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10384 22420091151167 Studies of melatonin in the mudskipper Boleophthalmus pectinirostris

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Contents Contens Chinese Abstract...1 English Abstract...3 Chapter 1 General Introduction...5 1 Advances in the study on Boleophthalmus pectinirostris...5 1.1 Biological characteristics and habits of Boleophthalmus pectinirostris...5 1.2 Semilunar periodicity spawning phenomenon of Boleophthalmus pectinirostris...7 1.3 Advances in the study on pineal gland of Boleophthalmus pectinirostris...8 2 Advances in the study on melatonin...9 2.1 The Physicochemical property of melatonin...9 2.2 Synthesis and release of melatonin...10 2.3 The effects of melatonin on reproduction in fish...12 3 Advances in the study on sex steroids...14 3.1 Biosynthesis tissue of sex steroids...14 3.2 The effects of sex steroids...14 4 Objectives and significance of this study...16 Chapter 2 Semilunar and circadian rhythms of serum melatonin levels in Boleophthalmus pectinirostris...18 1 Materials and methods...19 1.1 Semilunar rhythm of serum melatonin levels in Boleophthalmus pectinirostris...19 1.2 Circadian variations of serum melatonin levels in Boleophthalmus pectinirostris...21 III

Contents 1.3 Effects of light intensities and tide on serum melatonin levels in Boleophthalmus pectinirostris...21 1.4 Effect of phase-shifted light cycles on serum melatonin levels in Boleophthalmus pectinirostris...23 2 Results...23 2.1 Semilunar rhythm of serum melatonin levels in Boleophthalmus pectinirostris...23 2.2 Circadian rhythm of serum melatonin levels in Boleophthalmus pectinirostris...24 2.3 Effects of light intensities and tide on serum melatonin levels in Boleophthalmus pectinirostris...25 2.4 Effects of phase-shifted light cycles on serum melatonin levels in Boleophthalmus pectinirostris...26 3 Discussion...27 Chapter 3 Effects of melatonin on the release of gonadal sex steroids in Boleophthalmus pectinirostris...30 1 Materials and methods...31 1.1 Effects of melatonin on the in vitro release of gonadal sex steroids from the gonad of Boleophthalmus pectinirostris...31 1.2 Effects of melatonin injection on the release of gonadal sex steroids in Boleophthalmus pectinirostris...34 1.3 Effects of melatonin injection at different doses on 17α,20β-DHP release in female Boleophthalmus pectinirostris...35 2 Results...36 2.1 Effects of melatonin on the in vitro release of gonadal sex steroids from the gonad of Boleophthalmus pectinirostris...36 2.2 Effects of melatonin injection on the release of gonadal sex steroids in Boleophthalmus pectinirostris...39 IV

Contents 2.3 Effects of melatonin injection at different doses on 17α,20β-DHP release in female Boleophthalmus pectinirostris...42 3 Discussion...43 Abbreviations and symbols...46 Conclusions and prospects...48 Characteristics and innovation...50 References...51 Publications during study...60 Research grants...60 Acknowledgements...61 V

Boleophthalmus pectinirostris 1. 1 2 1 1 2. 100pg/mL 17α,20β-DHPp<0.01 E 2 11-KT 17α,20β-DHP 50ng/g body wt1h 17α,20β-DHP p<0.05 1

17α,20β-DHP 4h E 2 11-KT17α,20β-DHP 17α,20β-DHP 17α,20β-DHP --Hypothalamus-Pituitary-Gonad 17α,20β-DHP 17α,20β-DHP 2

