scholarly journals Transcription factors protect from DNA re-methylation during reprograming of primordial germ cells and pre-implantation embryos

2019 ◽  
Author(s):  
Isaac Kremsky ◽  
Victor G. Corces
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Methylation Status ◽  
Primordial Germ Cell ◽  
Cell Mass ◽  
Phenotypic Traits ◽  
Epigenetic Information

AbstractA growing body of evidence suggests that certain phenotypic traits of epigenetic origin can be passed across generations via both the male and female germlines of mammals. These observations have been difficult to explain owing to a global loss of the majority of known epigenetic marks present in parental chromosomes during primordial germ cell development and after fertilization. By integrating previously published BS-seq, DNase-seq, ATAC-seq, and RNA-seq data collected during multiple stages of primordial germ cell and preimplantation development, we find that the methylation status of the majority of CpGs genome-wide is restored after global reprogramming, despite the fact that global CpG methylation drops to 10% in primordial germ cells and 20% in the inner cell mass of the blastocyst. We estimate the proportion of such CpGs with preserved methylation status to be 78%. Further, we find that CpGs at sites bound by transcription factors during the global re-methylation phases of germ line and embryonic development remain hypomethylated across all developmental stages observed. On the other hand, CpGs at sites not bound by transcription factors during the global re-methylation phase have high methylation levels prior to global de-methylation, become de-methylated during global de-methylation, and then become re-methylated. The results suggest that transcription factors can act as carriers of epigenetic information during germ cell and pre-implantation development by ensuring that the methylation status of CpGs is maintained after reprogramming of DNA methylation. Based on our findings, we propose a model in which transcription factor binding during the re-methylation phases of primordial germ cell and pre-implantation development allow epigenetic information to be maintained trans-generationally even at sites where DNA methylation is lost during global de-methylation.

Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens

2008 ◽  
Vol 20 (8) ◽  
pp. 900 ◽  
Author(s):  
Yoshiaki Nakamura ◽  
Yasuhiro Yamamoto ◽  
Fumitake Usui ◽  
Yusuke Atsumi ◽  
Yohei Ito ◽  
...  
Keyword(s):  
Sustained Release ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Sesame Oil ◽  
Chicken Embryos ◽  
Whole Mount ◽  
Phosphate Buffered Saline ◽  
Transfer Study

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.

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.

scholarly journals Purification of mouse primordial germ cells by Nycodenz

Reproduction ◽  
2003 ◽  
pp. 667-675 ◽  
Author(s):  
T Mayanagi ◽  
R Kurosawa ◽  
K Ohnuma ◽  
A Ueyama ◽  
K Ito ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Specific Antibody ◽  
Membrane Surface ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Specific Antigen ◽  
Cell Lineage ◽  
New Method ◽  
Purification Method

Primordial germ cells are important cells for the study of germ cell lineage. It has proved difficult to obtain highly purified primordial germ cells for preparation of a specific antibody. In the present study, a new method for purifying mouse primordial germ cells was developed using a Nycodenz gradient. Furthermore, the polyclonal anti-mouse primordial germ cells IgG derived from mouse primordial germ cells was prepared. As this IgG reacted only with primordial germ cells obtained at day 12.5 after mating, this antibody appeared to recognize the stage-specific antigen of primordial germ cells. One reason that a continuous primordial germ cell marker has not been obtained is because the purity of the primordial germ cells used has been too low to prepare the antibody. This new method represents a significant improvement in the purification of primordial germ cells; it is simpler than previous methods, and produced mouse primordial germ cells with a purity of more than 95%. In addition, the separation reagent Nycodenz is non-toxic and achieved separation of primordial germ cells without attachment of antibodies against the primordial germ cell membrane surface. This new purification method and stage-specific antibody will be useful for the analysis of the mechanisms of primordial germ cell migration.

Mouse embryonic germ (EG) cell lines: transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3197-3204 ◽  
Author(s):  
P.A. Labosky ◽  
D.P. Barlow ◽  
B.L. Hogan
Keyword(s):  
Growth Factor ◽  
Germ Cell ◽  
Cell Lines ◽  
Germ Cells ◽  
Coat Color ◽  
Primordial Germ Cells ◽  
Methylation Status ◽  
Embryonic Stem ◽  
Insulin Like Growth Factor ◽  
Significant Difference

