Complexities in Bombyx germ cell formation process revealed by Bm-nosO (a Bombyx homolog of nanos) knockout

et al. 2020

Preprint

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.

Section: Discussionmentioning

confidence: 99%

Section: Bmdsxm Knockout In Masc-r Females Induces the Expression Of mentioning

confidence: 99%

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

Yuzawa¹,

et al. 2020

Preprint

“…Bmovo-1 knockdown resulted in degenerated ovaries and markedly fewer oocytes [24]. Bm-nosO is required for the arrangement of eggs in the ovariole and normal morphogenesis of eggs [25]. BmDSX proteins act as transcription factors regulating the expression of genes involved in the development of sexually dimorphic traits [15].…”

Section: Discussionmentioning

confidence: 99%

Section: Bmdsxm Knockout In Masc-r Females Induces the Expression Of mentioning

confidence: 99%

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

Yuzawa¹,

et al. 2020

Preprint

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 of if it promotes male differentiation in germ cells indirectly by inducing the expression of BmdsxM. Results: In this study, we performed genetic analyses using a transgenic line that expressed Masc, as well as various Bmdsx knockout lines. Masc-expressing females express both BmdsxF and BmdsxM and have degenerated ovaries combined with testis-like tissues, which produce sperm. 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. In comparison, 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 can induce male development in germ cells and 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 gonads and 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 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.

“…To get more insight into the gene expression pro le in the gonads of Masc-R/+, Bmdsx MΔ7/ MΔ7 females, we performed qRT-PCR to quantify expression levels of Bmovo-1 and Bm-nosO, both of which are important for oogenesis in the silkworm [34,35]. While Masc-R expression in females suppressed the expression of these two genes (Fig.…”

Section: Bmdsxm Knockout In Masc-r Females Induces the Expression Of mentioning

confidence: 99%

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

Yuzawa¹,

et al. 2020

Preprint

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. However, the importance of Bmdsx in sexual differentiation in germ cells remains unclear. In Drosophila melanogaster, mechanisms regulating sexual differentiation differ between germ cells and somatic cells. dsx is not required within female or male germ cells for sexual development. However, it remains unclear whether this is also the case in other insect species. Results In this study, we performed genetic analyses using a transgenic line that expressed Masc, as well as various Bmdsx knockout lines. Masc-expressing females express both BmdsxF and BmdsxM and have degenerated ovaries combined with testis-like tissues, which produce sperm. 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. In comparison, 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 can induce male development in germ cells and 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 Bmdsx-AS1 lncRNA, which is a testis-specific factor involved in the male-specific splicing of Bmdsx pre-mRNA. Conclusions Combined, our findings suggest that Masc induces male differentiation in gonads and germ cells by interacting with Bmdsx-AS1 lncRNA and enhancing the production of BmdsxM. Unlike Drosophila dsx, BmdsxM was able to induce spermatogenesis in genetically female (ZW) germ cells. To our knowledge, this is the first report indicating the role of dsx in germ cell sexual development differs among insect species.