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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林劭品 | zh_TW |
| dc.contributor.advisor | Shau-Ping Lin | en |
| dc.contributor.author | 歐德仁 | zh_TW |
| dc.contributor.author | Dymbren Ochirov | en |
| dc.date.accessioned | 2026-03-18T16:09:36Z | - |
| dc.date.available | 2026-03-19 | - |
| dc.date.copyright | 2026-03-18 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2026-01-26 | - |
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Q., Li, T., Liu, X., Zhao, S., Chen, C., Li, J., Wang, X., Ding, D., & Liu, M. F. (2024). piRNA loading triggers MIWI translocation from the intermitochondrial cement to chromatoid body during mouse spermatogenesis. Nat Commun, 15(1), 2343. https://doi.org/10.1038/s41467-024-46664-3 Wood, G. S., & Warnke, R. (1981). Suppression of endogenous avidin-binding activity in tissues and its relevance to biotin-avidin detection systems. J Histochem Cytochem, 29(10), 1196-1204. https://doi.org/10.1177/29.10.7028859 Wu, C.-H. (2024). DNA Methyltransferase 3-Like Adilt Testis Isoform (DNMT3L_AT) is Essential for Mouse Male Germ Cell Development [Master Thesis, National Taiwan University]. Yan Cheng, C., & Mruk, D. D. (2015). Biochemistry of Sertoli cell/germ cell junctions, germ cell transport, and spermiation in the seminiferous epithelium. In M. D. Griswold (Ed.), Sertoli Cell Biology (pp. 333-383). 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/102177 | - |
| dc.description.abstract | DNA methyltransferase 3-like「Adult Testis」（DNMT3L_AT）是由 Dnmt3l 基因較短轉錄變異所編碼的新型蛋白異構體。它主要表現在減數分裂期與減數分裂後的雄性生殖細胞之細胞質中，其分布樣式有別於在胚胎期前精原細胞中促進 DNA 甲基化的典型「Stem cell」形式 DNMT3L_S。我以免疫螢光自早期 Pachytene 精母細胞起，直至精子發生的倒數第二階段，系統性描繪 DNMT3L_AT 的表現。於減數分裂期精母細胞中，我證實 DNMT3L_AT 與生殖顆粒（germ granule）共定位。此時期之生殖顆粒名為粒線體間膠；（Intermitochondrial Cement, IMC），包含VASA、MILI 與 TDRKH等組成份。雄鼠生殖細胞發育至圓形精細胞（round spermatid）時期，生殖顆粒凝聚為單一的 RNA-蛋白凝縮體 – chromatoid body 其中包含 VASA、MILI 與 MIWI等蛋白組成；然而 DNMT3L_AT 於圓形精細胞中仍分散於細胞質內，未被納入 chromatoid body。我發現 DNMT3L_AT 與粒線體在整個精子發生過程中持續共定位，直至伸長型精細胞 (elongated) 時期於成形中之精子尾部中段出現粒線體互鎖為止。結構預測顯示 DNMT3L_AT 可能與精子形成（spermiogenesis）期間，與粒線體在精子尾中斷互鎖現象相關之ARMC12 蛋白具有強結合潛力。由於ARMC12 與 TBC1D21 及 VDAC2/3 於精子尾部形成階段共同促成粒線體互鎖，且結構分析預測 ARMC12 與 DNMT3L_AT 或與 TBC1D21‑VDAC2/3 的結合呈互斥，我們推測 DNMT3L_AT 可暫時「扣留」ARMC12，以防止粒線體過早互鎖。我觀察到 Dnmt3l_AT 特異性剔除（E8-11Fusion）小鼠一些細精管（seminiferous tubules）的精子發生的嚴重缺陷，包括 Leptotene/Zygotene 期生殖細胞異常增加、晚期 Pachytene 精母細胞顯著減少，以及圓形精子細胞幾近全缺。進一步分析成鼠突變體之副睪尾部精子運動性，顯示總運動力與前向運動力皆下降。此外，在 Protamine‑Cre 誘導之條件性 Dnmt3l 剔除中，我確認伸長型精細胞的 DNMT3L_AT 部分下降，並可能導致精子中段（midpiece）之粒線體過早排列；其副睪尾部精子運動性同樣呈現總運動力與前向運動力的異常降低。
總結而言，我們的研究成果預測 DNMT3L_AT在雄鼠生殖細胞發育之不同階段具有不同的功能模式：其一，於減數分裂期促進生殖顆粒（IMC）中RNA與蛋白分子凝聚體結構之形成；其二，於整個精子發生過程中作為粒線體動態的調節者。 | zh_TW |
| dc.description.abstract | DNA methyltransferase 3-like “Adult Testis” (DNMT3L_AT) is a novel protein isoform encoded by shorter transcription variants of the Dnmt3l gene. It primarily expressed in the cytoplasm of meiotic and post-meiotic male germ cells, displaying a distribution pattern different from that of the canonical “Stem cell” DNMT3L_S form, which facilitates DNA methylation in embryonic prospermatogonia. I characterized DNMT3L_AT expression via immunofluorescent studies from early pachytene spermatocytes to the