are upregulated during reproductive periods when compared to non-reproductive periods. A stony coral Euphyllia ancora was used as the experimental animals. A SSH-enriched cDNA library was generated by subtracting common cDNA from E. ancora corresponding reproductive period (April). By combining the methods differential screening of the library and semi-quantitative RT-PCR analysis, we could identify 4 clones that expression levels were significantly higher in the reproductive period (April) compared with that of the non-reproductive period (June). Furthermore, it was conformed that these 4 clones were highly expressed in the putative ovarian tissue as determined by tissue distribution analysis. These 4 clones obtained in the present study may be related to germ cells/gonadal development of the coral.

Key words: coral (Euphyllia ancora), sexual reproduction, suppression subtractive hybridization (SHH)

105 Poster Presentation 7‐1

Dose 11-KT Increase GnRH Neurons by Enhancing Neurogenesis in Female Mozambique Tilapia

Narita Y.1*, Okada N.1, Tsutiya A.1, Kaneko T.2, Ohtani-Kaneko R.1 1Department of Life Sciences, Faculty of Life Sciences, Toyo University, Gunma, Japan 2Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan e-mail address (R. Ohtani-Kaneko): [email protected]

In our previous study (Kuramochi et al., 2011), we found sexual dimorphism of gonadotropin-releasing hormone type III (GnRH3) neurons in Mozambique tilapia Oreochromis mossambicus; males have a greater number of GnRH3 neurons in the terminal ganglion than females. Treatment with androgen (11-ketotestosterone (11-KT) or methyltestosterone (MT)) induced male-specific nest-building behavior in 70% of females within two weeks after the onset of the treatment. The androgen treatment increased the number of GnRH3 neurons in females to a level similar to that in males. However, the mechanism underlying androgen-induced increase of GnRH3 neurons reminds unsolved. The adult neurogenesis has recently been shown to occur in various vertebrate species. The aim of the present study is to investigate whether the increase of GnRH3neurons in androgen-treated female tilapia is mediated by the adult neurogenesis. To examine this, we first examined whether androgen increased the proliferating cells in adult females by immunohistocemically detecting cells with the expression of a proliferation-related marker, proliferating cell nuclear antigen (PCNA). Next, we studied the fate of newly generated cells by double labeling with antibodies against 5-bromo-2-deoxyuridine (BrdU) and one of markers of interest including GnRH3. After the treatment with 11-KT or MT, we found that PCNA-positive cells were increased in various regions of the adult female brain, compared to control females injected with oil. We further studied Hu-positive immature neurons, GFAP-positive radial glias/progenitors and GnRH3-positive cells in combination with BrdU labeling.

Keyword: GnRH3, sex reversal, androgen, brain, neurogenesis

Reference: Kuramochi et al., Sexual dimorphism of gonadotropin-releasing hormone type-III (GnRH3) neurons and hormonal sex reversal of male reproductive behavior in Mozambique tilapia. Zoological Science 28 (2011): 733–739.

106 Poster Presentation 7‐2

Sexual bipotentiality of the brain for olfactory processing and sexual behavior in goldfish

Shinohara Y.* and Kobayashi M. Department of Life Science, International Christian University, Tokyo 181-8585, Japan.

It is known that brain sex differentiation occurs in mammals and in some other vertebrates, and these species do not normally perform heterotypical sexual behavior (sexual behavior of opposite sex). However, in goldfish, Carassius auratus, heterotypical sexual behavior can be induced by hormonal treatments in sexually mature fish. These facts led us to propose a hypothesis that brain sexual differentiation does not occur in goldfish, unlike mammals, and the brain is sexually bipotential and can regulate both male- and female-typical sexual behavior. In the present study, we further examined this hypothesis by observing the effects of olfactory blockage on sexual behavior in goldfish. Male sexual behavior is triggered by sex pheromone from females, and the behavior is suppressed by blockage of pheromone reception (nasal occlusion, NaOcc), and blockage of olfactory information from the olfactory bulb (OB) to the telencephalon (olfactory tract section, OTX). In females, sexual behavior is triggered by ovarian prostaglandin. Blockage of reception of olfactants by NaOcc activates the inhibitory system of OB suppressing female sexual behavior, whereas OTX removes this inhibition, resulting in the resumption of the behavior. Female goldfish treated with androgen for the induction of male-typical behavior showed the same responses as in males by the olfactory blockage. Male goldfish treated with prostaglandin for the induction of female-typical behavior showed the same responses as shown in females by the olfactory blockage. These results indicate that both male and female possess the same bisexual brain system for olfactory processing and sexual behavior.

Key words: brain bisexuality, goldfish, olfaction, sexual behavior

107 Poster Presentation 7‐3

Activation of brain steroidogenesis and neurogenesis at the time of gonadal sex differentiation in protandrous black porgy, Acanthopagrus schlegelii

Lin C.J.1*, FanChiang Y.C.1, Dufour S.2, Chang C.F.1* 1Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan. 2Research Unit BOREA, Biology of Aquatic Organisms and Ecosystems, CNRS 7208/IRD 207/UPMC, Muséum National d’Histoire Naturelle, 7 rue Cuvier, 75231 Paris Cedex 05, France * E-mail: [email protected]

The relationship between brain development and gonad sex differentiation is an important issue in vertebrates but it still remains unknown. We applied black porgy as a model animal to study molecular and cellular mechanisms during gonadal differentiation. A peak expression of neurosteroidogenesis related genes cyp19a1b and increased concentration of estradiol (E2) were found in the early brain during gonadal differentiation, and more brain cells were observed in preoptic area (POA). Immunohistochemical analysis in POA showed higher cell proliferation activity during this period. Significantly higher expressions of radial glial cell marker blbp (a brain lipid binding protein) and neuron marker bdnf (a brain-derived neurotrophic factor) were further detected in the brain during gonadal differentiation. Strong immunohistochemical staining of Blbp as well as extended cellular projections was found in the POA during gonadal differentiation. The data demonstrated that the early brain during the gonadal differentiation had higher activity of E2 synthesis, cells proliferation, and genes expression as compared to other periods.

To investigate the function of E2 in the early brain, E2 was given to undifferentiated fish. And we found E2 stimulating progenitors including up-regulation of cyp19a1b in brain and blbp in hypothalamus, enhanced Cyp19a1b, Blbp, and also proliferation activity. The analytic results from castration experiment confirmed gonad removing has no influence on the peaked pattern of cyp19a1b, blbp, bdnf, and neither brain cells amounts. Our data clearly supported that brain is more active in genetic and cellular level during the period of gonadal differentiation, which were affected by locally biosynthesized estradiol.

