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

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。

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.