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

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Tomohisa Yuzawa ◽  
Misato Matsuoka ◽  
Megumi Sumitani ◽  
Fugaku Aoki ◽  
Hideki Sezutsu ◽  
...  
Keyword(s):  
Germ Cell ◽  
Bombyx Mori ◽  
Germ Cells ◽  
Sexual Development ◽  
Somatic Cells ◽  
Specific Factor ◽  
Physical Interaction ◽  
Homozygous Mutation ◽  
Rna Immunoprecipitation ◽  
Male Specific

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.

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

2020 ◽  
Author(s):  
Tomohisa Yuzawa ◽  
Misato Matsuoka ◽  
Megumi Sumitani ◽  
Fugaku Aoki ◽  
Hideki Sezutsu ◽  
...  
Keyword(s):  
Germ Cell ◽  
Bombyx Mori ◽  
Germ Cells ◽  
Sexual Development ◽  
Somatic Cells ◽  
Specific Factor ◽  
Physical Interaction ◽  
Homozygous Mutation ◽  
Male Specific ◽  
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 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.

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

2020 ◽  
Author(s):  
Tomohisa Yuzawa ◽  
Misato Matsuoka ◽  
Megumi Sumitani ◽  
Fugaku Aoki ◽  
Hideki Sezutsu ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Sexual Development ◽  
Somatic Cells ◽  
Specific Factor ◽  
Physical Interaction ◽  
Homozygous Mutation ◽  
Rna Immunoprecipitation ◽  
Male Specific

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.

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

2020 ◽  
Author(s):  
Tomohisa Yuzawa ◽  
Misato Matsuoka ◽  
Megumi Sumitani ◽  
Fugaku Aoki ◽  
Hideki Sezutsu ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Sexual Development ◽  
Somatic Cells ◽  
Specific Factor ◽  
Physical Interaction ◽  
Homozygous Mutation ◽  
Rna Immunoprecipitation ◽  
Male Specific

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.

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

2020 ◽  
Author(s):  
Tomohisa Yuzawa ◽  
Misato Matsuoka ◽  
Megumi Sumitani ◽  
Fugaku Aoki ◽  
Hideki Sezutsu ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Sexual Development ◽  
Sexual Differentiation ◽  
Somatic Cells ◽  
Insect Species ◽  
Specific Factor ◽  
Homozygous Mutation ◽  
Rna Immunoprecipitation ◽  
Male Specific

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. 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.

scholarly journals Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1813-1819 ◽  
Author(s):  
Eri Shiraishi ◽  
Norifumi Yoshinaga ◽  
Takeshi Miura ◽  
Hayato Yokoi ◽  
Yuko Wakamatsu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Germ Cells ◽  
Sex Differentiation ◽  
Somatic Cells ◽  
Oryzias Latipes ◽  
Cell Number ◽  
Gonadal Differentiation ◽  
Loss Of Function ◽  
Germ Cell Proliferation

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-β superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2′-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

scholarly journals Abnormal Meiosis Initiation in Germ Cell Caused by Aberrant Differentiation of Gonad Somatic Cell

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Min Chen ◽  
Min Chen ◽  
Suren Chen ◽  
Jingjing Zhou ◽  
Fangfang Dong ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Germ Cell ◽  
Somatic Cell ◽  
Germ Cells ◽  
Somatic Cells ◽  
Ectopic Expression ◽  
Genital Ridge ◽  
Male And Female ◽  
Germ Cell Differentiation ◽  
Exact Function