Keyword: brain development, gonad differentiation, aromatase, black porgy

108 Poster Presentation 8‐1

Suppression of NF-κB signaling by nme1 through direct interaction with IKKβ

You D.J.*, Park C.R., Lee H.B., Seong J.Y., Hwang J.I.* Laboratory of G Protein Coupled Receptors, Graduate School of Medicine, Korea University, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, Republic of Korea * Corresponding author. Tel.: +82 2 2286 1093; fax: +82 2 921 4355. e-mail address : [email protected]

TNFα-stimulated NF-κB (Nuclear factor-κB) and inhibitor of NF-κB kinase (IKK) complex proteins regulate many physiological processes including immune response, stress response, and tumor development. Recently the molecular processes of IKK activity regulation have been partially uncovered. Here, through yeast two-hybrid screening system, we identified NME1 (Nm23-H1) as a binding partner of IKK. NME1 which is a master regulator of tumor migration and metastasis is expressed as two distinct splicing variants endogenously, NME1 (177 a.a., long form of NME1) and NME1 (152 a.a., short form of NME1). A large number of studies in NME1 function have been limited on the short form of NME1. Here, we showed that the long form of NME1, not the short form, interacts with IKK without binding to IKK and NEMO. Domain mapping revealed that IKK interacts with NME1 through Leucine Zipper (LZ) domain. Overexpression of NME1 inhibited TNF -stimulated NF- B activation, and Down-regulation of NME1 expression by RNAi enhanced NF- B activity by TNF. IKK auto-phosphorylation stimulated by ectopic expression was blocked by NME1. Interestingly, the long form of NME1 dramatically suppress cell migration compared with the short form of NME1. These results indicate that long form of NME1 is likely to negatively regulate TNF -stimulated NF- B signaling by interacting with IKK, and suppress cancer cell migration more efficiently

109 Poster Presentation 8‐2

Analysis of Cholecystokinin, peptide Y and Ghrelin in the appetite and digestion of pacific bluefin tuna (Thunnus orientalis)

Kondo F.*, Iwai T., Miura C., Miura T. South Ehime Fisheries Research Center (SEFREC), 1289-1 Funakoshi, Ainan-town, 798-4292, Japan

Pacific bluefin tuna is known to have a high growth rate and considered to be a valuable food source, but under aquaculture conditions growth problem has been a concern in this species. Their body growth is affected by appetite and digestion, which are regulated by the peptide hormones such as cholecystokinin (cck), peptide Y (py) and ghrelin, among others. However, the roles and interaction of these hormones in teleost have not been understood in detail. Furthermore, there are no studies on the respective roles of CCK, PY and Ghrelin in pacific bluefin tuna. We observed the passage and digestion of food in the digestive tract. Intestinal contents were decreased in 10-15 hours after feeding. Quantitative PCR showed that the expression of cck, in intestine increased, while py expression in pyloric caeca decreased when food is digested with the intestine. In addition, ghrelin and gh expression both increased at the same time. In the brain, cck expression increased, whereas ghrelin expression decreased prior to initiation of intestinal digestion. These data suggest that appetite is suppressed while the food is being digested in the intestine, and raised the possibility that this is regulated by antagonistic effects of cck, py and ghrelin. Elucidation of the control mechanism in the expression of these hormones will enable the efficient production of pacific bluefin tuna under aquaculture conditions.

110 Poster Presentation 8‐3

Effect of β-Glucans on Diabetic Mouse Regarding to Natural Killer Cells

Cheng L.C.1*, Liang C.C.1, Kong Z.L.1 1Department of Food Science, National Taiwan Ocean University e-mail: [email protected]

Backgrounds: Diabetes mellitus is characterized by high blood glucose level. Hyperglycemia has been identified as the main factor contributing to the development of diseases associated with diabetes mellitus. Diabetes is widely believed to predispose to serious infections. Beta glucans are now recognized as potent immunological activators. Glucose metabolism disorders influence anticarcinogenic function of natural killer cells. On the other hand, both oat and fungal beta-glucans reduce blood glucose concentrations after oral administration in animal experiments and clinical trials. The aim of study is to investigate the beneficial effect of natural killer cells on diabetic mice. Materials and Methods: Female BALB/cByJNarl and C57Bl/6JNarl mice were induced diabetes by streptozotocin (65 mg/kg), and we used nicotinamide (230 mg/kg) as the protective agent. First, orally administrated with beta-glucans at the doses of 4, 8 and 16 mg/kg body weight for 6 weeks. Second, natural killer cells were cultivated in vitro and injected with different concentrations of natural killer cells into diabetic mice. Results: The results showed that the dose of 16 mg/kg beta-glucans significantly improved the hyperglycemia and decreased the levels of total cholesterol (TC), triglyceride (TG). Then, beta-glucans also increased natural killer cells activity. On the other hand, natural killer cells was apoptotic in high glucose medium. After treating with IL-15 and beta glucans natural killer cells were more viable. Furthermore, injecting natural killer cells back into diabetic mice can reduce high blood glucose level. Conclusion: This study demonstrated natural killer cells can protect mice from diabetes, and natural killer cells therapy may decrease diabetic symptoms in future.

Keyword: Natural killer cells, type 2 diabetes, beta-glucans

111 Poster Presentation 9‐1

Gonadotropins affected the in vitro expression of neuropeptide and steroidogenesis in the gonad of tilapia

Chou C.H.*, Chiu H.C., Wu L.T., Chen C.H., Kang Y.R., Chang Y.S., Lee T.L., Huang W.T. Department of Molecular Biotechnology, Da-Yeh University, Chang-Hwa, Taiwan e-mail: [email protected]

Reproduction including gonad development, gametogenesis and steroidogenesis in vertebrates are under the regulation of gonadotropins (GTHs) – follicle stimulating hormone (FSH) and luteinizing hormone (LH) secreted by pituitary gland. Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide, has diverse functions including the regulation of proliferation, differentiation, metabolism, endocrine and immune systems in animals. Our previous studies showed that tpacap38 and its type I receptor (tpac1-r) expressed in the gonads of tilapia (Oreochromis mossambicus) and were involved in the cAMP-protein kinase A signaling pathway. However, the relation among GTH, PACAP, and steroids in fish remains unclear. Aim of this study was to identify the effect of gonadotropins on the expressions of tpacap38 and tpac1-r, and steroidogenesis in tilapia gonads. Tilapia testes and ovarian follicles were cultured in DMEM with different concentrations of human chorionic gonadotropin (hCG; 5, 15, 50 IU) or pregnant mare's serum gonadotropin (PMSG; 5, 15, 50 and 100 IU) or combination of hCG and PMSG (5+5, 15+15, and 50+50) for 2 hours in a dose-dependent experiment or 0 to 8 hours in a time-course experiment, respectively. The expressions of tpacap38 and tpac1-r, and concentrations of gonadal steroids (estradiol, testosterone, 11-keto testosterone, and progesterone) were increased by gonadotropins in a dose- and time-dependent manner, and the inductive function could be suppressed by protein kinase A inhibitor H89. However, combination of hCG and PMSG had no synergetic effect on the elevation of either the expressions of tpacap38 and tpac1-r, and secretion of steroids. The present data indicated that gonadotropins affected the expression of tpacap38 and tpac1-r, and enhanced de novo synthesis of steroids in tilapia gonads.

