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1
Cherny, RA, TM Stokes, J. Merei, L. Lom, MR Brandon, and RL Williams. "Strategies for the isolation and characterization of bovine embryonic stem cells." Reproduction, Fertility and Development 6, no. 5 (1994): 569. http://dx.doi.org/10.1071/rd9940569.
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The practical application of advanced breeding technologies and genetic manipulation of domestic animals is dependent on the efficient and routine isolation of embryonic stem (ES) cell lines from these species. ES cell lines of proven totipotency have thus far been isolated only from the mouse. Murine ES cells can be identified by a number of criteria including morphology and characteristics in culture, the presence of specific markers, differentiative capacity and contribution to chimaeras. Reported cell lines derived from ruminant preimplantation embryos do not stably exhibit these characteristics. As demonstrated for the mouse, primordial germ cells may provide an alternative source for pluripotential cell lines. The isolation, culture and preliminary characterization of bovine primordial germ cell-derived (PGCd) cells are described in this paper. The PGCd cells are capable of differentiation in vitro and display murine ES cell markers including alkaline phosphatase. With farm animals, long generation intervals and small numbers of offspring make it important to develop techniques for evaluating chimaeric embryos in vitro before embarking on expensive in vivo programmes. A method for labelling putative pluripotential cells with a fluorochrome marker to follow the fate of such cells was developed. Labelled PGCd cells were injected into blastocysts and the chimaeric embryos were monitored in vitro. Preliminary results demonstrate that the labelled PGCd cells incorporate preferentially within the inner cell mass of the host blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)
2
Martin, Bruno, Christian Mauduit, and Joël Rivat. "PROPRIÉTÉS LOCALES DES CHIFFRES DES NOMBRES PREMIERS." Journal of the Institute of Mathematics of Jussieu 18, no. 1 (2017): 189–224. http://dx.doi.org/10.1017/s1474748017000044.
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Let $b$ be an integer larger than 1. We give an asymptotic formula for the exponential sum $$\begin{eqnarray}\mathop{\sum }_{\substack{ p\leqslant x \\ g(p)=k}}\exp \big(2\text{i}\unicode[STIX]{x1D70B}\unicode[STIX]{x1D6FD}p\big),\end{eqnarray}$$ where the summation runs over prime numbers $p$ and where $\unicode[STIX]{x1D6FD}\in \mathbb{R}$, $k\in \mathbb{Z}$, and $g:\mathbb{N}\rightarrow \mathbb{Z}$ is a strongly $b$-additive function such that $\operatorname{pgcd}(g(1),\ldots ,g(b-1))=1$.
3
Cheng, L., D. P. Gearing, L. S. White, D. L. Compton, K. Schooley, and P. J. Donovan. "Role of leukemia inhibitory factor and its receptor in mouse primordial germ cell growth." Development 120, no. 11 (1994): 3145–53. http://dx.doi.org/10.1242/dev.120.11.3145.
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The pleiotropic cytokine leukemia inhibitory factor (LIF) is able to promote the growth of mouse primordial germ cells (PGCs) in culture. It is unclear whether LIF acts directly on PGCs or indirectly via feeder cells or embryonic somatic cells. To understand the role of LIF in PGC growth, we have carried out molecular and cell culture analyses to investigate the role of both the LIF ligand and its receptor in PGC development. LIF is able to stimulate PGC growth independently of the presence of feeder cells supporting the hypothesis that LIF acts directly on PGCs to promote their growth. We show here that transcripts for the low-affinity LIF receptor (LIFR), an integral component of the functional LIF receptor complex, are expressed in the developing gonad. Fluorescence-activated cell sorter (FACS) analysis, using an anti-LIFR antiserum, demonstrates that LIFR is present on the surface of PGCs, suggesting that PGCs are likely to be a direct target of LIF action in culture. Signalling via LIFR is essential for PGC growth in culture since the anti-LIFR antiserum, which blocks LIF binding to its receptor, abolishes PGC survival in culture. Two LIF-related cytokines, namely oncostatin M and ciliary neurotrophic factor, can also promote PGC growth in culture in addition to LIF. Thus one or more of these LIFR-dependent cytokines may play an important role in PGC development in mice.
4
Yoshimizu, T., M. Obinata, and Y. Matsui. "Stage-specific tissue and cell interactions play key roles in mouse germ cell specification." Development 128, no. 4 (2001): 481–90. http://dx.doi.org/10.1242/dev.128.4.481.
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Primordial germ cells (PGCs) in mice have been recognized histologically as alkaline phosphatase (AP) activity-positive cells at 7.2 days post coitum (dpc) in the extra-embryonic mesoderm. However, mechanisms regulating PGC formation are unknown, and an appropriate in vitro system to study the mechanisms has not been established. Therefore, we have developed a primary culture of explanted embryos at pre- and early-streak stages, and have studied roles of cell and/or tissue interactions in PGC formation. The emergence of PGCs from 5.5 dpc epiblasts was observed only when they were co-cultured with extra-embryonic ectoderm, which may induce the conditions required for PGC formation within epiblasts. From 6.0 dpc onwards, PGCs emerged from whole epiblasts as did a fragment of proximal epiblast that corresponds to the area containing presumptive PGC precursors without neighboring extra-embryonic ectoderm and visceral endoderm. Dissociated epiblasts at these stages, however, did not give rise to PGCs, indicating that interactions among a cluster of a specific number of proximal epiblast cells is needed for PGC differentiation. In contrast, we observed that dissociated epiblast cells from a 6.5-b (6.5+15-16 hours) to 6.75 dpc embryo that had undergone gastrulation gave rise to PGCs. Our results demonstrate that stage-dependent tissue and cell interactions play key roles in PGC determination.
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Hayashi, Yohei, Kei Otsuka, Masayuki Ebina, et al. "Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells." Proceedings of the National Academy of Sciences 114, no. 31 (2017): 8289–94. http://dx.doi.org/10.1073/pnas.1620915114.
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Primordial germ cells (PGCs), undifferentiated embryonic germ cells, are the only cells that have the ability to become gametes and to reacquire totipotency upon fertilization. It is generally understood that the development of PGCs proceeds through the expression of germ cell-specific transcription factors and characteristic epigenomic changes. However, little is known about the properties of PGCs at the metabolite and protein levels, which are directly responsible for the control of cell function. Here, we report the distinct energy metabolism of PGCs compared with that of embryonic stem cells. Specifically, we observed remarkably enhanced oxidative phosphorylation (OXPHOS) and decreased glycolysis in embryonic day 13.5 (E13.5) PGCs, a pattern that was gradually established during PGC differentiation. We also demonstrate that glycolysis and OXPHOS are important for the control of PGC reprogramming and specification of pluripotent stem cells (PSCs) into PGCs in culture. Our findings about the unique metabolic property of PGCs provide insights into our understanding of the importance of distinct facets of energy metabolism for switching PGC and PSC status.
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Wang, Bei, Ai-Quan Jia, Hong-Mei Yang, Jing-Long Liu, and Qian-Feng Zhang. "Encapsulation of ferrocenes by hydrogen-bonded pyrogallol[4]arene dimers." Zeitschrift für Naturforschung B 73, no. 8 (2018): 597–600. http://dx.doi.org/10.1515/znb-2018-0083.
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AbstractThe treatment of C-iso-butylpyrogallarene (PgC4) or C-ethylpyrogallarene (PgC2) with ferrocene (FcH) in a 2:1 molar ratio under different reaction conditions afforded the host–guest compounds FcH@(PgC4)2·CH3OH·3H2O (1) and FcH@(PgC2)2·3EtOH·2H2O (2), respectively. Complexes 1 and 2 are both pyrogallarene dimers providing capsule-type voids. Single crystal X-ray crystallography was used to investigate the role of hydrogen bonding networks in the assembly of the two host–guest systems.
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De Felici, Massimo, Anna Di Carlo, and Maurizio Pesce. "Role of stem cell factor in somatic–germ cell interactions during prenatal oogenesis." Zygote 4, no. 04 (1996): 349–51. http://dx.doi.org/10.1017/s0967199400003373.
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During embryogenesis germ cells originate from primordial germ cells (PGCs). The development of mammalian PGCs involves a number of complex events (formation and segregation of PGC precursors, PGC migration and proliferation) which lead to the differentiation of oocytes or prospermatogonia (for a review see De Feliciet al., 1992). During recent years developments in methods for isolation, purification and culture of mouse PGCs have led to significant progress in the understanding of molecular mechanisms of migration, proliferation and differentiation of these cells (for reviews see De Felici, 1994; and De Felici & Pesce, 1994a). In this paper we describe the key role played by stem cell factor (SCF) in PGC development and early folliculogenesis.
8
Weidinger, Gilbert, Uta Wolke, Marion Köprunner, Christine Thisse, Bernard Thisse, and Erez Raz. "Regulation of zebrafish primordial germ cell migration by attraction towards an intermediate target." Development 129, no. 1 (2002): 25–36. http://dx.doi.org/10.1242/dev.129.1.25.
