scholarly journals Defining the expression of piRNA and transposable elements in Drosophila ovarian germline stem cells and somatic support cells

2019 ◽  
Vol 2 (5) ◽  
pp. e201800211 ◽  
Author(s):  
Benjamin Story ◽  
Xing Ma ◽  
Kazue Ishihara ◽  
Hua Li ◽  
Kathryn Hall ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transposable Elements ◽  
Germ Cell ◽  
Germ Cells ◽  
Genome Stability ◽  
Cell Function ◽  
Germline Stem Cells ◽  
Sequencing Data ◽  
Rich Information ◽  
The Rich

Piwi-interacting RNAs (piRNAs) are important for repressing transposable elements (TEs) and modulating gene expression in germ cells, thereby maintaining genome stability and germ cell function. Although they are also important for maintaining germline stem cells (GSCs) in the Drosophila ovary by repressing TEs and preventing DNA damage, piRNA expression has not been investigated in GSCs or their early progeny. Here, we show that the canonical piRNA clusters are more active in GSCs and their early progeny than late germ cells and also identify more than 3,000 new piRNA clusters from deep sequencing data. The increase in piRNAs in GSCs and early progeny can be attributed to both canonical and newly identified piRNA clusters. As expected, piRNA clusters in GSCs, but not those in somatic support cells (SCs), exhibit ping-pong signatures. Surprisingly, GSCs and early progeny express more TE transcripts than late germ cells, suggesting that the increase in piRNA levels may be related to the higher levels of TE transcripts in GSCs and early progeny. GSCs also have higher piRNA levels and lower TE levels than SCs. Furthermore, the 3′ UTRs of 171 mRNA transcripts may produce sense, antisense, or dual-stranded piRNAs. Finally, we show that alternative promoter usage and splicing are frequently used to modulate gene function in GSCs and SCs. Overall, this study has provided important insight into piRNA production and TE repression in GSCs and SCs. The rich information provided by this study will be a beneficial resource to the fields of piRNA biology and germ cell development.

scholarly journals The Controversy, Challenges, and Potential Benefits of Putative Female Germline Stem Cells Research in Mammals

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Zezheng Pan ◽  
Mengli Sun ◽  
Xia Liang ◽  
Jia Li ◽  
Fangyue Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Mammalian Species ◽  
Research Progress ◽  
Germline Stem Cells ◽  
Conventional View ◽  
Proliferation And Differentiation ◽  
Potential Benefits ◽  
Female Germline

The conventional view is that female mammals lose their ability to generate new germ cells after birth. However, in recent years, researchers have successfully isolated and cultured a type of germ cell from postnatal ovaries in a variety of mammalian species that have the abilities of self-proliferation and differentiation into oocytes, and this finding indicates that putative germline stem cells maybe exist in the postnatal mammalian ovaries. Herein, we review the research history and discovery of putative female germline stem cells, the concept that putative germline stem cells exist in the postnatal mammalian ovary, and the research progress, challenge, and application of putative germline stem cells in recent years.

A role for the Drosophila bag-of-marbles protein in the differentiation of cystoblasts from germline stem cells

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2937-2947 ◽  
Author(s):  
D. McKearin ◽  
B. Ohlstein
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Germ Cell ◽  
Germ Cells ◽  
Germline Stem Cell ◽  
Cytoplasmic Protein ◽  
Germline Stem Cells ◽  
Bag Of Marbles

Cell differentiation commonly dictates a change in the cell cycle of mitotic daughters. Previous investigations have suggested that the Drosophila bag of marbles (bam) gene is required for the differentiation of germline stem cell daughters (cystoblasts) from the mother stem cells, perhaps by altering the cell cycle. In this paper, we report the preparation of antibodies to the Bam protein and the use of those reagents to investigate how Bam is required for germ cell development. We find that Bam exists as both a fusome component and as cytoplasmic protein and that cytoplasmic and fusome Bam might have separable activities. We also show that bam mutant germ cells are blocked in differentiation and are trapped as mitotically active cells like stem cells. A model for how Bam might regulate cystocyte differentiation is presented.

scholarly journals Male germline stem cells in non-human primates

2017 ◽  
Vol 4 (2) ◽  
pp. 173-184 ◽  
Author(s):  
Swati Sharma ◽  
Joana M. D. Portela ◽  
Daniel Langenstroth-Röwer ◽  
Joachim Wistuba ◽  
Nina Neuhaus ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Regulatory Mechanisms ◽  
Primate Species ◽  
Germline Stem Cells ◽  
Germline Development ◽  
Human Primate

