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研究生：	廖怡雯 Liao, Yi-wen
論文名稱：	探討STK31在造精過程中所扮演的角色 Characterize the role of STK31 in spermatogenesis
指導教授：	郭保麟 Kuo, Pao-lin 洪良宜 Hong, Liangy-yi
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	73
中文關鍵詞：	不孕
外文關鍵詞：	stk31, centrosome
相關次數：	點閱：73 下載：1
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世界上約有百分之二至十二比例的夫妻受到生殖能力降低的影響。男性及女性因素所導致的不孕約各佔百分之五十的比例。而大部分的不孕症男性通常在造精過程中有所缺陷。許多基因牽連在哺乳類的生殖過程中，而大多數仍未被探索。在生殖過程中，配子中心粒會減半，受精後才能維持正確的數目。中心粒不正常的複製會破壞胚胎細胞中心粒數量的平衡。中心粒是細胞主要的微管生中心亦是個調控細胞週期的因子。為了研究男性生殖過程，我們執行 cDNA 微陣列技術分析造精缺陷患者的睪丸組織。STK31 是被選擇進一步研究下降調節基因的其中之一，在我們過去的研究指出stk31大量表現在老鼠的精原細胞及初級精母細胞。我們發現人類 STK31 確實在不具生殖細胞的病人睪丸組織當中是有意義的下降表現。由GC-1 spg 細胞的免疫螢光染色分析，無論是在細胞間期或細胞分裂時期stk31 與 γ-tubulin共同表現在紡錘體/中心體。我們利用核酸干擾試驗去探討stk31的功能，由實驗結果得知stk31可能參與中心粒的功能及成熟相關，且當利用核酸干擾實驗的方式去破壞GC-1 cell中stk31的表現會導致不正常的微管組織及細胞週期停留在G2/M時期。綜合以上，我們假設STK31為生殖細胞專有的細胞週期檢查點的蛋白，可能參與在中心粒功能及成熟，在細胞分裂時微管的集結所必需。

Between 2% and 12% of couples worldwide are affected by reduced fertility. Male factors and female factors accounts for approximately 50% of causes in infertile couples, respectively. In men, a large proportion of infertile men have defects in the process of spermatogenesis, but the cause of spermatogenic defect is not known for the majority of cases. Centrosome serves as the main microtubule organizing center (MTOC) of cell as well as a regulator of cell-cycle progression. Sexual reproduction requires reduction in the gametes of centrosome number so that upon fertilization, the correct number of centrosomes is restored. Abnormal centrosome duplication will disrupt balancing centrosome number in embryonic cells. In the previous study, we have identified STK31 as a candidate sterile gene by cDNA microarray analysis of testicular tissues from patients with spermatogenic defect. We also showed stk31 is abundantly expressed in spermatogonia and primary spermatocytes of mouse testis. In addition, the mRNA expression level of STK31 is significantly decreased in the testicular tissue of men with spermatogenic defect. Immunofluorescence staining showed that stk31 is colocalized with γ-tubulin at the interphase centrosome and the mitotic spindle poles in GC-1, a mouse spermatogonia cell line. In this study, we analyzed the physiological function of STK31 by using shRNA knockdown strategy. Our study suggests that STK31 may be involved in centrosome function and maturation. Disruption of stk31 in GC-1 cells resulted in abnormal microtubule organization and cell cycle arrest in G2/M phase. Taken together, we hypothesize that STK31 is a novel germ-cell specific centrosomal protein, which may be involved in centrosomal function and maturation, and may be required for nucleation of microtubule during cell division.