Primordial germ cells of the mouse cultured on feeder layers with leukemia inhibitory factor, Steel factor and basic fibroblast growth factor give rise to cells that resemble undifferentiated blastocyst-derived embryonic stem cells. These primordial germ cell-derived embryonic germ cells can be induced to differentiate extensively in culture, form teratocarcinomas when injected into nude mice and contribute to chimeras when injected into host blastocysts. Here, we report the derivation of multiple embryonic germ cell lines from 8.5 days post coitum embryos of C57BL/6 inbred mice. Four independent embryonic germ cell lines with normal male karyotypes have formed chimeras when injected into BALB/c host blastocysts and two of these lines have transmitted coat color markers through the germline. We also show that pluripotent cell lines capable of forming teratocarcinomas and coat color chimeras can be established from primordial germ cells of 8.0 days p.c. embryos and 12.5 days p.c. genital ridges. We have examined the methylation status of the putative imprinting box of the insulin-like growth factor type 2 receptor gene (Igf2r) in these embryonic germ cell lines. No correlation was found between methylation pattern and germline competence. A significant difference was observed between embryonic stem cell and embryonic germ cell lines in their ability to maintain the methylation imprint of the Igf2r gene in culture. This may illustrate a fundamental difference between these two cell types.

scholarly journals Sexual Differentiation and Primordial Germ Cell Distribution in the Early Horse Fetus

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2422
Author(s):  
Dragos Scarlet ◽  
Stephan Handschuh ◽  
Ursula Reichart ◽  
Giorgia Podico ◽  
Robyn E. Ellerbrock ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Sexual Differentiation ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Tubular Structures ◽  
Micro Computed Tomography ◽  
Computed Tomography Scanning ◽  
Fetal Ovary ◽  
Well Differentiated

It was the aim of this study to characterize the development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localization of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size 23-fold from 45 to 60 days of gestation, and an even greater increase was observed in the metanephros volume. Signs of mesonephros atrophy were detected during this time. Tubular structures of the fetal testes were present from day 50 onwards, whereas cell clusters dominated in the fetal ovary. The genital ducts were well-differentiated and presented a lumen in all samples. No sign of mesonephric or paramesonephric duct degeneration was detected. Expression of AMH was strong in the fetal testes but absent in ovaries. Irrespective of sex, primordial germ cells selectively expressed LIN28. Migration of primordial germ cells from the mesonephros to the gonad was detected at 45 days, but not at 60 days of development. Their number and distribution within the gonad were influenced (p < 0.05) by fetal sex. Most primordial germ cells (86.8 ± 3.2% in females and 84.6 ± 4.7% in males) were characterized as pluripotent according to co-localization with CD117. However, only a very small percentage of primordial germ cells were proliferating (7.5 ± 1.7% in females and 3.2 ± 1.2% in males) based on co-localization with Ki67. It can be concluded that gonadal sexual differentiation in the horse occurs asynchronously with regard to sex but already before 45 days of gestation.

Effects of 2,2',5,5'-tetrachlorobiphenyl (PCB52) on migration of chicken primordial germ cells

2005 ◽  
Vol 17 (5) ◽  
pp. 587 ◽  
Author(s):  
Yixiang Zhang ◽  
Xiumei Jin ◽  
Haitang Han ◽  
Zandong Li
Keyword(s):  
Cell Migration ◽  
Germ Cell ◽  
Germ Cells ◽  
Direct Action ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Initial Stage ◽  
Germ Cell Migration ◽  
Cell Migration And Proliferation ◽  
Migration And Development

Polychlorinated biphenyls cause developmental and physiological anomalies in the reproductive system. This study investigated the effects of 2,2′,5,5′-tetrachlorobiphenyl (PCB52), which can produce oestrogenic effects on the homeostasis of chicken primordial germ cells from the initial stage until completion of their settlement in the gonadal primordium. The blastoderm of chicken embryos was injected with 1 μL PCB52 (10 µmol/L) and oestradiol (100 µmol/L) before incubation, and the number of primordial germ cells was determined during their migration and development. The number of primordial germ cells in germinal crescents in PCB52-treated groups was slightly decreased (P = 0.068), but it was reduced significantly at stages 13–15 and 28–30 (P < 0.01, respectively) compared with controls. No obvious effects on primordial germ cell migration were observed with oestradiol treatments. The present results suggest that the influence of PCB52 on chicken primordial germ cell migration and proliferation may be via its toxic effect, not its oestrogen-mimicking effect, and provide information on the sensitivity of primordial germ cells to the direct action of PCB52.

