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 [smRNA-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Vasa（DEAD-box RNA helicase Vasa）是多种动物性腺发育与生育能力维持所必需的蛋白。在果蝇中，Vasa在卵子发生过程中发挥核心功能，包括生殖系干细胞（GSCs）的维持、可移动遗传元件的piRNA沉默、翻译调控以及原始生殖细胞特化。尽管Vasa的重要性不言而喻，但其作用的机制基础以及在精子发生中的精确功能却仍难以阐明。此前多项研究证实，携带vasa突变的雄性个体仍可保持生育能力；但也有研究表明，Vasa对于piRNA介导的星状基因（Stellate genes）抑制是必需的，而该抑制过程是维持雄性生育能力的关键。本研究发现，功能丧失型vasa突变会导致果蝇睾丸内生殖系干细胞的维持能力快速下降，总生殖细胞数量大幅减少，并随时间推移显著降低雄性生育能力。通过对小RNA文库的分析，我们证实当vasa表达缺失时，piRNA的生物发生过程会发生全面崩溃。尽管如此，我们并未在vasa突变体睾丸的早期生殖细胞中观察到细胞死亡事件增多的现象。在vasa突变体背景中引入编码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，由于缺乏对应的piRNA，星状基因出现强烈的去抑制，导致精母细胞的减数分裂进程以及经rhino转基因挽救的雄性个体的生育能力受到破坏。我们的研究表明，Vasa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与增殖，二是在精母细胞阶段抑制星状基因的表达。实验设计：为更深入地探究Vasa在星状基因沉默中的作用机制，我们从vasa突变体果蝇、对照杂合雄性个体，以及携带vasa突变背景下rhi-GFP转基因构建体的雄性个体的睾丸中分离提取总转录组RNA与18~29 nt的小RNA，并利用Illumina测序平台完成文库构建与高通量测序。