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Migration of primordial germ cells (PGCs) from their site of specification towards the developing gonad is controlled by directional cues from somatic tissues. Although in several animals the PGCs are attracted by signals emanating from their final target, the gonadal mesoderm, little is known about the mechanisms that control earlier steps of migration. We provide evidence that a key step of zebrafish PGC migration, in which the PGCs become organized into bilateral clusters in the anterior trunk, is regulated by attraction of PGCs towards an intermediate target. Time-lapse observations of wild-type and mutant embryos reveal that bilateral clusters are formed at early somitogenesis, owing to migration of PGCs towards the clustering position from medial, posterior and anterior regions. Furthermore, PGCs migrate actively relative to their somatic neighbors and they do so as individual cells. Using mutants that exhibit defects in mesoderm development, we show that the ability to form PGC clusters depends on proper differentiation of the somatic cells present at the clustering position. Based on these findings, we propose that these somatic cells produce signals that attract PGCs. Interestingly, fate-mapping shows that these cells do not give rise to the somatic tissues of the gonad, but rather contribute to the formation of the pronephros. Thus, the putative PGC attraction center serves as an intermediate target for PGCs, which later actively migrate towards a more posterior position. This final step of PGC migration is defective in hands off mutants, where the intermediate mesoderm of the presumptive gonadal region is mispatterned. Our results indicate that zebrafish PGCs are guided by attraction towards two signaling centers, one of which may represent the somatic tissues of the gonad.Movies available on-line
9
Ying, Ying, Xiao-Ming Liu, Amy Marble, Kirstie A. Lawson, and Guang-Quan Zhao. "Requirement of Bmp8b for the Generation of Primordial Germ Cells in the Mouse." Molecular Endocrinology 14, no. 7 (2000): 1053–63. http://dx.doi.org/10.1210/mend.14.7.0479.
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Abstract In the mouse embryo, the generation of primordial germ cells (PGCs) from the epiblast requires a bone morphogenetic protein-4 (BMP4) signal from the adjacent extraembryonic ectoderm. In this study, we report that Bmp8b, a member of the Gbb-60A class of the BMP superfamily, is expressed in the extraembryonic ectoderm in pregastrula and gastrula stage mouse embryos and is required for PGC generation. A mutation in Bmp8b on a mixed genetic background results in the absence of PGCs in 43% null mutant embryos and severe reduction in PGC number in the remainder. The heterozygotes are unaffected. On a largely C57BL/6 background, Bmp8b null mutants completely lack PGCs, and Bmp8b heterozygotes have a reduced number of PGCs. In addition, Bmp8b homozygous null embryos on both genetic backgrounds have a short allantois, and this organ is missing in some more severe mutants. Since Bmp4 heterozygote embryos have reduced numbers of PGCs, we used a genetic approach to generate double-mutant embryos to study interactions of Bmp8b and Bmp4. Embryos that are double heterozygotes for the Bmp8b and Bmp4 mutations have similar defects in PGC number as Bmp4 heterozygotes, indicating that the effects of the two BMPs are not additive. These findings suggest that BMP4 and BMP8B function as heterodimers and homodimers in PGC specification in the mouse.
10
Xin-Yan, Tang, Zeng Wei-Dong, Mi Yu-Ling, Liu Hong-Yun, and Zhang Cai-Qiao. "Isolation, culture and characterization of chicken primordial germ cells." Chinese Journal of Agricultural Biotechnology 3, no. 3 (2006): 183–88. http://dx.doi.org/10.1079/cjb2006107.
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AbstractPrimordial germ cells (PGCs) were isolated from the genital ridges of chicken (Gallus domesticus) embryos at the 19th stage and purified by Ficoll density-gradient centrifugation. PGCs were co-cultured with somatic cells in preliminary culture and subcultured. Identification of PGCs was carried out by histochemical methods, including alkaline phosphatase (AKP) and periodic acid–Schiff (PAS). The proliferating activity of PGCs in subculture was demonstrated by immunocytochemistry of proliferating cell nuclear antigen (PCNA). Meanwhile, proliferating PGCs were compared under different culture conditions of 5–20% fetal cattle serum (FCS), insulin–transferrin–selenite (ITS) medium, conditioned medium (CM), 15% FCS+ITS, 15% FCS+40% CM. The results showed that the cultured PGCs were positive for AKP and PAS staining and displayed intensive proliferating activity by PCNA. The PGCs without centrifugation grew better than those with centrifugation. The PGCs formed larger colonies in media with 5% FCS or ITS than other media, indicating that 5% FCS or ITS supplemented media could be an ideal culture system for PGC proliferation in the PGC-somatic cell co-culture, in addition to the embryonic fibroblast feeder layer.
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Sang, Xianpeng, Matthew S. Curran, and Antony W. Wood. "Paracrine Insulin-Like Growth Factor Signaling Influences Primordial Germ Cell Migration: In Vivo Evidence from the Zebrafish Model." Endocrinology 149, no. 10 (2008): 5035–42. http://dx.doi.org/10.1210/en.2008-0534.
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IGF signaling has been shown to stimulate migration of multiple cell types in vitro, but few studies have confirmed an equivalent function for IGF signaling in vivo. We recently showed that suppression of IGF receptors in the zebrafish embryo disrupts primordial germ cell (PGC) migration, but the mechanism underlying these effects has not been elucidated. We hypothesized that PGCs are intrinsically dependent upon IGF signaling during the migratory phase of development. To test this hypothesis, we first examined the spatial expression patterns of IGF ligand genes (igf1, igf2a, and igf2b) in the zebrafish embryo. In situ analyses revealed distinct expression patterns for each IGF ligand gene, with igf2b mRNA expressed in a spatial pattern that correlates strongly with PGC migration. To determine whether PGC migration is responsive to IGF signaling in vivo, we synthesized gene hybrid expression constructs that permit conditional overexpression of IGF ligands by PGCs into the PGC microenvironment. Conditional overexpression of IGF ligands consistently disrupted PGC migration, confirming that PGC migration is sensitive to local aberrations in IGF signaling. Finally, we show that conditional suppression of IGF signaling, via PGC-specific overexpression of a mutant IGF-I receptor, disrupts PGC migration, confirming that zebrafish PGCs intrinsically require IGF signaling for directional migration in vivo. Collectively, these studies confirm an in vivo role for IGF signaling in cell migration and identify a candidate ligand gene (igf2b) regulating PGC migration in the zebrafish.
12
Sano, Hiroko, Andrew D. Renault, and Ruth Lehmann. "Control of lateral migration and germ cell elimination by the Drosophila melanogaster lipid phosphate phosphatases Wunen and Wunen 2." Journal of Cell Biology 171, no. 4 (2005): 675–83. http://dx.doi.org/10.1083/jcb.200506038.
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In most organisms, primordial germ cells (PGCs) arise far from the region where somatic gonadal precursors (SGPs) are specified. Although PGCs in general originate as a single cluster of cells, the somatic parts of the gonad form on each site of the embryo. Thus, to reach the gonad, PGCs not only migrate from their site of origin but also split into two groups. Taking advantage of high-resolution real-time imaging, we show that in Drosophila melanogaster PGCs are polarized and migrate directionally toward the SGPs, avoiding the midline. Unexpectedly, neither PGC attractants synthesized in the SGPs nor known midline repellents for axon guidance were required to sort PGCs bilaterally. Repellent activity provided by wunen (wun) and wunen-2 (wun-2) expressed in the central nervous system, however, is essential in this migration process and controls PGC survival. Our results suggest that expression of wun/wun-2 repellents along the migratory paths provides faithful control over the sorting of PGCs into two gonads and eliminates PGCs left in the middle of the embryo.
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Subramaniam, K., and G. Seydoux. "nos-1 and nos-2, two genes related to Drosophila nanos, regulate primordial germ cell development and survival in Caenorhabditis elegans." Development 126, no. 21 (1999): 4861–71. http://dx.doi.org/10.1242/dev.126.21.4861.
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In Drosophila, the posterior determinant nanos is required for embryonic patterning and for primordial germ cell (PGC) development. We have identified three genes in Caenorhabditis elegans that contain a putative zinc-binding domain similar to the one found in nanos, and show that two of these genes function during PGC development. Like Drosophila nanos, C. elegans nos-1 and nos-2 are not generally required for PGC fate specification, but instead regulate specific aspects of PGC development. nos-2 is expressed in PGCs around the time of gastrulation from a maternal RNA associated with P granules, and is required for the efficient incorporation of PGCs into the somatic gonad. nos-1 is expressed in PGCs after gastrulation, and is required redundantly with nos-2 to prevent PGCs from dividing in starved animals and to maintain germ cell viability during larval development. In the absence of nos-1 and nos-2, germ cells cease proliferation at the end of the second larval stage, and die in a manner that is partially dependent on the apoptosis gene ced-4. Our results also indicate that putative RNA-binding proteins related to Drosophila Pumilio are required for the same PGC processes as nos-1 and nos-2. These studies demonstrate that evolutionarily distant organisms utilize conserved factors to regulate early germ cell development and survival, and that these factors include members of the nanos and pumilio gene families.
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Güralp, H., K. Pocherniaieva, M. Blecha, T. Policar, M. Pšenička, and T. Saito. "Migration of primordial germ cells during late embryogenesis of pikeperch Sander lucioperca relative to blastomere transplantation." Czech Journal of Animal Science 62, No. 3 (2017): 121–29. http://dx.doi.org/10.17221/40/2016-cjas.
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Pikeperch Sander lucioperca is a valuable fish in Europe, and basic information about its embryonic development, especially primordial germ cell (PGC) migration, is important for use in biotechnology. We categorized pikeperch embryonic development into six stages as in other fish species: zygote, cleavage, blastula, gastrula, segmentation, and hatching and described PGC migration. PGCs were visualized by injection of synthesized green fluorescent protein (GFP) within the 3’untranslated region (UTR) mRNA of nanos3. GFP-positive PGCs appeared in all embryos at approximately 100% epiboly. Time-lapse imaging revealed the PGC migration pattern from their initial appearance to location at the gonadal ridge. We conducted blastomere transplantation (BT) at the blastula stage. Donor embryos were labelled with GFP-nos3 3’UTR mRNA and tetramethylrhodamine dextran to label PGCs and somatic cells, respectively. Twelve BT chimeras were produced, with eight surviving to hatching. All exhibited donor-derived somatic cells in the developing body. The PGCs from donor embryos were observed to migrate towards the gonad region of the host embryos. Our results indicated that BT can be successfully applied in pikeperch, and these findings may be useful to produce germline chimeras in percids.