Keyword: tilapia, gonads, gonadotropins, pituitary adenylate cyclase-activating polypeptide (PACAP), steroidogenesis

112 Poster Presentation 9‐2

Effects of edcs on sex as demonstrated by feminization of artificially propagated southern catfish by feeding of Limnodilus spp

Dong R. *, Yang S., Fang L., Feng R., Sun Y., Zhang Y., Xie X., Wang D. Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Science, Southwest University, Chongqing, 400715, China. e-mail: [email protected]

Endocrine disrupting chemicals (EDCs) as environmental contaminants have received considerable attention since the early 1990s. Feminization of animals derived from areas polluted by EDCs has been observed in all classes of vertebrates including fish, amphibians, reptiles, birds and mammals. However, feminization of artificially propagated offspring by feeding of specific live organisms has never been reported. Here we report that feeding of Limnodilus spp resulted in complete feminization of southern catfish (Silurus meridionalis), which has a 1:1 sex ratio in wild conditions, for the first time. Furthermore, HPLC analysis showed that the extraction of Limnodilus spp contained EDCs, such as diethylstilbestrol (DES), bisphenol A (BPA), 4-Nonylphenol (4-NP) and 4-tert-octylphenol (4-t-OP), which were further confirmed via LC-MS. Limnodilus spp fed fish displayed similar serum estradiol-17β levels and gonadal Sf1, Dmrt1, Foxl2, Cyp19a1a expression levels to those of female control. Taken together, our study demonstrated that EDCs in Limnodilus spp cause catfish feminization by upregulation of aromatase expression and endogenous estrogen level. Therefore, more attention should be act to the impacts of EDCs on wildlife and human beings.

Keywords: Feminization, Catfish, EDCs, Limnodilus spp, Cyp19a1a and estrogen level

113 Poster Presentation 9‐3

Neuropeptide affected protein expression in the gonad of tilapia in vitro

Chiu H.C.1*, Chou C.H.1, Chen J.H.1, Chang Y.S.1, Lee T.L.1, Tang P.C.2, Huang W.T.1# 1 Department of Molecular Biotechnology, Da-Yeh University, Chang-Hwa, Taiwan 2 Department of Animal Science, National Chung-Hsing University, Taichung, Taiwan e-mail: [email protected]

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide which can elevate the concentration of intracellular cAMP and calcium in the gonado- and somatotroph cells, and regulates the physiological function of the organisms. Tilapia is one of the most common commercial fish in Taiwan, but literature concerning the mechanism of proteins expression in tilapia gonads is still scant. Our previous results showed that PACAP and its receptor expressed in tilapia (Oreochromis mossambicus) gonads and was involved in the cAMP-protein kinase A (PKA) signaling pathway and could regulate reproduction system in an autocrine/paracrine manner. In present study effect of PACAP on the protein expression profiles in tilapia gonads in vitro was studied. Protein extracted from tilapia testes and ovarian follicles was divided into 7 groups including fresh tissue, Pre-culture (cultured in DMEM for 8 hours), 4 experimental groups (PACAP, Forskolin [adenylate cyclase activator], H89 [PKA inhibitor], and PACAPForskolin, all groups with 10 M) for additional 2 hours after Pre-culture, and control (DMEM), and were analyzed by two-dimensional gel electrophoresis. Spot detection and matching were performed using the Phoretix 2D software. The present data showed that PACAP treated groups in both gender had significantly higher protein spot number than those of H89 treated groups, but no significant difference was observed when compared with other groups. This revealed that the expression of tilapia gonadal protein in vitro was affected by PACAP and specific proteins were identified. Similar results were observed in those previously treated by steroids and gonadotropins. The results revealed the relationship among gonadotropins, steroids, neuropeptide, and related proteins in tilapia gonads, and could be helpful in fish reproductive endocrine research.

Keyword: tilapia, gonads, pituitary adenylate cyclase activating peptide (PACAP), proteomics

114 Poster Presentation 9‐4

Morphological and molecular approaches on pituitary gland of the cloudy catshark, Scyliorhinus torazame

Uchida K.1*, Hyodo S.2, Watanabe T.2, Kagawa H.1 1Department of Marine Biology and Environmental Sciences, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan. 2Atmosphere and Ocean Research Institute, University of Tokyo, Kashiwa 277-8564, Japan. e-mail: k-uchida @cc.miyazaki-u.ac.jp

Cartilaginous fishes (Chondrichthyes) are ancient groups of jawed-vertebrates, which have emerged during Silurian period. Chondrichthyes have unique reproductive patterns such as oviparity, ovoviviparity and viviparity. However, little information is available on endocrine control of their manifold reproductive strategies at physiological levels. In the present study, to clarify the endocrine mechanisms of their reproductive physiology, we first observed the morphology of pituitary gland of cloudy catshark, Scyliorhinus torazame. The histological observations have revealed that the adenohypophysis was divided into three major parts, rostral and proximal pars distalis and pars intermedia. In addition, there was an additional part, the ventral lobe, beneath the adenohypophysis. The ventral lobe was attached to the cartilaginous palate and covered entirely with connective tissue of the palate. In the second experiment, specific polyclonal antibodies against catshark gonadotropins (GTHs), follicle stimulating hormone (FSH) and luteinizing hormone (LH), was raised in rabbits. Immunocytochemical staining revealed that FSH and LH are produced specifically in the ventral lobe of pituitary gland. In the third experiment, transcriptome analysis of the hypothalamus-pituitary organs was carried out by the next generation sequencing technology (Roche). We obtained a total of 27,6951 reads and de novo assembly yielded a total of 6,252 contigs with an average length of 683 bp. Based on homology searching by BLAST, 57.8% of contig sequences (total 3,616 contigs) were hit with significant similarity and identified as functional molecules. Reproductive endocrine factors such as gonadotropin-releasing hormone and glycoprotein hormone alpha subunit were found in the molecular profiles, whereas the beta subunits of GTHs were not included. We are now trying to identify the whole gene structure of the beta subunit of two GTHs by PCR cloning technique.

Keyword: elasmobranch, pituitary, hypothalamus, GTHs, De novo assembly

115 Poster Presentation 9‐5

Characterization of two duplicated star genes in a teleost, Nile tilapia (Oreochromis niloticus)

Yu X., Wu L., Xie L., Yang S., Shi H., Wang D., Zhou L.* Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Science, Southwest University, Chongqing 400715, P.R. China. e-mail:[email protected]

Steroidogenic acute regulatory protein (Star) mediates translocation of cholesterol, the substrate for steroid synthesis, to the inner membranes of mitochondria. It is well known that estrogen is essential for female sex determination/differentiation in fish. However, no reports showed that the conventional star, which is essential for estrogen production, was expressed in gonads during the critical timing of female sex determination/differentiation. In this study, two different star isoforms, named as star1 and star2, were characterized from the gonads of Nile tilapia (Oreochromis niloticus). Phylogenetic analysis revealed that duplication of star gene existed only in teleosts. Real-time PCR revealed that star1 was dominantly expressed in the testis, head kidney and kidney; while star2 was expressed exclusively in the gonads. In situ hybridization and immunohistochemistry demonstrated that Star1 was expressed in the interrenal cells of the head kidney and Leydig cells of the testis; while Star2 was expressed in both the Leydig cells of the testis and the interstitial cells of the ovary. Ontogenic analysis demonstrated that Star2 was expressed abundantly from 5 day after hatching in the somatic cells in XX gonads, whereas in XY gonads, both Stars could be detected only from 30 dah until adulthood. Intraperitoneal injection of human chorionic gonadotropin resulted in significant increase of Star2 and 1 at 4 h and 8 h after injection in the testis, respectively. Taken together, our data demonstrated that Star1 is likely required in cortisol production in the head kidney, while Star2 is probably involved in estrogen production during early sex differentiation in XX gonads. Both Stars might play important roles in the production of androgen and gametes maturation inducing hormone during spermatogenesis and sperm maturation. For the first time, our data demonstrated that two fish Stars might be involved in steroidogenesis in a tissue and developmental stage dependent manner.

