Essential functions of RNA helicase Vasa in Drosophila spermatogenesis, from maintenance of germline stem cells to passage through meiosis between Drosophila melanogaster and Drosophila simulans [RNA-seq]. Essential functions of RNA helicase Vasa in Drosophila spermatogenesis, from maintenance of germline stem cells to passage through meiosis between Drosophila melanogaster and Drosophila simulans [RNA-seq]

DEAD-box RNA helicase Vasa is required for gonad development and fertility in multiple animals. In Drosophila, Vasa performs essential functions in oogenesis, including the maintenance of germline stem cells (GSCs), piRNA silencing of mobile elements, translation regulation, and primordial germ cell specification. Despite its evident significance, the mechanistic basis of Vasa action and its precise role in spermatogenesis become incomprehensible. Several papers affirm the fertility of males carrying vasa mutations. However, it is also shown that Vasa is essential for piRNA-mediated repression of Stellate genes needed for the maintenance of male fertility.Here we found that loss-of-function vasa mutations led to a rapid decline in GSC maintenance in the testes, a severe loss of total germ cell content, and a strong decrease in male fertility over time. With the aid of analysis of small RNA libraries, we revealed that collapse of piRNA biogenesis in the absence of vasa expression. Despite that, we did not reveal increasing cell death events in the early germ cells of vasa mutant testes. The introduction of the transgene rhino copy, encoding a nuclear component of the piRNA pathway, in vasa mutant background allowed us to rescue premeiotic stages of spermatogenesis, including GSC maintenance and the development of spermatogonia and spermatocytes. However, the progression of spermatocytes through meiosis and the fertility of the rhino transgene-rescued males were disrupted by strong Stellate gene derepression owing to the absence of corresponding piRNAs. We have shown that Vasa functions in spermatogenesis are essential at two separate developmental stages: in GSCs for their maintenance and in spermatocytes for the repression of Stellate genes. Overall design: To study the contribution of Vasa in Stellate silencing in more detail, we prepared and sequenced on the Illumina platform whole-transcriptome libraries and 18–29 nt small RNAs isolated from the testes of vasa mutants, control heterozygous males, and males with the transgenic construct rhi-GFP in the background of vasa mutations.

DEAD盒RNA解旋酶（DEAD-box RNA helicase）瓦萨蛋白（Vasa）在多种动物的性腺发育与生育维持过程中发挥必需作用。在果蝇（Drosophila）中，瓦萨蛋白在卵子发生过程中执行核心功能，包括维持生殖系干细胞（GSCs）、对转座因子进行PIWI互作RNA（piRNA）沉默、翻译调控以及原始生殖细胞特化。尽管其功能重要性不言而喻，但瓦萨蛋白作用的分子机制及其在精子发生中的精确功能长期以来难以阐明。已有多项研究证实携带vasa基因突变的雄性果蝇仍可保持生育能力，然而也有研究表明，瓦萨蛋白对于piRNA介导的星状基因（Stellate genes）沉默是必需的，而该沉默过程对雄性生育维持至关重要。
本研究发现，功能丧失型vasa基因突变会导致睾丸内生殖系干细胞的维持能力快速下降，总生殖细胞数量严重减少，并随时间推移显著降低雄性生育力。通过对小RNA文库的分析，我们揭示了在瓦萨蛋白表达缺失的情况下piRNA生物发生过程发生崩溃。尽管如此，我们并未在vasa突变体睾丸的早期生殖细胞中检测到细胞死亡事件的增加。
在vasa突变背景下引入编码piRNA通路核组分的rhino转基因拷贝，可挽救精子发生的减数分裂前阶段，包括生殖系干细胞的维持以及精原细胞和精母细胞的发育。然而，由于缺乏对应piRNA，星状基因出现严重去抑制，这破坏了精母细胞的减数分裂进程以及携带rhino转基因挽救株的雄性生育能力。我们证实，瓦萨蛋白在精子发生中的功能在两个独立的发育阶段发挥关键作用：一是在生殖系干细胞阶段维持其存活，二是在精母细胞阶段对星状基因进行沉默。
整体实验设计：为更深入研究瓦萨蛋白在星状基因沉默中的作用，我们从vasa突变体果蝇、对照杂合雄性果蝇，以及携带vasa突变背景且整合rhi-GFP转基因构建体的雄性果蝇的睾丸中分离总RNA，构建了全转录组文库与18–29 nt小RNA文库，并在Illumina测序平台上完成测序。