scholarly journals Maternal Piwi Regulates Primordial Germ Cell Development to Ensure the Fertility of Female Progeny in Drosophila

Genetics ◽  
2021 ◽  
Author(s):  
Lauren E Gonzalez ◽  
Xiongzhuo Tang ◽  
Haifan Lin
Keyword(s):  
Germ Cell ◽  
Primordial Germ Cell ◽  
Early Embryo ◽  
Null Alleles ◽  
Germline Stem Cell ◽  
Germline Development ◽  
Female Progeny ◽  
Adult Ovary ◽  
Pirna Pathway ◽  
Germline Sex Determination

Abstract In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.

scholarly journals Maternal Piwi Regulates Primordial Germ Cell Development to Ensure the Fertility of Female Progeny in Drosophila

2021 ◽  
Author(s):  
Lauren E Gonzalez ◽  
Xiongzhuo Tang ◽  
Haifan Lin
Keyword(s):  
Germ Cell ◽  
Primordial Germ Cell ◽  
Early Embryo ◽  
Null Alleles ◽  
Germline Stem Cell ◽  
Germline Development ◽  
Female Progeny ◽  
Adult Ovary ◽  
Pirna Pathway ◽  
Germline Sex Determination

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the early embryo in Drosophila. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.

scholarly journals Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3099
Author(s):  
Aline Fernanda de Souza ◽  
Fabiana Fernandes Bressan ◽  
Naira Caroline Godoy Pieri ◽  
Ramon Cesar Botigelli ◽  
Tamas Revay ◽  
...  
Keyword(s):  
Germ Cell ◽  
Turner Syndrome ◽  
Genetic Disorder ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Cell Formation ◽  
In Vitro Models ◽  
Germline Development ◽  
Germ Cell Specification

Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.

scholarly journals Testing the role of SOX15 in human primordial germ cell fate

2019 ◽  
Vol 4 ◽  
pp. 122
Author(s):  
Merrick Pierson Smela ◽  
Anastasiya Sybirna ◽  
Frederick C.K. Wong ◽  
M. Azim Surani
Keyword(s):  
Flow Cytometry ◽  
Germ Cell ◽  
Primordial Germ Cell ◽  
Mechanistic Study ◽  
Germline Development ◽  
Cell Identity ◽  
Protein Levels ◽  
Germ Cell Specification ◽  
The Impact ◽  
Early Human

Background: Potentially novel regulators of early human germline development have been identified recently, including SOX15 and SOX17, both of which show specific expression in human primordial germ cells. SOX17 is now known to be a critical specifier of human germ cell identity. There have been suggestions, as yet without evidence, that SOX15 might also play a prominent role. The early human germline is inaccessible for direct study, but an in vitro model of human primordial germ cell-like cell (hPGCLC) specification from human embryonic stem cells (hESCs) has been developed. This enables mechanistic study of human germ cell specification using genetic tools to manipulate the levels of SOX15 and SOX17 proteins to explore their roles in hPGCLC specification. Methods: SOX15 and SOX17 proteins were depleted during hPGCLC specification from hESCs using the auxin-inducible degron system, combined with a fluorescent reporter for tracking protein levels. Additionally, SOX15 protein was overexpressed using the ProteoTuner system. Protein-level expression changes were confirmed by immunofluorescence. The impact on hPGCLC specification efficiency was determined by flow cytometry at various time points. qPCR experiments were performed to determine some transcriptional effects of SOX15 perturbations. Results: We observed specific SOX15 expression in hPGCLCs by using immunofluorescence and flow cytometry analysis. Depletion of SOX15 had no significant effect on hPGCLC specification efficiency on day 4 after induction, but there was a significant and progressive decrease in hPGCLCs on days 6 and 8. By contrast, depletion of SOX17 completely abrogated hPGCLC specification. Furthermore, SOX15 overexpression resulted in a significant increase in hPGCLC fraction on day 8. qPCR analysis revealed a possible role for the germ cell and pluripotency regulator PRDM14 in compensating for changes to SOX15 protein levels. Conclusions: SOX17 is essential for hPGCLC specification, yet SOX15 is dispensable. However, SOX15 may have a role in maintaining germ cell identity.

scholarly journals Extensive nuclear gyration and pervasive non-genic transcription during primordial germ cell development in zebrafish