Abstract. Over the past few decades, several studies have attempted to decipher the biology of mammalian germline stem cells (GSCs). These studies provide evidence that regulatory mechanisms for germ cell specification and migration are evolutionarily conserved across species. The characteristics and functions of primate GSCs are highly distinct from rodent species; therefore the findings from rodent models cannot be extrapolated to primates. Due to limited availability of human embryonic and testicular samples for research purposes, two non-human primate models (marmoset and macaque monkeys) are extensively employed to understand human germline development and differentiation. This review provides a broader introduction to the in vivo and in vitro germline stem cell terminology from primordial to differentiating germ cells. Primordial germ cells (PGCs) are the most immature germ cells colonizing the gonad prior to sex differentiation into testes or ovaries. PGC specification and migratory patterns among different primate species are compared in the review. It also reports the distinctions and similarities in expression patterns of pluripotency markers (OCT4A, NANOG, SALL4 and LIN28) during embryonic developmental stages, among marmosets, macaques and humans. This review presents a comparative summary with immunohistochemical and molecular evidence of germ cell marker expression patterns during postnatal developmental stages, among humans and non-human primates. Furthermore, it reports findings from the recent literature investigating the plasticity behavior of germ cells and stem cells in other organs of humans and monkeys. The use of non-human primate models would enable bridging the knowledge gap in primate GSC research and understanding the mechanisms involved in germline development. Reported similarities in regulatory mechanisms and germ cell expression profile in primates demonstrate the preclinical significance of monkey models for development of human fertility preservation strategies.

scholarly journals Krüppel-like factor 4 is required for development and regeneration of germline and yolk cells from somatic stem cells in planarians

2021 ◽  
Author(s):  
Melanie Issigonis ◽  
Akshada Redkar ◽  
Tania Rozario ◽  
Umair Khan ◽  
Rosa Mejia-Sanchez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
System Development ◽  
Primordial Germ Cells ◽  
Reproductive Organs ◽  
Germline Stem Cells ◽  
Yolk Cell ◽  
Evolutionary Innovation ◽  
Parasitic Flatworm

Sexually reproducing animals segregate their germline from their soma. In addition to gamete-producing gonads, planarian and parasitic flatworm reproduction relies on yolk-cell-generating accessory reproductive organs (vitellaria) supporting development of yolkless oocytes. Despite the importance of vitellaria for flatworm reproduction (and parasite transmission), little is known about this unique evolutionary innovation. Here we examine reproductive system development in the planarian Schmidtea mediterranea, in which pluripotent stem cells generate both somatic and germ cell lineages. We show that a homolog of the pluripotency factor Klf4 is expressed in primordial germ cells, presumptive germline stem cells, and yolk-cell progenitors. klf4 knockdown animals fail to specify or maintain germ cells; surprisingly, they also fail to maintain yolk cells. We find that yolk cells display germ-cell-like attributes and that vitellaria are structurally analogous to gonads. In addition to identifying a new proliferative cell population in planarians (yolk cell progenitors) and defining its niche, our work provides evidence supporting the hypothesis that flatworm germ cells and yolk cells share a common evolutionary origin.

Ectopic expression of the Drosophila Bam protein eliminates oogenic germline stem cells

Development ◽  
1997 ◽  
Vol 124 (18) ◽  
pp. 3651-3662 ◽  
Author(s):  
B. Ohlstein ◽  
D. McKearin
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Germ Cell ◽  
Cell Fate ◽  
Germ Cells ◽  
Ectopic Expression ◽  
Cell Lineage ◽  
Limiting Factor ◽  
Germline Stem Cells ◽  
Stem Cell Divisions

The Drosophila germ-cell lineage has emerged as a remarkable system for identifying genes required for changes in cell fate from stem cells into more specialized cells. Previous work indicates that bam expression is necessary for cystoblast differentiation; bam mutant germ cells fail to differentiate, but instead proliferate like stem cells. This paper reports that ectopic expression of bam is sufficient to extinguish stem cell divisions. Heat-induced bam+ expression specifically eliminated oogenic stem cells while somatic stem cell populations were not affected. Together with previous studies of the timing of bam mRNA and protein expression and the state of arrest in bam mutant cells, these data implicate Bam as a direct regulator of the switch from stem cell to cystoblast. Surprisingly, ectopic bam+ had no deleterious consequences for male germline cells suggesting that Bam may regulate somewhat different steps of germ-cell development in oogenesis and spermatogenesis. We discuss a model for how bam+ could direct differentiation based on our data (McKearin and Ohlstein, 1995) that Bam protein is essential to assemble part of the germ-cell-specific organelle, the fusome. We propose that fusome biogenesis is an obligate step for cystoblast cell fate and that Bam is the limiting factor for fusome maturation in female germ cells.

scholarly journals Phase Transitioned Nuclear Oskar Promotes Cell Division of Drosophila Primordial Germ Cells

