ホシガレイの卵母細胞の最終成熟過程と排卵周期

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1 ホシガレイの卵母細胞の最終成熟過程と排卵周期 誌名 水産増殖 = The aquiculture ISSN 著者 巻 / 号 澤口, 小有美大久保, 信幸有瀧, 真人ほか2 名, 54 巻 4 号 掲載ページ p 発行年月 2006 年 12 月 農林水産省農林水産技術会議事務局筑波産学連携支援センター Tsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research Council Secretariat

2 Aquaculture Science Process of Final Oocyte Maturation and Ovulation Cycle in Captive Spotted Halibut, Verasper variegatus Sayumi SAWAGUCHI 1, Nobuyuki OHKUBO 1, Masato ARITAKI 2, Kengo OHTA 3 and Takahiro MATSUBARA 1 Abstract: Acquisition of high-quality eggs showing high fertility is essential for the success of artificial production of juvenile spotted halibut Verasper variegatus. The objective of this study is to clarify ovulation cycles and time course changes of maturating oocytes of this species for improvement of the method of egg collection. By cannulating a fragment of ovigerous lamellae using a syringe connected to a polyvinyl tube inserted into the ovary from the genital pore, oocytes with follicle layers and eggs were obtained. The oocytes and eggs at different maturational phases were categorized into six maturational stages based on their morphological appearances. With the progression of the maturational stages, the transparency of oocytes increased due to yolk granule fusion and the oocytes hydrated through marked water influx. Time course changes in the maturational stages based on daily cannulation of the same individuals demonstrated that oocyte maturation was completed within five or six days and ovulation occurred at cycles of three days. Results from comparison of fertility of eggs from females with or without removing formerly ovulated eggs coupled with the ovulation cycle suggested the effectiveness of periodical removal of deposited overripe eggs to improve the method of collecting eggs for artificial fertilization in spotted halibut. Key words: Spotted halibut; Ovulation cycle; Oocyte maturation; Egg quality Verasper variegatus 60 cm 6 kg 1984; 1998 Paralichthys olivaceus ,000 /kg 1999 Verasper moseri Hokkaido National Fisheries Research Institute, Kushiro, Hokkaido , Japan 2 Miyako Station of the National Center for Stock Enhancement, Miyako, Iwate , Japan 3 Hakatajima Station of the National Center for Stock Enhancement, Imabari, Ehime , Japan

3 group-synchronous oocyte development Koya et al Matsubara and Koya Koya et al t kg 3.37 kg kg 0.83 kg cm 30 cm 10 ml 1 ml 4 Hirano et al Matsubara and Koya mg = 100 / ml l

4 1 g 0.1 ml l Fig Fig. 1-Stage I Matsubara and Koya 1997 Stage II Stage III Stage IV Stage V OV 6 Stage I : Stage II : Stage I Fig. 1. Changes in the appearance of oocytes during oocyte maturation in spotted halibut. Photomicrographs of oocytes at different maturational stages and an ovulated egg are shown. Stage I: Post vitellogenic stage (I), Stage II: (II), Stage III: (III), Stage IV: (IV), Stage V: (V), Stage OV: Ovulated egg (OV). Bar = 1 mm.

5 Stage III : Stage IV : Stage V : Stage OV : Fig. 2 Stage I 0.96 mm Stage III Stage III Stage IV 1.63 mm Fig. 2A Stage I III 0.54 mg 0.80 mg Stage IV 2.04 mg 2.53 mg Fig. 2B 0.2 mg Fig. 2B 90 Fig. 2C Fig No. 1 No. 3 1 Stage I Stage III Stage III 2 Stage IV 3 Stage V 5 2 Stage I Stage II Stage II Stage V 3 Stage II 4 Fig. 2. Time course of changes in oocyte and egg diameter (A), wet and dry weight (B), and water percentage (C) during oocyte maturation of spotted halibut. Each maturational stage corresponds to those illustrated in Figure 1. Different letters indicate significant differences (p 0.05) from each other.

6 Fig. 4A Fig. 4B Matsubara and Koya Fig. 3. Patterns in daily progressions of maturational stages of oocytes and eggs cannulated from five females of spotted halibut. Each maturational stage corresponds to the stages illustrated in Figure 1. Open arrowheads indicate ovulation. Fig. 4. Comparison of eggs from females with or without removing formerly ovulated eggs. A: percentages of fertility, B: percentages of hatchability. Int: intact group, Exp: experimental group. Females in experimental group received periodical removal of eggs. Different letters indicate significant differences (p 0.05) from each other.

7 Koya et al Matsubara and Koya Stage I 1 2 Stage V Stage II 2 Matsubara and Koya 1997 Fig. 5. Schematic illustration demonstrating a possible mechanism in the deterioration of egg quality in spotted halibut. Egg fertility decreases due to the deposition of overripe eggs in females which repeatedly ovulate but do not spontaneously spawned females. Stage I Stage I in vitro 1 17, 20 dihydroxy-4-pregnen-3-one DHP: see Nagahama et al Stage II Kayaba et al Fig t 5 5 Koya et al

8 in vitro Koya et al Matsubara et al Matsubara et al Carnevali et al Matsubara et al LaFleur et al Carnevali, O., F. Centonze, S. Brooks, I. Marota and J. P. Sumpter (1999) Molecular cloning and expression of ovarian cathepsin D in seabrem, Sparus aurata. Biol. Reprod., 61, Hirano, T., D. W. Johnson and H. A. Bern (1971) Control of water movement in flounder urinary bladder by prolactin. Nature, 230, Kayaba, T., T. Sugimoto, T. Mori, N. Satoh, S. Adachi and K. Yamauchi (2003) Induced spontaneous spawning using an increased temperature stimulus in the cultured barfin flounder Verasper moseri. Fish. Sci., 69, Koya, Y., T. Matsubara and T. Nakagawa (1994) Efficient artificial fertilization method based on the ovulation cycle in barfin flounder Verasper moseri. Fish. Sci., 60, LaFleur, G. J., Jr, D. Raldua, M. Fabra, O. Carnevali, N. Denslow, R. A. Wallace and J. Cerda (2005) Derivation of major yolk proteins from parental vitellogenins and alternative processing during oocyte maturation in Fundulus heteroclitus. Biol. Reprod., 73, Matsubara, T., A. Hara and K. Takano (1985) Immunochemical identification and purification of coelomic fluid-specific protein in chum salmon (Oncorhynchus keta). Comp. Biochem. Physiol., 81B, Matsubara, T., A. Hara and K. Takano (1995) Cationic constituents, protein concentration, and osmolality of coelomic fluid in Salmonid fish. Fish. Sci., 61, Matsubara, T. and Y. Koya (1997) Course of proteolytic cleavage in three classes of yolk proteins during oocyte maturation in barfin flounder Verasper moseri, a marine teleost

9 spawning pelagic eggs. J. Exp. Zool., 278, Matsubara, T., M. Nagae, N. Ohkubo, T. Andoh, S. Sawaguchi, N. Hiramatsu, C. V. Sullivan and A. Hara (2003) Multiple vitellogenins and their unique roles in marine teleosts. Fish Physiol. Biochem., 28, pp Nagahama, Y., M. Yoshikuni, M. Yamashita, T. Tokumoto and Y. Katsu (1995) Regulation of oocyte growth and maturation in fish. Curr. Top. Develop. Biol., 30, pp Oncorhynchus keta