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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 鍾德憲,邱智賢 | |
| dc.contributor.author | Yue-Jia Lee | en |
| dc.contributor.author | 李月嘉 | zh_TW |
| dc.date.accessioned | 2021-05-15T17:51:00Z | - |
| dc.date.available | 2017-12-31 | |
| dc.date.available | 2021-05-15T17:51:00Z | - |
| dc.date.copyright | 2014-09-05 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-18 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5018 | - |
| dc.description.abstract | 近年研究指出，Kisspeptin與其受器KISS1R在動物生殖生理的調控機制中扮演相當重要的角色。Kisspeptin是由Kiss1基因轉譯而來的蛋白質產物，而KISS1R則是典型的穿膜蛋白。當kisspeptin與促性腺激素釋放素神經元 (GnRH neuron) 上的KISS1R結合時，便會刺激GnRH neuron興奮，進而調控下視丘-腦垂腺-性腺軸 (HPG axis)，促使性腺發育成熟並開始表現生殖能力。除了下視丘之外，先前研究也顯示人類和嚙齒類的睪丸組織皆有Kiss1和Kiss1r基因表現其中。然而，對於kisseptin和KISS1R在睪丸中的位置分佈和所執行的功能目前仍不是很清楚。
首先，為了獲得高專一性的抗體來偵測KISS1R在睪丸中的位置分佈，我們選用母雞作為抗體生產的宿主。成功獲得具專一性之抗體後，我們更進一步利用免疫組織染色法證實了，kisspeptin主要表現在萊迪氏細胞中，而KISS1R則主要分布在精細胞細胞膜上。此外，透過即時定量聚合酶鏈鎖反應，我們也發現睪丸中的Kiss1和Kiss1r基因表現量隨著時間進展而有不同的變化。自小鼠出生後，睪丸中的Kiss1r基因表現量便維持恆定，然而，Kiss1基因卻是從小鼠出生後第四周才開始大量表現。有鑑於Kiss1基因表現量有所轉變且其時間點與小鼠性成熟初始時間重疊，因此我們進一步比較Kiss1基因與其他睪丸發育相關基因之表現情形。有趣的是，Insl3 (參與生精作用與睪丸沉降) 以及Cyp19a1 (負責雌二醇生成) 兩個基因表現型態皆與Kiss1基因相似，皆是從小鼠出生後第四周才開始被大量表現。另一基因，Lhcgr，雖同樣有漸趨上升的基因表現形態，卻比前述三個基因早約一周於睪丸中表現。此結果顯示，睪丸中的kisspeptin/KISS1R系統可能受到LH和其受器下游訊號所調控。為了證實這項假設，我們分別將初代萊迪氏細胞處理綿羊排卵素 (ovine luteinizing hormone, oLH) 以及將MA-10細胞株處理LH下游第二訊息傳遞因子，Br-cAMP。結果顯示，不論是oLH或Br-cAMP皆會增加萊迪氏細胞內的Kiss1基因表現量 (p < 0.05)。此外，在Br-cAMP及RP-cAMPS (PKA抑制劑)共處理之下，萊迪氏細胞內的Kiss1基因表現量明顯受到抑制 (p < 0.05)。 綜觀上述，小鼠萊迪式細胞中可觀察到kisspeptin的表現，且Kiss1基因表現量可透過排卵素的刺激及下游訊息傳遞而有所增加；反之，主要位於細精管內部的Kiss1r其基因表現量則不受排卵素調控。此外，由於發現可接受kisspeptin訊號的KISS1R表現位置以及kiss1基因表現時間點與生精作用相關之基因表現時間相似，故我們猜測kisspeptin可能會協同其他負責生精作用的相關因子一起調控生精作用的運作。然而，關於kisspeptin於睪丸中所扮有的實際角色仍須進行更深入的實驗才能證實。 | zh_TW |
| dc.description.abstract | Kisspeptin and KISS1R had been considered as essential regulators of animal reproductive functions in recent year. Kisspeptin, peptide encoded by the Kiss1 gene, is synthesized in the discrete neurons of hypothalamus. When kisspeptin binds with its receptor, KISS1R, the following signals induce GnRH secretion to regulate the HPG axis. Interestingly, not only in the hypothalamus, previous data has already revealed the presence of Kiss1 and Kiss1r mRNAs in the testis of human beings and rodents. However, the precise location and possible physiological role of kisspeptin/KISS1R system in testis remain unclear.
