Gonadal Primordial Germ Cells Research Articles

Research Note: Diethylstilbestrol reduces primordial germ cells in male Japanese quail

This study investigated the detrimental effects of diethylstilbestrol (DES), an estrogenic endocrine-disrupting chemical, on the viability of primordial germ cells (PGCs), embryonic precursors of germ cells, in Japanese quail. We injected 50 or 100 nmol DES solubilized in sesame oil into the yolk of stage X embryos and assessed changes in the population and cell cycle properties of circulating PGCs in blood vessels and gonadal PGCs after 2.5- and 7-day incubations, respectively. Liquid chromatography tandem mass spectrometer and Western blotting analyses identified DEAD-box polypeptide 4 (DDX4) and proliferating cell nuclear antigen (PCNA) as a stem cell marker and proliferation marker of quail PGCs, respectively. Immunochemical analyses revealed significant decreases in the number of DDX4- and PCNA-positive blood-circulating PGCs in males treated with 50 and 100 nmol DES than in the oil-treated control group. These reductions were not observed in females. Furthermore, the number of DDX4-positive gonadal PGCs was smaller in males treated with 50 and 100 nmol DES than in the control group, and these reductions were not observed in females. The protein expression of the Sertoli cell marker showed normal testis development in DES-treated embryos on d 7. These results demonstrate the potentially cytotoxic effects of DES on male germ cells, namely, the inhibition of cell cycle progression and induction of apoptosis in Japanese quail.

Comparative single-cell transcriptomic analysis reveals differences in signaling pathways in gonadal primordial germ cells between chicken (Gallus gallus) and zebra finch (Taeniopygia guttata).

Primordial germ cells (PGCs) have been used in avian genetic resource conservation and transgenic animal production. Despite their potential applications to numerous avian taxa facing extinction due to habitat loss and degradation, research has largely focused on poultry, such as chickens, in part owing to the difficulty in obtaining intact PGCs from other species. Recently, phenotypic differences between PGCs of chicken and zebra finch, a wild bird with vocal learning, in early embryonic development have been reported. In this study, we used advanced single-cell RNA sequencing (scRNA-seq) technology to evaluate zebra finch and chicken PGCs and surrounding cells, and to identify species-specific characteristics. We constructed single-cell transcriptome landscapes of chicken gonadal PGCs for a comparison with previously reported scRNA-seq data for zebra finch. We identified interspecific differences in several signaling pathways in gonadal PGCs and somatic cells. In particular, NODAL and insulin signaling pathway activity levels were higher in zebra finch than in chickens, whereas activity levels of the downstream FGF signaling pathway, involved in the proliferation of chicken PGCs, were higher in chickens. This study is the first cross-species single-cell transcriptomic analysis targeting birds, revealing differences in germ cell development between phylogenetically distant Galliformes and Passeriformes. Our results provide a basis for understanding the reproductive physiology of avian germ cells and for utilizing PGCs in the restoration of endangered birds and the production of transgenic birds.

Prediction of sex-determination mechanisms in avian primordial germ cells using RNA-seq analysis

In birds, sex is determined through cell-autonomous mechanisms and various factors, such as the dosage of DMRT1. While the sex-determination mechanism in gonads is well known, the mechanism in germ cells remains unclear. In this study, we explored the gene expression profiles of male and female primordial germ cells (PGCs) during embryogenesis in chickens to predict the mechanism underlying sex determination. Male and female PGCs were isolated from blood and gonads with a purity > 96% using flow cytometry and analyzed using RNA-seq. Prior to settlement in the gonads, female circulating PGCs (cPGCs) obtained from blood displayed sex-biased expression. Gonadal PGCs (gPGCs) also exhibited sex-biased expression, and the number of female-biased genes detected was higher than that of male-biased genes. The female-biased genes in gPGCs were enriched in some metabolic processes. To reveal the mechanisms underlying the transcriptional regulation of female-biased genes in gPGCs, we performed stimulation tests. Retinoic acid stimulation of cultured gPGCs derived from male embryos resulted in the upregulation of several female-biased genes. Overall, our results suggest that sex determination in avian PGCs involves aspects of both cell-autonomous and somatic-cell regulation. Moreover, it appears that sex determination occurs earlier in females than in males.