Keywords: two star genes; sexual differentiation; hCG induction; cortisol biosynthesis

116 Poster Presentation 9‐6

Transdifferentiation of differentiated ovary into functional testis by long term treatment of aromatase inhibitor in Nile tilapia

Sun L.1, Jiang X.1, Xie Q.1, Zhou L. 1, Nagahama Y.2,3,4 , Wang D.1* 1Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), Key Laboratory of Aquatic Science of Chongqing, School of Life Science, Southwest University, Chongqing, 400715, China. Fax: +86-2368253005, E-mail：[email protected]. 2Solution-Oriented Research for Science and Technology (SORST); 3Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan; 4South Ehime Fisheries Research Center, Ehime University, Matsuyama 790-8577, Japan.

Females with differentiated ovary of a gonochoristic fish, Nile tilapia, were masculinized by long term treatment with an aromatase inhibitor (Fadrozole) in the present study. The reversed gonads developed into functional testes with fertile sperm. The longer the fish experienced sex differentiation, the longer treatment time was needed for successful sex reversal. Furthermore, Fadrozole induced sex reversal, designated as secondary sex reversal (SSR), was successfully rescued by supplement of exogenous 17β-estradiol. Gonadal histology, immunohistochemistry, transcriptome and serum steroid level were analyzed during SSR. The results indicated that spermatogonia were transformed from oogonia or germline stem cell-like cells distributed in germinal epithelium; while Leydig and Sertoli cells probably came from the interstitial cells and granulosa cells of the ovarian tissue, respectively. The transdifferentiation of somatic cells, as indicated by the appearance of Dmrt1 (pre-Sertoli cells) and Cyp11b2 (pre-Leydig cells) positive cells in the ovary, provided micro-niche for the transdifferentiation of germ cells. Decrease of serum 17β-estradiol was detected earlier than increase of serum 11-ketotestosterone, indicating decrease of estrogen was the cause while increase of androgen was the consequence of SSR. The sex reversed gonad displayed more similarity in morphology and histology with a testis while the global gene expression profiles remained closer to the female control. Detailed analysis indicated that transdifferentiation was driven by suppression of female pathway genes and activation of male pathway genes. In short, SSR provides a good model for study of sex reversal in teleosts, and for understanding of sex determination and differentiation in non-mammalian vertebrates.

Keywords: transdifferentiation; differentiated ovary; functional testis; global gene expression profiles; aromatase inhibitor

117 Poster Presentation 9‐7

Molecular cloning and gene expression of Foxl2 in spotted scat (Scatophagus argus)

Li G.L.*, Zhang M.Z., Deng S.P., Zhu C.H. Fisheries College, Guangdong Ocean University, Zhanjiang, 524088, China e-mail: [email protected]

Foxl2 is known to be involved in ovarian development and the maintenance of ovarian function in some vertebrates. In this study, the complete Foxl2 cDNA sequence was isolated by SMARTer RACE. Two-year-old female spotted scat, Scatophagus argus, were reared at different temperatures (23°C, 26°C and 29°C) for 6 weeks, or fed with different concentrations of dietary fish oil (0, 2%, or 6%) for 8 weeks, respectively. Ovarian development was examined by histology and ovarian Foxl2 expression was measured with quantitative real-time reverse-transcription PCR. Results showed that the full-length S. argus Foxl2 cDNA was 2024bp and encoded a protein with 305 amino acids with a predicted molecular weight of 36.365kDa. Molecular phylogenetic analysis indicated that S. argus Foxl2 shared the highest sequence identity of 98.4% with that of Epinephelus merra, and was consistent with those analyzed by traditional morphology and biochemistry. At the end of experiment, ovarian Foxl2 mRNA expression in fish reared at 23°C and 26°C was significantly higher than that in fish reared at 29°C, and that in 2% and 6% fish oil groups was also significantly higher than that in control group (P < 0.05). There was a significant positive correlation between ovarian development and Foxl2 expressions. Results showed that the optimal temperature for ovarian development in S. argus ranged from 23°C to 26°C, and 6% fish oil supplementation could effectively promote ovarian development. Optimal temperature and fish oil supplement could increase ovarian Foxl2 mRNA expressions to promote ovarian development in S. argus.

Keywords: Scatophagus argus; temperature; Fish oil; Foxl2 cloning; Foxl2 expression; Ovarian development

118 Poster Presentation 9‐8

Genetic sex determination in pejerrey: evidence from the testis-determining gene, AMHY

Zhang Y.*, Yamamoto Y., Sarida M., Hattori R.S. Strüssmann C.A. Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 108-8477 Tokyo, Japan. e-mail: [email protected]

The fate of gonadal differentiation in pejerrey Odontesthes bonariensis is highly dependent on the water temperature experienced by an individual early in life and genotypic determinants of sex are considered to be virtually inexistent in this species. In this study, we examined the presence of a homologue of the testis-determining gene amhy (Y-chromosome-linked, anti-Müllerian hormone) found in the congeneric species O. hatcheri in pejerrey and its functionality at an intermediate, mixed sex-producing temperature (MixPT).To examine the presence of amhy in pejerrey, genomic DNA from laboratory-reared broodstock was extracted, subjected to amplification by PCR using amhy-specific primers, and then sequenced. Larvae derived from a cross between an amhy-/- female and an amhy+/- male were reared at MixPT (25ºC) for up to 14 weeks after hatching (wah) and sampled weekly for the analyses of amhy and autosomal amh (amha) mRNA expression by real-time PCR and in situ hybridization (ISH) in the gonads. The remaining larvae were sampled at 14 wah for the determination of sex ratios by light microscopy. Approximately 50% of broodstock fish tested in this study possessed amhy and most were males; the autosomal amha was present in all fish. The sex ratio in the amhy+/-/amhy-/- full sibling progeny reared at 25ºC was 69% male: 31% female. Amhy-based genotyping revealed that all amhy+/- fish developed as males whereas about 2/3 and 1/3 of the amhy-/- were females and males, respectively. Amhy expression showed a transient increase before the critical period of sex determination whereas amha expression increased concomitantly with the amhy decrease and remained high during sex determination. Amha expression was detected in approximately 1/3 of the amhy-/- fish during sex determination. All gonads of amhy-/- animals with amha ISH signals after histological differentiation were identified as testes and those without it as ovaries. These results reinforce the importance of amha for testis formation in pejerrey and suggest a leading role for amhy, alone or in combination with temperature, in directing animals to differentiate as males. These findings represent the first clear evidence of a genotypic sex trend in pejerrey and suggest that genetic and environmental sex determinants can coexist even in species with marked temperature-dependent sex determination.

