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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林劭品(Shau-Ping Lin) | |
| dc.contributor.author | Jen-Yun Chang | en |
| dc.contributor.author | 張人允 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:40:23Z | - |
| dc.date.copyright | 2020-08-24 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18943 | - |
| dc.description.abstract | 類3號DNA甲基化轉移酶（DNA methyltransferase 3-like, DNMT3L）為表觀遺傳調節分子，協助胚胎發育中新生DNA甲基化之建立。在DNMT3L 缺失的小鼠中，轉座子表現失調並且導致生精作用中斷，造成雄性不育。本研究室發現新穎的DNMT3L蛋白異構物，由於此異構物之RNA最初於成年小鼠睪丸被證實，同樣地此蛋白異構物大量被表現於成年小鼠睪丸中，故命名為DNMT3L_AT (adult testis)。DNMT3L_AT 主要座落在精母細胞 (spermatocytes) 與精細胞 (spermatids) 之細胞質中，不同於典型DNMT3L_S (stem cell) 多存在於前精原細胞 (prospermatogonia) 與精原細胞 (spermatogonia) 之細胞核當中。鑑於其胞內位置與表現時期的差異，我們推論此DNMT3L_AT具有不同於典型構型DNMT3L_S的生物功能。本研究試圖利用in vivo以及in vitro 的策略來驗證此蛋白並且探索其交互作用因子以及潛在功能探討。研究結果顯示在5%-40%蔗糖密度梯度離心分離中，發現DNMT3L_AT 座落於germ granule的成員如VASA 與PIWI 聚集的分層當中。免疫染色也得到DNMT3L_AT與在intermitochondrial cement (IMC) 中表現的 MILI以及chromatoid body (CB)之標記 VASA具有高度訊號重疊，而與MIWI的交互作用經由免疫沈澱實驗佐證。因此我推測其參與germ granule的功能如調節piRNA生成與作用、信使RNA儲存以及其他後轉錄修飾功能。 | zh_TW |
| dc.description.abstract | DNA methyltransferase 3-like (DNMT3L) is an important epigenetic modulator which facilitates de novo DNA methylation by cooperating with other DNA methyltransferases (DNMTs). In Dnmt3l knockout mice, males are infertile because of the spermatogenic arrest in early meiosis and derepression of transposons elements (TEs). Strikingly, our lab identifies a novel protein isoform of DNMT3L, DNMT3L adult testis form (DNMT3L_AT), in mouse testes. It is a much smaller protein compared with canonical DNMT3L stem cell form (DNMT3L_S). I confirmed the transcriptional variants and protein expression in postnatal testes of different developmental stages. Spermatocytes and spermatids are the major developmental stages with high DNMT3L_AT expression level. Besides, the subcellular pattern of DNMT3L_AT displays granule-like structures in the cytoplasm, which is distinct from DNMT3L_S that mainly resides in the nucleus. Based on the characteristic protein size, subcellular localization, and the expression period, we deduced that DNMT3L_AT has functions beyond epigenomic modulation during spermatogenesis. I analyzed male germ cell lysates separated in the 5%-40% sucrose density gradient. DNMT3L_AT coexists in the fractions enriched with germ granule’s proteins. Co-immunoprecipitation assay shows potential interaction of DNMT3L_AT with MIWI, the only PIWI protein expressed at later spermatogenesis. In spermatocytes and spermatids, there are substantial overlaps between the immunofluorescent signal of DNMT3L_AT with that of MILI or VASA, marking germ granules, i.e., intermitochondrial cement (IMC) and chromatoid body (CB). IMC or CB has been suggested as the subcellular membrane-less compartment accommodating mRNA regulation and small RNA mediated processes. With potential interaction between DNMT3L_AT and germ granules, we propose that DNMT3L_AT may play roles in post-transcriptional gene regulation for later male germ cell development. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:40:23Z (GMT). No. of bitstreams: 1 U0001-1808202001273600.pdf: 27289942 bytes, checksum: d46a95e6640ec1da6f24db5609a88d65 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書 # 誌謝 i 中文摘要 ii ABSTRACT iii CONTENTS v LIST OF FIGURES viii LIST OF TABLES x Abbreviations xi Introduction Predicament of infertility 1 Male germ cell development 2 DNA (cytosine-5)-methyltransferase-3-like 4 Piwi-interacting RNA system 7 Germ granule 10 Significance and aim of this research 11 Materials and Methods Dnmt3l mutant mouse model 14 RNA extraction 15 Reverse transcription 16 Male germ cell enrichment from testes 17 Protein extraction 18 Western blotting 19 Immunocytochemistry staining (ICC staining) 21 Immunohistochemistry (IHC staining) 22 Immunoprecipitation 23 Cell culture 24 Plasmid construction 25 Density gradient ultracentrifugation 26 Results Transcripts and protein of Dnmt3l isoform are detected in postnatal mouse testes 27 DNMT3L_AT is expressed in late spermatocytes and spermatids, and the signal is mainly located in cytoplasm 28 DNMT3L_AT protein and antibody specificity were validated by expressing ectopic recombinant tag-DNMT3L_AT in 3T3 cells 29 DNMT3L_AT existing in the fraction enriched with components of germ granule, and interaction between DNMT3L_AT and germ granule 30 DNMT3L_AT showing colocalization with MILI and VASA in spermatocytes, yet limited in round spermatids 31 Potential interaction between MIWI and DNMT3L_AT 32 Discussion Features, expressing timing, and potential functions of DNMT3L isoform in postnatal male germ cells 35 Exploring DNMT3L_AT function by revealing its interacting partners 37 Mouse model of DNMT3L_AT specific knockout 39 Other potential protein isoforms of DNMT3L in mouse and human 41 The roles of DNMT3L_S in postnatal testes 42 Conclusion and perspectives 44 Appendix 74 REFERENCES 78 | |
| dc.language.iso | en | |
| dc.subject | 生精作用 | zh_TW |
| dc.subject | 生殖顆粒 | zh_TW |
| dc.subject | 類3號甲基化轉移酶異構物 | zh_TW |
| dc.subject | DNMT3L isoform | en |
| dc.subject | Spermatogenesis | en |
| dc.subject | germ granule | en |
| dc.title | 雄鼠減數分裂生殖細胞表現之 新型類3號DNA甲基酶異構體研究 | zh_TW |
| dc.title | Novel DNA Methyltransferase 3-Like Isoforms in Meiotic Male Germ Cells: Beyond Epigenomic Modulation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 宋麗英(Li-ying Sung),陳全木(Chuan-Mu Chen),靖永皓(Yung-Hao Ching),張原翊(Yuan-I Chang) | |
| dc.subject.keyword | 生精作用,類3號甲基化轉移酶異構物,生殖顆粒, | zh_TW |
| dc.subject.keyword | Spermatogenesis,DNMT3L isoform,germ granule, | en |
| dc.relation.page | 86 | |
| dc.identifier.doi | 10.6342/NTU202003902 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-20 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 生物科技研究所 | zh_TW |
| 顯示於系所單位： | 生物科技研究所 | |
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