Finer resolution analysis of transcriptional programming during the active migration of chicken primordial germ cells

Primordial germ cells (PGCs) in chickens polarize and move passively toward the anterior region by the morphogenetic movement of the embryo. Further migration of PGCs towards the genital ridge via the germinal crescent region and blood vessels occurs actively through the chemoattractive signals. The mechanisms of initiation of PGCs migration, lodging the PGCs in the vascular system, and colonization of PGCs in the gonads are well-studied. However, transcriptome sequencing-based cues directing the migration of the PGCs towards gonads, some of the relevant molecules, biological processes, and transcription factors (TFs) are less studied in chickens. The current study comprehensively interprets the transcriptional programming of PGCs during their active migration (E2.5 to E8). Current results revealed several vital understandings, including a set of genes that upregulated male-specifically (XPA, GNG10, RPL17, RPS23, and NDUFS4) or female-specifically (HINTW, NIPBL, TERAL2, ATP5F1AW, and SMAD2W) in migrating PGCs, and transcriptionally distinct PGCs, particularly in the gonadal environment. We identified DNA methylation and histone modification-associated genes that are novel in chicken PGCs and show a time-dependent enrichment in migrating PGCs. We further identified a large number of differentially expressed genes (DEGs, including TFs) in blood PGCs (at E2.5) compared to gonadal PGCs (at E8) in both sexes; however, this difference was greater in males. We also revealed the enriched biological processes and signaling pathways of significant DEGs identified commonly, male-specifically, or female-specifically between the PGCs isolated at E2.5, E6, and E8. Collectively, these analyses provide molecular insights into chicken PGCs during their active migration phase.

Single-Cell RNA Sequencing Revealed the Heterogeneity of Gonadal Primordial Germ Cells in Zebra Finch (Taeniopygia guttata).

Primordial germ cells (PGCs) are undifferentiated gametes with heterogeneity, an evolutionarily conserved characteristic across various organisms. Although dynamic selection at the level of early germ cell populations is an important biological feature linked to fertility, the heterogeneity of PGCs in avian species has not been characterized. In this study, we sought to evaluate PGC heterogeneity in zebra finch using a single-cell RNA sequencing (scRNA-seq) approach. Using scRNA-seq of embryonic gonadal cells from male and female zebra finches at Hamburger and Hamilton (HH) stage 28, we annotated nine cell types from 20 cell clusters. We found that PGCs previously considered a single population can be separated into three subtypes showing differences in apoptosis, proliferation, and other biological processes. The three PGC subtypes were specifically enriched for genes showing expression patterns related to germness or pluripotency, suggesting functional differences in PGCs according to the three subtypes. Additionally, we discovered a novel biomarker, SMC1B, for gonadal PGCs in zebra finch. The results provide the first evidence of substantial heterogeneity in PGCs previously considered a single population in birds. This discovery expands our understanding of PGCs to avian species, and provides a basis for further research.

Sex-specific chromatin remodelling safeguards transcription in germ cells.

Stability of the epigenetic landscape underpins maintenance of the cell-type-specific transcriptional profile. As one of the main repressive epigenetic systems, DNA methylation has been shown to be important for long-term gene silencing; its loss leads to ectopic and aberrant transcription in differentiated cells and cancer1. The developing mouse germ line endures global changes in DNA methylation in the absence of widespread transcriptional activation. Here, using an ultra-low-input native chromatin immunoprecipitation approach, we show that following DNA demethylation the gonadal primordial germ cells undergo remodelling of repressive histone modifications, resulting in a sex-specific signature in mice. We further demonstrate that Polycomb has a central role in transcriptional control in the newly hypomethylated germline genome as the genetic loss of Ezh2 leads to aberrant transcriptional activation, retrotransposon derepression and dramatic loss of developing female germ cells. This sex-specific effect of Ezh2 deletion is explained by the distinct landscape of repressive modifications observed in male and female germ cells. Overall, our study provides insight into the dynamic interplay between repressive chromatin modifications in the context of a developmental reprogramming system.

Comparison of the Transcriptomic and Epigenetic Profiles of Gonadal Primordial Germ Cells of White Leghorn and Green-Legged Partridgelike Chicken Embryos.