Keywords: Odontesthes bonariensis, amhy, amha, sex determination

119 Poster Presentation 9‐9

Effects of basswood culture Antrodia cinnamomea ethanol extracts on reproductive function in streptozotocin-nicotinamide induced diabetic male rats

Cheng S.C.1, Ting T.L.1, Kong Z.L.1* 1Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan e-mail: [email protected]

Backgrounds: Diabetes mellitus with the subsequent generation of reactive oxygen species can affect spermatogenesis, which can cause male infertility. Reproductive dysfunction is a consequence of diabetes, but the underlying mechanisms are poorly understood. Antrodia cinnamomea (A. cinnamomea) is a unique mushroom in Taiwan for the treatment of several types of cancers and inﬂammatory disorders. The aim of this study was to investigate the capability of anti-inflammatory and anti-oxidative for A. cinnamomea ethanol extract (ACEE), and to evaluate the ameliorating effect on male reproduction in diabetic rats. Materials and Methods: Male Sprague-Dawley rats were induced into diabetes by nicotinamide (230 mg/kg) and streptozotocin (65 mg/kg). A. cinnamomea ethanol extract was tested in three doses (385, 770, 1540 mg/kg per day, p.o. daily) for 4 weeks. Besides, rosiglitazone (RSG) was administrated (0.571 mg/kg) as the positive control. Results: The results indicate that ACEE cannot improve the STZ-NA induced hyperglycemia. But after the dose of 770 mg/kg ACEE administration, epididymal sperm abnormalities were decreased, whereas sperm count and motility were increased. In addition, ACEE can increase the activity of superoxide dismutase, catalase and glutathione and decrease the lipid peroxidation levels on testes. Furthermore, there was a higher level of testosterone. Treatment with ACEE restored the relative weights of testes, activities of antioxidant enzymes, sperm and hormonal index of the diabetic animals. Conclusion: Our study indicated that A. cinnamomea have anti-inflammation properties to prevent diabetes-induced male reproductive damage from oxidative stress.

Keywords: diabetes, male fertility, Antrodia cinnamomea

120 Poster Presentation 9‐10

Egg shell formation in the oviducal gland of the cloudy catshark, Scyliorhinus torazame, elasmobranchs

Matsumoto M. 1*, Uchida K. 1, Watanabe T. 2, Hyodo S. 2, Nakamura M.3, Sato K.3, Kagawa H. 1 1Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan; 2Atmosphere and Ocean Research Institute, the University of Tokyo, Kashiwa 277-8564, Japan; 3Okinawa Churashima Foundation, Okinawa 905-0206, Japan e-mail: [email protected]

Accumulation of basic knowledge on shark reproduction is essential to recover and conserve shark resources. However, detailed information on the endocrine control of their reproduction at the molecular level has not been reported. Therefore, in the present study, to clarify the mechanisms of shark gametogenesis at the molecular level, we first investigated a unique organ in the elasmobranchs, the oviducal gland, which forms eggshell during the process of egg formation. We initially made anatomical and histological observations of the oviducal gland of the cloudy catshark, Scyliorhinus torazame, used as a model fish. Anatomical observations showed that a single ovary, containing ovarian follicles at various developmental stages, is located in the centre of the body cavity, and that oviducts (oviducal funnel, oviducal gland, uterus, and urogenital pore) are located on both sides of the dorsal body cavity. Histological and histochemical analysis with periodic acid-Schiff and alcian blue staining showed that the oviducal gland consists of four different secretory zones (club, papillary, baffle, and terminal zones), suggesting that the baffle zone is responsible for the production of the egg capsule, and that other zones may be involved in the production of neutral and sulphated acid mucins. Transcriptome analysis was carried out using next-generation sequencing technology. We obtained a total of 159,335 reads for a part of the oviducal gland (baffle zone) from the cloudy catshark. De novo assembly yielded 1,741 contigs with an average length of 391 bp. On the basis of a sequence similarity search using the BLAST programme, a total of 954 (54.8%) contigs were identified as functional molecules. Of these, 16 contigs, which were classified as structural proteins such as keratin and collagen, are predicted to be involved in egg capsule formation. These results will contribute to an understanding of the molecular mechanism of eggshell formation in elasmobranchs.

Keyword: elasmobranch, oviducal gland, histology, De novo assembly

121 Poster Presentation 9‐11

The characters of final oocyte maturation in largescale mullet, Chelon macrolepis

Yueh W.S. 1*, Jeng S.R.1, Haung P.H. 1, Chiang S.D.1, Chang C.F.2,3 1 Department of Aquaculture, National Kaohsiung Marine University, Kaohsiung, Taiwan 2 Department of Aquaculture, 3 Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan e-mail: [email protected]

Largescale mullet, Chelon macrolepis, is one of the economically important aquaculture species in south of Taiwan. In farmed female fish, the spawning season is from January to February at one year old when hepatosomatic index and gonadosomatic index were increased to 2.9% and 11.8%, respectively. The full grown oocyte diameter were around 600μm which could be induced into final oocyte maturation stage (FOM) with oocyte diameters increased to 850 μm at germinal vesicle breakdown (GVBD) stage, transparent ooplasm and single clear oil droplet. During FOM, the development of the oocyte maturation could be divided into hormone-insensitive stage (insensitive to gonadotropin and maturation inducing steroid (MIS), 0-6 h after treatment), MIS-sensitive (respond to MIS, 6-12 h after treatment) and spontaneous stage (GVBD in the hormone-free condition, 18 h after treatment), respectively. On MIS-sensitive stage, in the response of various hormone in vitro studies, both 17, 20, 21-trihydorxy-4-pregnen-3-one (20S), and 17,20-dihydroxy- 4-pregnen-3-one (DHP) were the most effective steroids to induce FOM. 20S has a best potency than DHP in inducing FOM; 20S might be the MIS in largescale mullet. 17-hydroxyprogesterone and 21-hydroxy- 4-pregnen-3, 20-dione also could significantly induce oocyte maturation at higher concentrations. In contrast, high dose of E2 resulted in a decrease of GVBD while co-cultured in vitro with 20S. Fish treated with LHRH-A (D-Ala6，des-Gly10 LHRH ethylamide analog)，LHRH-A with dopamine antagonist, HCG were not effect in induce FOM even in high dose. Catfish pituitary homogenates (CPH) treatments could significantly induce FOM, but with highly fragmented oil droplets, and low fertilized successes. Only treated by CPH, MIS and dopamine antagonist together had a better oocyte quality in the fish.

