Kisspeptin與GPR54在小鼠生殖功能之探討

The Novel Role of Kisspeptin and GPR54 in Mice Reproduction

10.6342/NTU.2011.00871

許孟傑

kisspeptin ； GPR54 ； 睪丸 ； 精子 ； 授精作用 ； kisspeptin ； GPR54 ； testis ； sperm ； fertilization

國立臺灣大學動物科學技術學系學位論文

2011年

碩士

邱智賢

繁體中文

近年研究指出Kisspeptin與其受體GPR54在動物的生殖生理調控扮演重要的角色。Kisspeptin為Kiss1基因的蛋白質產物，其生殖生理之功能主在刺激GnRH神經元興奮進而調控下游的下視丘-腦垂腺-性腺軸及其所引發的相關生理調控。目前已有報告提出Kiss-1與Gpr54基因在動物多種器官組織均有表現，然而其所調控的生理功能則尚未完全被瞭解。因此，本研究將探討kisspeptin與GPR54在小鼠睪丸與卵巢組織之表現性狀及其可能扮演之功能。 本研究以成年ICR小鼠做為實驗動物，將睪丸組織分離並進行半定量RT-PCR分析，結果顯示Kiss1基因主要表現於萊迪氏細胞，而Gpr54基因則表現於細精管組織中；進一步以免疫組織染色法證實Kisspeptin與GPR54在睪丸中的表現，結果顯示Kisspeptin僅分佈於管間細胞中，而GPR54則存在於細精管組織並且特異性地表現於精細胞中，GPR54且隨著精細胞的發育而有分佈型態的變化，故推測其可能位於頂體區域。透過免疫螢光染色法，將睪丸切片進行GPR54、SP56(頂體標記)抗體與Hoechst33342進行雜合染色，結果確認GPR54表現於精細胞之頂體區域，鑑於睪丸之血睪障壁結構，萊迪氏細胞所分泌之kisspeptin並無法作用於精細胞而另有其功能。為進一步探討GPR54在精細胞的功能，取自附睪的成熟精子進行相同螢光染色步驟，結果亦顯示GPR54存在於頂體區域，此結果顯示GPR54可能用於調控成熟的精子。同時，我們亦發現kisspeptin會被表現在輸卵管之柱狀上皮細胞、卵丘細胞及卵子上，因此我們推測在授精過程中，其表現之kisspeptin與GPR54可能扮演一定的功能。透過體外授精試驗，結果顯示經kisspeptin拮抗劑peptide-234處理之精子，其授精率自80%下降至50%(P < 0.05)。 總結上述結果，在本研究發現在雌性小鼠之輸卵管、卵子及其周圍卵丘細胞會表現kisspeptin，而其受體GPR54則會在成熟精子表現並且位在頂體區域，透過體外授精實驗證實kisspeptin參與授精作用。然而，其更詳細的的調控機制有待未來進一步的實驗證實之。

In recent studies, it has been reported that kisspeptin act as an upstream regulator of hypothalamus-pituitary-gonad axis (HPG axis), the main regulatory system of animal reproduction. Kisspeptin, a short peptide encoded from Kiss1 gene, is a ligand of G protein-coupled receptor 54 (GPR54) that is expressed in GnRH neurons. Kisspeptin is able to stimulate GnRH neurons through GPR54 and its downstream signals, and elicit the hormone transductions in HPG axis. Kiss1 and Gpr54 have been found expressed in various tissues including testis and ovary, but their functions are still not well understood. The purpose of this study is to investigate the expression profiles and functions of kisspeptin and GPR54 in mice testis and ovary. Tissue samples were collected from adult ICR strain mice and extracted total RNA for semi-quantitative RT-PCR. The results showed that Gpr54 gene was highly expressed in testis and other tissues. Also, we used collagenase and filter for testis tissues dissociation and found that Gpr54 mRNA was mainly expressed in seminiferous tubule cells but not Leydig cell. For identifying the possible location of GPR54 in seminiferous tubules, the testis tissues were analyzed by immunohistochemistry staining method. On further examination, GPR54 was specifically expressed in spermatid, but not spermatogonia, spermatocyte, or sertoli cell. Because of GPR54 expressed pattern was closed to the site of acrosome, the testis sections were stained with GPR54, SP56 (an acrosome marker) antibodies and Hoechst 33342, as triple-label immunofluorescense staining. The results showed that GPR54 was located in acrosome area of both spermatids and mature sperms. Interestingly, we also observed that GPR54’s ligand kisspeptin was expressed in culumus-oocyte complex and oviduct’s epithelium on ovary and oviduct section. Based on above results, we hypothesized kisspeptin and GPR54 might be involved in fertilization controls when sperm reaches female reproductive ducts. Through in vitro fertilization (IVF) method, it was revealed that blocking kisspeptin signal by antagonist peptide-234 decreased the fertilization rate. To summarize, the present studies indicated that kisspeptin and GPR54 were existed in both male and female gametes and other surrounding cells, suggested that they were related to the process of fertilization. Owing to fertilization includes multiple control mechanisms, the exact physiological role of the kisspeptin-GPR54 system in regulating sperm function remains to be elucidated.