Developmental potential of mouse primordial germ cells

Development ◽  
1999 ◽  
Vol 126 (9) ◽  
pp. 1823-1832 ◽  
Author(s):  
Y. Kato ◽  
W.M. Rideout ◽  
K. Hilton ◽  
S.C. Barton ◽  
Y. Tsunoda ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Germ Line ◽  
Gene Dosage ◽  
Primordial Germ Cells ◽  
Cell Mass ◽  
Monoallelic Expression ◽  
Imprinted Genes ◽  
Developmental Potential ◽  
Allele Specific

There are distinctive and characteristic genomic modifications in primordial germ cells that distinguish the germ cell lineage from somatic cells. These modifications include, genome-wide demethylation, erasure of allele-specific methylation associated with imprinted genes, and the re-activation of the X chromosome. The allele-specific differential methylation is involved in regulating the monoallelic expression, and thus the gene dosage, of imprinted genes, which underlies functional differences between parental genomes. However, when the imprints are erased in the germ line, the parental genomes acquire an equivalent epigenetic and functional state. Therefore, one of the reasons why primordial germ cells are unique is because this is the only time in mammals when the distinction between parental genomes ceases to exist. To test how the potentially imprint-free primordial germ cell nuclei affect embryonic development, we transplanted them into enucleated oocytes. Here we show that the reconstituted oocyte developed to day 9.5 of gestation, consistently as a small embryo and a characteristic abnormal placenta. The embryo proper also did not progress much further even when the inner cell mass was ‘rescued’ from the abnormal placenta by transfer into a tetraploid host blastocyst. We found that development of the experimental conceptus was affected, at least in part, by a lack of gametic imprints, as judged by DNA methylation and expression analysis of several imprinted genes. The evidence suggests that gametic imprints are essential for normal development, and that they can neither be initiated nor erased in mature oocytes; these properties are unique to the developing germ line.

Production of Clone-like Chicken by Primordial Germ Cells Induced From Somatic Cells

2021 ◽  
Author(s):  
Ruifeng Zhao ◽  
Qisheng Zuo ◽  
Xia Yuan ◽  
Kai Jin ◽  
Yani Zhang ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Somatic Cells ◽  
Bird Species ◽  
Unique Characteristic ◽  
Practical Application ◽  
Reprogramming Factors ◽  
First Time

Abstract The chicken primordial germ cell (PGCs) has the unique characteristic of settling in gonad through blood migration, which was the only way to realize the recovery of bird species. However, the PGCs obtained from a single embryo was far from enough to meet the practical application, while somatic cells can be obtained in large quantities. Therefore, the problem of insufficient PGCs can be solved by the induction of somatic cells into PGCs. Here, we successfully transdifferentiate somatic cells into PGCs, which can be transplanted to the recipient to produce offspring. In detail, The CEF of Black-Feathered Langshan Chicken was reprogrammed into iPS by reprogramming factors Oct4, Sox2, Nanog and Lin28, then was induced into PGCs by BMP4/BMP8b/EGF system. The induced PGCs has similar biological characteristics to the primary PGCs, which was transplanted into White Plymouth Rock Chicken, which self-crossed to produce clone-like offspring. It was the the first time to demonstrate the feasibility of avian cloning from somatic cells.

Mouse primordial germ cells lacking beta1 integrins enter the germline but fail to migrate normally to the gonads

Development ◽  
1999 ◽  
Vol 126 (8) ◽  
pp. 1655-1664 ◽  
Author(s):  
R. Anderson ◽  
R. Fassler ◽  
E. Georges-Labouesse ◽  
R.O. Hynes ◽  
B.L. Bader ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Migration ◽  
Germ Cell ◽  
Germ Cells ◽  
Fluorescent Protein ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Integrin Subunit ◽  
Integrin Beta1 ◽  
Germ Cell Migration

Primordial germ cells are the founder cells of the gametes. They are set aside at the initial stages of gastrulation in mammals, become embedded in the hind-gut endoderm, then actively migrate to the sites of gonad formation. The molecular basis of this migration is poorly understood. Here we sought to determine if members of the integrin family of cell surface receptors are required for primordial germ cell migration, as integrins have been implicated in the migration of several other motile cell types. We have established a line of mice which express green fluorescent protein in germline cells that has enabled us to efficiently purify primordial germ cells at different stages by flow cytometry. We have catalogued the spectrum of integrin subunit expression by primordial germ cells during and after migration, using flow cytometry, immunocytochemistry and RT-PCR. Through analysis of integrin beta1(−/−)--&gt;wild-type chimeras, we show that embryonic cells lacking beta1 integrins can enter the germline. However, integrin beta1(−/−) primordial germ cells do not colonize the gonad efficiently. Embryos with targeted deletion of integrin subunit alpha3, alpha6, or alphaV show no major defects in primordial germ cell migration. These results demonstrate a role for beta1-containing integrins in the development of the germline, although an equivalent role for * integrin subunit(s) has yet to be established.

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