Development ◽  
2020 ◽  
pp. dev.193060
Author(s):  
Stefan Redl ◽  
Antonio M. de Jesus Domingues ◽  
Edoardo Caspani ◽  
Stefanie Möckel ◽  
Willi Salvenmoser ◽  
...  
Keyword(s):  
Germ Cell ◽  
Cell Fate ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Genital Ridge ◽  
Non Coding Rna ◽  
Pathway Activation ◽  
Pirna Pathway ◽  
Time Point

Primordial germ cells (PGCs) are the precursors of germ cells, which migrate to the genital ridge during early development. Relatively little is known about PGCs after their migration. We studied this post-migratory stage using microscopy and sequencing techniques, and found that many PGC-specific genes, including genes known to induce PGC fate in the mouse, are only activated several days after migration. At this same time point, PGC nuclei become extremely gyrated, displaying general broad opening of chromatin and high levels of intergenic transcription. This is accompanied by changes in nuage morphology, expression of large loci (PGC-Expressed non-coding RNA Loci, PERLs) that are enriched for retro-transposons and piRNAs, and a rise in piRNA biogenesis signatures. Interestingly, no nuclear Piwi protein could be detected at any time point, indicating that the zebrafish piRNA pathway is fully cytoplasmic. Our data show that the post-migratory stage of zebrafish PGCs holds many cues to both germ cell fate establishment and piRNA pathway activation.

scholarly journals The Drosophila pumilio Gene Encodes Two Functional Protein Isoforms That Play Multiple Roles in Germline Development, Gonadogenesis,Oogenesis and Embryogenesis

Genetics ◽  
1999 ◽  
Vol 153 (1) ◽  
pp. 235-250 ◽  
Author(s):  
Michael Parisi ◽  
Haifan Lin
Keyword(s):  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Protein Isoforms ◽  
Germline Stem Cell ◽  
Molecular Evidence ◽  
Dependent Manner ◽  
Embryonic Patterning ◽  
Phenotypic Analysis ◽  
Functional Protein ◽  
Germline Development

Abstract The pumilio (pum) gene plays an essential role in embryonic patterning and germline stem cell (GSC) maintenance during oogenesis in Drosophila. Here we report on a phenotypic analysis using pumovarette mutations, which reveals multiple functions of pum in primordial germ cell proliferation, larval ovary formation, GSC division, and subsequent oogenic processes, as well as in oviposition. Specifically, by inducing pum– GSC clones at the onset of oogenesis, we show that pum is directly involved in GSC division, a function that is distinct from its requirement in primordial germ cells. Furthermore, we show that pum encodes 156- and 130-kD proteins, both of which are functional isoforms. Among pumovarette mutations, pum1688 specifically eliminates the 156-kD isoform but not the 130-kD isoform, while pum2003 and pum4277 specifically affect the 130-kD isoform but not the 156-kD isoform. Normal doses of both isoforms are required for the zygotic function of pum, yet either isoform alone at a normal dose is sufficient for the maternal effect function of pum. A pum cDNA transgene that contains the known open reading frame encodes only the 156-kD isoform and rescues the phenotype of both pum1688 and pum2003 mutants. These observations suggest that the 156- and 130-kD isoforms can compensate for each other's function in a dosage-dependent manner. Finally, we present molecular evidence suggesting that the two PUM isoforms share some of their primary structures.

scholarly journals The Study of Primordial Germ Cell Development as a Tool for Gene Transfer in Chickens

1991 ◽  
Author(s):  
James Petitte ◽  
Hefzibah Eyal-Giladi ◽  
Malka Ginsburg
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cell ◽  
Genetic Material ◽  
Early Embryo ◽  
Avian Embryo ◽  
Stage Of Development ◽  
Germ Cell Differentiation ◽  
Donor Cells ◽  
Foreign Dna

