scholarly journals Male-biased protein expression in primordial germ cells, identified through a comparative study of UAS vectors in Drosophila

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masaki Masukawa ◽  
Yuki Ishizaki ◽  
Hiroki Miura ◽  
Makoto Hayashi ◽  
Ryoma Ota ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Expression ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Translational Activity ◽  
Male Germline ◽  
Germline Cells ◽  
Female Germline ◽  
Go Terms ◽  
Mrna Expression Levels

AbstractIn Drosophila, three types of UAS vectors (UASt, UASp, and UASz) are currently available for use with the Gal4-UAS system. They have been used successfully in somatic cells and germline cells from ovaries. However, it remains unclear whether they are functional in the germline cells of embryos, larvae, and adult testes. In this study, we found that all three types of UAS vectors were functional in the germline cells of embryos and larvae and that the UASt and UASz vectors were active in the germline of the distal tip region in adult testes. Moreover, we observed that protein expression from the UAS vectors was male-biased in germline cells of late embryos, whereas their respective mRNA expression levels were not. Furthermore, O-propargyl-puromycin (OPP) staining revealed that protein synthesis was male-biased in these germline cells. In addition, GO terms related to translation and ribosomal maturation were significantly enriched in the male germline. These observations show that translational activity is higher in male than in female germline cells. Therefore, we propose that male-biased protein synthesis may be responsible for the sex differences observed in the early germline.

scholarly journals The Mammalian Ovary from Genesis to Revelation

2009 ◽  
Vol 30 (6) ◽  
pp. 624-712 ◽  
Author(s):  
Mark A. Edson ◽  
Ankur K. Nagaraja ◽  
Martin M. Matzuk
Keyword(s):  
Germ Cells ◽  
Ovarian Function ◽  
Primordial Germ Cells ◽  
Bioactive Molecules ◽  
Sex Characteristics ◽  
Germline Development ◽  
Peptide Growth Factors ◽  
Development And Differentiation ◽  
Common Destiny ◽  
Female Germline

Abstract Two major functions of the mammalian ovary are the production of germ cells (oocytes), which allow continuation of the species, and the generation of bioactive molecules, primarily steroids (mainly estrogens and progestins) and peptide growth factors, which are critical for ovarian function, regulation of the hypothalamic-pituitary-ovarian axis, and development of secondary sex characteristics. The female germline is created during embryogenesis when the precursors of primordial germ cells differentiate from somatic lineages of the embryo and take a unique route to reach the urogenital ridge. This undifferentiated gonad will differentiate along a female pathway, and the newly formed oocytes will proliferate and subsequently enter meiosis. At this point, the oocyte has two alternative fates: die, a common destiny of millions of oocytes, or be fertilized, a fate of at most approximately 100 oocytes, depending on the species. At every step from germline development and ovary formation to oogenesis and ovarian development and differentiation, there are coordinated interactions of hundreds of proteins and small RNAs. These studies have helped reproductive biologists to understand not only the normal functioning of the ovary but also the pathophysiology and genetics of diseases such as infertility and ovarian cancer. Over the last two decades, parallel progress has been made in the assisted reproductive technology clinic including better hormonal preparations, prenatal genetic testing, and optimal oocyte and embryo analysis and cryopreservation. Clearly, we have learned much about the mammalian ovary and manipulating its most important cargo, the oocyte, since the birth of Louise Brown over 30 yr ago.

scholarly journals An autoregulatory switch in sex-specific phf7 transcription causes loss of sexual identity and tumors in the Drosophila female germline

2020 ◽  
Author(s):  
Anne E. Smolko ◽  
Laura Shapiro-Kulnane ◽  
Helen K. Salz
Keyword(s):  
Germ Cell ◽  
Sexual Identity ◽  
Germ Cells ◽  
Transcriptional Repression ◽  
Feedback Mechanism ◽  
Phd Finger ◽  
Transcriptional Reprogramming ◽  
Male Germline ◽  
Regulatory Circuit ◽  
Female Germline

ABSTRACTMaintenance of germ cell sexual identity is essential for reproduction. Entry into the spermatogenesis or oogenesis pathway requires that the appropriate gene network is activated and the antagonist network is silenced. For example, in Drosophila female germ cells, forced expression of the testis-specific PHD finger protein 7 (PHF7) disrupts oogenesis leading to either an agametic or germ cell tumor phenotype. Here we show that PHF7 expressing ovarian germ cells inappropriately express hundreds of genes, many of which are male germline genes. We find that the majority of genes under PHF7 control in female germ cells are not under PHF7 control in male germ cells, suggesting that PHF7 is acting in a tissue-specific manner. Remarkably, transcriptional reprogramming includes a positive autoregulatory feedback mechanism in which ectopic PHF7 overcomes its own transcriptional repression through promoter switching. Furthermore, we find that tumorigenic capacity is dependent on the dosage of phf7. This study reveals that high levels of ectopic PHF7 in female germ cells leads to a loss of sexual identity and promotion of a regulatory circuit beneficial for tumor initiation and progression.