The Green-legged Partridgelike fowl is a native, dual-purpose Polish chicken. The White Leghorn has been intensively selected for several decades to mainly improve reproductive traits. Primordial germ cells (PGCs) represent the germline stem cells in chickens and are the only cells that can transfer the information stored in the genetic material from generation to generation. The aim of the study was to carry out a transcriptomic and an epigenetic comparison of the White Leghorn and Green-legged Partridgelike gonadal PGCs (gPGCs) at three developmental stages: days 4.5, 8, and 12 of the embryonic development. RNA and DNA were isolated from collected gPGCs. The RNA was further subjected to microarray analysis. An epigenetic analysis was performed based on the global methylation analysis and qMSP method for the particular silenced genes demonstrated in transcriptomic analysis. Statistically significant differences between the gPGCs from both breeds were detected on the day 8 of embryonic development. Global methylation analysis showed significant changes at the methylation level in the White Leghorn gPGCs on day 8 of embryonic development. The results suggest faster development of Green-legged Partridgelike embryos as compared to White Leghorn embryos. Changes in the levels of gene expression during embryonic development are determined by genetic and environmental factors, and this variability is influenced by breed and gender.

Characterization of Alternative Splicing (AS) Events during Chicken (Gallus gallus) Male Germ-Line Stem Cell Differentiation with Single-Cell RNA-seq.

Simple SummaryStudies have shown that alternative splicing (AS) has been utilized in a wide variety of life processes. However, there are very few studies on AS during germ cell development. In this study, we preliminarily investigated the variation of variable shear events during the formation of chicken germ cells through the RNA-seq data analysis of embryonic stem cells (ESCs), gonad PGCs (gPGCs), and spermatogonia stem cells (SSCs), and the critical AS mode for several crucial stage-specific genes, which were identified during germ cell development. The results of this study lay a theoretical foundation for further analysis of the regulation mechanism of key genes involved in germ cell formation.Alternative splicing (AS) is a ubiquitous, co-transcriptional, and post-transcriptional regulation mechanism during certain developmental processes, such as germ cell differentiation. A thorough understanding of germ cell differentiation will help us to open new avenues for avian reproduction, stem cell biology, and advances in medicines for human consumption. Here, based on single-cell RNA-seq, we characterized genome-wide AS events in manifold chicken male germ cells: embryonic stem cells (ESCs), gonad primordial germ cells (gPGCs), and spermatogonia stem cells (SSCs). A total of 38,494 AS events from 15,338 genes were detected in ESCs, with a total of 48,955 events from 14,783 genes and 49,900 events from 15,089 genes observed in gPGCs and SSCs, respectively. Moreover, this distribution of AS events suggests the diverse splicing feature of ESCs, gPGCs, and SSCs. Finally, several crucial stage-specific genes, such as NANOG, POU5F3, LIN28B, BMP4, STRA8, and LHX9, were identified in AS events that were transmitted in ESCs, gPGCs, and SSCs. The gene expression results of the RNA-seq data were validated by qRT-PCR. In summary, we provided a comprehensive atlas of the genome-wide scale of the AS event landscape in male chicken germ-line cells and presented its distribution for the first time. This research may someday improve treatment options for men suffering from male infertility.

Paternal reprogramming-escape histone H3K4me3 marks located within promoters of RNA splicing genes.

MotivationExposure of mouse embryos to atrazine decreased histone tri-methylation at lysine 4 (H3K4me3) and increased expression of alternatively spliced RNA in the third generation. Specificity protein (SP) family motifs were enriched in the promoters of genes encoding differentially expressed alternative transcripts.ResultsH3K4me3 chromatin immunoprecipitation sequencing (ChIP-seq) of mouse sperm, preimplantation embryo development and male gonad primordial germ cells (PGCs) were analysed to identify the paternal reprogramming-escape H3K4me3 regions (RERs). In total, 251 RERs selected harbour H3K4me3 marks in sperm, with signals occurring in the paternal genome during early development and in male gonad PGCs, and 179 genes had RERs within 1 kb of transcription start sites (TSSs). These genes were significantly enriched in the gene ontology term ‘RNA splicing’, and SP1/SP2/SP3 motifs were enriched in RER-associated H3K4me3 peaks. Overall, the H3K4me3 marks within TSSs of RNA splicing genes survived two rounds of the epigenetic reprogramming process.Supplementary information Supplementary data are available at Bioinformatics online.

In Vitro Culture of Chicken Circulating and Gonadal Primordial Germ Cells on a Somatic Feeder Layer of Avian Origin.

