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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳瑞菁(Jui-ching Wu) | |
| dc.contributor.author | Hsiao-Fang Peng | en |
| dc.contributor.author | 彭筱芳 | zh_TW |
| dc.date.accessioned | 2021-06-17T01:16:37Z | - |
| dc.date.available | 2027-08-14 | |
| dc.date.copyright | 2017-08-28 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-14 | |
| dc.identifier.citation | 1.Wistuba, J.S., J.B. and C.M. Luetjens, Mammalian Spermatogenesis. Functional Development and Embryology, 2007. 1: p. 99-117.
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J Cell Sci, 2000. 113 ( Pt 1): p. 103-12. 27.Brenner, S., The genetics of Caenorhabditis elegans. Genetics, 1974. 77(1): p. 71. 28.Fernandez, A.G., et al., High-throughput fluorescence-based isolation of live C. elegans larvae. Nat. Protocols, 2012. 7(8): p. 1502-1510. 29.Kosinski, M., et al., C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes. Development, 2005. 132(15): p. 3357. 30.Altschul, S.F., et al., Basic local alignment search tool. Journal of Molecular Biology, 1990. 215(3): p. 403-410. 31.Schultz, J., et al., SMART: a web-based tool for the study of genetically mobile domains. Nucleic Acids Research, 2000. 28(1): p. 231-234. 32.Nelson, G.A., K.K. Lew, and S. Ward, Intersex, a temperature-sensitive mutant of the nematode Caenorhabditis elegans. Developmental Biology, 1978. 66(2): p. 386-409. 33.Barton, M.K., T.B. Schedl, and J. Kimble, Gain-of-function mutations of fem-3, a sex-determination gene in Caenorhabditis elegans. Genetics, 1987. 115(1): p. 107-19. 34.Kuwabara, P.E., The multifaceted C. elegans major sperm protein: an ephrin signaling antagonist in oocyte maturation. Genes Dev, 2003. 17(2): p. 155-61. 35.Großhans, J., et al., RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly during Drosophila cellularisation. Development, 2005. 132(5): p. 1009. 36.Padash Barmchi, M., S. Rogers, and U. Häcker, DRhoGEF2 regulates actin organization and contractility in the Drosophila blastoderm embryo. The Journal of Cell Biology, 2005. 168: p. 575-585. 37.Mazumdar, A. and M. Mazumdar, How one becomes many: Blastoderm cellularization in Drosophila melanogaster. BioEssays, 2002. 24(11): p. 1012-1022. 38.Wenzl, C., et al., Localization of RhoGEF2 during Drosophila cellularization is developmentally controlled by slam. Mechanisms of Development, 2010. 127(7): p. 371-384. 39.Thomas, Y., et al., Cdk1 Phosphorylates SPAT-1/Bora to Promote Plk1 Activation in C. elegans and Human Cells. Cell Reports, 2016. 15(3): p. 510-518. 40.Boxem, M., Cyclin-dependent kinases in C. elegans. Cell Division, 2006. 1: p. 6-6. 41.Nguyen, M.M., et al., Requirement of PDZ-containing proteins for cell cycle regulation and differentiation in the mouse lens epithelium. Mol Cell Biol, 2003. 23(24): p. 8970-81. 42.Maiato, H., P. Sampaio, and C.E. Sunkel, Microtubule-Associated Proteins and Their Essential Roles During Mitosis. International Review of Cytology, 2004. 241: p. 53-153. 43.Carazo-Salas, R.E., et al., Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. Nature, 1999. 400(6740): p. 178-181. 44.Mattaj, I.W. and L. Englmeier, Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem, 1998. 67: p. 265-306. 45.Cesario, J. and K.S. McKim, RanGTP is required for meiotic spindle organization and the initiation of embryonic development in Drosophila. Journal of Cell Science, 2011. 124(22): p. 3797-3810. 46.Ciciarello, M., R. Mangiacasale, and P. Lavia, Spatial control of mitosis by the GTPase Ran. Cellular and Molecular Life Sciences, 2007. 64(15): p. 1891-1914. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66993 | - |
| dc.description.abstract | 在有性生殖的動物中，精子產生於高度特異性的雄性生殖系統，因此在雄性生殖系統中，必定有特殊的調控者參與精子製造的過程。利用線蟲進行蛋白質體分析，SMZ-1及SMZ-2兩個帶有PDZ domain且極為相像的蛋白質，被發現大量表現於精子的染色質。儘管在過去帶有PDZ domain的蛋白質已經被知道參與在細胞訊息傳遞的過程中，然而PDZ domain在成精作用中所扮演的角色尚未被發掘。
為了探討SMZ-1及SMZ-2兩者在線蟲成精作用中扮演的角色，利用CRISPR/Cas9技術，獲得了兩隻基因型不一樣且不會表現SMZ-2的線蟲株，接著藉由交配產生smz-1、smz-2雙重基因剔除線蟲株。首先透過計算雌雄同體線蟲後代產生率和孵化率來分析SMZ-1、SMZ-2是否參與在線蟲生殖發育的過程之中，發現僅剔除smz-1或smz-2其中一基因的線蟲株可以正常產生後代，然而在smz-1; smz-2基因雙剔除的線蟲株無法產生可計數的後代。表示SMZ-1和SMZ-2參與線蟲後代生殖發育並且帶有相同的功能。 接著觀察smz-1;smz-2線蟲株的子宮，發現充滿了未受精的卵，代表著在沒有SMZ-1和SMZ-2的情況下線蟲無法正常受精，而無法受精的原因可能源於精子或卵子的缺陷。為了區分無法受精的原因，我們先將不孕的smz-1;smz-2雌雄同體線蟲株與帶有正常smz-1、smz-2基因的雄性線蟲進行交配，此時smz-1;smz-2線蟲株可以正常受孕，暗示著SMZ-1和SMZ-2僅在精子扮演功能。以性腺只製造精子或卵子的線蟲株進行西方墨點法分析SMZ-1、SMZ-2表現，發現SMZ-1、SMZ-2只表現在製造精子的性腺。由以上實驗結果可以知道SMZ-1、SMZ-2的功能在於製造具有功能的正常精子。 為了瞭解SMZ-1、SMZ-2缺失的情況下對於精子形成過程的影響，我們將smz-1;smz-2雄蟲性腺固定染色，發現與野生株相比smz-1;smz-2在性腺中沒有精子形成。接著透過曠時攝影的方式觀察smz-1;smz-2的精原細胞，發現細胞不僅無法進到第一次中期，同時smz-1;smz-2也製造出體積較小的精原細胞。由於細胞進入中期需要紡錘絲形成，再利用固定染色及曠時攝影的方式，發現紡錘絲在smz-1;smz-2精原細胞中無法正常維持結構。透過以上實驗結果，同時缺乏SMZ-1和SMZ-2使得細胞無法開始減數分裂，進而影響精子形成。 | zh_TW |