Keywords: largescale mullet, final oocyte maturation, spawning

122 Poster Presentation 9‐12

Effects of the Cilostazol (a specific phosphodiesterase type Ⅲ inhibitor) on primary-secondary oocyte development in the induced Japanese eel

Zhu C.-W. 1, Lee Y.-H. 2, Chang C.-F. 3 , Huang Y.-S.1* 1Depart. Life Sci., Natl. Univ. of Kaoshiung, Taiwan. 2 Tungkang Biotech. Res. Center, TFRI., Taiwan. 3 Depart, Aquaculture, Natl. Taiwan Ocean Univ., Keelung, Taiwan. e-mail: [email protected]

The stimulatory effects of gonaotropins upon ovarian development are provoked by the intracellular cAMP production in mammals. Cilostazol (Pletal®) is a selective inhibitor of phosphodiesterase type III (PDE3, mainly located in mammalian ovarian follicle cells) and mediates its effects by increasing cellular cAMP levels. Furthermore, cilostazol-induced vasculo-angiogenesis effects have also been shown, but there is no data on its effects in fish. We address this question in the Japanese eel, since its ovarian development could be manipulated to study if cilostazol can ameliorate induced ovarian development or reinforce ovarian angiogenesis. Eels were treated with salmon pituitary extracts (SPE), cilostazol (cilo), or the combination (SPE+cilo) twice a week for 6 weeks. In the end, the ovarian tissues, gas glands, and blood of treated eels were sampled. The data showed that the GSI (gonadal index) and RMSI (gas glands index) were both stimulated by SPE while the levels of blood glucose (BG) significantly (x2.5) stimulated in the SPE+cilo group uniquely as compared with the controls; the positive effect of SPE or cilo alone on RMSI was showed, an added positive effect on RMSI was seen in the SPE+cilo group; the positive effect of SPE on GSI seemed inhibited by cilo, more interesting, cilo alone inhibited GSI significantly (x0.75) as compared with the controls. The distributions of oocyte diameters in each group were tested, cilo alone inhibited maximal oocyte diameters as compared with the controls, and the positive effect of SPE on maximal oocyte diameters was also inhibited by cilo. Our results suggested that a continuing administration of PDE3 inhibitor had inhibitory effects on the induced ovarian development in the Japanese eel, and intracellular cAMP levels seem to be temporal and spatial regulated in the eel ovarian follicles.

Keyword: Anguilla japonica, development, oocyte, phosphodiesterase inhibitor,

123 Poster Presentation 9‐13

The role of germ cell differentiation genes during testicular development and germ cell proliferation in black porgy, Acanthopagrusschlegelii

Chen C.*, Wu G.C., Chang C.F. Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan e-mail:[email protected]

Black porgy is a protandrous hermaphroditism. The ovotestis is separated by connective tissue. The sexual phase can be changed from male to female with long-term estradiol-17b (E2) treatment. After E2 termination, the testicular part would be regenerated in the ovotestis. Interestingly, the ectopic oocytes are broadly observed in the newly regenerated testicular part of ovotestis. How ectopic oocyte affected the growth and proliferation of the neighbor? We intend to demonstrate the expression pattern of early germ cells derived growth factors: gonadal soma-derived factor (gsdf) and glial cell-line derived neurotrophic factor (gdnf) in testis and in regenerated testis (with ectopic oocyte). Gsdf had a higher expression in spermatogonia A and decreased gradually within the spermatogonia differentiation. Gdnf had a higher expression in primary oocytes. These data revealed that gsdf and gdnf have an adverse function in the testicular and ovarian development, respectively. Consequently, we using recombinant Gsdf and Gdnfin vitro organ culture to demonstrate the role of gsdf and gdnf during Sertoli cells undergo autonomous transdifferentiation into follicle-like cells.

Keywords：ectopic oocyte, gsdf, gdnf

124 Poster Presentation 9‐14

The soma fate alter by the ectopic oocytes in the regenerated testis in black porgy, Acanthopagrus schlegelii

Luo J.W.＊, Wu G.C., Chang C.F. Department of Aquaculture, National Taiwan Ocean University E-mail:[email protected]

In the protandrous black porgy, the ovotestis is separated by connective tissue, and no intersex (ectopic located germ cells) characteristics are observed in either part. We generated the abnormal testicular part of the ovotestis with estradiol-17beta (E2) treatment, in which newly regenerated testis have ectopic oocytes. In the previously study, the cells surrounded the oocytes are Sertoli cells with high figla expression in the oocytes at an early stage and then switch to follicle cell with low figla expression in the oocytes at a later stage. The downstream genes of figla are not clear yet. In this study, we establish an vitro oocytes culture system to investigate the role of figla on altering the fate of soma cells. Compared with the control group, the expression levels of folliculogenesis-inducing factor (gdf9 and bmp15) no difference in the figla overexpression group. This data reveal that gdf9 and bmp15 are not the downstream gene of figla. Therefore, we performed a transcriptome analysis of these figla overexpression oocytes to find the target genes of soma cells transdifferentiation.

Key words：gdf9,bmp15,figla

125 Poster Presentation 9‐15

The interaction between brain and gonad during gonadal differentiation in black porgy, Acanthopagrus schlegelii

Fan C.H.＊, Wu G.C., Chang C.F. Department of Aquaculture, National Taiwan Ocean University E-mail:[email protected]

Black porgy is a protandrous hermaphroditic fish with bisexual gonads. During gonadal sex differentiation, the aromatase (cyp19a1b) had highly expression and the concentration of estradiol (E2) in the hypothalamus. However, the relationship between hypothalamus and gonad is still unknown. In this study, we use transcriptome to analyze the different stage of hypothalamus, including maleness (with bisexual gonads), femaleness (testis excised) and no sexuality (fish with no gonad). We obtain many genes (85 genes) have dimorphic expression in different groups. Therefore, we use these dimorphic expression genes to understand the brain sexuality during gonadal sex differentiation. According to the gonadal differentiation stage, the expression of target genes have three different patterns, including (i) lower expression after differentiation, (ii) highly expression after differentiation, and (iii) highly expression at differentiation time. Following these result, we are going to understanding the role of candidate genes of brain sexuality formation. For these reasons, we performed a E2-induced female (ovarian development) to understand the possibility of brain sexuality.

Keywords：transcriptome, E2(estradiol), aromatase

126 Poster Presentation 9‐16

The functional change of germ and soma during sex reversal in Orange-spotted grouper.

Tey W.G.*, Wu G.C., Chang C.F. Institute of Aquaculture, National Taiwan Ocean University E-mail：[email protected]

Orange-spotted grouper (Epinephelus coioides), a protogynous hermaphroditic fish, could be manipulated their sexuality by exogenous steroid treatment. Previous studies have shown that male fish can induce by androgen treatments. In addition, androgen-induced male are reversible after treatments termination. This study focuses on the germ cells and it surrounding cells functional changes and gene expression during reversible sex change after terminated of MT (methyltestosterone) and AI (aromatase inhibiter) treatments. We performed an immunohistochemistry analysis of BrdU of these regenerating ovarian tissues to find the sexual fate of BrdU negative cells. Moreover, the male-specific and female-specific markers were be selected to analyze the gene expression pattern during sex reversal (male-specific-dmrt1, sox9, cyp11b; female-specific -figla, foxl2, cyp19a1a).Fish showed male function(spermatids) after three month treatments with MT and AI at the same period. Sex reversal was initiated after terminated treatment in first month. However, the female function (primary oocyte) presents one month earlier in AI terminated group (forth month) than MT terminated group (fifth month). Regard to gene analysis with Q-PCR, the expression levels of male-related genes was coincident with male gonad development. Male-related genes were reduced following degradation of male gonad development. To further understand the processes, this study will continue analyze the changes in germ cells and it surrounding cells during sex reversal by immunohistochemistry staining.

