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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 吳瑞菁(Jui-Ching Wu) | |
| dc.contributor.author | Ya-Chen Chiu | en |
| dc.contributor.author | 邱雅貞 | zh_TW |
| dc.date.accessioned | 2023-03-19T22:37:08Z | - |
| dc.date.copyright | 2022-10-13 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-09-27 | |
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Wolf, Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells. STAR Protoc, 2021. 2(4): p. 100949. 40. Macdonald, L., et al., Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons. Elife, 2022. 11. 41. Bence, M., et al., Combining the auxin-inducible degradation system with CRISPR/Cas9-based genome editing for the conditional depletion of endogenous Drosophila melanogaster proteins. FEBS J, 2017. 284(7): p. 1056-1069. 42. Trost, M., A.C. Blattner, and C.F. Lehner, Regulated protein depletion by the auxin-inducible degradation system in Drosophila melanogaster. Fly (Austin), 2016. 10(1): p. 35-46. 43. Stiernagle, T., Maintenance of C. elegans. WormBook, 2006: p. 1-11. 44. Brenner, S., The genetics of Caenorhabditis elegans. Genetics, 1974. 77(1): p. 71-94. 45. Martinez, M.A.Q. and D.Q. Matus, Auxin-mediated Protein Degradation in Caenorhabditis elegans. Bio Protoc, 2020. 10(8). 46. Dokshin, G.A., et al., Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis elegans. Genetics, 2018. 210(3): p. 781-787. 47. Mello, C.C., et al., Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J, 1991. 10(12): p. 3959-70. 48. Wu, J.C., et al., Sperm development and motility are regulated by PP1 phosphatases in Caenorhabditis elegans. Genetics, 2012. 190(1): p. 143-57. 49. Jaramillo-Lambert, A., et al., Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line. Dev Biol, 2007. 308(1): p. 206-21. 50. Huang, Y., et al., Autophagy Participates in Lysosomal Vacuolation-Mediated Cell Death in RGNNV-Infected Cells. Front Microbiol, 2020. 11: p. 790. 51. Jin, Y. and L.S. Weisman, The vacuole/lysosome is required for cell-cycle progression. Elife, 2015. 4. 52. Wang, J.Y., DNA damage and apoptosis. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84996 | - |
| dc.description.abstract | 精子生成是雄性性腺中產生成熟精子的獨特過程。先前研究發現，在秀麗隱桿線蟲的精母細胞開始減數分裂前，轉錄就停止了，代表該過程主要依賴轉錄後及轉譯調節，這與我們先前研究發現抑制轉譯會阻止減數分裂之進行相互呼應。 本研究探討精子特異性轉譯調節因子CPB-1在秀麗隱桿線蟲精子生成中的作用。CPB-1是一種細胞質聚腺苷酸化元件結合蛋白 (Cytoplasmic polyadenylation element binding, CPEB)，其被認為可以透過與mRNA 3’端非翻譯區中的特定序列結合來調節轉譯。 為了研究CPB-1在精子生成中的作用，我們透過CRISPR技術將帶有AID*標記的序列嵌入cpb-1基因，以利用生長素誘導降解系統 (Auxin-inducible degradation, AID) 使內源性CPB-1降解。 我們發現在雄性的生殖系統中，CPB-1可在幾小時內被誘導降解，且此降解具有可逆性。此外，缺乏CPB-1的雌雄同體線蟲生育力大幅削弱，但其生育能力在提供正常精子後得以恢復，此結果表明CPB-1對雄性生育能力是不可或缺的，但不影響雌性。 利用免疫螢光觀察CPB-1在雄性生殖腺中的分佈，我們發現CPB-1在雄性減數分裂前期 (prophase) 的細胞質中表達，而消失在減數分裂後期和分裂的精母細胞中，此外，缺乏CPB-1的精母細胞染色體無法正確進行染色質濃縮，而且在性腺中堆積未能正常分裂的精母細胞。縮時影像攝影觀察的結果發現缺乏CPB-1的初級精母細胞大量停滯並伴隨嚴重的染色體分離缺陷，且通常含有非溶酶體的細胞質液泡狀結構。綜合以上，我們發現CPB-1在減數分裂前期調控精子第一次減數分裂時所需之蛋白質生成，以協助配對後的同源染色體再次濃縮及促使精子減數分裂。 最後，為了找出由CPB-1調節的目標mRNA及其下游影響的蛋白質，我們比較正常及CPB-1缺乏的雄性線蟲蛋白質萃取物，初步分析顯示，CPB-1缺乏對雄性線蟲整體蛋白質組成沒有顯著影響，但有數個未知蛋白質的表現明顯低下。這些結果將有助於其後蛋白質體分析之研究，以暸解受CPB-1調控之蛋白質如何促進雄性生育能力。 | zh_TW |