15
Houston, D. W., and M. L. King. "A critical role for Xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus." Development 127, no. 3 (2000): 447–56. http://dx.doi.org/10.1242/dev.127.3.447.
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Xdazl is an RNA component of Xenopus germ plasm and encodes an RNA-binding protein that can act as a functional homologue of Drosophila boule. boule is required for entry into meiotic cell division during fly spermatogenesis. Both Xdazl and boule are related to the human DAZ and DAZL, and murine Dazl genes, which are also involved in gamete differentiation. As suggested from its germ plasm localization, we show here that Xdazl is critically involved in PGC development in Xenopus. Xdazl protein is expressed in the cytoplasm, specifically in the germ plasm, from blastula to early tailbud stages. Specific depletion of maternal Xdazl RNA results in tadpoles lacking, or severely deficient in, primordial germ cells (PGCs). In the absence of Xdazl, PGCs do not successfully migrate from the ventral to the dorsal endoderm and do not reach the dorsal mesentery. Germ plasm aggregation and intracellular movements are normal indicating that the defect occurs after PGC formation. We propose that Xdazl is required for early PGC differentiation and is indirectly necessary for the migration of PGCs through the endoderm. As an RNA-binding protein, Xdazl may regulate translation or expression of factors that mediate migration of PGCs.
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Copp, Andrew J., Heather M. Roberts, and Paul E. Polani. "Chimaerism of primordial germ cells in the early postimplantation mouse embryo following microsurgical grafting of posterior primitive streak cells in vitro." Development 95, no. 1 (1986): 95–115. http://dx.doi.org/10.1242/dev.95.1.95.
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A microsurgical grafting technique has been used to introduce primordial germ cell (PGC) precursors into intact primitive-streak-stage mouse embryos in vitro. Operated embryos were cultured for 36–40 h and then analysed by a combined histochemical and autoradiographic method. PGC chimaerism occurred in embryos that received grafts of caudal primitive streak cells but not adjacent embryonic endoderm or anterolateral ectoderm/mesoderm cells. Graftderived PGCs were found to be migrating through the gut endoderm alongside host-derived PGCs in approximately half of the chimaeric embryos whereas in the other 50% of cases PGCs remained at the site of grafting in association with graft-derived somatic cells. A similar pattern of somatic chimaerism was produced by primitive streak and anterolateral ectoderm/mesoderm grafts: the allantois was colonized predominantly, with, in addition, formation of amnion, surface ectoderm and caudal mesoderm in a few embryos. The majority of embryonic endoderm grafts failed to incorporate into host embryos and formed yolk-sac-like vesicles. The findings of this study indicate that (a) PGCs originate from the embryonic ectoderm via the primitive streak during development of the mouse embryo, and (b) anterolateral ectoderm and mesoderm cells are unable to form PGCs after heterotopic grafting to the posterior primitive streak site. The combined microsurgical and embryo culture methods provide an experimental system for the analysis of PGC development in intact mouse embryos.
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Higaki, Shogo, Yoshiki Eto, Yutaka Kawakami, et al. "Production of fertile zebrafish (Danio rerio) possessing germ cells (gametes) originated from primordial germ cells recovered from vitrified embryos." REPRODUCTION 139, no. 4 (2010): 733–40. http://dx.doi.org/10.1530/rep-09-0549.
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This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos.
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Zuo, Qisheng, Jing Zhou, Man Wang, Yani Zhang, Guohong Chen, and Bichun Li. "Study on the Function and Mechanism of Lin28B in the Formation of Chicken Primordial Germ Cells." Animals 11, no. 1 (2020): 43. http://dx.doi.org/10.3390/ani11010043.
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Lin28A and Lin28B are two homologues of the same family of RNA binding proteins (RBPs). The function and molecular mechanism of Lin28A in the formation of primordial germ cells (PGCs) are very clear, but the related research on Lin28B is rarely reported. Here, we found that the overexpression of Lin28B can promote the formation of PGC in vivo. Furthermore, the overexpression of Lin28B also resulted in the inhibition of totipotency gene expression and upregulated the PGCs marker genes, and a significant increase in the number of PGCs in genital ridge, as detected by Periodic Acid-Schiff(PAS) staining. However, the inhibited Lin28B expression showed completely opposite results, which were confirmed on the PGC induction model in vitro. Mechanistically, we found that the overexpression of Lin28B can inhibit the maturation of let-7a-3p, and the results of high-throughput sequencing indicated that let-7a-3p was a negative regulator of the formation process of PGCs. Therefore, we conclude that our results determine that Lin28B participates in the formation of PGCs through let-7a-3p, which set a theoretical foundation for improving the function and mechanism of Lin28 family in the formation of PGCs.
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Ffrench-Constant, C., A. Hollingsworth, J. Heasman, and C. C. Wylie. "Response to fibronectin of mouse primordial germ cells before, during and after migration." Development 113, no. 4 (1991): 1365–73. http://dx.doi.org/10.1242/dev.113.4.1365.
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The adhesive extracellular matrix glycoprotein fibronectin is thought to play a central role in cell migration during embryogenesis. In order to define this role, we have examined the response to fibronectin in cell culture of mouse primordial germ cells (PGCs) before, during and after their migration from the hindgut into their target tissue, the genital ridges. Using an explant culture system, we show that PGCs will emigrate from tissue fragments containing hindgut, and that fibronectin stimulates this migration. Adhesion assays show that the start of PGC migration is associated with a fall in adhesion to fibronectin. Double-labelling studies using in situ hybridization and histochemistry demonstrate that migrating PGCs do not contain detectable fibronectin mRNA, suggesting that they do not synthesize and secrete the fibronectin within their migratory substratum. Taken together, these findings are consistent with an important role for fibronectin in stimulating PGC migration. In addition, however, they suggest that the interaction between PGCs and fibronectin may be important in timing the start of migration, with the fall in adhesion allowing the PGCs to commence their migration towards the genital ridges.
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Kobolak, J., E. Deak, and A. Dinnyes. "172 PRIMORDIAL GERM CELL DIFFERENTIATION FROM ES CELLS IN VITRO IN MOUSE." Reproduction, Fertility and Development 17, no. 2 (2005): 236. http://dx.doi.org/10.1071/rdv17n2ab172.
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The primordial germ cells (PGC) in the genital ridge of the embryo are the progenitors of sperm and eggs. The goal of the present study was to derive PGC cells from embryonic stem (ES) cells and compare their gene expression with that of primary PGC cultures. R1 (Nagy A et al. 1990 Development 110, 815–821) and Oct4-GiP (Ying QL et al. 2002 Nature 416, 545–548) ES cell lines were differentiated into PGCs. For in vitro differentiation, the modified method of Geijsen N et al. (2004 Nature 427, 148–154) was used. In brief, ES cell suspension was put into hanging drops (400 cells per drop) for two days, where they formed embryoid bodies (EBs). The medium consisted of Iscove's Modified Dulbecco's Media (Gibco) supplemented with 10% FBS (Hyclone, Logan, UT, USA), 30 μg mL−1 iron saturated transferrin (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Sigma, Hungary), non-essential amino acids (Sigma), 4.5 mM monothioglycerol (Sigma), 50 μg mL−1 ascorbic acid (Sigma), 2 mM glutamine (Gibco), and antibiotics. The EB clumps were differentiated in suspension culture for 2 or 5 days, and then dissociated with collagenase treatment. Cells positive for SSEA-1 were isolated from dissociated EBs by immunomagnetic bead sorting and plated into gelatinized plates in the presence of 2 μM retinoic acid (Sigma). After 7 days of culture, individual PGC colonies were isolated and subcloned. The subcloned PGCs were cultured in PGC medium consisting of Dulbecco's Modified Eagle Media (Gibco) supplemented with 15% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1000 U recombinant mouse leukaemia inhibitor factor (ESGRO®, Chemicon International, Inc., Temecula, CA, USA), 20 ng mL−1 basic fibroblast growth factor (Sigma), 60 ng mL−1 soluble mouse stem cell factor (Sigma), and antibiotics. The gene expression profile was monitored using semi-quantitative RT-PCR. The gene expression of Oct4, Nanog, Stella, Piwil2, Rnf17, and Tex14 were analyzed during the differentiation. Primary PGC cultures were also isolated from (C57BL/6 × DBA)F1 embryos of age 8.5 and 11.5 days post-coitum, and differentiated in vitro. The previously described PGC medium was used to proliferate the isolated cells. The gene expression profile of PGCs and ES-derived PGC lines were compared. There were no great differences between the gene expression profiles of PGCs and ES cell-derived PGC cells. SSEA-1 and alkaline phosphatase staining of cells did not show differences between the two cell populations. We have shown here the two PGC populations do not differ from each other in gene expression of the selected genes. Further investigation is needed to differentiate the PGCs into gametes and to analyze the gene expression of other genes involved in gamete differentiation. The authors would like to acknowledge Gyorgyi Kungl for the technical help. This research was supported by OTKA T046171 grant.