Simple SummaryChicken primordial germ cells are specialized cells that are formed outside the developing embryo, from where they migrate into the gonad, and give rise to the gametes. There are two types of those cells: Circulating blood primordial germ cells and gonadal primordial germ cells. They can be isolated only in low numbers from the bloodstream or gonads of donor embryos. Hence, efficient in vitro cultivation systems are required to increase their quantity through proliferation. Here, we provide a single culture system that can be used to cultivate both cell types. We also present a novel, easy-to-train, and non-invasive method to identify live primordial germ cells in the culture. In this analysis, chicken primordial germ cells obtained from embryonic blood or gonadal regions were cultured in vitro on feeder cell layers derived from embryonic chick cells. The use of the chicken origin feeder layer allowed reducing the xenogenic animal factors in the culture. We demonstrated a feasible and cost-efficient technique to routinely assess the cultivated primordial germ cells on the basis of their morphological characteristics and using the optical features of cells in darkfield illumination. This method is especially useful to distinguish primordial germ cells during co-cultivation with other cell types.The present study had two aims: (1) To develop a culture system that imitates a normal physiological environment of primordial germ cells (PGCs). There are two types of PGCs in chicken: Circulating blood (cPGCs) and gonadal (gPGCs). The culture condition must support the proliferation of both cPGCs and gPGCs, without affecting their migratory properties and must be deprived of xenobiotic factors, and (2) to propose an easy-to-train, nonlabeling optical technique for the routine identification of live PGCs. To address the first aim, early chicken embryo’s feeder cells were examined instead of using feeder cells from mammalian species. The KAv-1 medium at pH 8.0 with the addition of bFGF (basic fibroblast growth factor) was used instead of a conventional culture medium (pH approximately 7.2). Both cPGCs and gPGCs proliferated in vitro and retained their migratory ability after 2 weeks of culture. The cultivated cPGCs and gPGCs colonized the right and/or left gonads of the recipient male and female embryos. To address the second aim, we demonstrated a simple and rapid method to identify live PGCs as bright cells under darkfield illumination. The PGCs rich in lipid droplets in their cytoplasm highly contrasted with the co-cultured feeder layer and other cell populations in the culture.

95 Obtaining birds with chimeric gonads using invitro lentiviral transduction of primordial germ cells

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.

In vitro culture and characterization of duck primordial germ cells

ABSTRACTThis study aimed to isolate, culture, and characterize duck primordial germ cells (PGCs) and to compare these cells with chicken PGCs. We first cultured Muscovy duck (Cairina moschata) circulating PGCs and gonadal PGCs (gPGCs) in the modified serum-containing medium used to amplify chicken PGCs. gPGCs were found to proliferate better in serum-free chemically defined medium than in serum-containing medium. Thereafter, gPGCs were similarly isolated from 2 other duck breeds, the Pekin duck (Anas platyrhynchos) and the hybrid mule duck (C. moschata × A. platyrhynchos), and amplified for a limited period of time in the chemically defined culture condition, but sufficiently to be characterized and transplanted. Cultured gPGCs of all 3 duck breeds were characterized by Periodic acid-Schiff staining, immunocytochemical staining, and expression analysis of germline-specific and pluripotency genes. Cultured duck gPGCs colonized the gonads after being genetically labeled and injected into recipient embryos. Taken together, these results demonstrate that duck PGCs retain their germline characteristics after being isolated, expanded in vitro, and genetically modified. Further studies are required to establish the optimal conditions for long-term culture of duck PGCs, which may involve supplementing the culture medium with other growth factors or compounds.

Long-term in vitro culture and preliminary establishment of chicken primordial germ cell lines.

Primordial germ cells (PGCs) are precursors of functional gametes and can be used as efficient transgenic tools and carriers in bioreactors. Few methods for long-term culture of PGCs are available. In this study, we tested various culture conditions for PGCs, and used the optimum culture system to culture chicken gonad PGCs for about three hundred days. Long-term-cultured PGCs were detected and characterized by karyotype analysis, immunocytochemical staining of SSEA-1, c-kit, Sox2, cDAZL, and quantitative RT-PCR for specific genes like Tert, DAZL, POUV, and NANOG. Cultured PGCs labeled with PKH26 were reinjected into Stage X recipient embryos and into the dorsal aorta of Stage 14–17 embryos to assay their ability of migration into the germinal crescent and gonads, respectively. In conclusion, the most suitable culture system for PGCs is as follows: feeder layer cells treated with 20 μg/mL mitomycin C for 2 hours, and with 50% conditioned medium added to the factor culture medium. PGCs cultured in this system retain their pluripotency and the unique ability of migration without transformation, indicating the successful preliminary establishment of chicken primordial germ cell lines and these PGCs can be considered for use as carriers in transgenic bioreactors.