scholarly journals Germline masculinization by Phf7 in D. melanogaster requires its evolutionarily novel C-terminus and the HP1-family protein HP1D3csd

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shu Yuan Yang
Keyword(s):  
Sex Determination ◽  
Germ Cells ◽  
Transcriptome Profiling ◽  
Phd Finger ◽  
Intrinsic Factors ◽  
Male Germline ◽  
C Terminus ◽  
Family Protein ◽  
Key Factor ◽  
Female Germline

AbstractGerm cells inDrosophila melanogasterneed intrinsic factors along with somatic signals to activate proper sexual programs. A key factor for male germline sex determination is PHD finger protein 7 (Phf7), a histone reader expressed in the male germline that can trigger sex reversal in female germ cells and is also important for efficient spermatogenesis. Here we find that the evolutionarily novel C-terminus in Phf7 is necessary to turn on the complete male program in the early germline ofD. melanogaster, suggesting that this domain may have been uniquely acquired to regulate sexual differentiation. We further looked for genes regulated byPhf7related to sex determination in the embryonic germline by transcriptome profiling of FACS-purified embryonic gonads. One of the genes positively-regulated by Phf7 in the embryonic germline was anHP1family member,Heterochromatin Protein 1D3 chromoshadow domain (HP1D3csd).We find that this gene is needed for Phf7 to induce male-like development in the female germline, indicating that HP1D3csd is an important factor acting downstream of Phf7 to regulate germline masculinization.

scholarly journals Efficient expression of genes in the Drosophila germline using a UAS-promoter free of interference by Hsp70 piRNAs

2018 ◽  
Author(s):  
Steven Z. DeLuca ◽  
Allan C. Spradling
Keyword(s):  
Germ Cells ◽  
Somatic Cells ◽  
Developmental Genetics ◽  
Technical Limitation ◽  
Promoter Sequences ◽  
Expression Of Genes ◽  
Gal4 Expression ◽  
Efficient Expression ◽  
Germline Cells ◽  
Female Germline

ABSTRACTControlling the expression of genes using a binary system involving the yeast GAL4 transcription factor has been a mainstay of Drosophila melanogaster developmental genetics for twenty-five years. However, most existing GAL4 expression constructs only function effectively in somatic cells, but not in germ cells during oogenesis, for unknown reasons. A special UAS promoter, UASp was created that does express during oogenesis, but the need to use different constructs for somatic and female germline cells has remained a significant technical limitation. Here we show that the expression problem of UASt and many other Drosophila molecular tools in germline cells is caused by their core Hsp70 promoter sequences, which are targeted in female germ cells by Hsp70-directed piRNAs generated from endogenous Hsp70 gene sequences. In a genetic background lacking genomic Hsp70 genes and associated piRNAs, UASt-based constructs function effectively during oogenesis. By reducing Hsp70 sequences targeted by piRNAs, we created UASz, which functions better than UASp in the germline and like UASt in somatic cells.

scholarly journals RDC complex executes a dynamic piRNA program during Drosophila spermatogenesis to safeguard male fertility

2020 ◽  
Author(s):  
Peiwei Chen ◽  
Yicheng Luo ◽  
Alexei A. Aravin
Keyword(s):  
Germ Cells ◽  
Male Fertility ◽  
Germline Stem Cells ◽  
Male Germline ◽  
Pirna Cluster ◽  
Transposon Silencing ◽  
Normal Spermatogenesis ◽  
Non Coding Rnas ◽  
Female Germline ◽  
Active Genes

SUMMARYpiRNAs are small non-coding RNAs that guide the silencing of transposons and other targets in animal gonads. In Drosophila female germline, many piRNA source loci dubbed ‘piRNA clusters’ lack hallmarks of active genes and exploit an alternative path for transcription, which relies on the Rhino-Deadlock-Cutoff (RDC) complex. It remains to date unknown how piRNA cluster transcription is regulated in the male germline. We found that components of RDC complex are expressed in male germ cells during early spermatogenesis, from germline stem cells (GSCs) to early spermatocytes. RDC is essential for expression of dual-strand piRNA clusters and transposon silencing in testis; however, it is dispensable for expression of Y-linked Suppressor of Stellate piRNAs and therefore Stellate silencing. Despite intact Stellate repression, rhi mutant males exhibited compromised fertility accompanied by germline DNA damage and GSC loss. Thus, piRNA-guided repression is essential for normal spermatogenesis beyond Stellate silencing. While RDC associates with multiple piRNA clusters in GSCs and early spermatogonia, its localization changes in later stages as RDC concentrates on a single X-linked locus, AT-chX. Dynamic RDC localization is paralleled by changes in piRNA cluster expression, indicating that RDC executes a fluid piRNA program during different stages of spermatogenesis.