The interaction between germ cell and somatic cell plays important roles in germ cell development. However, the exact function of gonad somatic cell in germ cell differentiation is unclear. In the present study, the function of gonad somatic cell in germ cell meiosis was examined by using mouse models with aberrant somatic cell differentiation. In Wt1R394W/R394W mice, the genital ridge is absent due to the apoptosis of coelomic epithelial cells. Interestingly, in both male and female Wt1R394W/R394W germ cells, STRA8 was detected at E12.5 and the scattered SYCP3 foci were observed at E13.5 which was consistent with control females. In Wt1-/flox; Cre-ERTM mice, Wt1 was inactivated by the injection of tamoxifen at E9.5 and the differentiation of Sertoli and granulosa cells was completely blocked. We found that most germ cells were located outside of genital ridge after Wt1 inactivation. STRA8, SYCP3, and γH2AX proteins were detected in germ cells of both male and female Wt1-/flox; Cre-ERTM gonads, whereas no thread-like SYCP3 signal was observed. Our study demonstrates that aberrant development of gonad somatic cells leads to ectopic expression of meiosis-associated genes in germ cells, but meiosis was arrested before prophase I. These results suggest that the proper differentiation of gonad somatic cells is essential for germ cell meiosis.

scholarly journals Optimization of In Vitro Culture Conditions of Sturgeon Germ Cells for Purpose of Surrogate Production

Animals ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 106 ◽  
Author(s):  
Xuan Xie ◽  
Ping Li ◽  
Martin Pšenička ◽  
Huan Ye ◽  
Christoph Steinbach ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Somatic Cells ◽  
Culture Conditions ◽  
Specific Gene ◽  
Acipenser Gueldenstaedtii ◽  
Acipenser Ruthenus ◽  
Before And After ◽  
Optimal Culture Condition

To expand germ cell populations and provide a consistent supply for transplantation, we established basal culture conditions for sturgeon germ cells and subsequently increased their mitotic activity by eliminating gonad somatic cells, supplementing with growth factor, and replacing fetal bovine serum (FBS). The initial basal culture conditions were Leibovitz’s L-15 medium (pH 8.0) supplemented with 5% FBS (p < 0.001) at 21 °C. Proliferation of germ cells was significantly enhanced and maintained for longer periods by elimination of gonad somatic cells and culture under feeder-cell free conditions, with addition of leukemia inhibitory factor and glial-cell-derived neurotrophic factor (p < 0.001). A serum-free culture medium improved germ cell proliferation compared to the L-15 with FBS (p < 0.05). Morphology remained similar to that of fresh germ cells for at least 40 d culture. Germline-specific gene expression analysis revealed no significant changes to germ cells before and after culture. Sterlet Acipenser ruthenus germ cells cultured more than 40 days showed development after transplant into Russian sturgeon Acipenser gueldenstaedtii. Polymerase chain reaction showed 33.3% of recipient gonads to contain sterlet cells after four months. This study developed optimal culture condition for sturgeon germ cells. Germ cells after 40 d culture developed in recipient gonads. This study provided useful information for culture of sturgeon germ cells.

scholarly journals NANOS2 suppresses the cell cycle by repressing mTORC1 activators in embryonic male germ cells

2020 ◽  
Author(s):  
Ryuki Shimada ◽  
Hiroko Koike ◽  
Takamasa Hirano ◽  
Yumiko Saga
Keyword(s):  
Cell Cycle ◽  
Germ Cell ◽  
Germ Cells ◽  
Rna Binding ◽  
Initial Step ◽  
Sexually Dimorphic ◽  
Wild Type ◽  
Male Germ Cells ◽  
Cycle Arrest ◽  
Male Specific

AbstractDuring murine germ cell development, male germ cells enter the mitotically arrested G0 stage, which is an initial step of sexually dimorphic differentiation. The male specific RNA-binding protein NANOS2 has a key role in suppressing the cell cycle in germ cells. However, the detailed mechanism of how NANOS2 regulates the cell cycle remains unclear. Using single-cell RNA sequencing (scRNA-seq), we extracted the cell cycle state of each germ cell in wild-type and Nanos2-KO testes, and revealed that Nanos2 expression starts in mitotic cells and induces mitotic arrest. We also found that NANOS2 and p38 MAPK work in parallel to regulate the cell cycle, suggesting that several different cascades are involved in the induction of cell cycle arrest. Furthermore, we identified Rheb, a regulator of mTORC1, and Ptma as possible targets of NANOS2. We propose that the repression of the cell cycle is a primary function of NANOS2 and that it is mediated via the suppression of mTORC1 activity by repressing Rheb in a post-transcriptional manner.

