Expression of biologically active red seabream gonadotropins in a baculovirus system

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 16580163
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: General fisheries
• Research Institution: National Research Institute of Aquaculture, Fisheries Agency
• Principal Investigator: GEN Koichiro National Research Institute of Aquaculture Fisheries Agency, Aquaculture Biology Division, Senior Researcher, 養殖研究所・生産技術部, 主任研究員 (80372051)
• Co-Investigator(Kenkyū-buntansha): KAGAWA Hirohiko Miyazaki University, Department of Biological Production and Environmental Science, Professor, 農学部生物環境科学科, 教授 (60169381)
• Project Period (FY): 2004 – 2005
• Keywords: gonadotropins / baculovirus expression system / recombinant protein / red seabream / steroidogenesis

Research Abstract

Recent advances in recombinant gene technology have enabled the production of biologically active fish gonadotropins (GTHs : FSH and LH). However, an efficient expression method for the production of GTHs has not yet been developed. In this study, we attempted to produce red seabream GTHs in silkworm larvae using two different baculovirus expression systems. To synthesize recombinant red seabream heterodimeric GTHs, two strategies were employed. The first utilized a dual vector expression strategy, in which each GTH subunit (α, FSHβ and LHβ) was expressed from a separate comparative vector in a single host. The second method used a bicistronic expression strategy, in which both GTH subunit cDNAs were expressed from the vector. Either a dual or a bicistronic expression vector inoculated into silkworm larvae of the 5th instar early stage. Five days after inoculation, the hemolymph of the larvae containing recombinant FSH and LH was collected, and examined of biochemical property and biological activity. Western blot analysis revealed that by using a dual vector expression system, each subunit of FSH and LH was successfully synthesized and secreted into the hemolymph, allowing the synthesis of heterodimeric molecules. In the case of the bicistronic vector coexpressing GTH subunits, the yields of FSHα and LHα subunits was very low, whereas those of both FSHβ and LHβ subunits were high. The biological activities of GTHs produced by a dual vector system were comparable to those of native GTHs in in vitro steroid hormone production. Both recombinant FSH and LH stimulated the in vitro 11-ketotestosterone production in sliced testis of red seabream. Furthermore, recombinant LH stimulated the in vitro production of estradiol-17β by ovarian follicles in a concentration-dependent manner. Conversely, GTHs derived from a bicistronic vector system had no effect on in vitro steroid hormone production.

Research Products

• [Journal Article] Influence of estradiol-17β, testosterone, and 11-ketotestosterone on testicular development, serum steroid hormone, and gonadotropin secretion in male red seabream Pagrus maior. (2006)
  • Author(s): Yamaguchi, S., Gen, K., Okuzawa, K., Matsuyama, M., Kagawa, H.
  • Journal Title: Fisheries Science 72 Pages: 835-845
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Influence of estradiol-17β, testosterone, and 11-ketotestosterone on testicular development, serum steroid hormone, and gonadotropin secretion in male red seabream Pagrus major. (2006)
  • Author(s): Yamaguchi, S., Gen, K., Okuzawa, K., Matsuyama, M., Kagawa, H.
  • Journal Title: Fisheries Science 72 Pages: 835-845
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Effects of 11-ketotestosterone and gonadotropin-releasing hormone on follicle-stimulating hormone and luteinizing hormone gene expression in castrated and sham-operated male red seabream Pagrus major. (2005)
  • Author(s): Yamaguchi, S., Gen, K., Okuzawa, K., Kumakura, N., Matsuyama, M., Kagawa, H
  • Journal Title: Fisheries Science 71 Pages: 1049-1058
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Expression of gonadotropin subunit genes following 4-nonylphenol exposure in masu salmon : Effects on transcript levels and promoter activities via estrogen receptor alpha. (2005)
  • Author(s): Maeng, S., Jung, Y., Choi, E., Jeon, J-K., Kim, S., Gen, K., Sohn, YC.
  • Journal Title: Comparative Biochemistry and Physiology Part B 142 Pages: 383-390
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Effects of 11-ketotestosterone and gonadotropin-releasing hormone on follicle-stimulating hormone and luteinizing hormone gene expression in castrated and sham-operated male red seabream Pagrus major. (2005)
  • Author(s): Yamaguchi, S., Gen, K., Okuzawa, K., Kumakura, N., Matsuyama, M., Kagawa, H.
  • Journal Title: Fisheries Science 71 Pages: 1049-1058
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Physiological roles of FSH and LH in red seabream, Pagrus major. (2004)
  • Author(s): Gen, K., Yamaguchi, S., Okuzawa, K., Kumakura, N., Tanaka, H., Kagawa, H.
  • Journal Title: Fish Physiology and Biochemistry 28 Pages: 77-80
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Regulation of gonadotropin subunit genes expression by 11-ketotestosterone during early spermatogenesis in male red seabream, Pagrus major. (2004)
  • Author(s): Yamaguchi, S., Gen, K., Okuzawa, K., Kumakura, N., Kagawa, H., Matsuyama, M.
  • Journal Title: Fish Physiology and Biochemistry 28 Pages: 111-112
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Effect of gonadotropin-releasing hormone on pituitary-ovarian axis of one-year old pre-pubertal red seabream. (2004)
  • Author(s): Kumakura, N., Okuzawa, K., Gen, K., Yamaguchi, S., Lim, BS., Kagawa, H.
  • Journal Title: General and Comparative Endocrinology, 138 Pages: 105-112
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Silicone implants for delivery of estradiol-17β and 11-ketotestosterone to red seabream, Pagrus major. (2004)
  • Author(s): Yamaguchi, S., Kagawa, H., Gen, K., Okuzawa, K., Matsuyama, M.
  • Journal Title: Aquaculture 239 Pages: 489-496
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] 卵や精子の形成に働くホルモン-魚類の生殖腺刺激ホルモンの生合成機構 (2004)
  • Author(s): 玄 浩一郎
  • Journal Title: NIASアグリバイオサイエンスシリーズ 1 Pages: 28-31
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Effects of gonadotropin-releasing hormone agonist and testosterone on gonadotropin subunit gene expression in female red seabream (Pagrus major). (2004)
  • Author(s): Kumakura, N., Okuzawa, K., Yamaguchi, S., Gen, K., Lim, BS., Kagawa, H.
  • Journal Title: Fish Physiology and Biochemistry 28 Pages: 99-100
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Effect of gonadotropin-releasing hormone on pituitary-ovarian axis of one-year old pre-pubertal red seabream. (2004)
  • Author(s): Kumakura, N., Okuzawa, K., Gen, K., Yamaguchi, S., Lim, BS., Kagawa, H.
  • Journal Title: General and Comparative Endocrinology 138 Pages: 105-112
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] 魚類の生殖内分泌系における作用機構 : 環境ホルモン (2006)
  • Author(s): 香川浩彦, 奥澤公一, 玄 清一郎, 松山倫也
  • Total Pages: 124-142
  • Publisher: 恒星社厚生閣
  • Description: 「研究成果報告書概要(和文)」より