scholarly journals An autoregulatory switch in sex-specific phf7 transcription causes loss of sexual identity and tumors in the Drosophila female germline

Development ◽  
2020 ◽  
Vol 147 (17) ◽  
pp. dev192856
Author(s):  
Anne E. Smolko ◽  
Laura Shapiro-Kulnane ◽  
Helen K. Salz
Keyword(s):  
Germ Cell ◽  
Sexual Identity ◽  
Germ Cells ◽  
Transcriptional Repression ◽  
Feedback Mechanism ◽  
Phd Finger ◽  
Transcriptional Reprogramming ◽  
Male Germline ◽  
Regulatory Circuit ◽  
Female Germline

ABSTRACTMaintenance of germ cell sexual identity is essential for reproduction. Entry into the spermatogenesis or oogenesis pathway requires that the appropriate gene network is activated and the antagonist network is silenced. For example, in Drosophila female germ cells, forced expression of the testis-specific PHD finger protein 7 (PHF7) disrupts oogenesis, leading to either an agametic or germ cell tumor phenotype. Here, we show that PHF7-expressing ovarian germ cells inappropriately express hundreds of genes, many of which are male germline genes. We find that the majority of genes under PHF7 control in female germ cells are not under PHF7 control in male germ cells, suggesting that PHF7 is acting in a tissue-specific manner. Remarkably, transcriptional reprogramming includes a positive autoregulatory feedback mechanism in which ectopic PHF7 overcomes its own transcriptional repression through promoter switching. Furthermore, we find that tumorigenic capacity is dependent on the dosage of phf7. This study reveals that ectopic PHF7 in female germ cells leads to a loss of sexual identity and the promotion of a regulatory circuit that is beneficial for tumor initiation and progression.

Conservation of chicken male germline by orthotopic transplantation of primordial germ cells from genetically distant donors†

2019 ◽  
Vol 101 (1) ◽  
pp. 200-207 ◽  
Author(s):  
Jitka Mucksová ◽  
Markéta Reinišová ◽  
Jiří Kalina ◽  
Barbora Lejčková ◽  
Jiří Hejnar ◽  
...  
Keyword(s):  
Genetic Variability ◽  
Genome Editing ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Avian Species ◽  
Domestic Chicken ◽  
White Leghorn ◽  
Orthotopic Transplantation ◽  
Viable Offspring ◽  
Male Germline

Abstract Successful derivation and cultivation of primordial germ cells (PGCs) opened the way to efficient transgenesis and genome editing in the chicken. Furthermore, implantation of male PGCs from non-chicken galliform species into the chicken embryos resulted in cross-species germline chimeras and viable offspring. We have recently improved the PGC technology by demonstrating that chicken male PGCs transplanted into the testes of adult cockerel recipients mature into functional sperms. However, the availability of this orthotopic transplantation for cross-species transfer remains to be explored. Here we tested the capacity of genetically distant male PGCs to mature in the microenvironment of adult testes. We derived PGCs from the Chinese black-bone Silkie and transplanted them into infertile White Leghorn cockerels. Within 15–18 weeks after transplantation, we observed restoration of spermatogenesis in recipient cockerels and production of healthy progeny derived from the transplanted PGCs. Our findings also indicate the possibility of cross-species orthotopic transplantation of PGCs. Thus, our results might contribute to the preservation of endangered avian species and maintaining the genetic variability of the domestic chicken.

scholarly journals stand still, a Drosophila Gene Involved in the Female Germline for Proper Survival, Sex Determination and Differentiation

Genetics ◽  
1997 ◽  
Vol 145 (4) ◽  
pp. 975-987
Author(s):  
Giuseppa Pennetta ◽  
Daniel Pauli
Keyword(s):  
Sex Determination ◽  
Germ Cells ◽  
Loss Of Function ◽  
Germline Development ◽  
Survival Sex ◽  
Male Germline ◽  
New Gene ◽  
Mutant Phenotypes ◽  
Female Germline ◽  
Novel Protein

We identified a new gene, stand still (stil), required in the female germline for proper survival, sex determination and differentiation. Three strong loss-of-function alleles were isolated. The strongest phenotype exhibited by ovaries dissected from adult females is the complete absence of germ cells. In other ovaries, the few surviving germ cells frequently show a morphology typical of primary spermatocytes. stil is not required either for fly viability or for male germline development. The gene was cloned and found to encode a novel protein. stil is strongly expressed in the female germ cells. Using P[stil  +] transgenes, we show that stil and a closely localized gene are involved in the modification of the ovarian phenotypes of the dominant alleles of ovo caused by heterozygosity of region 49 A-D. The similarity of the mutant phenotypes of stil to that of otu and ovo suggests that the three genes function in a common or in parallel pathways necessary in the female germline for its survival, sex determination and differentiation.

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