2018 ◽  
Author(s):  
Kathryn E. Kistler ◽  
Tatjana Trcek ◽  
Thomas R. Hurd ◽  
Ruoyu Chen ◽  
Feng-Xia Liang ◽  
...  
Keyword(s):  
Germ Cell ◽  
Cell Fate ◽  
Germ Cells ◽  
Cell Function ◽  
Primordial Germ Cells ◽  
Nuclear Localization Sequence ◽  
Cell Systems ◽  
Germ Granules ◽  
Localization Sequence ◽  
Transcriptional Gene Regulation

ABSTRACTGerm granules are non-membranous ribonucleoprotein granules deemed the hubs for post-transcriptional gene regulation and functionally linked to germ cell fate across species. Little is known about the physical properties of germ granules and how these relate to germ cell function. Here we study two types of germ granules in the Drosophila embryo: cytoplasmic germ granules that instruct primordial germ cells (PGCs) formation and nuclear germ granules within early PGCs with unknown function. We show that cytoplasmic and nuclear germ granules are phase transitioned condensates nucleated by Oskar protein that display liquid as well as hydrogel-like properties. Focusing on nuclear granules, we find that Oskar drives their formation in heterologous cell systems. Multiple, independent Oskar protein domains synergize to promote granule phase separation. Deletion of Oskar’s nuclear localization sequence specifically ablates nuclear granules in cell systems. In the embryo, nuclear germ granules promote germ cell divisions thereby increasing PGC number for the next generation.

217 PROTEIN-INDUCED TRANSDIFFERENTIATION INTO MALE GERM CELL-LIKE LINEAGE FROM CHICKEN FETAL BONE MARROW STEM CELLS

2012 ◽  
Vol 24 (1) ◽  
pp. 220
Author(s):  
J. M. Yoo ◽  
J. J. Park ◽  
K. Gobianand ◽  
J. Y. Ji ◽  
J. S. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Retinoic Acid ◽  
Germ Cell ◽  
Germ Cells ◽  
Cultured Cells ◽  
Male Germ Cell ◽  
Male Germ Cells ◽  
Germ Cell Differentiation ◽  
Transgenic Chickens

Bone marrow (BM)-derived stem cells are capable of transdifferentiation into multilineage cells like muscle, bone, cartilage, fat and nerve cells. In this study, we investigated the capability of mesenchymal stem cells (MSC) derived from BM into germ cell differentiation in the chicken. Chicken MSCs were isolated from BM of day 20 fertilized fetal chicken with Ficoll-Paque Plus. Isolated cells were cultured in advance-DMEM (ADMEM) supplemented with 10% fetal bovine serum and antibiotics. Once confluent, cells were subcultured until five passages. The cultured cells showed fibroblast-like morphology. The cells had positive expressions of Oct4, Sox2 and Nanog. Two induction methods were conducted to examine the ability of transdifferentation into male germ cells. In group 1, MSC were cultured in ADMEM containing retinoic acid and chicken testicular extracts proteins for 10 to 15 days. In group 2, MSC were permeabilized by streptolysin O and treated with chicken testicular protein extracts. In both treatment groups, MSC were cultured in ADMEM containing retinoic acid for 10 to 15 days. We found that chicken MSC had a positive expression of pluripotent proteins such as Oct4, Sox2, Nanog and a small population of chicken MSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers and male germ-cell-specific markers (Dazl, C-kit, Stra8 and DDX4) as analysed by reverse transcription-PCR and immunohistochemistry. These results demonstrated that chicken MSC may differentiate into male germ cells and the same might be used as a potential source of cells for production of transgenic chickens. This study was carried out with the support of Agenda Program (Project No. PJ0064692011), RDA and Republic of Korea.

154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 207
Author(s):  
J. Galiguis ◽  
C. E. Pope ◽  
C. Dumas ◽  
G. Wang ◽  
R. A. MacLean ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Transcript Abundance ◽  
Domestic Cat ◽  
Sample Type