Key words: Sex reversal, Cellular function conversion, Protogynous

127 Poster Presentation 10‐1

Identification of molecular signatures related to ischemic heart failure by gene expression analysis on human myocardial autopsy materials

Kim J.Y.1*, Kim Y.1, Chung S.1, Jung H.2, Park S.H.1, Son G.H.1 1Department of Legal Medicine, College of Medicine, Korea University, Seoul 136-705, KOREA; 2Department of Anatomy, College of Medicine, Yonsei University, Seoul 120-749, KOREA; e-mail : [email protected]

Myocardial ischemia mainly caused by coronary artery disease is the most common type of heart disease and the leading cause of sudden cardiac death (SCD). However little is known about the molecular signatures, particularly related to the severity of myocardial damage and susceptibility to fatal heart dysfunction. In the present study, we analyzed genome-wide expression profiles in human myocardial autopsy tissues from subjects with ischemic heart disease without recent infarction (IHD) and acute myocardial infarction (AMI) in comparison with their controls with non-cardiac cause of death. A group of genes under the control of insulin receptors (INSRs), and targets of several nuclear receptors such as estrogen-related receptors (ERRs) and peroxisome proliferator-activated receptors (PPARs) were extensively and concurrently reduced in their mRNA expression in the AMI cases. On the other hand, downstream genes of nuclear receptor subfamily 3, group C, member 2 (NR3C2) receptor signaling were suggested to be enhanced in an AMI-specific manner. In contrast, mRNA expression pattern in the IHD subjects showed modest alterations in several genes implicated in impaired heart contractibility and increased susceptibility to myocardial cell death. Genes involved in the regulation of lipid metabolism and energy production commonly changed in both cases. In conclusion, the present study revealed key molecular features underlying two distinct modes of sudden cardiac death associated with myocardial ischemia.

Keyword : ischemic heart failure, myocardial ischemia, fatal heart dysfunction, receptor signaling, myocardial cell death

128 Poster Presentation 10‐2

Cell death-associated 28S ribosomal rna cleavage in postmortem tissues

Kim Y.*, Kim J.Y., Kim J.W., Hwang J.J., Park S.H., Son G.H. Department of Legal Medicine, College of Medicine, Korea University, Seoul 136-705, KOREA e-mail: [email protected]

It is well established that a subset of RNA species including 28S ribosomal RNA (rRNA) is specifically cleaved into distinct fragments during acepoptotic cell death. D8 variable domain of mammalian 28S rRNA is considered to be the most susceptible to the cell death-associated cleavage. Interestingly, the cleavage site in the D8 domain is well conserved among different species although this domain is one of the most divergent domains of 28S rRNA, suggesting its functional relevance to turn translation off promptly. Since activation of apoptotic pathway is widely observed during the early postmortem period, the present study intended to establish the postmortem profiles of 28S rRNA degradation. As expected, the D8 domain containing region of 28S rRNA was rapidly removed in all of examined mouse tissues in parallel with overall postmortem RNA degradation. By contrast, the 5’-end of 28S rRNA was remarkably stable during postmortem RNA degradation, presumably due to their primary sequences as well as structural features. Indeed, the stable 5’-end region of eukaryotic 28S rRNA is known to form tight stem-loop structures and then associate with 5.8S rRNA. Importantly, the differences in degradation rates between the 5’-end and D8 domains were highly proportional to postmortem interval (PMI) with a linear correlation even up to ~4 days in both mouse and human tissues; the slopes were distinct among tissues depending on their ribonuclease contents. In conclusion, we demonstrate that cell death-associated cleavage patterns of 28s rRNA in relation to the degradation-resistant domain serves as a simple, but reliable indication for the postmortem RNA integrity, which is potentially applicable for the forensic estimation of PMI as well as the postmortem gene expression profiling.

Keyword: Cell death, Ribosomal RNA cleavage, D8 domain, Postmortem interval

129 Author Index

Ahn C. 87 Chen T.Y. 56 Feng B.Y. 85 Alimuddin 63 Chen Y.C. 83 Feng R. 113 Amano H. 103 Chen Y.F. 81 Fukuda Y. 89 Andriyanto 73 Chen Y.-J. 32 Ge W. 31 Aruna A. 50, 60, 96, 101 Cheng L.C. 111 Gong H.Y. 68 Astuti P. 73 Cheng S.C. 120 Gueguen M.M. 15 Bae S-H. 57 Cheon S. 49 Guh Y.J. 94 Balasubramanian C. P. 69 Chiang S.D. 122 Gwo J.C. 81 Bao C.C. 85 Chiewchanchai M. 93 Hamid N.K.A. 24 Belsham D.D. 89 Chin H.Y. 68 Han K.-N. 90 Bhattacharya S. 5 Chiu H.C. 112, 114 Handarini R. 73 Boediono A. 73 Chiu Y.L. 105 Hara S. 100 Cano-Nicolau J. 15 Cho E.B. 58, 98 Haraguchi S. 25, 100 Chang C.F. 17, 41, Chou C.H. 112, 114 Harikumar K.G. 22 50, 53, Chow B.K. 22 Hasunuma I. 89 54, 60, Chu J.Y. 22 Hattori R.S. 119 67, 79, Chun S.K. 47 Haung P.H. 122 81, 96, Chung A.Y. 18 Hayashi D. 65 99, 101, Chung B.C. 2 Hazarika J.K. 51 102, 104, Chung H.Y. 61 He F. 59 105, 108, Chung S. 45, 47, He M. 12 122-127 128 Higuchi M. 27 Chang C.T. 61 Chung Y.J. 41, 104 Hong L. 71 Chang Y.S. 112, 114 Dahms H.-U. 36, 90, Hong W. 71 Chang Y.-S. 32 95 Hou G.J. 66 Charoenphandhu N. 34 Deng S.P. 74, 118 Hsieh Y.J. 46 Chaudhuri M.K. 75 Diotel, N. 15 Hu C.H. 61 Chen C. 124 Donald J.A. 24 Hu G. 12 Chen C.H. 112 Dong R. 113 Hu H.J. 97 Chen C.J. 41, 53 Dufour S. 11, 54, Hu M.Y. 83 Chen H. 70, 77 102, 108 Hu W. 13 Chen H.l. 66 Fan C.H. 126 Huang S.C. 68 Chen J.H. 114 Fan-Chiang Y.C. 108 Huang W.-S. 32 Chen L. 80 Fang L. 113 Huang W.T. 112, 114 Chen S. 71, 80 Fatimah R. 62 Huang Y.S. 81, 123

