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研究生：	李威德 Li, Wei-De
論文名稱：	SEPT14絲狀結構在精子生成過程中的組成及功能 Organization and function of SEPT14 filament in spermatogenesis
指導教授：	郭保麟 Kuo, Pao-Lin
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：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	49
中文關鍵詞：	SEPT14 、SEPT7 、SEPT2 、精子生成、SEPT絲狀結構、環體
外文關鍵詞：	SEPT14, SEPT7, SEPT2, spermatogenesis, SEPT filament, annulus
相關次數：	點閱：193 下載：1
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精子生成 (spermatogenesis) 為精子成熟的過程，過程包括：細胞有絲分裂、減數分裂、精子尾部的發育及環體 (annulus) 形成。在過去研究發現環體為SEPT所形成的高級環狀結構，環體缺陷對於精子結構、活動力及男性生殖造成影響，SEPT絲狀結構對於細胞分裂、神經生成、和精子生成是重要的，近年來發現SEPT家族中新穎成員SEPT14 蛋白，過去研究已知SEPT14會表現在精巢和腦組織中，然而對於SEPT14在男性生殖方面所扮演的角色仍不清楚。
在本篇研究中，我們發現SEPT14表現在精子尾部中的環體，環體過去被證實是由SEPT絲狀結構所形成，因此我們推測SEPT14可能在環體的位置形成絲狀結構，幫助環體的形成。我們藉由免疫螢光染色以及免疫沉澱法觀察在細胞及精子環體中SEPT14絲狀結構的組成，實驗結果顯示SEPT14可以和SEPT7、 SEPT2形成鍵結並且組成細小的絲狀結構，然而SEPT14和SEPT4不會形成絲狀結構。根據以上的結果證明SEPT14主要是和SEPT7跟SEPT2形成絲狀結構。接著我們透過免疫沉澱法發現SEPT14複合體組成的排列順序，結果顯示SEPT14複合體是由SEPT7-14-2-2-14-7的排列組合。我們更進一步發現在SEPT14複合體中，SEPT14是透過GTP結合區和SEPT2 GTP結合區形成鍵結，SEPT14 和SEPT7則是透過C端區域鍵結。進一步我們發現 SEPT14絲狀結構有表現在老鼠及人類精子上的環體，並且從環體在精子頸部形成到環體移動到最終定位點，SEPT7-14-2皆會表現在環體，總結以上，我們發現在精子發育過程中，SEPT14絲狀結構的組成對於環體生成可能扮演一個重要的角色。

Spermatogenesis, a process of mature sperm production, involves cell mitosis, cell meiosis, sperm tail development, and annulus formation. Sperm annulus, a SEPT-organized filamentous ring in sperm tail, is necessary for sperm motility and male fertility. It has known that SEPT filament is required for numerous biological processes such as cytokinesis, neurogenesis and spermatogenesis. SEPT14, a novel protein in SEPT family, is specifically expressed in testis and brain; however, the role of SEPT14 in male reproductive system remains unknown.
In this study, we found that SEPT14 located at sperm annulus, a SEPT-organized filament in the sperm tail, suggesting SEPT14 may form filament in annulus formation. To examine SEPT14 filament formation in the cell and at sperm annulus, the immunoprecipitation analyses were conducted. These findings showed that SEPT14 interacted with the SEPT2、SEPT4、SEPT7, and SEPT14 is co-localized with SEPT2, SEPT7 to form thin and spindly filament. However, SEPT14 formed aggregates but not filament with SEPT4. Taken together, these data suggested that major SEPT14 filament consists of SEPT2, SEPT7 and SEPT14. We also explored the assembly order of the SEPT14 complexes by immunoprecipitation that used HA–SEPT2 as a bait to capture SEPT14 and SEPT7. The result revealed that the SEPT14 complex structured in the order SEPT7–SEPT14–SEPT2. Additionally, we found SEPT14 interacted with SEPT2 through the GTP-binding domain and interacted with SEPT7 through C-terminal region. In line with above evidence, SEPT14 is co-localized with SEPT7 and SEPT2 at both human and mouse sperm annulus. Importantly, the expression pattern of SEPT14 filament is consistent with annulus biogenesis including annulus establishment at sperm neck, annulus migration along axoneme and final position of annulus in sperm tail. The result suggests that this SEPT14 filament is involved in annulus establishment. In conclusion, these results indicate the potential role and filament organization of SEPT14 in spermatogenesis.

