類3號DNA甲基轉移酶成年睪丸異構體對小鼠雄性生殖細胞發育的重要性

Abstract

男性生殖細胞的分化成熟受到許多分子機制的精細調控。生殖顆粒，是在生殖細胞的細胞質中不斷變化的生物分子凝聚物，其對於生殖細胞的發育和物種的生育能力扮演舉足輕重的角色。在小鼠中，其中一種生殖顆粒為粒線體間水泥（intermitochondrial cement, IMC），也稱為pi-body，是PIWI-piRNA途徑和piRNA生成的主要場所，特別是在減數分裂的精母細胞 (spermatocyte) 中。piRNA是一類與PIWI蛋白結合的小非編碼RNA，主要功能是抑制轉座子（transposable element, TE）的表達。此外，piRNA也通過降解mRNA或啟動轉譯起始來調控編碼蛋白基因。在我們的研究中，我們發現了由Dnmt3l較短的轉錄變體編碼的成年睾丸異構體 (DNA methyltransferase 3-like adult testis isoform, DNMT3L_AT），主要定位於精母細胞和精子細胞 (spermatid) 的細胞質中。隨後，DNMT3L_AT與多餘的細胞質殘餘物一起包裹在殘留體 (residual body) 中並脱去。值得注意的是，DNMT3L_AT與粒線體及粒線體間水泥組成成分中的MILI（PIWI-like 2）和VASA共定位。透過對DNMT3L_AT鄰近的蛋白質分析，我們假設其可能參與piRNA生物合成及其他次細胞RNA富集複合物 (subcelluler RNA-enriched complex) 中的功能。為了進一步探討這一點，我們使用條件式基因剔除 (conditional knockout, cKO) 的方式在減數分裂期間剔除Dnmt3l-at 第11號外顯子，儘管我們懷疑條件式基因剔除中因FVB 品系的Stra8-Cre在 C57BL/6JNarl 背景品系中有不完全剔除的現象，但仍導致小鼠睾丸和精子發育受到缺陷，並且其生殖細胞轉座子失去抑制，也表現出較高的細胞凋亡信號。初步的精子品質檢測顯示，Dnmt3l 條件式剔除附睾中的精子活動力和數量均低於對照組，且形態異常。此外，我們也使用另一種敲除方法是透過刪除Dnmt3l-at特有的外顯子和其潛在的啟動子和增強子，也改變其第11號外顯子中的潛在起始密碼子。在這樣的基因編輯小鼠中，我們發現其睾丸比對照組小，並在組織切片中觀察到生精小管中的生殖細胞耗盡。這些結果表明DNMT3L_AT對小鼠出生後的精子發育至關重要。

Male germ cell differentiation is intricately regulated by multiple biochemical processes. Germ granules, dynamic biomolecular condensates in the cytoplasm of germ cells, are crucial for germ cell development and fertility. In mice, a germ granule–intermitochondrial cement (IMC), also known as pi-body, serves as a primary site where the PIWI-piRNA pathway takes place and piRNAs are generated, especially in meiotic spermatocytes. In our study, we identified DNMT3L_AT (adult testis isoform), encoded by the shorter transcript variants of Dnmt3l, predominantly localized within the cytoplasm of spermatocytes and spermatids. Subsequently, DNMT3L_AT, along with cytoplasmic remnants, is encapsulated within residual bodies and released. Notably, DNMT3L_AT is highly colocalized with mitochondria and the IMC components MILI (PIWIL2), and VASA. Through the analysis of proteins proximal to DNMT3L_AT, we hypothesized its potential involvement in piRNA biogenesis and other functions within subcellular RNA-rich complexes. To investigate this further, the conditional knockout (cKO) method was employed by removing loxP-flanked exon 11 of Dnmt3l during meiosis to nullify Dnmt3l-at translation. This resulted in Dnmt3l cKO mice having smaller testes and spermatogenic defects with LINE-1 derepression and high apoptotic signals, even though incomplete recombination was suspected as the Stra8-Cre transgene generated from the FVB/NJ taking effects in the C57BL/6JNarl background. Under the regional Dnmt3l knockout model, we still demonstrated lower sperm motility and quantity, as well as abnormal sperm morphology such as abrupt bending, in the Dnmt3l cKO epididymis compared to the control littermate. Additionally, an alternative knockout method was applied by removing Dnmt3l-at-specific exons and its potential promoters and enhancers, as well as by editing eight potential start codons in exon 11. In this exon 8~11 fusion model, a complete germ cell depletion in seminiferous tubules of the much smaller testes was observed. These results suggest that DNMT3L_AT is critical for mouse postnatal spermatogenesis.