21
Hickford, D., A. Pask, G. Shaw, and M. B. Renfree. "264. Primordial germ cell specification in a marsupial, the tammar wallaby." Reproduction, Fertility and Development 20, no. 9 (2008): 64. http://dx.doi.org/10.1071/srb08abs264.
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Primordial germ cells (PGCs) are the precursors of the gametes. In the mouse, PGCs are specified within the proximal epiblast in response to signals from the extraembryonic membranes during early gastrulation. Epiblast cells competent to form PGCs express Ifitm3. A subset of these cells then express Blimp1, a marker of PGC precursors. Once lineage-restricted, PGCs express Stella. Germ cells entering the gonads express VASA protein, which is a component of the germ plasm in animals in which germ cells are specified by the inheritance of maternal determinatives. Almost all of the research on mammalian PGC specification has used the mouse as a model and it is tacitly assumed that findings in the mouse will apply to mammals in general. We are using the tammar wallaby as a marsupial model for PGC specification. Eutherians and marsupials diverged 125–148 million years ago, so comparisons between the two will provide insights into the evolution of the control of mammalian PGC specification. There are IFITM clusters in both the human (chromosome 11) and mouse (chromosome 7). In the mouse, IFITM1, 2 and 3 are expressed in PGCs, whereas IFITM4 and 5 are not (1). Only one IFITM member, IFITM5, is annotated in the opossum Ensemble database. We have cloned one tammar IFITM member and identified at least one other putative member in the tammar trace archive database. We have also cloned tammar BLIMP1 and VASA, both of which show high sequence conservation with other mammals. RT–PCR profiles for both genes during tammar gastrulation are similar to those for the mouse. In contrast, no marsupial STELLA orthologueue has been identified in either the opossum or tammar genomes. These findings suggest that some but not all of the signals and mechanisms involved in eutherian PGC specification are also applicable to marsupials. (1) Lange UC, Saitou M, Western P, Barton SC and Surani MA (2003) BMC Dev. Biol. Epub 2003 Mar 19
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Anand, Mahek, Bence Lázár, Roland Tóth, et al. "Enhancement of chicken primordial germ cell in vitro maintenance using an automated cell image analyser." Acta Veterinaria Hungarica 66, no. 4 (2018): 518–29. http://dx.doi.org/10.1556/004.2018.046.
Full textAbstract:
Primordial germ cells (PGCs) were isolated from blood samples of chicken embryos. We established four PGC lines: two males (FS-ZZ-101, GFP-ZZ-4ZP) and two females (FS-ZW-111, GFP-ZW-5ZP). We could not detect a significant difference in the marker expression profile, but there was a remarkable difference between the proliferation rates of these PGC lines. We monitored the number of PGCs throughout a three-day period using a high-content screening cell imaging and analysing system (HCS). We compared three different initial cell concentrations in the wells: ~1000 cells (1×, ~4000 (4× and ~8000 (8×. For the GFPZW- 5ZP, FS-ZZ-101 and FS-ZW-111 PGC lines the lowest doubling time was observed at 4× concentration, while for GFP-ZZ-4ZP we found the lowest doubling time at 1× concentration. At 8× initial concentration, the growth rate was high during the first two days for all cell lines, but this was followed by the appearance of cell aggregates decreasing the cell growth rate. We could conclude that the difference in proliferation rate could mainly be attributed to genotypic variation in the established PGC lines, but external factors such as cell concentration and quality of the culture medium also affect the growth rate of PGCs.
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Durcova-Hills, Gabriela, Katja Prelle, Sigrid Müller, et al. "Primary culture of porcine PGCs requires LIF and porcine membrane-bound stem cell factor." Zygote 6, no. 3 (1998): 271–75. http://dx.doi.org/10.1017/s0967199498000215.
Full textAbstract:
We studied the effect of murine leukaemia inhibitory factor (LIF), human basic fibroblast growth factor (bFGF) and porcine stem cell factor (SCF) on the survival and/or proliferation of porcine primordial germ cells (PGCs) obtained from 27-day-old embryos in vitro. PGCs were cultured in embryonic stem cell (ESC) medium supplemented with or without either LIF (1000 IU/ml) alone or LIF together with bFGF (10 ng/ml). They were seeded on mitotically inactivated feeder cells, either STO or transfected STO cells (STO#8), expressing the membrane-bound form of porcine SCF. PGCs were identified by their alkaline phosphatase (AP) activity and counted after 1, 3 and 5 days in culture. After 1 day of culture, PGCs cultured on STO#8 cells showed significantly higher survival than PGCs cultured on STO cells (p < 0.05). The combined effect of SCF and LIF caused a significant increase in PGC number by day 3 of culture when PGCs were cultured on either STO cells (p < 0.01) or STO#8 (p < 0.001). When SCF and LIF were used together with bFGF no increase in the PGC number was observed. Our results suggest that the membrane-bound form of porcine SCF plays a pivotal role in the primary culture of porcine PGCs and that bFGF is not required in vitro.
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Nakamura, Yoshiaki, Yasuhiro Yamamoto, Fumitake Usui, et al. "Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens." Reproduction, Fertility and Development 20, no. 8 (2008): 900. http://dx.doi.org/10.1071/rd08138.
Full textAbstract:
The aim of the present study was to improve the efficiency of endogenous primordial germ cell (PGC) depletion and to increase the ratio of donor PGCs in the gonads of recipient chicken embryos. A sustained-release emulsion was prepared by emulsifying equal amounts of Ca2+- and Mg2+-free phosphate-buffered saline containing 10% busulfan solubilised in N,N-dimethylformamide and sesame oil, using a filter. Then, 75 μg per 50 μL busulfan sustained-release emulsion was injected into the yolk. To determine the depletion and repopulation of PGCs in the gonads after 6 days incubation, whole-mount immunostaining was performed. The busulfan sustained-release emulsion significantly reduced the number of endogenous PGCs compared with control (P < 0.05). Moreover, the busulfan sustained-release emulsion significantly depleted endogenous PGCs compared with other previously reported busulfan delivery systems (P < 0.05), but with less variation, suggesting that the sustained-release emulsion delivered a consistent amount of busulfan to the developing chicken embryos. The PGC transfer study showed that the proportion of donor PGCs in the gonads of busulfan sustained-release emulsion-treated embryos after 6 days incubation increased 28-fold compared with control. In conclusion, the results demonstrate that exogenous PGCs are capable of migrating and settling in gonads from which endogenous PGCs have been removed using a busulfan sustained-release emulsion.
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Jiang, Jingyi, Chen Chen, Shaoze Cheng, et al. "Long Noncoding RNA LncPGCR Mediated by TCF7L2 Regulates Primordial Germ Cell Formation in Chickens." Animals 11, no. 2 (2021): 292. http://dx.doi.org/10.3390/ani11020292.
Full textAbstract:
Although lncRNAs have been identified as playing critical roles in the development of germ cells, their potential involvement in the development of PGCs in chickens remains poorly understood. Differentially expressed lncRNAs (DELs) from previous RNA-seq of embryonic stem cells (ESCs), PGCs, and spermatogonial stem cells (SSCs) were analyzed by K-means clustering, from which a key candidate, lncRNA (lncRNA PGC regulator, LncPGCR) was obtained. We confirmed that LncPGCR plays a positive role in the development of PGCs by increasing the expression of the PGC marker gene (Cvh and C-kit), while downregulating the pluripotency-associated gene (Nanog) in vitro and in vivo. The activation and expression of LncPGCR are regulated by histone acetylation, and transcription factor TCF7L2. Mechanistically, a rescue assay was performed to further confirm that LncPGCR contributed to the development of PGCs by regulating the gga-miR-6577-5p/Btrc signaling pathway. Adsorption of gga-miR-6577-5p activated the WNT signaling cascade by relieving the gga-miR-6577-5p-dependent inhibition of Btrc expression. Taken together, our study discovered the growth-expedited role of LncPGCR in PGCs development, showing the potential LncPGCR/miR-6577-5p/Btrc pathway. The results and findings provide a novel insight into the development of PGCs.
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Jin, Yilin, Wei Liu, Yangxi Xiang, et al. "Maternal miR-202-5p is required for zebrafish primordial germ cell migration by protecting small GTPase Cdc42." Journal of Molecular Cell Biology 12, no. 7 (2019): 530–42. http://dx.doi.org/10.1093/jmcb/mjz103.
Full textAbstract:
Abstract In many lower animals, germ cell formation, migration, and maintenance depend on maternally provided determinants in germ plasm. In zebrafish, these processes have been extensively studied in terms of RNA-binding proteins and other coding genes. The role of small non-coding RNAs in the regulation of primordial germ cell (PGC) development remains largely unknown and poorly investigated, even though growing interests for the importance of miRNAs involved in a wide variety of biological processes. Here, we reported the role and mechanism of the germ plasm-specific miRNA miR-202-5p in PGC migration: (i) both maternal loss and knockdown of miR-202-5p impaired PGC migration indicated by the mislocalization and reduced number of PGCs; (ii) cdc42se1 was a direct target gene of miR-202-5p, and overexpression of Cdc42se1 in PGCs caused PGC migration defects similar to those observed in loss of miR-202-5p mutants; (iii) Cdc42se1 not only interacted with Cdc42 but also inhibited cdc42 transcription, and overexpression of Cdc42 could rescue PGC migration defects in Cdc42se1 overexpressed embryos. Thus, miR-202-5p regulates PGC migration by directly targeting and repressing Cdc42se1 to protect the expression of Cdc42, which interacts with actin to direct PGC migration.
