Transgenic and knockout analyses of Masculinizer and doublesex illuminated the unique functions of doublesex in germ cell sexual development of the silkworm, Bombyx mori

Abstract

Background 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. Results 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. Conclusions 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.

Transcriptome analysis in the silkworm Bombyx mori overexpressing piRNA-resistant Masculinizer gene

“…In D. melanogaster , the master-feminizing gene Sxl also functioned in germ cell meiosis and differentiation [37]. According to the B. mori genome database (SilkDB 3.0 [38]) and a previous study [39], Masc is expressed in both somatic and germ tissues. Additionally, Masc is involved in developing the female external genitalia [40], suggesting that Masc has some unknown functions in both sexes.…”

In Vitro Comparison of Sex-Specific Splicing Efficiencies of fem Pre-mRNA under Monoallelic and Heteroallelic Conditions of csd, a Master Sex-Determining Gene in the Honeybee

“…BmN cells transfected with the fem minigene and the empty vector (pIEx-1) served as negative controls. Three days after transfection, the cells were harvested and whole cell lysates were prepared according to a previously described protocol [ 21 , 22 ]. Then, 20 μL of Dynabeads Protein G (Invitrogen, Waltham, MA, USA) was incubated with 3.5 μg of anti-V5 tag antibody (ProteinTech Group, Rosemont, IL, USA) at room temperature for 10 min with gentle agitations.…”