penultimate spermatogenesis stages and confirmed its colocalization with germ granule (intermitochondrial cement; IMC) components VASA, MILI and TDRKH in meiotic spermatocytes. In round spermatids, the germ granule is composed of one single condensed RNA-protein condenstate called the chromatoid body, which includes VASA, MILI and MIWI. DNMT3L_AT remained dispersed in the cytoplasm and excluded from the chromatoid body in round spermatids. I discovered a consistent colocalization of DNMT3L_AT with mitochondria throughout spermatogenesis until mitochondria interlocking in elongating spermatozoa. Structural prediction suggested a strong binding between DNMT3L_AT with ARMC12, a factor required for mitochondrial interlocking with TBC1D21 and VDAC2/3 during spermiogenesis. Since ARMC12 binding to DNMT3L_AT or TBC1D21‑VDAC2/3 is predicted to be mutually exclusive, we suggest that DNMT3L_AT sequesters ARMC12 to prevent premature interlocking. I characterized Dnmt3l_AT-specific knockout (E8-11Fusion) mice and observed profound defects in spermatogenesis, including aberrantly increased Leptotene/Zygotene germ cell population, significantly decreased late Pachytene spermatocytes, and near‑complete absence of round spermatids in affected seminiferous tubules. Further analysis of adult mutants’ caudal epididymal sperm motility showed reduction in total and progressive motility. Furthermore, in conditional Dnmt3l knockout under Protamine‑Cre induction, I confirmed partial reduction of DNMT3L_AT in elongating spermatids with potential premature alignment of mitochondria at the midpiece of spermatozoa. Caudal epididymis sperm motility showed abnormal decrease in total and progressive motility. In conclusion, we discovered different modes of DNMT3L_AT’s functionality: a potential structural facilitator in meiotic germ granules (IMC), and a mitochondrial dynamics regulator throughout spermatogenesis. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2026-03-18T16:09:36Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2026-03-18T16:09:36Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | ACKNOWLEDGEMENTS I
摘要 II ABSTRACT IV CONTENTS VI LIST OF FIGURES X LIST OF TABLES XI ABBREVIATIONS XII CHAPTER I INTRODUCTION 1 1.1. SPERMATOGENESIS 1 1.1.1. Primordial germ cell specification during embryogenesis 2 1.1.2. Postnatal spermatogenesis 2 1.1.2.1. Postnatal reactivation and stem cell pool establishment 2 1.1.2.2. Meiotic initiation and spermatogenesis progression 3 1.1.2.3. Spermiogenesis 4 1.2. DNA METHYLTRANSFERASE 3-LIKE (DNMT3L) 6 1.2.1. DNMT3L overview 7 1.2.2. Embryonic gonocytes development 7 1.2.3. Butterfly DNMT3L-DNMT3A2 heterotetramer for proper DNA methylation in prospermatogonia 7 1.2.4. Postnatal expression dynamics 8 1.2.5. Male DNMT3L_S loss-of-function 8 1.2.6. Sex-specific Dnmt3l promoters, transcripts and proteins 8 1.2.7. Elucidating functions of DNMT3L_AT 9 1.2.7.1. In-house “S”-specific α-DNMT3L_SN distinguishes isoforms of DNMT3L 9 1.2.7.2. DNMT3L_AT associates with germ granule components in spermatocytes, but remains in cytoplasm and excluded from chromatoid body in spermatids 10 1.2.7.3. Generation of the Stra8-Cre mediated Dnmt3l conditional knockout model 11 1.2.7.4. Germ cell