In vitro improvement of quail primordial germ cell expansion through activation of TGF-beta signaling pathway.

Avian primordial germ cells (PGCs) have valuable potentials to cell-based approaches for transgenic bird production. In this regard, improvement of avian PGC expansion in vitro is necessary. Among experimental avian species, quail is a good model for transgenic technology, especially due to its short generation time. In the present study, we have examined the proliferative effects of transforming growth factor β (TGF-β) on the quail PGCs. After isolation of quail PGCs from blood (Hamburger-Hamilton [HH stages 13-15]) and gonads (HH stages 28-30), these cells were cultured on quail embryonic fibroblasts (QEF). Our results indicated th at cultured gonadal-derived PGCs proliferated 400 times in comparison to 100 times for blood PGCs over 40-50 days. Upon in vitro exposure to TGF-β inducers by Activin or the inducer of definitive endoderm 1 (IDE1) small molecule, the number of gonad PGCs significantly increased to 26% and 64%, respectively. In contrast, inhibition of the TGF-β signaling pathway by SB431542 resulted in a significant reduction in the numbers of PGCs (P < 0.001). Moreover, Phosphorylation of SMAD2/3 in the IDE1 group was higher compared to the Activin-treated ones. We confirmed the PGC identification with periodic acid-Schiff (PAS) staining, anti-SSEA1, β-catenin, β-integrin, and Nanog immunofluorescence staining. Exogenously IDE1 treated-PGCs migrated toward the embryonic gonads after transplantation into the heart of the recipient embryo at HH stages 13-15. Our results suggested that the application of IDE1 small molecule into the culture of quail PGCs represented a step toward achieving efficient expansion of the avian PGCs.

Epigenetic reprogramming enables the transition from primordial germ cell to gonocyte.

Gametes are highly specialised cells that can give rise to the next generation through their ability to generate a totipotent zygote. In mouse, germ cells are first specified in the developing embryo as primordial germ cells (PGCs) starting around embryonic day (E) 6.251 (Fig. 1a). Following subsequent migration into the developing gonad, PGCs undergo a wave of extensive epigenetic reprogramming at E10.5/E11.52–11, including genome-wide loss of 5-methylcytosine (5mC)2–5,7–11 (Fig. 1a). The underlying molecular mechanisms of this process have remained enigmatic leading to our inability to recapitulate this step of germline development in vitro12–14. Using an integrative approach, we show that this complex reprogramming process involves the coordinated interplay between promoter sequence characteristics, DNA (de)methylation, Polycomb (PRC1) complex and both DNA demethylation-dependent and -independent functions of Tet1 to enable the activation of a critical set of germline reprogramming responsive (GRR) genes involved in gamete generation and meiosis. Our results also unexpectedly reveal a role for Tet1 in safeguarding but not driving DNA demethylation in gonadal PGCs. Collectively, our work uncovers a fundamental biological role for gonadal germline reprogramming and identifies the epigenetic principles of the PGC-to-gonocyte transition that will be instructive towards recapitulating complete gametogenesis in vitro.

Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice.

The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells invitro under a defined condition without the use of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC-like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression invivo faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence-most typically, DNA demethylation of relevant genes-which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans.

Isolation and Number of Gonadal Primordial Germ Cells (Gonadal PGCs) on the Stages of Early Embryonic Development of KUB Chicken

Primordial germ cells (PGCs) are cells that will differentiate themselves into spermatogonia in the testis or oogonia in the ovary. Primordial germ cells arise from epiblast and circulate through the bloodstream and finally entering gonadal anlage. The aim of this study was to determine the number of gonadal PGCs of KUB chicken at different development stages. Sixty KUB chicken fertile eggs were divided into four groups (6, 7, 8, and 9 days incubation periods), and incubated at 38 oC with a humidity of 60%. Harvesting was synchronized to the embryonic development at 6-9 d. Gonads were collected using sharp tweezers, and were placed in Eppendorf tube 1.5 mL containing 500 µL PBS [-]. Gonadal PGCs were purified using PBS [-]. The results showed that the average number of gonadal PGCs at 6, 7, 8, and 9 d were 113.7; 143.5; 92.9; and 85.7 cells per embryo, respectively. Number of gonadal PGCs per embryo of KUB chicken were significantly affected by stage of embryonic development (P<0.05), which reached a peak at day 7 of incubation, so that the isolation and collection of PGCs from the gonads were recommended at day 7 of incubation. This information is useful in production of germline chimera of other Indonesian local chickens.

