鍾德憲Chiu, Chih-Hsien;Jong, De-Shien臺灣大學:動物科學技術學研究所曾泰祥Tseng, Tai-HsiangTai-HsiangTseng2010-05-112018-06-292010-05-112018-06-292009U0001-2007200914233700http://ntur.lib.ntu.edu.tw//handle/246246/182086發身(puberty)是動物啟動生殖周期的發軔，並進入成熟生理的變化；不論雄性或雌性動物達發身年齡後，體內生殖內分泌開始運轉，此時下視丘會分泌促性腺激素釋放激素(Gonadotropin release hormone, GnRH)，進而刺激腦垂腺之排卵素(LH)、濾泡激素(FSH)等激性腺素分泌，使性腺發育成熟。此為現今熟知能調控動物生殖生理機制的HPG軸(Hypothalamic-pituitary-gonad axis) 。近年來發現，人類和小鼠下視丘上GPR54 (G protein-coupled receptor 54)受體可調控GnRH神經元分泌GnRH，進而促使腦垂腺分泌LH及FSH以調控HPG軸的運作。 GPR54是一種G蛋白偶合受體，最初在1999年被發現時，並未找到其配體(ligand)，故被分類為新型孤兒G蛋白偶合受體(new orphan GPCR)。直至2001年GPR54的配體kisspeptin才隨之發現。Kisspeptin 蛋白為145個胺基酸的親水性蛋白，在不同物種間Kiss1蛋白的序列都有其相似性，譬如在胜汰前的分泌序列(secretory signal sequence)，中間的PEST序列及共同的磷酸化位置。KiSS-1相對應的受體為GPR54，又稱作hOT7T175或是AXOR12，是GPCR家族的成員之一。在動物組織分佈的方面，GPR54和kisspeptin兩者經常於同一位置表現，而胎盤中兩者的表現量都很高，可能參與催產功能(Horikoshi et al., 2003)；此外，KiSS-1和GPR54兩者在神經系統的分佈相對廣泛，尤其是在下視丘和腦垂體。除了神經系統外，KiSS-1也在睪丸、胰臟、肝臟和小腸有表現，而GPR54則會在胰臟大量表現，另外在脾臟、淋巴結及脂肪組織中也有少量表現。 雖已知kisspeptin與GPR54表現於許多動物組織中，但科學家截至目前只對其與HPG軸之上游較為明瞭，而其他組織表現之kisspeptin與GPR54尚需更多研究證實其功能。因此，本試驗利用十二週齡之ICR小鼠，取其組織並萃取組織之RNA，並以RT-PCR技術、免疫組織染色技術與西方墨染法技術，瞭解KiSS-1與GPR54於睪丸之表現情形。 試驗結果顯示KiSS-1與GPR54在下視丘、腎臟和睪丸表現量較其他組織高，此外，GPR54另外在小腸、胰臟、卵巢和白色脂肪組織也有表現。而後也利用西方墨染法確認這項mRNA的表現結果。 而GPR54在表現位置與功能研究方面，除腦組織及癌細胞外，其它如睪丸、胰臟和小腸等組織，尚無研究證實。又因睪丸之生精作用功能與類固醇生成特性，且相關文獻並無提及kisspeptin、GPR54與睪丸之關連性，因此本試驗將以睪丸為主要研究組織，探討kisspeptin與GPR54在睪丸中之定位與功能。為了定義出kisspeptin與GPR54在睪丸中的位置，本試驗利用免疫組織染色(IHC)進行定位之實驗。結果發現kisspeptin與GPR54都高度表現於睪丸之萊吉氏細胞中，由此推測kisspptin可能分泌自萊吉氏細胞並作用於萊吉氏細胞本身而形成自我分泌(autocrine)路徑。此外，以相同試驗技術實驗於四週齡之未發身小鼠，發現未發身小鼠萊吉氏細胞之kisspeptin與GPR54表現量顯著低於發身後之小鼠。 綜上所述，HPG軸在哺乳類動物繁殖功能的維持扮演相當重要的角色，且KiSS-1基因與kisspeptin已被證明參與了繁殖系統之活化與功能。但截至目前為止只能推測其功能，而kisspeptin/GPR54系統在睪丸中更精確之生理作用尚需更多實驗證實解答，這些研究結果應能對季節性生殖的調控機制有更深入的瞭解，也將解開生殖研究領域長久存在的問題。藉由此一重大研究進展，在未來生殖生理學上將是相當重要之突破發展。The function of animal reproduction is mainly regulated by the hypothalamic-pituitary-gonadal (HPG) axis. As animals reach puberty, the reproductive hormones start to work. The gonadotropin releasing hormone (GnRH) from hypothalamus would provoke to increase gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH) which are released from anterior pituitary. Gonadotropins stimulate gonads via the blood circulatory system to regulate the development and functions of sexual gonads. In recent years, researchers have found that GPR54, which is located in hypothalamus of human and mice can stimulate GnRH neuron to secret GnRH and subsequently activate HPG axis. KiSS-1 gene is a cancer cell metastasis suppressor. Recently, KiSS-1 has been identified as the upregulatory gene for the HPG axis. The KiSS-1 product, kisspeptin, has strong affinity to GPR54 receptor on the membrane of GnRH neuron. It can tightly bind to GPR54, and stimulate GnRH neurons to release GnRH and then activate downstream genes regulating the onset of puberty and reproductive function. So far kisspeptin and GPR54 have been identified expressing in various tissues, but