Abstract Abstract Mudskipper (Boleophthalmus pectinirostris) is one of the major aquaculture species in the southeast coast of China. Our previous study found that mudskipper is a restricted lunar-synchronized spawner, during the spawning season it spawns at an interval of about 15 days, which synchronizes with the semilunar periodicity. The periodicity of animal spawning is a physiological rhythm, this rhythm is mainly affected by environmental factors. The environment factors are mainly perceived by certain sensor, such as pineal gland. Melatonin synthesis and release of the pineal gland are obvious rhythmic. Melatonin is an important transmitter of environmental cues, notably photic information,in vertebrates and it involves in the synchronization of many rhythmic biological functions such as growth, development, sexual maturation, reproduction, aging, and cellular immunity through the photoneuroendocrine transduction of daily and annual photoperiodic signals. In order to understand the mechanism of spawning with a semilunar periodicity in the muddskipper, the serum melatonin levels were measured during the semilunar periodicity and normal day-night cycle. In addition, the effects of melatonin on the realease of gonadal sex steroids were also investigated (in vivo and in vitro) by using methods of ELISA, Intraperitoneal injection and static incubation. The main results and conclusions are as follows: 1. Variation in the production of the serum melatonin was investigated in the mudskipper within the two complete semilunar cycles and the LD cycles during the spawning season. The effects of light intensities, tide and phase-shifted light cycles on serum melatonin levels were also investigated by using method of ELISA. The results from the present study showed that in the mudskipper the serum concentrations of melatonin appeared to have a cyclic pattern associated with semilunar periodicity. The melatonin level exhibited two cycles, each cycle with a peak. The peaks of melatonin production coincide with the timing of spawning in this species in Funing Bay, the 3

Abstract first peak of serum melatonin was observed around the last quarter moon, and the second one around the first quarter moon. Our results also showed that serum melatonin levels exhibited a daily variation. Melatonin levels during the dark phase were significantly (p<0.05) higher than those during the light phase, and it is suggested that this profile should be controlled by internal clocks. 2. The in vitro experiment showed that, the low concentration melatonin (100pg/mL) elicited a significant increase in culture medium 17α,20β-DHP levels p<0.01. Both the low and high melatonin concentrations have no effects on the release of E 2 from the ovary, and 11-KT and 17α,20β-DHP from the testis. The results from the in vivo experiment agreed with the in vitro experiment. Intraperitoneal administration of melatonin (50ng/g body wt) significantly increased the serum 17α,20β-DHP levels (p<0.05), 1h post injection. Thereafter, the serum 17α,20β-DHP levels gradually decreased to the base content, 4h post injection. In conclusion, the information gathered in the present study promotes the idea that the mudskipper can perceive the cues from the moon by the pineal gland, and translate the environmental signals into hormone information (melatonin). Then the melatonin act on the HPG axis or directly act on the ovary, to modulate the release of 17α,20β-DHP, which can make the mudskipper spawning at the certain lunar phase by inducing the final maturation of oocyte. Keywords: Boleophthalmus pectinirostris; melatonin; sex steroids; semilular periodity 4

1 1.1 Boleophthalmus pectinirostrischordata VertebrateOcteichthyesTeleostomi PerciformesGobioideiOxudercinae Boleophthalmus 1020cm 2050g 2 3 1 2 1 3 2 5 6 1 5 3 2 2 3 1.1 [1,2] 5

12 3 5~9 5~7 15d 6d [3] 16 8 [4] [5] 20 60 1970 1980 20 80 1990 13000 1.1 Fig. 1.1 Boleophthalmus pectinirostris 6

1.2 Semilunar Cycle15d Lunar Cycle30d Siganus guttatussiganus canaliculatuslates calcarifer [6-8] Fundulus grandispomacentrus taeniometopon Apogon amboinensis [9-11] [8] Hong 2007 1 2 15d 1 2 [12] Wang 20082006 5 31 6 30 (E 2 )17α-17α-PT T 11-11-KT 1 2 1 3 1 3d 2 4d 2 1 3d 2 [13] 7

[14,15] Apogon amboinensis [10] 1.3 Pineal Gland Pineal Complex [16] [17-19] Dodt1963 Oncorhynchus mykiss [20] [21] Paraphysis [22] [23,24] Van 1980 Gasterosteus aculeatus Photoreceptor Cell 8

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