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Ying, Ying, Xiaoxia Qi, and Guang-Quan Zhao. "Induction of Primordial Germ Cells from Pluripotent Epiblast." Scientific World JOURNAL 2 (2002): 801–10. http://dx.doi.org/10.1100/tsw.2002.155.
Full textAbstract:
The formation of germ cells during embryogenesis bears the ultimate importance for the continuation of every species. It becomes evident that mechanisms governing germ cell fate specification are not well conserved across the animal kingdom. In most of the invertebrate and nonmammalian vertebrate species, certain maternally derived factors are key to the establishment of germ cell lineage. In contrast, mouse primordial germ cells (PGCs) are induced from the pluripotent epiblast cells before and during gastrulation by the extraembryonic cell-derived signals. The molecular identity for some of these signals has recently been revealed by genetic and epiblast culture experiments. Both bone morphogenetic proteins 4 (Bmp4) and 8b (Bmp8b) are expressed in the extraembryonic ectoderm and are required for PGC formation. Furthermore, BMP4 or BMP8B alone are unable to induce PGCs from cultured epiblasts, while they can in combination, indicating they signal through separate receptor complexes. In addition, Bmp4 homozygous embryos cannot be induced to form PGCs by the synergistic action of BMP4 and BMP8B, suggesting that BMP4 proteins produced by pregastrula embryos are required for epiblast cells to maintain pluripotency. Moreover, Bmp2, a close relative of Bmp4, is expressed in visceral endoderm at the time of PGC specification, and inactivation of Bmp2 results in a reduction in PGC number, revealing a novel function of visceral endoderm in PGC generation in the mouse.
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Babich, Bridget, George Roba, Siti Sarah Safura, Kevin Callahan, and Edward Freeman. "Transcription of nanos-1 in Zebrafish Embryos is not Affected by Bisphenol A: Evaluated Using Quantitative Real-Time PCR." American Journal of Undergraduate Research 16, no. 1 (2019): 15–21. http://dx.doi.org/10.33697/ajur.2019.012.
Full textAbstract:
The presence of primordial germ cells (PGCs) is crucial for proper gonad formation in zebrafish (Danio rerio). The many aspects of PGC migration that allow these cells to reach the proper location at the gonadal ridge include receptors, ligands, germ plasm components, and internal maintenance of PGCs. Any one of these factors could be affected by endocrine-disrupting chemicals (EDCs), which have been shown to alter the directed migration of these cells during early embryonic development. Based on recent research wherein the EDC bisphenol A (BPA) inhibited normal PGC migration, we have used the same dose of BPA to determine the impact of BPA on a gene central to proper germ cell migration. Zebrafish embryos were exposed to BPA, and the levels of the target gene nanos-1 were analyzed using quantitative real-time PCR (q-PCR). The target gene nanos-1 is a critically important germplasm component that allows for survival and proper migration of PGCs. The q-PCR results showed that BPA did not affect the transcription level of nanos-1 in zebrafish embryos. KEYWORDS: Zebrafish; Zebrafish Embryos; nanos-1; Primordial Germ Cells; PGC Migration; Gonad Development; Endocrine-Disrupting Chemicals; Bisphenol A; Sex Determination
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Glumakova, K. A., O. N. Mityaeva, E. N. Antonova, O. V. Glazova, A. S. Komarchev, and P. Y. Volchkov. "95 Obtaining birds with chimeric gonads using invitro lentiviral transduction of primordial germ cells." Reproduction, Fertility and Development 32, no. 2 (2020): 173. http://dx.doi.org/10.1071/rdv32n2ab95.
Full textAbstract:
Avian primordial germ cells (PGCs) have unique migration capacity towards the gonads via the bloodstream. Therefore, PGCs invitro genome modification is a dominant approach for poultry genetic modification. The aim of this study was to improve cultivation conditions of PGCs in terms of proliferation activity and further analyse their migration abilities. We isolated PGCs from whole blood cells collected from the embryonic dorsal aorta (Hamburger-Hamilton (HH) stage 14-17) and cultured them without feeder cells in customized avian knockout Dulbecco's modified Eagle's medium basal medium supplemented with fibroblast growth factor 2, Activin A and B-27 supplement containing insulin. We analysed the number of cells during the month of culture. Proliferation of PGCs increased in the first 7d of culture when both BMP4 and Activin A growth factors were added to the medium (four out of seven samples). Kinetic of stemness and PGC-marker genes (Nanog, PouV, DAZL) were similar to expression in mature gonadal PGCs (gPGCs). Expression levels of marker genes were significantly greater in the freshly isolated PGCs compared to the gPGCs and decreased during cultivation. In order to confirm the migration activity of the cultivated PGCs, cells were labelled with lentivirus (ZsGreen) and injected into the embryo blood stream (HH 14-17). Gonads from the recipient embryos were retrieved (HH 20-26) and analysed. Due to the low level of fluorescence in mature cells, the presence of transgenic PGC in the gonads was detected by ZsGreen-specific quantitative PCR. We confirmed that the cultured cells maintained migration activity, and efficient engraftment was detected in 75% of embryos.
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Lee, Jiyoung, Kimiko Inoue, Ryuichi Ono, et al. "Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells." Development 129, no. 8 (2002): 1807–17. http://dx.doi.org/10.1242/dev.129.8.1807.
Full textAbstract:
Genomic imprinting is an epigenetic mechanism that causes functional differences between paternal and maternal genomes, and plays an essential role in mammalian development. Stage-specific changes in the DNA methylation patterns of imprinted genes suggest that their imprints are erased some time during the primordial germ cell (PGC) stage, before their gametic patterns are re-established during gametogenesis according to the sex of individuals. To define the exact timing and pattern of the erasure process, we have analyzed parental-origin-specific expression of imprinted genes and DNA methylation patterns of differentially methylated regions (DMRs) in embryos, each derived from a single day 11.5 to day 13.5 PGC by nuclear transfer. Cloned embryos produced from day 12.5 to day 13.5 PGCs showed growth retardation and early embryonic lethality around day 9.5. Imprinted genes lost their parental-origin-specific expression patterns completely and became biallelic or silenced. We confirmed that clones derived from both male and female PGCs gave the same result, demonstrating the existence of a common default state of genomic imprinting to male and female germlines. When we produced clone embryos from day 11.5 PGCs, their development was significantly improved, allowing them to survive until at least the day 11.5 embryonic stage. Interestingly, several intermediate states of genomic imprinting between somatic cell states and the default states were seen in these embryos. Loss of the monoallelic expression of imprinted genes proceeded in a step-wise manner coordinated specifically for each imprinted gene. DNA demethylation of the DMRs of the imprinted genes in exact accordance with the loss of their imprinted monoallelic expression was also observed. Analysis of DNA methylation in day 10.5 to day 12.5 PGCs demonstrated that PGC clones represented the DNA methylation status of donor PGCs well. These findings provide strong evidence that the erasure process of genomic imprinting memory proceeds in the day 10.5 to day 11.5 PGCs, with the timing precisely controlled for each imprinted gene. The nuclear transfer technique enabled us to analyze the imprinting status of each PGC and clearly demonstrated a close relationship between expression and DNA methylation patterns and the ability of imprinted genes to support development.
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Liu, Yifan, Siyan Liu, Ziyi Liu, et al. "Porous quasi-graphitic carbon sheets for unprecedented sodium storage." Inorganic Chemistry Frontiers 7, no. 13 (2020): 2443–50. http://dx.doi.org/10.1039/d0qi00325e.
Full textAbstract:
Water-soluble KCl as the catalyst and template to produce PGC sheets in CCVD. Abundant channels on the surface of PGCs helps the transportation and diffusion of Na<sup>+</sup>. PGC sheets deliver a high reversible specific capacity of 237 mA h g<sup>−1</sup> at 0.1 A g<sup>−1</sup>.
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Pesce, M., M. G. Farrace, M. Piacentini, S. Dolci, and M. De Felici. "Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis)." Development 118, no. 4 (1993): 1089–94. http://dx.doi.org/10.1242/dev.118.4.1089.
Full textAbstract:
Proliferating primordial germ cells (PGCs) isolated from mouse embryos soon after their arrival in the genital ridges would only survive in vitro at temperature of less than 30 degrees C (De Felici, M. and McLaren, A. (1983). Exp. Cell. Res. 144, 417–427; Wabik-Sliz, B. and McLaren, A. (1984). Exp. Cell. Res. 154, 530–536) or when co-cultured on cell feeder layers (Donovan, P. J., Stott, D., Godin, I., Heasman, J. and Wylie, C. C. (1986). Cell 44, 831–838; De Felici, M. and Dolci, S. (1991). Dev. Biol. 147, 281–284). In the present paper we report that mouse PGC death in vitro occurs with all the hallmarks of programmed cell death or apoptosis. We found that after 4–5 hours in culture many PGCs isolated from 12.5 dpc fetal gonads assumed a nuclear morphology and produced membrane bound fragments (apoptotic bodies) typical of apoptotic cells. In addition, PGCs in culture accumulated high level of tissue transglutaminase (tTGase; an enzyme that is induced and activated during apoptosis) and showed extensive degradation of DNA to oligonucleosomal fragments, which is characteristic of apoptosis. The physiological relevance of this mechanism of PGC death is supported by the finding that some PGCs undergoing apoptosis, as revealed by the high level of tTGase expression, were detected in the embryo. Most importantly, we show that the addition of stem cell factor (SCF) or leukemia inhibitory factor (LIF) to the culture medium, two cytokines known to favour PGC survival and/or proliferation in vitro, markedly reduced the occurrence of apoptosis in PGCs during the first hours in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
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Yu, Minli, Dongfeng Li, Wanyan Cao, Xiaolu Chen, and Wenxing Du. "Effects of ten–eleven translocation 1 (Tet1) on DNA methylation and gene expression in chicken primordial germ cells." Reproduction, Fertility and Development 31, no. 3 (2019): 509. http://dx.doi.org/10.1071/rd18145.