| dc.description.abstract | Spermatogenesis is a unique process in the male gonads to produce mature spermatozoa. Previous studies show that transcription ceases before spermatocyte begins meiotic divisions in nematode Caenorhabditis elegans, indicating the process is mainly regulated at the post-transcriptional and translational levels. Consistently, our previous study showed that inhibition of translation prevented the progression of meiotic division. In this study, we aim to investigate the roles of spermatogenic-specific translation regulator CPB-1 in C. elegans spermatogenesis. CPB-1 is a cytoplasmic polyadenylation element binding (CPEB) protein that is predicted to regulate translation of mRNAs through binding to specific sequences in the 3' untranslated region. To investigate the roles of CPB-1 in spermatogenesis, we CRISPR-engineered an AID* tag inserted into genomic cpb-1, allowing Auxin-inducible degradation of endogenous CPB-1. We showed that CPB-1 degradation can be reversibly induced in both male germline within a few hours. CPB-1-depleted hermaphrodites showed significant sterility that could be rescued with wild-type sperm, suggesting that CPB-1 is essential in male but not female fertility. Results from immunofluorescence staining showed that CPB-1 is expressed in the cytoplasm of male meiotic prophase, and is absent from late meiotic and dividing spermatocytes. Also, paired homologous chromosomes failed to undergo proper condensation stages, leading to accumulation of immature spermatocytes in CPB-1-depleted males. Time-lapse recordings revealed that CPB-1-depleted spermatocytes exhibited severe chromosomal segregation defects and failed to initiate the first meiotic division. Additionally, CPB-1-depleted spermatocytes often contain cytoplasmic vacuole-like structures that were not lysosomal origin. Taken together, we hypothesize that during male meiotic prophase, CPB-1 regulates the production of proteins essential for late meiotic chromosome condensation and initiation of male meiotic division. To identify the target mRNAs regulated by CPB-1 and their downstream proteins, we compared male worm extracts generated from wild-type and CPB-1-depleted male worms. Preliminary analysis revealed that CPB-1 depletion does not impact global proteome in male worms, however, a few proteins were significantly reduced. These results facilitate further proteomic analyses to elucidate how CPB-1-dependent proteostasis contributes to male fertility. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T22:37:08Z (GMT). No. of bitstreams: 1 U0001-2309202214190300.pdf: 11503156 bytes, checksum: 2131c02bd9cd7c4da9f85484c0464759 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 口試委員審定書 i 誌謝 ii 摘要 iii Abstract v Chapter 1 -Introduction 1 Chapter 2 -Materials and Methods 10 2.1 C. elegans strains and maintenance 10 2.2 AID*::3xFLAG::CPB-1 strain construction 10 2.3 Auxin-inducible degradation (AID) experiment 11 2.4 Brood size and hatch rate assay 12 2.5 Immunofluorescence staining 13 2.6 Time-lapse microscopy 14 2.7 LysoTracker labeling 15 2.8 Preparation of whole male worm extraction 15 2.9 Biochemical protein analyses 16 2.9.1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 16 2.9.2 Silver staining 16 2.9.3 Immunoblotting assay 16 2.10 Image processing and statistical analyses 17 Chapter 3 -Results 18 3.1 Generation of inducible CPB-1 depleted strain. 18 3.2 Optimization of Auxin-inducible degradation (AID) in the germline. 19 3.3 CPB-1 functions in sperm production contributing to fertility. 