scholarly journals Testes of DAZL null sheep lack spermatogonia and maintain normal somatic cells

2019 ◽  
Author(s):  
Zachariah McLean ◽  
Sarah Jane Appleby ◽  
Jingwei Wei ◽  
Russell Grant Snell ◽  
Björn Oback
Keyword(s):  
Germ Cell ◽  
Somatic Cell ◽  
Germ Cells ◽  
Genetic Improvement ◽  
Rna Binding ◽  
Somatic Cells ◽  
Marker Genes ◽  
Specific Marker ◽  
Loss Of Function ◽  
Fetal Fibroblasts

AbstractMultiplying the germline would increase the number of offspring that can be produced from selected animals, accelerating genetic improvement for livestock breeding. This could be achieved by producing multiple chimaeric animals, each carrying a mix of donor and host germ cells in their gonads. However, such chimaeric germlines would produce offspring from both donor and host genotypes, limiting the rate of genetic improvement. To resolve this problem and produce chimaeras with absolute donor germline transmission, we have disrupted the RNA-binding protein DAZL and generated germ cell-deficient host animals. Using Cas9 mediated homology-directed repair (HDR), we introduced a DAZL loss-of-function mutation in male ovine fetal fibroblasts. Following manual single-cell isolation, 4/48 (8.3%) of donor cell strains were homozygously HDR-edited. Sequence-validated strains were used as nuclear donors for somatic cell cloning to generate three lambs, which died at birth. All DAZL-null male neonatal sheep lacked germ cells. Somatic cells within their testes were morphologically intact and expressed normal levels of somatic cell-specific marker genes, indicating that the germ cell niche remained intact. This extends the DAZL-mutant phenotype beyond mice into agriculturally relevant ruminants, providing a pathway for using absolute transmitters in rapid livestock improvement.

scholarly journals A transgenic DND1GFP fusion allele reports in vivo expression and RNA binding targets in undifferentiated mouse germ cells

2021 ◽  
Author(s):  
Victor A Ruthig ◽  
Tetsuhiro Yokonishi ◽  
Matthew B Friedersdorf ◽  
Sofia Batchvarova ◽  
Josiah Hardy ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Rna Binding ◽  
Cell Transformation ◽  
Primordial Germ Cell ◽  
Fetal Life ◽  
Mouse Line ◽  
Transgenic Mouse Line ◽  
Rna Immunoprecipitation

Abstract In vertebrates, the RNA binding protein (RBP) Dead End 1 (DND1) is essential for primordial germ cell (PGC) survival and maintenance of cell identity. In multiple species, Dnd1 loss or mutation leads to severe PGC loss soon after specification or, in some species, germ cell transformation to somatic lineages. Our investigations into the role of DND1 in PGC specification and differentiation have been limited by the absence of an available antibody. To address this problem, we used CRISPR/Cas9 gene editing to establish a transgenic mouse line carrying a DND1GFP fusion allele. We present imaging analysis of DND1GFP expression showing that DND1GFP expression is heterogeneous among male germ cells (MGCs) and female germ cells (FGCs). DND1GFP was detected in MGCs throughout fetal life but lost from FGCs at meiotic entry. In postnatal and adult testes, DND1GFP expression correlated with classic markers for the pre-meiotic spermatogonial population. Utilizing the GFP-tag for RNA immunoprecipitation (RIP) analysis in MGCs validated this transgenic as a tool for identifying in vivo transcript targets of DND1. The DND1GFP mouse line is a novel tool for isolation and analysis of embryonic and fetal germ cells, and the spermatogonial population of the postnatal and adult testis.

Sign in / Sign up

Export Citation Format

Share Document