Full textAbstract:
Ten–eleven translocation 1 (Tet1) is involved in DNA demethylation in primordial germ cells (PGCs); however, the precise regulatory mechanism remains unclear. In the present study the dynamics of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing PGCs and the role of Tet1 in PGC demethylation were analysed. Results show that 5mC levels dropped significantly after embryonic Day 4 (E4) and 5hmC levels increased reaching a peak at E5–E5.5. Interestingly, TET1 protein was highly expressed during E5 to E5.5, which showed a consistent trend with 5hmC. The expression of pluripotency-associated genes (Nanog, PouV and SRY-box 2 (Sox2)) and germ cell-specific genes (caveolin 1 (Cav1), piwi-like RNA-mediated gene silencing 1 (Piwi1) and deleted in azoospermia-like (Dazl)) was upregulated after E5, whereas the expression of genes from the DNA methyltransferase family was decreased. Moreover, the Dazl gene was highly methylated in early PGCs and then gradually hypomethylated. Knockdown of Tet1 showed impaired survival and proliferation of PGCs, as well as increased 5mC levels and reduced 5hmC levels. Further analysis showed that knockdown of Tet1 led to elevated DNA methylation levels of Dazl and downregulated gene expression including Dazl. Thus, this study reveals the dynamic epigenetic reprogramming of chicken PGCs invivo and the molecular mechanism of Tet1 in regulating genomic DNA demethylation and hypomethylation of Dazl during PGC development.
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Gomperts, M., M. Garcia-Castro, C. Wylie, and J. Heasman. "Interactions between primordial germ cells play a role in their migration in mouse embryos." Development 120, no. 1 (1994): 135–41. http://dx.doi.org/10.1242/dev.120.1.135.
Full textAbstract:
Primordial germ cells (PGCs) are the founder cell population of the gametes which form during the sexually mature stage of the life cycle. In the mouse, they arise early in embryogenesis, first becoming visible in the extraembryonic mesoderm, posterior to the primitive streak, at 7.5 days post coitum (d.p.c.). They subsequently become incorporated into the epithelium of the hind gut, from which they emigrate (9.5 d.p.c.) and move first into the dorsal mesentery (10.5 d.p.c.), and then into the genital ridges that lie on the dorsal body wall (11.5 d.p.c.). We have used confocal microscopy to study PGCs stained with an antibody that reacts with a carbohydrate antigen (Stage-Specific Embryonic Antigen-1, SSEA-1) carried on the PGC surface. This allows the study of the whole PGC surface, at different stages of their migration. The appearance of PGCs in tissue sections has given rise to the conventional view that they migrate as individuals, each arriving in turn at the genital ridge. In this paper, we show that PGCs leave the hind gut independently, but then extend long (up to 40 microns) processes, with which they link up to each other to form extensive networks. During the 10.5-11.5 d.p.c. period, these networks of PGCs aggregate into groups of tightly apposed cells in the genital ridges. As this occurs, their processes are lost, and their appearance suggests they are now non-motile. Furthermore, we find that PGCs taken from the dorsal mesentery at 10.5 d.p.c. perform the same sequence of movements in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
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Aflatoonian, Behrouz, and Harry Moore. "Germ cells from mouse and human embryonic stem cells." Reproduction 132, no. 5 (2006): 699–707. http://dx.doi.org/10.1530/rep-06-0022.
Full textAbstract:
Mammalian gametes are derived from a founder population of primordial germ cells (PGCs) that are determined early in embryogenesis and set aside for unique development. Understanding the mechanisms of PGC determination and differentiation is important for elucidating causes of infertility and how endocrine disrupting chemicals may potentially increase susceptibility to congenital reproductive abnormalities and conditions such as testicular cancer in adulthood (testicular dysgenesis syndrome). Primordial germ cells are closely related to embryonic stem cells (ESCs) and embryonic germ (EG) cells and comparisons between these cell types are providing new information about pluripotency and epigenetic processes. Murine ESCs can differentiate to PGCs, gametes and even blastocysts – recently live mouse pups were born from sperm generated from mESCs. Although investigations are still preliminary, human embryonic stem cells (hESCs) apparently display a similar developmental capacity to generate PGCs and immature gametes. Exactly how such gamete-like cells are generated during stem cell culture remains unclear especially as in vitro conditions are ill-defined. The findings are discussed in relation to the mechanisms of human PGC and gamete development and the biotechnology of hESCs and hEG cells.
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Barnett, Austen A., Taro Nakamura, and Cassandra G. Extavour. "Hox genes limit germ cell formation in the short germ insect Gryllus bimaculatus." Proceedings of the National Academy of Sciences 116, no. 33 (2019): 16430–35. http://dx.doi.org/10.1073/pnas.1816024116.
Full textAbstract:
Hox genes are conserved transcription factor-encoding genes that specify the identity of body regions in bilaterally symmetrical animals. In the cricket Gryllus bimaculatus, a member of the hemimetabolous insect group Orthoptera, the induction of a subset of mesodermal cells to form the primordial germ cells (PGCs) is restricted to the second through the fourth abdominal segments (A2 to A4). In numerous insect species, the Hox genes Sex-combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), and abdominal-A (abd-A) jointly regulate the identities of middle and posterior body segments, suggesting that these genes may restrict PGC formation to specific abdominal segments in G. bimaculatus. Here we show that reducing transcript levels of some or all of these Hox genes results in supernumerary and/or ectopic PGCs, either individually or in segment-specific combinations, suggesting that the role of these Hox genes is to limit PGC development with respect to their number, segmental location, or both. These data provide evidence of a role for this ancient group of genes in PGC development.
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Cantú, Andrea V., Svetlana Altshuler-Keylin, and Diana J. Laird. "Discrete somatic niches coordinate proliferation and migration of primordial germ cells via Wnt signaling." Journal of Cell Biology 214, no. 2 (2016): 215–29. http://dx.doi.org/10.1083/jcb.201511061.
Full textAbstract:
Inheritance depends on the expansion of a small number of primordial germ cells (PGCs) in the early embryo. Proliferation of mammalian PGCs is concurrent with their movement through changing microenvironments; however, mechanisms coordinating these conflicting processes remain unclear. Here, we find that PGC proliferation varies by location rather than embryonic age. Ror2 and Wnt5a mutants with mislocalized PGCs corroborate the microenvironmental regulation of the cell cycle, except in the hindgut, where Wnt5a is highly expressed. Molecular and genetic evidence suggests that Wnt5a acts via Ror2 to suppress β-catenin–dependent Wnt signaling in PGCs and limit their proliferation in specific locations, which we validate by overactivating β-catenin in PGCs. Our results suggest that the balance between expansion and movement of migratory PGCs is fine-tuned in different niches by the opposing β-catenin–dependent and Ror2-mediated pathways through Wnt5a. This could serve as a selective mechanism to favor early and efficient migrators with clonal dominance in the ensuing germ cell pool while penalizing stragglers.
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Mucksová, Jitka, Markéta Reinišová, Jiří Kalina, Barbora Lejčková, Jiří Hejnar, and Pavel Trefil. "Conservation of chicken male germline by orthotopic transplantation of primordial germ cells from genetically distant donors†." Biology of Reproduction 101, no. 1 (2019): 200–207. http://dx.doi.org/10.1093/biolre/ioz064.
Full textAbstract:
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.
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Soo Kang, Kyung, Hyung Chul Lee, Hyun Jeong Kim, et al. "Spatial and temporal action of chicken primordial germ cells during initial migration." REPRODUCTION 149, no. 2 (2015): 179–87. http://dx.doi.org/10.1530/rep-14-0433.
Full textAbstract:
In most animals, primordial germ cells (PGCs) originate from an extragonadal region and migrate across the embryo to the gonads, where they differentiate and function. During their migration, PGCs move passively by morphogenetic movement of the embryo or move actively through signaling molecules. To uncover the underlying mechanism of first-phase PGC migration toward the germinal crescent in chickens, we investigated the spatial and temporal action of PGCs during primitive streak formation. Exogenously transplanted PGCs migrated toward the anterior region of the embryo and the embryonic gonads when they were transplanted into the subgerminal cavity, but not into the posterior marginal zone, in Eyal–Giladi and Kochav stage X embryos. These results indicate that for passive migration toward the anterior region the initial location of PGCs should be the central region. Notably, although PGCs and DF-1 cells migrated passively toward the anterior region, only PGCs migrated to the germinal crescent, where endogenous PGCs mainly reside, by active movement. In a live-imaging experiment with green fluorescence protein-expressing transgenic embryos, exogenous PGCs demonstrated markedly faster migration when they reached the anterior one-third of the embryo, while somatic cells showed epiblast movement with constant speed. Also, migrating PGCs exhibited successive contraction and expansion indicating their active migration. Our results suggest that chicken PGCs use sequential passive and active forces to migrate toward the germinal crescent.
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Popovic, Mina, Monika Bialecka, Maria Gomes Fernandes, et al. "Human blastocyst outgrowths recapitulate primordial germ cell specification events." Molecular Human Reproduction 25, no. 9 (2019): 519–26. http://dx.doi.org/10.1093/molehr/gaz035.