21 3.4 CPB-1 localizes in the cytoplasm of the pachytene zone before meiotic division in the male germline. 22 3.5 CPB-1 depleted spermatocytes lack karyosome and accumulate at the metaphase-like stage. 23 3.6 Spermatogonia lacking CPB-1 were mostly stuck in the primary spermatocyte stage and unable to undergo meiotic division. 24 3.7 CPB-1 depletion caused vacuolization of spermatocyte cytoplasm. 26 3.8 CPB-1 depletion does not impact global proteome in male worms. 27 Chapter 4 -Discussion 29 4.1 CPB-1 is expressed in the cytoplasm of the pachytene zone, and forms the granular aggregates around the nucleus in the male germline. 29 4.2 The non-specific binding of anti-FLAG antibody showed in immunofluorescence staining and Western blotting. 30 4.3 CPB-1-depleted spermatocytes lacked tubular structure and showed vacuole-like structure in the cytoplasm. 31 4.4 The hypothetical mechanism of CPB-1 regulates translation and contributes to abnormal male meiosis. 32 4.5 Potential target mRNAs regulated by CPB-1. 33 Figure 35 Figure 1. Overview of the study: CPB-1 modulates translation of key proteins during spermatogenesis. 35 Figure 2. Construction of AID*::3xFLAG::cpb-1 transgenic strain. 36 Figure 3. AID-tagged proteins degradation optimizes in both hermaphrodite and male C. elegans germline. 38 Figure 4. CPB-1 depletion hermaphrodites reduced progeny dues to sperm defects. 40 Figure 5. CPB-1 could be degraded by 1 mM K-NAA after 2 hours treatment in male germline. 41 Figure 6. CPB-1 localizes in the cytoplasm of the pachytene zone in the male gonad. 42 Figure 7. CPB-1 depletion causes absence of karyosome stage and accumulation of metaphase-like spermatocytes in the male germline. 45 Figure 8. CPB-1 depletion caused various phenotypes during spermatogenesis. 48 Figure 9. Comparison of protein extracts from control and CPB-1-depleted male worms. 50 Table 51 Table 1. The C. elegans strain used in this study. 51 Table 2. Plasmid used in this study. 52 Table 3. Oligonucleotides used in this study. 53 Reference 54 Appendix 62 | |
| dc.language.iso | en | |
| dc.subject | CPB-1 | zh_TW |
| dc.subject | 精子生成 | zh_TW |
| dc.subject | 生長素誘導蛋白質降解 | zh_TW |
| dc.subject | 減數分裂 | zh_TW |
| dc.subject | CPEB | zh_TW |
| dc.subject | Auxin-induced degradation | en |
| dc.subject | Spermatogenesis | en |
| dc.subject | CPEB | en |
| dc.subject | CPB-1 | en |
| dc.subject | meiosis | en |
| dc.title | 以線蟲為模型探討雄性減數分裂特異性CPEB調控蛋白CPB-1在精子生成過程扮演的角色 | zh_TW |
| dc.title | Investigating the roles of male meiosis-specific CPEB protein CPB-1 during spermatogenesis in Caenorhabditis elegans. | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 薛雁冰(Yen-Ping Hsueh),王齡玉(Ling-Yu Wang),蔡欣祐(Hsin-Yue Tsai),郭靜穎(Ching-Ying Kuo) | |
| dc.subject.keyword | 精子生成,CPEB,CPB-1,減數分裂,生長素誘導蛋白質降解, | zh_TW |
| dc.subject.keyword | Spermatogenesis,CPEB,CPB-1,meiosis,Auxin-induced degradation, | en |
| dc.relation.page | 62 | |
| dc.identifier.doi | 10.6342/NTU202203909 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-09-28 | |
| 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 |
| dc.date.embargo-lift | 2022-10-13 | - |
| Appears in Collections: | 醫學檢驗暨生物技術學系 | |
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| U0001-2309202214190300.pdf Access limited in NTU ip range | 11.23 MB | Adobe PDF |
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