The ability to introduce novel genetic material into the genome of commercial poultry has been impeded by a lack of kowledge regarding the origin in the early embryo of the target cell of interest, namely, the germ cell. Hence, this project investigated the emergence of primordial germ cells (PGCs) during the early development of the avian embryo to aid in efforts to produce transgenic poultry on a routine basis. The strategy was to introduce foreign DNA into the area of the unincubated embryo that is destined to give rise to the germ line. The objectives of this project were: 1) to identify and localize a subpopulation of cells in the early embryo which will give rise to PGCs, 2) to determine the best location and stage of development to transfer donor cells for efficient germline chimerism, and 3) to transfect donor cells to produce transgenic/germline chimeric embryos. We show that by using the monoclonal antibody SSEA-1 and by various cell culture techniques that germ cells appear to segregate from the somatic lineages at St. X., a process that is gradual and continues through St. XIV. Using microsurgical transplantation between quail and chick embryos, we demonstrated that the inner 1/3 of the area pellucida between states X-XII gives rise to about 2/3 of the germ cell population at the time of their residence in the germinal crescent. Because of the non-localized emergence of PGCs, attempts to introduce foreign DNA into clonal precursors of germ cells through liposome-mediated transfection yielded unacceptable levels of efficiency. However, through our investigation of germ cell origins, an in vitro model of germ cell differentiation was developed that could offer a means of determining the factors required for the long term culture of avian PGCs thereby providing a convenient means of manipulating the avian genome.

57 Primordial germ cell distribution in the horse fetal gonad

2020 ◽  
Vol 32 (2) ◽  
pp. 154
Author(s):  
D. Scarlet ◽  
U. Reichart ◽  
G. Podico ◽  
R. Ellerbrock ◽  
I. Canisso ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Germ Cell ◽  
Primordial Germ Cell ◽  
Temporal Distribution ◽  
Germline Stem Cell ◽  
Female Gonad ◽  
Specific Marker ◽  
Cortical Region ◽  
Germ Cell Differentiation

Germ cell development and differentiation is a complex process associated with down-regulation of stem cell-associated genes and up-regulation of markers of germ cell differentiation and meiosis. In horses, putative primordial germ cells (PGCs) were identified outside the gonads starting 20 days after ovulation (Curran et al. 1997 Equine Vet. J. Suppl. 25, 72-76). However, no information is available after the time when these cells enter the gonad. The aim of this study was to identify, localise, and quantify PGCs in fetal male and female gonads. Twelve (5 males and 7 females) equine fetuses were collected transcervically 60 days after ovulation. Fetuses were entirely fixed in formaldehyde and gonads were subsequently localised. Fetal gonads underwent multiplex immunofluorescence staining for pre-meiotic germ cell marker LIN28, stem-cell associated marker CD117, and cell proliferation marker Ki67. Specificity of the primary antibodies for equine samples has been first validated. Quantification of fluorescence positive areas for LIN28, CD117, and Ki67 in the fetal gonads was done using a macro for ImageJ. Furthermore, we analysed the co-localization of above-mentioned markers using the same macro. Statistical comparison for differences between males and females was performed using non-parametric tests. In both sexes, PGCs proliferated as determined by double immunofluorescence of Ki67 and LIN28. Protein expression of LIN28 and Ki67 was highly correlated (r=0.92; P=0.003). In the fetal female gonad, PGCs were organised in cord-like structures localised in the cortical region, but there were also LIN28+ cells in the surface germinal epithelium. In the fetal male gonad, PGCs were restricted to the already developed tubular structures. Fewer LIN28+ cells (3.0±0.4% vs. 4.5±0.3%; P<0.05) were present in female than in male gonads. The distribution pattern of the stem cell factor receptor CD117 was similar to LIN28, as 86.8±3.2% of LIN28+ cells in females and 84.6±4.7% in males were also CD117+. However, immunofluorescent co-localization analysis revealed a subpopulation of CD117+ cells (43.1±8.1% in females and 46.1±6.1% in males), which did not show an overlap with LIN28. These were presumably stem cells localised in the medullar area of the gonad. In summary, we analysed for the first time spatial distribution of PGCs in fetal equine gonads. We demonstrated LIN28 to be a specific marker for PGCs also in the horse gonad, which is in agreement with the situation in human and other species. Furthermore, we identified stem cells and described their localization in the fetal equine gonad. Nevertheless, the temporal distribution of PGC and stem cells in the developing horse gonad and the role of LIN28 in the maintenance of the germline stem cell state still need to be investigated.

scholarly journals A noncanonical role of NOD-like receptor NLRP14 in PGCLC differentiation and spermatogenesis