researchers were devoted to understand their role in upstream HPG axis. Therefore, their roles need to be identified in other tissues, especially in sex organ. The purpose of this study was to elucidate the expression of KiSS-1 and GPR54 in testis of mice. For this reason, thirteen-week old ICR strain mice were used in this study. The tissue samples were collected from these mice and RNA were isolated. The reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) staining and Western blotting were used in this study. The results showed that Kiss-1 and GPR54 gene were highly expressed in hypothalamus, kidney and testis. KiSS-1 and GPR54 were also expressed in intestine, pancreas, ovary and adipose tissue. This mRNA expression data was confirmed by Western blot analysis. Due to steroidogenic and spermatogenic properties of testis, the testis was chosen for the further study. In order to identify the location of kisspeptin and GPR54 in testis, the following experiment was designed to identify the localization of kisspeptin and GPR54. From the IHC staining results, we found that both kisspeptin and GPR54 were highly expressed in interstitial cells (Leydig cells) of testis. We speculated that Leydig cells might secret kisspeptin and have autocrine characteristic. Besides, four-week old juvenile mice were taken to do the same experiments. However, less kisspeptin and GPR54 expression were found via IHC analysis. In conclusion, the HPG axis is very important in maintenance of reproduction function in mammals. The KiSS-1 and kisspeptin have been identified involved in this system. Although kisspeptin may have direct effects on the testis through autocrine route, the precise physiological role of the kisspeptin-GPR54 system in the regulation of reproductive function remains to be elucidated.目 錄 頁次試委員會審定書...........................................i辭......................................................ii文摘要.................................................iii文摘要...................................................v錄....................................................viii次......................................................ix次.......................................................x一章 文獻檢討........................................1一節 前言...........................................1二節 哺乳動物發身定義與生殖生理調控機制.............3三節 Kisspeptin-GPR54之生理功能概論.................6四節 Kisspeptin-GPR54系統在生殖生理的作用機制......17五節 Kisspeptin-GPR54在生殖系統的研究進展..........23二章 動機與目的.....................................24三章 材料與方法.....................................25四章 結果...........................................32五章 討論...........................................36六章 結論...........................................40考文獻.................................................55application/pdf4477048 bytesapplication/pdfen-USkisspeptinGPR54發身HPG軸ICR鼠萊吉氏細胞pubertyHPG axisICR strain mouseLeydig cell[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182086/1/ntu-98-R96626011-1.pdf