defects are observed in Dnmt3l_AT cKO mutants 12 1.3. MITOCHONDRIA AND GERM GRANULES DYNAMICS 13 1.3.1. Mitochondria in embryonic prospermatogonia 14 1.3.2. Postnatal mitochondrial dynamics 14 1.3.2.1. Mitochondria in spermatogonia stem cells 15 1.3.2.2. Mitochondria in meiotic spermatocytes 16 1.3.2.3. Mitochondria in spermiogenesis 17 1.4. AIMS OF MY RESEARCH 19 CHAPTER II MATERIALS AND METHODS 20 2.1. Mouse strains and breeding schemes 20 2.1.1. Transgenes and targeted alleles 20 2.1.2. Breeding strategies 21 2.1.3. Genotyping 22 2.2. Immunohistochemistry (Cryosection) 23 2.2.1. Tissue preparation 23 2.2.2. Indirect Immunohistochemistry 23 2.2.3. Direct immunohistochemistry 24 2.2.4. Indirect immunofluorescent staining for super-resolution STED microscopy 26 2.3. Confocal imaging 27 2.4. Spermatogenesis stages identification 28 2.5. Western Blot Analysis 29 2.5.1. Protein extraction 29 2.5.2. Western Blot and Transfer 30 2.6. Sperm motility test 31 2.7. Protein-protein conformation modeling on BioID dataset 32 CHAPTER III RESULTS 33 3.1. Stage-specific differential expression and subcellular localization of DNMT3L isoforms during male germ cell development 33 3.2. Colocalization and potential functional interplay between DNMT3L_AT and mitochondria during male germ cell differentiation 35 3.3. Potential Role of DNMT3L_AT in Mitochondrial Alignment During Mouse Spermiogenesis 38 3.4. DNMT3L_AT is necessary during the pachytene stage of meiosis, potentially acting as scaffolding protein that tethers intermitochondrial cement (IMC) ribonuclear components 40 3.5. Conditional knockout of Dnmt3l_AT reveals its critical function during spermiogenesis 43 CHAPTER IV DISCUSSION 46 4.1. DNMT3L_AT as a Molecular Scaffold for Intermitochondrial Cement (IMC) and RNA Regulation 46 4.2. DNMT3L_AT's Potential Role in Mitochondrial Dynamics and Sperm Motility 48 4.3. Broader Implications and Future Directions 50 4.4. Conclusion 52 REFERENCES 108 | - |
| dc.language.iso | en | - |
| dc.subject | 類3號DNA 甲基轉移酶異構體 | - |
| dc.subject | 精子發生 | - |
| dc.subject | 精子形成 | - |
| dc.subject | 粒線體 | - |
| dc.subject | 生殖顆粒 | - |
| dc.subject | DNMT3L_AT | - |
| dc.subject | spermatogenesis | - |
| dc.subject | spermiogenesis | - |
| dc.subject | mitochondria | - |
| dc.subject | germ granules | - |
| dc.title | 類3號DNA 甲基轉移酶異構體 – 減數分裂及精子生成過程中粒線體重組的關鍵調控中介者 | zh_TW |
| dc.title | DNA Methyltransferase 3-like Adult Testis Isoform – A Potential Key Regulator of Meiosis and Mediator of Mitochondrial Reorganization in Spermiogenesis | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 114-1 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 蔡沛學;張原翊;林盈宏 | zh_TW |
| dc.contributor.oralexamcommittee | Pei-Shiue Tsai;Yuan-I Chang;Ying-Hung Lin | en |
| dc.subject.keyword | 類3號DNA 甲基轉移酶異構體,精子發生精子形成粒線體生殖顆粒 | zh_TW |
| dc.subject.keyword | DNMT3L_AT,spermatogenesisspermiogenesismitochondriagerm granules | en |
| dc.relation.page | 117 | - |
| dc.identifier.doi | 10.6342/NTU202600314 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2026-01-27 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 生物科技研究所 | - |
| dc.date.embargo-lift | N/A | - |
| 顯示於系所單位： | 生物科技研究所 | |
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