2020 ◽  
Vol 117 (36) ◽  
pp. 22237-22248
Author(s):  
Yike Yin ◽  
Shiyu Cao ◽  
Huancheng Fu ◽  
Xueying Fan ◽  
Jingfei Xiong ◽  
...  
Keyword(s):  
Germ Cell ◽  
Nuclear Translocation ◽  
Primordial Germ Cell ◽  
Male Mice ◽  
Germline Development ◽  
Severe Oligozoospermia ◽  
Knock Out ◽  
Germ Cell Differentiation

NOD-like receptors (NLRs) are traditionally recognized as major inflammasome components. The role of NLRs in germ cell differentiation and reproduction is not known. Here, we identified the gonad-specific Nlrp14 as a pivotal regulator in primordial germ cell-like cell (PGCLC) differentiation in vitro. Physiologically, knock out of Nlrp14 resulted in reproductive failure in both female and male mice. In adult male mice, Nlrp14 knockout (KO) inhibited differentiation of spermatogonial stem cells (SSCs) and meiosis, resulting in trapped SSCs in early stages, severe oligozoospermia, and sperm abnormality. Mechanistically, NLRP14 promoted spermatogenesis by recruiting a chaperone cofactor, BAG2, to bind with HSPA2 and form the NLRP14−HSPA2−BAG2 complex, which strongly inhibited ChIP-mediated HSPA2 polyubiquitination and promoted its nuclear translocation. Finally, loss of HSPA2 protection and BAG2 recruitment by NLRP14 was confirmed in a human nonsense germline variant associated with male sterility. Together, our data highlight a unique proteasome-mediated, noncanonical function of NLRP14 in PGCLC differentiation and spermatogenesis, providing mechanistic insights of gonad-specific NLRs in mammalian germline development.

scholarly journals Testing the role of SOX15 in human primordial germ cell fate

2019 ◽  
Vol 4 ◽  
pp. 122 ◽  
Author(s):  
Merrick Pierson Smela ◽  
Anastasiya Sybirna ◽  
Frederick C.K. Wong ◽  
M. Azim Surani
Keyword(s):  
Flow Cytometry ◽  
Germ Cell ◽  
Primordial Germ Cell ◽  
Mechanistic Study ◽  
Germline Development ◽  
Cell Identity ◽  
Protein Levels ◽  
Germ Cell Specification ◽  
The Impact ◽  
Early Human

Background: Potentially novel regulators of early human germline development have been identified recently, including SOX15 and SOX17, both of which show specific expression in human primordial germ cells. SOX17 is now known to be a critical specifier of human germ cell identity. There have been suggestions, as yet without evidence, that SOX15 might also play a prominent role. The early human germline is inaccessible for direct study, but an in vitro model of human primordial germ cell-like cell (hPGCLC) specification from human embryonic stem cells (hESCs) has been developed. This enables mechanistic study of human germ cell specification using genetic tools to manipulate the levels of SOX15 and SOX17 proteins to explore their roles in hPGCLC specification. Methods: SOX15 and SOX17 proteins were depleted during hPGCLC specification from hESCs using the auxin-inducible degron system, combined with a fluorescent reporter for tracking protein levels. Additionally, SOX15 protein was overexpressed using the ProteoTuner system. Protein-level expression changes were confirmed by immunofluorescence. The impact on hPGCLC specification efficiency was determined by flow cytometry at various time points. qPCR experiments were performed to determine some transcriptional effects of SOX15 perturbations. Results: We observed specific SOX15 expression in hPGCLCs by using immunofluorescence and flow cytometry analysis. Depletion of SOX15 had no significant effect on hPGCLC specification efficiency on day 4 after induction, but there was a significant and progressive decrease in hPGCLCs on days 6 and 8. By contrast, depletion of SOX17 completely abrogated hPGCLC specification. Furthermore, SOX15 overexpression resulted in a significant increase in hPGCLC fraction on day 8. qPCR analysis revealed a possible role for the germ cell and pluripotency regulator PRDM14 in compensating for changes to SOX15 protein levels. Conclusions: SOX17 is essential for hPGCLC specification, yet SOX15 is dispensable. However, SOX15 may have a role in maintaining germ cell identity.

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