Development and Conservation of Gonadal Primordial Germ Cells for Preservation of Local Chicken in Indonesia

One of the &lt;em&gt;ex situ&lt;/em&gt; conservation techniques for poultry that recently developed was to collect primordial germ cell (PGC) or gonadal primordial germ cell (gPGC) that isolated from embryo development. Primordial germ cells (PGC) are embryonic cells that migrate to the gonads and form the precursors of gametes. The unique nature and accessibility of PGC during the early development provides an opportunity to manipulate the poultry germplasm, for example by forming germline chimeras. There are some stages that must be done through isolation and collection of PGC from its resources i.e. blastoderm, embryonic circulation blood and gonad. PGC collection originating from the gonads is one of existing PGC resources and technologies. gonadal PGC have advantages compared with other sources, namely (1) A large number of gonadal PGC can be taken from an embryo; and (2) A collection of gonadal PGC can be used in developing management systems of local avian germplasm conservation. This review is intended to describe the usefulness of isolation and collection technology of gonadal PGC as the local poultry germplasm conservation in Indonesia.

Characterization of the Epigenetic Changes During Human Gonadal Primordial Germ Cells Reprogramming.

Epigenetic reprogramming is a central process during mammalian germline development. Genome-wide DNA demethylation in primordial germ cells (PGCs) is a prerequisite for the erasure of epigenetic memory, preventing the transmission of epimutations to the next generation. Apart from DNA demethylation, germline reprogramming has been shown to entail reprogramming of histone marks and chromatin remodelling. Contrary to other animal models, there is limited information about the epigenetic dynamics during early germ cell development in humans. Here, we provide further characterization of the epigenetic configuration of the early human gonadal PGCs. We show that early gonadal human PGCs are DNA hypomethylated and their chromatin is characterized by low H3K9me2 and high H3K27me3 marks. Similarly to previous observations in mice, human gonadal PGCs undergo dynamic chromatin changes concomitant with the erasure of genomic imprints. Interestingly, and contrary to mouse early germ cells, expression of BLIMP1/PRDM1 persists in through all gestational stages in human gonadal PGCs and is associated with nuclear lysine-specific demethylase-1. Our work provides important additional information regarding the chromatin changes associated with human PGCs development between 6 and 13 weeks of gestation in male and female gonads. Stem Cells 2016;34:2418-2428.

MEK inhibitor PD0325901 and vitamin C synergistically induce hypomethylation of mouse embryonic stem cells.

A rationally selected combination of small-molecule chemicals can affect cell plasticity and fate, suggesting an open chemistry way to manipulate cells to achieve a specific goal. Here we for the first time demonstrate that a combination of vitamin C (Vc) and PD0325901 can achieve about 90% erasure of 5-methylcytosine (5mC) within 5 days (decreasing from 3.2 to ~ 0.3 5mC per 100 C) in mouse embryonic stem cells (ESCs). The hypomethylated level is comparable to that of gonadal primordial germ cells (PGCs), whose pluripotency is closely associated with the global DNA hypomethylation. In contrast, Vc or PD0325901 alone only induces a moderately reduced level of global DNA methylation. Our mechanistic study suggested that PD0325901 elevated expression of Prdm14, which repressed de novo methyltransferase Dnmt3b and its cofactor Dnmt3l at levels of protein, via the mode to eliminate 5mC from de novo synthesis. By further addition of Vc, the oxidation of 5mC as catalyzed by Tet1/Tet2 dioxygenases was significantly increased as manifested by the elevated level of 5-hydroxymethylcytosine. However, by the depletion of Tet1/Tet2, Vc failed to enhance PD0325901-stimulated hypomethylation of ESCs’ genomic DNA. Furthermore, mouse ESCs in Vc/PD0325901-supplemented medium show great morphology and pluripotency. Therefore, we demonstrate a novel and synergistic chemical approach for promoting hypomethylation and sustaining pluripotency of ESCs.