Table of contents
Chinese abstract	i
Abstract	ii
Acknowledgement	iv
Table of contents	v
List of abbreviation	viii
1. Introduction	1
1.1 Spermatogenesis	1
1.2 Annulus, a ring-like filamentous structure, locates at junction of midpiece and the principal piece in the sperm tail	1
1.3 SEPT family	2
1.4 Assemble rule of SEPT core complex and filament	4
1.5 SEPT14, a novel protein of SEPT family	5
1.6 Objective and specific aims	5
2. Materials and methods	7
2.1 Preparation of sperm	7
2.2 Immmunofluorescent assay, IFA	7
2.3 Cell culture	11
2.4 Total protein extraction	13
2.5 DNA cloning	13
2.6 Co-Immunoprecipitation analysis (Co-IP)	16
2.7 Western blot analysis	18
2.8 Transfection	20
3. Results	21
3.1 SEPT14 is located at sperm annulus.	21
3.2 SEPT14 interacted with SEPT2, SEPT4 and SEPT7, but not SEPT12.	21
3.3 SEPT14 formed thin filamentous structure with SEPT7 and SEPT2.	22
3.4 SEPT14 complexes assembled in the orders of SEPT7–SEPT14–SEPT2.	22
3.5 Knockdown of SEPT14 disrupted SEPT14 filament formation.	23
3.6 SEPT14 interacted with SEPT2 through GTP-binding domain and associated with SEPT7 via C-terminal region.	23
3.7 The expression pattern of SEPT14 complex was consistent with annulus biogenesis.	24
4. Discussion	26
4.1 A novel SEPT filament consists of SEPT7-SEPT14-SPET2	26
4.2 The possible roles of SEPT14 filament in sperm annulus	26
4.3 The possible role of SEPT14 function in sperm head and neck	27
4.4 Other possible composition of SEPT14 filament	28
4.5 The possible difference between SEPT7-6-2-2-6-7 complexes and SEPT7-14-2-2-14-7 complexes.	28
5. Conclusion	30
References	31
Figures	34
Figure 1. SEPT14 was located at sperm annulus.	34
Figure 2. SEPT14 interacted with SEPT2, SEPT4 and SEPT7, but not SEPT12.	35
Figure 3. SEPT14 formed thin filament with SEPT7 and SEPT2, but not with SEPT4.	37
Figure 4. SEPT14 was co-localized with SEPT7 and SEPT2 in a filament pattern in NT2/D1 cell.	39
Figure5. Knockdown of SEPT14 disrupted SEPT14 filament formation.	41
Figure 6. SEPT14 complex was assembled in the order of SEPT7–SEPT14–SEPT2.	42
Figure 7. Diagrams of different truncated SEPT14, SEPT7 and SEPT2.	43
Figure 8. The interaction between SEPT14 and SEPT2 was via GTP-binding domain, and between SEPT14 and SEPT7 was through C-terminal region.	44
Figure 9. SEPT14 is co-localized with SEPT7 and SEPT2 at the annulus in mouse and human spermatozoa.	45
Figure 10. SEPT14 expression pattern correlated with annulus biogenesis during the spermiogenesis.	46
Figure 11. SEPT14 was co-localized with SEPT7 and SEPT2 during sperm annulus biogenesis.	48
Figure 12. The working model of SEPT14 filament during annulus formation.	49



                                    