As precursors to germline stem cells and gametes, there are many potential applications for primordial germ cells (PGC). Primordial germ cell-like cells have been generated from mouse embryonic stem cells and induced pluripotent stem cells, which subsequently were used to produce functional spermatozoa, oocytes, and healthy offspring (Hayashi et al. 2012 Science 338(6109), 971–975). Applying this approach to generate sperm and oocytes of endangered species is an appealing prospect. Detection of molecular markers associated with PGC is essential to optimizing the process of PGC induction. In the current study, in vitro-derived domestic cat embryos were assessed at various developmental stages to characterise the expression of markers related to the specification process of cat PGC. In vivo-matured, IVF oocytes were cultured until Days 7, 9, and 12 post-insemination. Then, embryos were assessed by RT-qPCR to determine relative transcript abundance of the pluripotency markers NANOG, POU5F1, and SOX2; the epiblast marker DNMT3B; the primitive endoderm marker GATA4; the PGC marker PRDM14; and the germ cell marker VASA; RPS19 was used as the internal reference gene. To validate the qPCR results, fibroblasts served as the negative control cells, whereas spermatogonial stem cells (SSC) served as the positive control cells for GATA4, PRDM14, and VASA. Total mRNA were isolated using the Cells-to-cDNA™ II Kit (Ambion/Thermo Fisher Scientific, Waltham, MA, USA) from either pools of 2 to 6 embryos or ~25 000 fibroblasts/SSC. A minimum of 2 biological replicates for each sample type was analysed, with transcript abundance detected in 2 technical replicates by SYBR Green chemistry. Student’s t-tests were performed on the ΔCts for statistical analysis. PRDM14, specific to the germ cell lineage, was detected as early as Day 7, suggesting the presence of PGC precursor cells. Compared with their levels at Day 7, PRDM14 expression was 0.34-fold lower in SSC (P < 0.05), whereas expression of VASA and GATA4 were 1964-fold and 144-fold higher, respectively (P < 0.05). This seems to emphasise the relative importance of PRDM14 in pre-germ cell stages. In general, all genes analysed were up-regulated from Day 7 to Day 9. This up-regulation was statistically significant for SOX2 and GATA4 (P < 0.05). Relative to that at Day 9, all transcripts were relatively less abundant at Day 12 (P < 0.05 for NANOG, POU5F1, SOX2, DNMT3B, and PRDM14). The data suggest that PGC specification takes place near Day 9, with peak specification activity concluding by Day 12. Although much needs be explored about PGC specification in the cat before applying induction and in vitro germ cell production techniques, these findings represent the first step towards a new potential strategy for preserving endangered and threatened felids.

428 PRODUCTION OF GERM-LINEAGE CELL AND FUNCTIONAL GAMETES FROM MULTI-POTENT SPERMATOGONIAL STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 371
Author(s):  
J. E. Lim ◽  
J. H. Eum ◽  
H. J. Kim ◽  
H. S. Lee ◽  
J. H. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Culture Medium ◽  
Genetically Modified ◽  
Spermatogonial Stem Cells ◽  
Specific Gene ◽  
Specific Marker ◽  
Male Gametes ◽  
Genetically Modified Mice

Multi-potent spermatogonial stem cells (mSSC), derived from uni-potent SSC, are a type of reprogrammed cells with similar characteristics to embryonic stem cells (ESC). Similar to ESC, mSSC are capable of differentiating into 3-germ layers in vitro and teratoma formation in vivo. Additionally, mSSC proliferate rapidly and can be transfected more easily than SSC. In contrast to previous reports, we have found that mSSC also have germ-cell-specific micro (mi)RNA and gene expression profiles. Therefore, the aims of this study were to compare the efficiency of mSSC v. ESC to differentiate into germ lineage and produce male gametes, as well as to develop a novel system for the production of genetically modified mice. Mouse mSSC were transfected with a lentiviral vector expressing green fluorescent protein (GFP) and testis-specific gene and maintained in the ESC-culture medium containing leukemia inhibitory factor (LIF). Embryonic bodies (EB) were formed after the cells were detached from the feeder cells. Bone morphogenetic protein (BMP)-4 (10 ng mL˜1) and retinoic acid (RA, 0.1 μM) were added to the ESC-culture medium for 3 days in order to induce differentiation into germ lineage cells. Then, these cells were changed to germ cell-culture medium (Stem-Pro™ containing GDNF; Invitrogen, Carlsbad, CA, USA) and cultured for 3 days. After 6 days, cultured cells were sorted by magnetic activating cell sorting system using specific marker for germ cells, CD-9. Isolated germ lineage cells were transplanted into a busulfan-treated mouse testis for the production of male germ cells. Three to 6 weeks later, the testis and epididymis were collected, and half of the sample was used to perform histological analysis and the other half for the production of intracytoplasmic sperm injection (ICSI)-derived embryos. The statistical significance of differences between the 2 groups was evaluated by Student’s t-test Immunocytochemical and flow cytometrical analysis performed 6 days after differentiation showed that the ratio of germ cell-specific markers in EB derived from mSSC was higher than those from ESC. Moreover, after 3 to 6 weeks of transplantation the testis produced sperms and germ cells expressing GFP. We have successfully produced embryos by ICSI and offspring by embryo transfer into uteri of poster mothers. These results demonstrate that mSSC can be easily differentiated into germ lineage cells compared with ESC and have the potential to generate functional gametes. Therefore, the differentiation and transgenesis of mSSC may be a useful model for production of genetically modified mice. This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084923).

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