Full textAbstract:
Abstract Our current knowledge of the mechanisms leading to human primordial germ cell (PGC) specification stems solely from differentiation experiments starting from human pluripotent stem cells. However, information regarding the origin of PGCs in vivo remains obscure. Here we apply an improved system for extended in vitro culture of human embryos to investigate the presence of PGC-like cells (PGCLCs) 12 days post fertilization (dpf). Good quality blastocysts (n = 141) were plated at 6 dpf and maintained in hypoxia, in medium supplemented with Activin A until 12 dpf. We primarily reveal that 12 dpf outgrowths recapitulate human peri-implantation events and demonstrate that blastocyst quality significantly impacts both embryo viability at 12 dpf, as well as the presence of POU5F1+ cells within viable outgrowths. Moreover, detailed examination of 12 dpf blastocyst outgrowths revealed a population of POU5F1+, SOX2– and SOX17+ cells that may correspond to PGCLCs, alongside POU5F1+ epiblast-like cells and GATA6+ endoderm-like cells. Our findings suggest that, in human, PGC precursors may become specified within the epiblast and migrate either transiently to the extra-embryonic mesoderm or directly to the dorsal part of the yolk sac endoderm around 12 dpf. This is a descriptive analysis and as such the conclusion that POU5F1+ and SOX17+ cells represent bona fide PGCs can only be considered as preliminary. In the future, other PGC markers may be used to further validate the observed cell populations. Overall, our findings provide insights into the origin of the human germline and may serve as a foundation to further unravel the molecular mechanisms governing PGC specification in human.
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Kucia, Magdalena, Kasia Mierzejewska, Dong-Myung Shin, Yupo Ma, and Mariusz Z. Ratajczak. "Most Primitive Murine Bone Marrow Hematopoietic Stem Cells Express Several Primordial Germline Cells (PGCs) Markers, Including SALL4 - a Proposed Developmental Link Between Hematopoietic and Primordial Germ Cell Lineages." Blood 120, no. 21 (2012): 4745. http://dx.doi.org/10.1182/blood.v120.21.4745.4745.
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Abstract Abstract 4745 Background. Sal-like protein-4 (SALL4), a member of the SALL gene family, is one of the most important transcriptional regulators for primitive stem cells. The SALL4/Oct4/Nanog core transcriptional network governs the self-renewal and pluripotent properties of human and murine ESCs. Sall4 has been demonstrated to be expressed in normal hematopoietic cells (HSCs) and leukemic cells. What is intriguing, in addition to the hematopoietic lineage, Sall4 is expressed in the germline lineage, which includes primordial germ cells (PGCs). Evidence accumulates that hematopoietic stem cells (HSCs) could become specified from a population of migrating PGCs during embryogenesis. In support of this intriguing possibility, HSCs and PGCs are highly migratory populations of stem cells and specification of the first primitive HSCs in yolk sac blood islands as well as the origin of definitive HSCs in the aorta-gonado-mesonephros (AGM) region are chronologically and anatomically correlated with the developmental migration of PGCs. Furthermore, sharing of chromosomal aberrations between germline tumors and leukemias or lymphomas has also been described, which suggests their clonal origin. Our recent work demonstrated the presence of small Oct-4+Nanog+Sca-1+Lin–CD45– stem cells in adult murine BM and small Oct-4+Nanog+CD133+Lin–CD45– stem cells in human umbilical cord blood that can be specified into the hematopoietic lineage (Leukemia 2012;25,1278, Exp. Hematology 2011;39:225). These cells were named very small embryonic-like stem cells (VSELs). Our initial observations suggested that murine VSELs express several markers shared with PGCs (Leukemia 2010;24:1450). Hypothesis. The aim of our study was to test the hypothesis that VSELs are related to PGCs, which would support an alternative explanation of the origin of HSPCs and a potential developmental link between hematopoiesis and the germ line. Experimental strategies. Murine Sca-1+Lin–CD45– VSELs and Sca-1+Lin–CD45+ HSCs were purified by double FACS sorting from murine BM. We employed RQ-PCR analysis, immunohistochemical staining, and promoter methylation analysis to evaluate the expression of genes characteristic of PGC specification and expressed in early PGCs, migrating PGCs, and post-migrating PGCs. In parallel, we also analyzed Sall4 expression. Expression of these genes in BM-purified VSELs and HSCs was studied under steady-state conditions as well as in a model of hematological stress due to prolonged bleeding. Results. We found that murine BM-VSELs highly express Sall4 (700-fold more) compared with BM mononuclear cells. Sall4 expression in VSELs has been subsequently confirmed at the protein level. Sall4 mRNA expression was also detected in murine BM-derived HSCs, but at a much lower level. Of note, the expression of Sall4, like Oct-4 and Nanog, was downregulated in VSELs during their hematopoietic specification in the prolonged-bleeding model. Furthermore, we found that VSELs isolated under steady-state conditions from BM highly express, at the mRNA and protein levels, genes involved in specification of the epiblast (e.g., Stella, Prdm14, Fragilis, Blimp1, Nanos3, and Dnd1). The expression of Stella in VSELs has been evaluated by analysis of histone modifications of this locus by ChIP assay and revealed that the Stella promoter chromatin structure is characteristic of an actively transcribed gene. Next, we focused on the expression of genes specific to various PGC developmental stages and, in addition to genes involved in PGC specification, VSELs highly express Dppa2, Dppa4, and Mvh, which characterize late migratory PGCs. By contrast, however, they do not express Sycp3, Dazl, and LINE1 genes, which are highly expressed in post-migratory PGCs. Conclusions. Our results suggest that VSELs deposited into murine BM express Sall4 and several genes characteristic for epiblast-derived migratory PGCs. In toto, our data support the alternative concept that HSCs could be specified during development from migrating PGCs, and VSELs, as the most primitive population of HSCs in BM, express several germline unique markers, including Sall4. Disclosures: Ratajczak: Neostem Inc: Member of SAB Other.
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García-Castro, Martín I., Robert Anderson, Janet Heasman, and Christopher Wylie. "Interactions between Germ Cells and Extracellular Matrix Glycoproteins during Migration and Gonad Assembly in the Mouse Embryo." Journal of Cell Biology 138, no. 2 (1997): 471–80. http://dx.doi.org/10.1083/jcb.138.2.471.
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Cells are known to bind to individual extracellular matrix glycoproteins in a complex and poorly understood way. Overall strength of adhesion is thought to be mediated by a combinatorial mechanism, involving adhesion of a cell to a variety of binding sites on the target glycoproteins. During migration in embryos, cells must alter their overall adhesiveness to the substrate to allow locomotion. The mechanism by which this is accomplished is not well understood. During early development, the cells destined to form the gametes, the primordial germ cells (PGCs), migrate from the developing hind gut to the site where the gonad will form. We have used whole-mount immunocytochemistry to study the changing distribution of three extracellular matrix glycoproteins, collagen IV, fibronectin, and laminin, during PGC migration and correlated this with quantitative assays of adhesiveness of PGCs to each of these. We show that PGCs change their strength of adhesion to each glycoprotein differentially during these stages. Furthermore, we show that PGCs interact with a discrete tract of laminin at the end of migration. Closer analysis of the adhesion of PGCs to laminin revealed that PGCs adhere particularly strongly to the E3 domain of laminin, and blocking experiments in vitro suggest that they adhere to this domain using a cell surface proteoglycan.
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Godin, I., C. Wylie, and J. Heasman. "Genital ridges exert long-range effects on mouse primordial germ cell numbers and direction of migration in culture." Development 108, no. 2 (1990): 357–63. http://dx.doi.org/10.1242/dev.108.2.357.
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The functional gametes of all vertebrates first arise in the early embryo as a migratory population of cells, the primordial germ cells (PGCs). These migrate to, and colonise, the genital ridges (GR) during the early organogenesis period, giving rise to the complete differentiating gonad. PGCs first become visible by alkaline phosphatase staining in the root of the developing allantois at 8.5 days post coitum (dpc). At 9.5 dpc they are found in the wall of the hind-gut and, during the following three days, they migrate along the hind-gut mesentery to the dorsal body wall, and then to the genital ridges. By 12.5 dpc, the great majority of PGCs have colonised the genital ridges. During this period the number of PGCs increases from less than 100 to approximately 4000. In a previous paper (Donovan et al. 1986), we showed that 10.5 dpc PGCs can be explanted from the hind-gut mesentery, and will spread and migrate on feeder cell layers. We showed also that the intrinsic ability of PGCs to spread and migrate changes as they colonise the genital ridges. In this paper, we examine extrinsic factors that control PGC behaviour in vitro. Using PGCs taken from 8.5 dpc embryos, at the beginning of their migratory phase, we show that culture medium conditioned by 10.5 dpc genital ridges causes an increase in the number of PGCs in these cultures. We also show that PGCs migrate towards 10.5 dpc genital ridges in preference to other explanted organs. These experiments show that genital ridges exert long-range effects on the migrating population of PGCs.(ABSTRACT TRUNCATED AT 250 WORDS)
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Günesdogan, Ufuk, Erna Magnúsdóttir, and M. Azim Surani. "Primoridal germ cell specification: a context-dependent cellular differentiation event." Philosophical Transactions of the Royal Society B: Biological Sciences 369, no. 1657 (2014): 20130543. http://dx.doi.org/10.1098/rstb.2013.0543.