To further address the exact site of KISS1R in testis, we successfully produced an anti-KISS1R IgY antibody. Later, by conducting IHC assays, we detected that kisspeptin was expressed in Leydig cells, while KISS1R localized in seminiferous tubules, especially on spermatid membrane. Besides, real-time PCR results showed that mouse testis constantly expressed Kiss1r mRNA from birth but it didn’t express Kiss1 mRNA until postnatal fourth week. With comparing testicular gene expression patterns in the first 12 weeks after birth, we found the mRNA expression pattern of Kiss1 was synchronized with Insl3 and Cyp19a1 which are genes important for testis descent and spermatogenesis, and estrogen synthesis. Moreover, Lhcgr (LH receptor encoding gene) expression was increased one week earlier than Kiss1 expression, which indicated that the Kisspeptin/KISS1R system in testis might be under control of LH and its receptor-mediated signaling such as cAMP pathway. To confirm this hypothesis, we treated primary Leydig cell with LH and MA-10 (Leydig cell line) with Br-cAMP, respectively. Indeed, these treatments obviously increased Kiss1 mRNA expression in both two types of cells (p < 0.05). In addition, co-treatment of Br-cAMP and RP-cAMPS, a PKA inhibitor, significantly suppressed 50% Br-cAMP-induced Kiss1 expression (p < 0.05). From our present study, it is concluded that LH act as an upstream initiator to induce Kiss1 expression in mouse Leydig cells through cAMP/PKA pathway. Since receptors for Kisspeptin are present on spermatids and the similar timing and location of gene expression between Kiss1 and several genes regulating spermatogenesis, it is tempting to assume the synergistic action of Kisspeptin and development-related factors on spermatogenesis. However, the definitive role for Kisspeptin/KISS1R system in testicular development needs more studies to elucidate. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-15T17:51:00Z (GMT). No. of bitstreams: 1 ntu-103-R01626005-1.pdf: 1504490 bytes, checksum: 526d44ca337f3e76a93149b72eabe0a0 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 致謝 i
中文摘要 ii Abstract iv Contents vi List of Figures viii List of Tables ix 1. Introduction 1 2. Literature Review 3 2.1 Male reproduction 3 2.2 Kisspeptin/KISS1R system 12 3. Materials and Methods 24 3.1 Synthetic peptides 24 3.2 Preparation of antibodies 24 3.3 Purification of egg yolk antibody 24 3.4 Enzyme-linked immunosorbent assay (ELSIA) 26 3.5 Animals and tissues collection 26 3.6 Primary Mouse Leydig cell culture 27 3.7 Cell line culture 28 3.8 Immunohistochemistry 29 3.9 Western Blot 30 3.10 RNA extraction and cDNA synthesis 31 3.11 Quantitative real-time PCR 31 3.12 Statistical analysis 32 4. Results 34 4.1 Titer of chicken-anti KISS1R antibody 34 4.2 Specificity of chicken-anti KISS1R antibody 34 4.3 Specific detection of KISS1R in various tissues of mouse 36 4.4 Immunohistochemical staining of kisspeptin and KISS1R in mouse testis 36 4.5 Gene expression of Kiss1 and Kiss1r during testicular development 37 4.6 Kiss1 gene expression is related to testicular development 38 4.7 Kiss1 gene expression depends on LH signaling 39 5. Discussion 41 5.1 The establishment of anti-KISS1R IgY antibody 41 5.2 Expression of KISS1R in mouse tissues 43 5.3 Expression of kisspeptin and KISS1R in mouse testis 44 5.4 Kiss1 gene expression is related to testicular development 45 5.5 Kiss1 gene expression depends on LH signaling 47 6. Conclusion 51 7. Figures 52 8. Reference 64 Appendix List of Abbreviations 81 | |
| dc.language.iso | en | |
| dc.title | Kisspeptin/KISS1R系統在小鼠睪丸發育過程中所扮演的角色 | zh_TW |
| dc.title | The Role of Kisspeptin/KISS1R System in Mice Testicular Development | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 梁耀仁,徐慶琳,吳兩新 | |
| dc.subject.keyword | kisspeptin,KISS1R,排卵素,生精作用,睪丸發育, | zh_TW |
| dc.subject.keyword | kisspeptin,KISS1R,luteinizing hormone,spermatogenesis,testicular development, | en |
| dc.relation.page | 82 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2014-08-19 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
| Appears in Collections: | 動物科學技術學系 | |
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