130 Hur S.P. 37 Kim E. 18 Li W. 76 58, 88, Kim H.-D. 45 Li X. 66 Hwang J.I. 109 Kim J.-H. 36, 90 Li Y. 80 Hwang J-.I. 87, 98 Kim J.W. 129 Liang C.C. 111 Hwang J.J. 129 Kim J.Y. 128, 129 Limjunyawong N. 72 40, 83, 6, 45, Lin C.H. 97 Hwang P.P 94, 97 Kim K. 47, 49 Lin C.J. 108 Hyodo S. 115, 121 Kim S. 18 8, 70, Ikegami T. 78 Kim Y. 128, 129 Lin H. 77 Iwai T. 110 Kim Y.J. 39 Lin L.-J. 32 Izumida D. 23 Kishida M. 52, 62, Lin M. 77 James N. 69 86 Liou J.Y. 41 Jang D. 29 Kitana J. 82, Liu X. 8, 70, Jang J. 47 91-93 77 Jannat M. 86 82, Lu J.K. 56 Jantawongsri K. 91 Kitana N. 91-93 Lu W. 38 Jaroenporn S. 72 Kittivanichkul D. 34, 64 Luo J.W. 125 Jeng S.R. 54, 79, Ko W.K.W. 12 Maehiro S. 48 102, 122 Kobayashi M. 107 Maheshwari H. 73 Jeong I. 18 Kobayashi Y. 14 Maitra S.K. 7 Jia J. 76 Kodera Y. 103 Maitrayee P. 75 Jiang X. 117 Kondo F. 110 Malaivijitnond S. 34, 64, Jiang Y.l. 66 Kong Z.L. 111, 120 72 Joy K. P. 35 Kuo S.F. 79 Maneein R. 82, 93 Jung H. 43, 128 Lafont A.G. 11 Matsuda K. 28 Kagawa H. 115, 121 Lau E.L. 67 Matsumoto M. 121 Kah O. 15, 102 Lee C. 87 Maugars G. 11 Kaifu K. 30 Lee C.-Y. 32 McLeod J.L. 24 Kaiya H. 30 Lee H.B. 109 Meesawat S. 93 Kaneko T. 106 Lee J.R. 83 Miller L.J. 22 Kang B.J. 57 Lee L.T. 22 Mita M. 25 Kang H.Y. 21 Lee M.F. 67 Miura C. 27, 65, Kang Y.R. 112 Lee T.L. 112, 114 110 Kawamura M. 103 Lee Y.H. 41, 123 27, 65, Khonsue W. 82, 93 Li G. 80 Miura T. 110 Kim D. 18 Li G.L. 74, 118 Mizusawa K. 14 Kim D.K. 58 Li M. 29 Moon M.J. 58, 87, Kim D-.K. 87 Li S. 8, 77 98

131 Moriyama S. 103 Ponniah A.G. 69 Strüssmann C.A. 119 Nagahama Y. 4, 117 Priyajit C. 75 Su H.M. 46 Nagaoka K. 72 Qi X. 8 Su H.T. 61 Nagarajan G. 50, 60, Qin C. 76 Subeer M. 75 96, 101 Rafiuddin M.A. 63 Subramoniam T. 69 Nakamachi T. 28 Rousseau K. 11 Sudrajat, Agus 63 Nakamura M. 23, 121 Roy P. 55 Oman Nalley W.M. 73 Saikrithi P. 69 Sugiyono 52 Narihiro M. 89 Samir B. 75 Suh Y.G. 47 Narita Y. 106 Sandip M. 75 Suharsono 63 Nasri H. 15 Sang Q. 70 Sun C. 76 Ng S.Y. 22 Sarida M. 119 Sun L. 29, 117 Nikhil K. 55 Satake H. 33 Sun S.F. 79 Nishitani C. 72 Sato K. 121 Sun W. 19 Ogawa S. 16 Sekar R. 22 Sun Y. 113 Oh S-.H. 87 Seong J.Y. 20, 58, Sung P.H. 83 Ohtani-Kaneko R. 106 87, 88, Sushmita B. 75 Oka Y. 48 98, 109 Tai T.Y. 68 Okada N. 106 Shao Z.F. 41, 99, Takahashi A. 14 Okubo K. 48 104 Takei Y. 1, 24 Okutsu T. 57 Sharan S. 55 Takemura A. 37, 78 Otta S. K. 69 Shi H. 116 Takeshige Y. 25 Oyama Y. 78 Shibata H. 28 Takeuchi, Y. 37 Pan H.H. 99 Shikina S. 41, 99, Tang P.C. 114 Parhar I.S. 3, 42 104, 105 Tao W. 29 Pariyanonth P. 93 Shim D.M.-C. 95 Tey W.G. 127 Park C.R. 58, 87, Shinohara Y. 107 Thammachoti P. 82 98, 109 Shinya S. 53 Thammachoti P. 93 Park H.C. 18 Si Y. 59 Thitiphuree T. 82, 93 Park N. 49 Sjahfirdi L. 73 Tien Y.-C. 32 Park S. 58, 88 Soga T. 42 Ting T.L. 120 Park S.H. 128, 129 Soma S. 75 Tiyasatkulkovit W. 34 Pasquier J. 11 Son G.H. 45, 47, Tomy S. 69 Pellegrini E. 15 129 Tseng H.P. 41, 61 Pen Y.T. 54 Son Y.L. 89 Tseng P.C. 68 Peter M.C. 26 Song J.X. 56 Tseng Y.C. 83, 94 Pewphong R. 92 Soumik A. 75 Tsukamoto K. 30 Phumsatitpong C. 72 Soyano K. 23 Tsutiya A. 106

132 Tsutsui K. 9, 25, Yoshihara Y. 65 89, 100 Yoshimura T. 44 Tzou W.S. 61 You D.J. 109 Ubuka T. 89, 100 You D-.J. 87 Uchida K. 103, 115, Yu X. 116 121 Yueh W.S. 54, 79, Uchiyama M. 84 102, 122 Urasopon N. 34 Yulnawati 73 Vaillant C. 15 Yun S. 98 Varanusupakul P. 82 Zhang H. 70 Wang B. 76 Zhang J. 66 Wang D. 29, 113, Zhang M.Z. 118 116, 117 Zhang Y. 8, 70, Wang l. 66 77, 113, Wang L. 95 119 Watanabe G. 72 Zhou L. 29, 116, Watanabe M. 25 117 Watanabe T. 115, 121 Zhu C. 80 Wen H. 59 Zhu C.H. 74, 118 Widyastuti U. 63 Zhu C.W. 123 Wilder M.N. 57 Zhu W. 71 Wong A.O.L. 12 Zhu Z.Y. 13 Wu Bo 74 Wu G.C. 17, 124-127 Wu L. 116 Wu L.T. 112 Xie L. 116 Xie Q. 117 Xie X. 113 Yada T. 30 Yamamoto K. 89 Yamamoto Y. 119 Yang C.Y. 94 Yang J. 95 Yang S. 113, 116 Yao K. 31 Ye H.H. 85

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