Masculinizer (Masc) plays a pivotal role in male sex determination in the silkworm, Bombyx mori. Masc is required for male-specific splicing of B. mori doublesex (Bmdsx) transcripts. The male isoform of Bmdsx (BmdsxM) induces male differentiation in somatic cells, while females express the female isoform of Bmdsx (BmdsxF), which promotes female differentiation in somatic cells. Our previous findings suggest that Masc could direct the differentiation of genetically female (ZW) germ cells into sperms. However, it remains unclear whether Masc directly induces spermatogenesis or if it promotes male differentiation in germ cells indirectly by inducing the expression of BmdsxM. In this study, we performed genetic analyses using the transgenic line that expressed Masc, as well as various Bmdsx knockout lines. We found that Masc-expressing females with a homozygous mutation in BmdsxM showed normal development in ovaries. The formation of testis-like tissues was abolished in these females. On the other hand, Masc-expressing females carrying a homozygous mutation in BmdsxF exhibited almost complete male-specific development in gonads and germ cells. These results suggest that BmdsxM has an ability to induce male development in germ cells as well as internal genital organs, while BmdsxF inhibits BmdsxM activity and represses male differentiation. To investigate whether MASC directly controls male-specific splicing of Bmdsx and identify RNAs that form complexes with MASC in testes, we performed RNA immunoprecipitation (RIP) using an anti-MASC antibody. We found that MASC formed a complex with AS1 lncRNA, which is a testis-specific factor involved in the male-specific splicing of Bmdsx pre-mRNA. Taken together, our findings suggest that Masc induces male differentiation in germ cells by enhancing the production of BmdsxM. Physical interaction between MASC and AS1 lncRNA may be important for the BmdsxM expression in the testis. Unlike in the Drosophila dsx, BmdsxM was able to induce spermatogenesis in genetically female (ZW) germ cells. To the best of our knowledge, this is the first report that the role of dsx in germ cell sexual development is different between insect species.

中文翻译：

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Masculinizer 和 doublesex 的转基因和敲除分析阐明了 doublesex 在蚕 Bombyx mori 生殖细胞性发育中的独特功能。

雄性化因子 (Masc) 在蚕 Bombyx mori 的雄性性别决定中起着关键作用。Masc 是 B. mori doublesex (Bmdsx) 转录本的雄性特异性剪接所必需的。Bmdsx 的雄性同种型 (BmdsxM) 诱导体细胞中的雄性分化，而雌性表达 Bmdsx 的雌性同种型 (BmdsxF)，从而促进体细胞中的雌性分化。我们之前的研究结果表明，Masc 可以指导遗传雌性 (ZW) 生殖细胞分化为精子。然而，目前尚不清楚 Masc 是直接诱导精子发生还是通过诱导 BmdsxM 的表达间接促进生殖细胞的雄性分化。在这项研究中，我们使用表达 Masc 的转基因系以及各种 Bmdsx 敲除系进行了遗传分析。我们发现在 BmdsxM 中具有纯合突变的表达 Masc 的雌性在卵巢中显示出正常发育。在这些雌性中，睾丸样组织的形成被消除了。另一方面，在 BmdsxF 中携带纯合突变的表达 Masc 的雌性在性腺和生殖细胞中表现出几乎完全的雄性特异性发育。这些结果表明 BmdsxM 具有诱导生殖细胞和内生殖器官雄性发育的能力，而 BmdsxF 抑制 BmdsxM 活性并抑制雄性分化。为了研究 MASC 是否直接控制 Bmdsx 的男性特异性剪接并鉴定在睾丸中与 MASC 形成复合物的 RNA，我们使用抗 MASC 抗体进行了 RNA 免疫沉淀 (RIP)。我们发现 MASC 与 AS1 lncRNA 形成复合物，这是参与 Bmdsx 前 mRNA 雄性特异性剪接的睾丸特异性因子。总之，我们的研究结果表明，Masc 通过增强 BmdsxM 的产生来诱导生殖细胞的雄性分化。MASC 和 AS1 lncRNA 之间的物理相互作用可能对睾丸中的 BmdsxM 表达很重要。与果蝇 dsx 不同，BmdsxM 能够在遗传雌性 (ZW) 生殖细胞中诱导精子发生。据我们所知，这是第一次报告 dsx 在生殖细胞性发育中的作用因昆虫物种而异。与果蝇 dsx 不同，BmdsxM 能够在遗传雌性 (ZW) 生殖细胞中诱导精子发生。据我们所知，这是第一次报告 dsx 在生殖细胞性发育中的作用因昆虫物种而异。与果蝇 dsx 不同，BmdsxM 能够在遗传雌性 (ZW) 生殖细胞中诱导精子发生。据我们所知，这是第一次报告 dsx 在生殖细胞性发育中的作用因昆虫物种而异。