Brinster, R. L. & Zimmermann, J. W. Spermatogenesis following male germ-cell transplantation. Proceedings of the National Academy of Sciences 91, 11298-11302 (1994).
2 Fishelson, L., Gon, O., Holdengreber, V. & Delarea, Y. Comparative spermatogenesis, spermatocytogenesis, and spermatozeugmata formation in males of viviparous species of clinid fishes (Teleostei: Clinidae, Blennioidei). The Anatomical Record 290, 311-323 (2007).
3 Kwitny, S., Klaus, A. V. & Hunnicutt, G. R. Annulus of the Mouse Sperm Tail Is Required to Establish a Membrane Diffusion Barrier That Is Engaged During the Late Steps of Spermiogenesis. Biology of reproduction (2010).
4 Phillips, D. M. Mitochondrial disposition in mammalian spermatozoa. Journal of ultrastructure research 58, 144-154 (1977).
5 Sugino, Y. et al. Septins as diagnostic markers for a subset of human asthenozoospermia. The Journal of urology 180, 2706-2709 (2008).
6 Kissel, H. et al. The Sept4 septin locus is required for sperm terminal differentiation in mice. Developmental cell 8, 353-364 (2005).
7 Kuo, Y. C. et al. SEPT12 mutations cause male infertility with defective sperm annulus. Human mutation 33, 710-719 (2012).
8 Kuo, Y.-C. et al. SEPT12 orchestrates the formation of mammalian sperm annulus by organizing core octameric complexes with other SEPT proteins. J Cell Sci 128, 923-934 (2015).
9 Myles, D. G., Primakoff, P. & Koppel, D. E. A localized surface protein of guinea pig sperm exhibits free diffusion in its domain. The Journal of cell biology 98, 1905-1909 (1984).
10 Cesario, M. M. & Bartles, J. R. Compartmentalization, processing and redistribution of the plasma membrane protein CE9 on rodent spermatozoa. Relationship of the annulus to domain boundaries in the plasma membrane of the tail. Journal of Cell Science 107, 561-570 (1994).
11 Touré, A. et al. The testis anion transporter 1 (Slc26a8) is required for sperm terminal differentiation and male fertility in the mouse. Human molecular genetics 16, 1783-1793 (2007).
12 Guan, J., Kinoshita, M. & Yuan, L. Spatiotemporal association of DNAJB13 with the annulus during mouse sperm flagellum development. BMC developmental biology 9, 23 (2009).
13 Mostowy, S. & Cossart, P. Septins: the fourth component of the cytoskeleton. Nature reviews. Molecular cell biology 13, 183 (2012).
14 Lin, Y.-H. et al. The expression level of septin12 is critical for spermiogenesis. The American journal of pathology 174, 1857-1868 (2009).
15 Shen, Y.-R. et al. SEPT12 phosphorylation results in loss of the septin ring/sperm annulus, defective sperm motility and poor male fertility. PLoS genetics 13, e1006631 (2017).
16 Kinoshita, M. Assembly of mammalian septins. Journal of biochemistry 134, 491-496 (2003).
17 Sellin, M. E., Sandblad, L., Stenmark, S. & Gullberg, M. Deciphering the rules governing assembly order of mammalian septin complexes. Molecular biology of the cell 22, 3152-3164 (2011).
18 Vrabioiu, A. M., Gerber, S. A., Gygi, S. P., Field, C. M. & Mitchison, T. J. The majority of the Saccharomyces cerevisiae septin complexes do not exchange guanine nucleotides. Journal of Biological Chemistry 279, 3111-3118 (2004).
19 Bertin, A. et al. Saccharomyces cerevisiae septins: supramolecular organization of heterooligomers and the mechanism of filament assembly. Proceedings of the National Academy of Sciences 105, 8274-8279 (2008).
20 Cao, L. et al. Phylogenetic and evolutionary analysis of the septin protein family in metazoan. FEBS letters 581, 5526-5532 (2007).
21 Shinoda, T. et al. Septin 14 is involved in cortical neuronal migration via interaction with Septin 4. Molecular biology of the cell 21, 1324-1334 (2010).
22 Rozenkrantz, L. et al. SEPT14 Is Associated with a Reduced Risk for Parkinson’s Disease and Expressed in Human Brain. Journal of Molecular Neuroscience 59, 343-350 (2016).
23 Peterson, E. A. et al. Characterization of a SEPT9 interacting protein, SEPT14, a novel testis-specific septin. Mammalian Genome 18, 796-807 (2007).
24 Sirajuddin, M. et al. Structural insight into filament formation by mammalian septins. Nature 449, 311-315 (2007).
25 Kim, M. S., Froese, C. D., Estey, M. P. & Trimble, W. S. SEPT9 occupies the terminal positions in septin octamers and mediates polymerization-dependent functions in abscission. J Cell Biol 195, 815-826 (2011).
26 Toure, A., Rode, B., Hunnicutt, G. R., Escalier, D. & Gacon, G. Septins at the annulus of mammalian sperm. Biological chemistry 392, 799-803 (2011).
27 Ihara, M. et al. Cortical organization by the septin cytoskeleton is essential for structural and mechanical integrity of mammalian spermatozoa. Developmental cell 8, 343-352 (2005).
28 Shinoda, T. et al. Septin 14 Is Involved in Cortical Neuronal Migration via Interaction with Septin 4. Mol Biol Cell 21, 1324-1334, doi:10.1091/mbc.E09-10-0869 (2010).
29 Kierszenbaum, A. L., Rivkin, E. & Tres, L. L. Acroplaxome, an F-actin–keratin-containing plate, anchors the acrosome to the nucleus during shaping of the spermatid head. Molecular biology of the cell 14, 4628-4640 (2003).
30 Zhou, J. et al. RIM-BP3 is a manchette-associated protein essential for spermiogenesis. Development 136, 373-382 (2009).
31 Schatten, H., Rawe, V. Y. & Sun, Q.-Y. The sperm centrosome: its role and significance in nature and human assisted reproduction. Journal of Reproductive and Stem Cell Biotechnology 2, 121-127 (2011).
32 Fawcett, D. W. & Phillips, D. M. The fine structure and development of the neck region of the mammalian spermatozoon. The Anatomical Record 165, 153-183 (1969).
33 Garcia, G. et al. Subunit-dependent modulation of septin assembly: budding yeast septin Shs1 promotes ring and gauze formation. J Cell Biol 195, 993-1004 (2011).

2022-08-24公開

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