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During embryonic development, the foundation of the germline is laid by the specification of primordial germ cells (PGCs) from the postimplantation epiblast via bone morphogenetic protein (BMP) and WNT signalling. While the majority of epiblast cells undergo differentiation towards somatic cell lineages, PGCs initiate a unique cellular programme driven by the cooperation of the transcription factors BLIMP1, PRDM14 and AP2γ. These factors synergistically suppress the ongoing somatic differentiation and drive the re-expression of pluripotency and germ cell-specific genes accompanied by global epigenetic changes. However, an unresolved question is how postimplantation epiblast cells acquire the developmental competence for the PGC fate downstream of BMP/WNT signalling. One emerging concept is that transcriptional enhancers might play a central role in the establishment of developmental competence and the execution of cell fate determination. Here, we discuss recent advances on the specification and reprogramming of PGCs thereby highlighting the concept of enhancer function.
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Miyoshi, Norikatsu, Jente M. Stel, Keiko Shioda, et al. "Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells." Proceedings of the National Academy of Sciences 113, no. 34 (2016): 9545–50. http://dx.doi.org/10.1073/pnas.1610259113.
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The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.
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Kostaman, Tatan, Tuti L. Yusuf, M. Fachrudin, and M. A. Setiadi. "Effectiveness of DMSO concentration on recovery rate and viability of primordial germ cell of Gaok chicken." Jurnal Ilmu Ternak dan Veteriner 22, no. 1 (2018): 30. http://dx.doi.org/10.14334/jitv.v22i1.1799.
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<p class="abstrak2">Recent technological developments to produce germ line chimeras with primordial germ cell (PGC) transfer into the recipient embryo provide an opportunity to conserve and retrieval of chicken genetic resources in complete form. The study was conducted to obtain the most effective DMSO percentage to recovery rate and viability of Gaok chicken PGC after freezing which will later be feasible to be transferred. In this study, the eggs of Gaok chicken were incubated for about 2.5 - 3 days to obtain embryos at stages 14 - 16. Blood retrieval was done through the dorsal aorta using micropipettes under microscope. The procedure of PGC isolation of Gaok chicken with centrifugation gradient was using nycodenz as a substance. Commercially available cryoprotectants (dimethyl sulfoxide = DMSO) were used for PGC freezing. Isolated and frozen PGCs of Gaok chicken were diluted with cryoprotectants containing 2.5; 5 and 10% DMSO in fetal bovine serum (FBS). The recovery rate of 2.5; 5 and 10% DMSO concentration were 36.4; 48.2 and 48 % respectively. The viability of PGC after freezing was significantly higher for 5% DMSO compared with 2.5% DMSO (P&lt;0.05), but not different from 10% DMSO. It can be concluded that the concentration DMSO of 5 % was effectve contration in freezing Gaok chicken PGC.</p>
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Fang, Changge, Caiqiao Zhang, Guoliang Xia, and Wei Yang. "Damaging effects of polychlorinated biphenyls on chicken primordial germ cells." Reproduction, Fertility and Development 14, no. 3 (2002): 177. http://dx.doi.org/10.1071/rd01118.
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This work describes the effects of a commercial polychlorinated biphenyl (PCB) mixture, Aroclor 1254, as well as 17β-oestradiol (E2) and testosterone on numbers and histomorphological changes of primordial germ cells (PGCs) in gonadal regions of Day 5 Hyline chicken embryos. The oestrogen receptor antagonist, clomiphen, alone or with PCBs was used in an attempt to protect the developing gonad from oestrogen-like effects of chemical PCBs. The results were as follows: (i) PCBs delayed embryonic development independently of dose (1�µg/eggP<0.05; 10 µg/eggP<0.01; 100 µg/eggP<0.001 v. the control) and caused a dose-independent increase in mortality compared with the control group (10 µg/egg P<0.01; 100 µg/eggP<0.05); maximal mortality was observed in the 1 µg/eggP<0.001); (ii) PCBs decreased PGC numbers dose dependently (P<0.001) and caused a swollen nucleus with hyperchromatism (pyknosis) or cytoplasm vacuolation as signs of gonadal PGC degeneration in all PCB groups, or even complete disappearance in the 100 µg/eggiii) after PCB treatment, the index of gonadal lesion increased significantly with the decrease of gonadal PGC number (1, 10 and 100 µg/eggP<0.001); (iv) there were no observed effects of E2, testosterone and clomiphen on PGCs in the experiments; and (v) clomiphen failed to block the damaging effects of PCBs. These results suggest that the adverse effects of PCBs on chicken gonadal and germ cell development were initiated during the early stages of incubation through direct toxic effects, rather than through oestrogen-mimicking actions. As PGC numbers in the gonads decrease and the index of gonadal lesion increases, one may expect reproductive function to be compromised.
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Alonso, Edurne, Francisco J. Sáez, Juan F. Madrid, and Francisco Hernández. "Lectin Histochemistry Shows Fucosylated Glycoconjugates in the Primordial Germ Cells of Xenopus Embryos." Journal of Histochemistry & Cytochemistry 51, no. 2 (2003): 239–43. http://dx.doi.org/10.1177/002215540305100212.
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Previous works have shown that glycoconjugates with terminal fucose (Fuc) are located in the primordial germ cells (PGCs) of some mammals and might play a role in the migration and adhesion processes during development. The aim of this work was to identify the terminal Fuc moieties of Xenopus PGCs by means of three Fuc-binding lectins: from asparagus pea (LTA), gorse seed (UEA-I), and orange peel fungus (AAA). The histochemical procedures were also carried out after deglycosylation pretreatments: β-elimination with NaOH to remove O-linked oligosaccharides; incubation with PNGase F to remove N-linked carbohydrate chains; and incubation with α(1,2)- and α(1,6)-fucosidase. The PGCs were always negative for LTA and UEA-I, two lectins that have the highest affinity for Fuc α(1,2)-linked. However, the PGCs were strongly labeled with AAA, which preferentially binds to Fuc with α(1,3) or α(1,4) linkages and to Fuc α(1,6)-linked to the proximal N-acetylglucosamine. There was fainter labeling with AAA when the sections were preincubated with α(1,6)-fucosidase, but the labeling remained strong when the sections were pretreated with α(1,2)fucosidase. When the β-elimination procedure was carried out, the PGC labeling with AAA was slight. If the PNGase F incubation was performed, the PGCs remained moderately positive for AAA. These data suggest that the Xenopus PGCs have Fuc moieties in O- and N-linked oligosaccharides, including Fuc α(1,6) linked to the innermost GlcNAc, and that the Fuc was not in α(1,2)-linkage.
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Ikenishi, K., and Y. Tsuzaki. "The positional effect of presumptive primordial germ cells (pPGCs) on their differentiation into PGCs in Xenopus." Development 102, no. 3 (1988): 527–35. http://dx.doi.org/10.1242/dev.102.3.527.
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To determine whether the location of ‘germ plasm’-bearing cells [presumptive primordial germ cells (pPGCs)] is crucial for their differentiation into PGCs in Xenopus, [3H]thymidine-labelled pPGCs were implanted into the anterior or posterior halves of the endoderm in unlabelled host neurulae. Labelled PGCs in the genital ridges of experimental tadpoles were investigated by autoradiography. When the labelled pPGCs were implanted into posterior halves of the endoderm where host pPGCs are situated, 65 and 77% of the experimental tadpoles (designated as p-tadpoles) had the labelled PGCs in series I and II, respectively. When implanted into the anterior halves, 20 and 27% of the experimental tadpoles (a- tadpoles) had the labelled PGCs in series I and II, respectively. In p-tadpoles, the average numbers of labelled PGCs per tadpole were 8á7 in series I and 10 in series II, whereas they were 2á0 in a-tadpoles of both series. Both the proportion and the average number in p-tadpoles of both series were significantly different from those in a-tadpoles. In both series, labelled PGCs in p-tadpoles were found to be distributed throughout the genital ridges while those in a-tadpoles were localized only in the anterior part of the ridges. These facts indicate that the location of pPGCs in the endoderm affects their successful migration into the genital ridges, and that not only the presence of the germ plasm but also the proper location in endoderm are prerequisites to PGC differentiation of the germ line cells.
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Woodcock, Mark E., Almas A. Gheyas, Andrew S. Mason, et al. "Reviving rare chicken breeds using genetically engineered sterility in surrogate host birds." Proceedings of the National Academy of Sciences 116, no. 42 (2019): 20930–37. http://dx.doi.org/10.1073/pnas.1906316116.
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In macrolecithal species, cryopreservation of the oocyte and zygote is not possible due to the large size and quantity of lipid deposited within the egg. For birds, this signifies that cryopreserving and regenerating a species from frozen cellular material are currently technically unfeasible. Diploid primordial germ cells (PGCs) are a potential means to freeze down the entire genome and reconstitute an avian species from frozen material. Here, we examine the use of genetically engineered (GE) sterile female layer chicken as surrogate hosts for the transplantation of cryopreserved avian PGCs from rare heritage breeds of chicken. We first amplified PGC numbers in culture before cryopreservation and subsequent transplantation into host GE embryos. We found that all hatched offspring from the chimera GE hens were derived from the donor rare heritage breed broiler PGCs, and using cryopreserved semen, we were able to produce pure offspring. Measurement of the mutation rate of PGCs in culture revealed that 2.7 × 10−10 de novo single-nucleotide variants (SNVs) were generated per cell division, which is comparable with other stem cell lineages. We also found that endogenous avian leukosis virus (ALV) retroviral insertions were not mobilized during in vitro propagation. Taken together, these results show that mutation rates are no higher than normal stem cells, essential if we are to conserve avian breeds. Thus, GE sterile avian surrogate hosts provide a viable platform to conserve and regenerate avian species using cryopreserved PGCs.