Abstract in Chinese 1
Abstract in English 2
Acknowledgements 4
Abbreviation 8
1.Introduction 9
1.1  Pathogenesis of male infertility 9
1.2  Process of spermatogenesis 9
1.3  Stage- and cell -specific genes expression during spermatogenesis 10
1.4  STK31 10
1.5  Centrosome - the microtubule organizing center (MTOC) 12
1.5.2  MTOC and the checkpoint response 12
1.5.3  Centrosome and male gametogenesis 13
1.6 Studies on spermatogonia-specific genes and their therapeutic implication 13
2.Materials and methods 15
2.1  Cell culture 15
2.1.1  Materials 15
2.1.2  Method 17
2.2  Total RNA isolation 18
2.2.1  Materials 18
2.2.2  Method 18
2.3  Reverse Transcription Polymerase Chain Reaction 19
2.3.1  Materials 19
2.3.1a  Reverse Transcription 19
2.3.1b  Polymer Chain Reaction 19
2.3.1c  Primer 20
2.3.2  Methods 21
2.4  SDS-Polyacrylamide Gel Electrophoresis 22
2.4.1  Materials 22
2.4.2  Method 23
2.5  Protein Concentration Detection 23
2.5.1  Materials 23
2.5.2  Method 24
2.6  Western Blot Analysis 24
2.6.1  Materials 24
2.6.2  Method 25
2.7  Cryopreservation of cultured cells 26
2.7.1  Materials 26
2.7.2  Method 26
2.8  Immunofluorescence staining 26
2.8.1  Materials 26
2.8.2  Method 27
2.9  Ttransfection 28
2.9.1  Materials: 28
2.9.2  Method: 28
2.10  Protein degradation assay 29
2.10.1  Materials: 29
2.10.2  Method: 29
2.11  Stable cell line selection 29
2.11.1  Material 30
2.11.2  Method 30
2.12  Flow cytometry 30
2.12.1  Material 30
2.12.2  Method 31
2.13  Cold treatment	 31
2.13.1  Material 31
2.13.2  Method 32
3. Result 33
3.1  Stk31 is a novel centrosomal component 33
3.2  Stk31 protein is very stable 33
3.3  Stk31 knockdown may reduce the recruitment of γ-tubulin to the centrosome 33
3.4  Stk31 may be involved in microtubule organization 34
3.5  Stk31 knockdown GC-1 stable cell line display abnormal microtubule organization. 34
3.6  Disruption of stk31 may lead to cell cycle arrest in G2/M phase 35
3.7  Stk31,a cancer/testis antigen,is also expressed in various cancer cell lines 35
3.8  Stk31 is co-localized with γ-tubulin in A549 cell line 36
3.9  Knockdown stk31 in A549 cell line also affects γ-tubulin recruit to the centrosome 36
3-10  Stk31 may be involved in microtubule organization in A549 cell line 36
4. Discussion 37
5.Reference 40
Resume 73
Figure 1.   Stk31 is a novel centrosomal protein. 45
Figure 2.   Stk31 protein is very stable 46
Figure 3A.  RT-PCR for stk31 knockdown by siRNA in GC-1 cells. 47
Figure 3B.  Western blot for stk31 knockdown by siRNA in GC-1 cells. 48
Figure 4.   Western Blot for stk31 knockdown by shRNA in 293T cells. 49
Figure 5A.  RT-PCR for stk31 knockdown by shRNA in GC-1 cells. 50
Figure 5B.  Westen blot for stk31 knockdown by shRNA in GC-1 cells. 51
Figure 6.   Construct of shRNA-GFP 52
Figure 7A.  RT-PCR for stk31 knockdown by shRNA-GFP in GC-1 cells. 53
Figure 7B.  Western Blot for stk31 knockdown by shRNA-GFP in GC-1 cells. 54
Figure 8.   Knockdown stk31 may reduce γ-tubulin recruitment to the centrosome 55
Figure 9.   Stk31 may be involved in microtubule organization 56
Figure 10A. Select stk31 knockdown stable line in GC-1 cell. 57
Figure11A.  GC-1 stk31 knockdown stable cell line show abnormal microtubule organization 59
Figure 11B.  Number of cells with abnormal microtubule organization. 60
Figure 12.  Disruption of stk31 results in cell cycle arrest at G2/M phase. 61
Figure 13.  GC-1 cell transfection efficiency 62
Figure 14.  Stk31 expression in A549 cell line. 63
Figure 15.  Stk31 is also localized to the centrosome in cancer cells 64
Figure 16.  RT-PCR for stk31 knockdown by shRNA in A549 cells. 65
Figure 17.  Knockdown stk31 in A549 cell also reducedγ-tubulin recruitment to the centrosome 66
Figure 18.  Stk31 may be involved in microtubule organization in cancer cells. 67
Appendix 1.  The process of spermatogenesis 68
Appendix 3.  Western Blot analysis of Stk31. 70
Appendix 4.  The diagram of cold treatment experiment. 71
Appendix 5.  Phylogenetic analysis of human protein kinase family. 72
                                    

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校內：2010-08-26公開
校外：2010-08-26公開

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