| dc.description.abstract | In sexually reproducing animals, sperm are generated in highly-specialized male germline. Therefore, male-specific regulators should be participated in functional sperm production. SMZ-1 and SMZ-2, two highly identical PDZ domain proteins, were found abundant in spermatogenic chromatin through proteomic study in C. elegans. While PDZ domain-containing proteins are known for acting as scaffolds for cell signal transduction, little is known about the roles of PDZ domain-containing proteins in sperm generation.
Since smz-2 deletion strain was not available, we first used CRISPR/Cas9 techniques to generate two smz-2 deletion strains. We then crossed smz-2 deletion worms with smz-1 deletion worms to generate double mutants. To test the roles of SMZ-1 and SMZ-2 in fertility, we compared total brood size and hatch rate of single and double mutant hermaphrodites. We found that worms bearing either smz-1 or smz-2 single deletion can normally generate offspring. However smz-1; smz-2 hermaphrodites do not have any countable progeny. The results show that SMZ-1 and SMZ-2 are functionally redundant in fertility. Through examination of smz-1; smz-2 hermaphrodite uterus revealed the uterus filled with unfertilized oocytes. This suggests fertilization defects happened to smz-1; smz-2 hermaphrodites, which can result from defects in sperm or oocyte function. To distinguish where the defects take place, we examined SMZ-1 and SMZ-2 protein expression in worms generating either sperm or oocytes. Immunoblotting analyses showed that SMZ-1 and SMZ-2 are highly expressed in sperm but not in oocytes or somatic cells. In agreement to this, the infertility in smz-1; smz-2 double mutant hermaphrodites can be rescued by mating with wild type males. These results indicate SMZ-1 and SMZ-2 only function in sperm. In order to define the origin defects of smz-1; smz-2 in sperm generation, we examined fixed smz-1; smz-2 male gonad. We found smz-1; smz-2 male germline failed to generate mature sperm comparing with wild type, suggesting there are meiotic division defects. Time-lapse recording of smz-1; smz-2 spermatocytes showed that chromosomes do not align and the cells are failed to progress into metaphase to enter meiotic division. Additionally, smz-1; smz-2 male tends to generate smaller spermatocytes than wild type male does. Since spindle is a key component for cell to proceed into metaphase, we examined the formation of spindles through staining and time-lapse recording. The results show that smz-1; smz-2 male failed to maintain spindle morphology. In conclusion, we proposed SMZ-1/2 are important to spermatogenesis, absence of the two proteins can result in the failure of generating sperm. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T01:16:37Z (GMT). No. of bitstreams: 1 ntu-106-R04424022-1.pdf: 2588966 bytes, checksum: 0a92ff2a0b193daf49f6c04ec7aef4de (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 謝辭 ................................................................................................................................... i
摘要 .................................................................................................................................. ii Abstract............................................................................................................................ iv Table of Content .............................................................................................................. vi Introduction ...................................................................................................................... 1 Material and Methods....................................................................................................... 5 Worm strains and maintenance................................................................................. 5 Generation of smz-2 deletion animals ...................................................................... 5 Construction of smz-1; smz-2 double mutant strain ................................................. 6 Fertility analyses....................................................................................................... 6 Immunoblotting assay .............................................................................................. 7 Immunostaining........................................................................................................ 8 Live imaging............................................................................................................. 9 Image processing and statistical analyses................................................................. 