|Abstract: ||摘要 在生殖調控的途徑中，下視丘分泌促性腺素釋放激素 (GnRH) 及促性腺激素抑制因子 (GnIF)，調控腦下垂體促性腺激素細胞合成及釋放GtHs，而促性腺素釋放激素受體 (gonadotropin-releasing hormone receptor, GnRH-R) 是針對GnRH反應的受器，在腦垂腺前葉中特異地表現在腦下垂體細胞 (gonadotropes) 的細胞膜上，GnRH與腦下垂體細胞表面的GnRH-R產生專一且高親和力的結合以開啟細胞內訊息傳導路徑，活化促性腺激素的生合成與釋出。 本研究之目的在於探討點帶石斑魚 Epinephelus coioides 促性腺素釋放激素受體 (GnRH-R) 在下視丘-腦下垂體-生殖線主軸中參與生殖內分泌的調控機制。利用RT-PCR 及 RACE 的方式，成功從腦下垂體中選殖出點帶石斑魚 GnRH receptor type I與GtH Iβ subunit全長以及GtH IIβ subunit之部分序列，所獲得之GnRH receptor cDNA 全長為1607 bp，內含轉譯讀窗 (open reading frame) 為1092 bp，可轉譯為364 個胺基酸，序列中具有7個穿透膜區域、N端及C端結構。GtH Iβ subunit (FSHβ subunit) cDNA全長為551 bp，內含轉譯讀窗為360 bp，可轉譯為120個胺基酸，序列中具有11個高度保守性cysteine位置。GtH IIβ subunit (LHβ subunit) cDNA部分序列為276 bp，可轉譯為92個胺基酸，序列中具有10個高度保守性cysteine位置及1個醣基結合位 (N-linked glycosylation site)。組織分佈方面，GnRH receptor type I 在腦下垂體的表現量遠高於其他組織，GtH Iβ subunit及GtH IIβ subunit基因其專一性的表現在腦下垂體。在活體注射不同型態GnRH處理，結果顯示，注射salmon GnRH會刺激GnRH-R type I、GtH Iβ subunit及GtH IIβ subunit 基因的表現。注射chicken GnRH II後，GnRH-R type I的表現量受到抑制，但是會刺激GtH Iβ subunit及GtH IIβ subunit基因的表現，推測可能與其他不同型態的GnRH-R結合。注射seabream GnRH後，會抑制GnRH-R type I、GtH Iβ subunit及GtH IIβ subunit基因的表現，推測尚未掌握到處理的劑量。本研究發現注射不同型態的GnRH後，會誘導GnRH-R基因的表現，而調控GtH Iβ subunit及GtH IIβ subunit基因的表現。|
Abstract In the regulation of the reproductive process in teleosts, the gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibiting factor (GnIF, known to be dopamine) are involved in the regulation of the synthesis and release of gonadotropin (GtHs) from the gonadotrophs in adenohypophysis. Furthermore, GnRH can activate multiple signal transduction pathways through binding to specific high affinity membrane GnRH receptors (GnRH-R) on the cell surface of pituitary gonadotropes. The objectives of this study were to investigate the molecular mechanisms of GnRH-R involved in the endocrine regulations on the reproduction in the Orange-spotted grouper, Epinephelus coioides. The full-length cDNA encoding GnRH-R type I and GtH Iβ subunit, and the partial cDNA encoding GtH IIβ subunit have been successfully cloned from the pituitary by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods in this grouper. The complete GnRH-R type I cDNA is 1607 bp, which includes an open reading frames of 1092 bp encoding a protein of 364 amino acids, seven alpha helix transmembrane domain, an N-terminal extracellular, and C-terminal cytoplasmic. The complete GtH Iβ subunit cDNA is 551 bp, which includes an open reading frames of 360 bp encoding a protein of 120 amino acids, and the putative eleven cysteine residues. The partial GtH IIβ subunit cDNA is 276 bp, which includes an open reading frames of 360 bp encoding a protein of 120 amino acids, the putative ten cysteine residues, and an N-linked glycosylation site. The expression of GnRH-R type I was found highest in the pituitary, while GtH Iβ subunit and GtH IIβ subunit gene expression are specific in pituitary. An intramuscular injection of various GnRH types in vivo was attempted. The expressions of GnRH-R type I, GtH Iβ- and GtH IIβ-subunits are all stimulated by a single injection of salman GnRH, while in the case of chicken GnRH II treatment, the expression of GnRH-R type I was inhibited, but those GtH Iβ- and GtH IIβ-subunits were stimulated. This suggests that the action of chick GnRH II is probably enhanced through the GnRH receptor of different form. Furthermore, none of them were expressed by an injection of seabream GnRH, and this is likely attributed to the injection dose below the threshold level, and remains to be further examined. In conclusion, GnRHs of various form are effectice in the expression of gonadotropins through the GnRH-R of various forms, and the multiple form of the receptor gene other than that of type likely exists in teleosts.