9 Online resource......................................................................................................... 9 Results .............................................................................................................................11 Generation of smz-2 deletion strains and construction of smz-1; smz-2 doublemutants. ...................................................................................................................11 SMZ-1 and SMZ-2 are functionally redundant for fertility.................................... 12 smz-1; smz-2 are defective in sperm function. ....................................................... 14 SMZ-1 and SMZ-2 are only expressed in spermatogenic germline....................... 14 smz-1; smz-2 hermaphrodite generate abnormal sperm in spermatheca. ............... 15 smz-1; smz-2 males failed to progress into metaphase to generate mature sperm. 16 smz-1; smz-2 spermatocytes failed to maintain meiotic spindle morphology. ....... 19 Discussion....................................................................................................................... 22 The localization and function of SMZ-1 and SMZ-2............................................. 22 Do SMZ-1 and SMZ-2 involve in spermatocytes cellularization?......................... 23 Do SMZ-1 and SMZ-2 regulate cell phase progression in spermatogenic gonad? 24 Do SMZ-1 and SMZ-2 participate in spindle morphology organization?.............. 24 Do SMZ-1 and SMZ-2 regulate chromatin-mediated spindle assembly? .............. 25 Defining the roles of PDZ domains in SMZ-1 and SMZ-2.................................... 25 Reference........................................................................................................................ 27 Figure.............................................................................................................................. 33 Figure 1 SMZ-1 and SMZ-2 are 99% identical...................................................... 33 Figure 2 Flow chart of smz-1; smz-2 double mutant construction ......................... 34 Figure 3 SMZ-1 and SMZ-2 are functionally redundant for fertility. .................... 35 Figure 4 smz-1; smz-2 hermaphrodites generate unfertilized oocytes.................... 36 Figure 5 smz-1; smz-2 hermaphrodites can be rescued by wild type sperm. ......... 37 Figure 6 SMZ-1 and SMZ-2 are specifically expressed in spermatogenic germline. ................................................................................................................................ 39 Figure 7 smz-1; smz-2 hermaphrodites generate abnormal sperm in spermatheca and can stimulate ovulation. ................................................................................... 40 Figure 8 smz-1; smz-2 male failed to generate sperm. ........................................... 42 Figure 9 smz-1; smz-2 spermatocytes fail to progress into metaphase................... 43 Figure 10 smz-1; smz-2 males generate smaller spermatocytes than wild type. .... 44 Figure 11 smz-1; smz-2 forms three different spindle microtubule morphology.... 45 Figure 12 smz-1; smz-2 spermatocytes generate defective spindle microtubules. . 47 Table ............................................................................................................................... 48 Table 1 Strains used in this study ........................................................................... 48 Table 2 Primers used in this study.......................................................................... 50 Appendix ........................................................................................................................ 51 | |
| dc.language.iso | en | |
| dc.subject | 成精作用 | zh_TW |
| dc.subject | PDZ domain | zh_TW |
| dc.subject | SMZ-1 | zh_TW |
| dc.subject | SMZ-2 | zh_TW |
| dc.subject | Spermatogenesis | en |
| dc.subject | SMZ-1 | en |
| dc.subject | PDZ domain | en |
| dc.subject | SMZ-2 | en |
| dc.title | 探討精子特異性蛋白質SMZ-1及SMZ-2調節線蟲成精作用 | zh_TW |
| dc.title | Spermatogenic-specific proteins SMZ-1 and SMZ-2 regulate spermatogenesis in the nematode Caenorhabditis elegans | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳昌熙,莊碧簪,蔡欣祐,楊雅倩 | |
| dc.subject.keyword | 成精作用,SMZ-1,SMZ-2,PDZ domain, | zh_TW |
| dc.subject.keyword | Spermatogenesis,SMZ-1,SMZ-2,PDZ domain, | en |
| dc.relation.page | 51 | |
| dc.identifier.doi | 10.6342/NTU201701351 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-14 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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