PRC2 is required for extensive reorganization of H3K27me3 during epigenetic reprogramming in mouse fetal germ cells
© The Author(s) 2017
Received: 9 December 2016
Accepted: 1 February 2017
Published: 20 February 2017
Defining how epigenetic information is established in the germline during fetal development is key to understanding how epigenetic information is inherited and impacts on evolution and human health and disease.
Here, we show that Polycomb Repressive Complex 2 is transiently localized in the nucleus of mouse fetal germ cells, while DNA methylation is removed from the germline. This coincides with significant enrichment of trimethylated lysine 27 on histone 3 near the nuclear lamina that is dependent on activity of the essential PRC2 catalytic proteins, Enhancer of Zeste 1 and/or 2.
Combined, these data reveal a role for Polycomb Repressive Complex 2 and trimethylated lysine 27 on histone 3 during germline epigenetic programming that we speculate is required to repress target sequences while DNA methylation is removed.
KeywordsEpigenetics Germ cells Epigenetic reprogramming Histone modifications H3K27me3
The sperm and oocyte convey epigenetic information to the offspring that is essential for development and postnatal life. To ensure that appropriate epigenetic information is transmitted, germ cells undergo extensive reprogramming during fetal development. This allows existing parent-specific information to be reset and potential epigenetic errors to be removed from the germline. The most evident change during germline epigenetic reprogramming is the extensive removal of DNA methylation between embryonic day (E) 8.0 and E13.5, after which new DNA methylation is established in the male and female germlines in a sex-specific manner. Removal of germline DNA methylation occurs during two developmental stages. The first occurs during proliferation and migration of germ cells in the early embryo and involves reduction of DNA methylation levels from 70 to 30% . This is followed by further reductions in DNA methylation as the germ cells enter the developing gonads and initiate sex-specific development. Combining this extensive epigenetic reprogramming results in global DNA methylation levels of ~14 and 7% in male and female germ cells at E13.5, respectively .
During this later period of reprogramming, DNA methylation is removed from imprint control regions (ICRs) of maternally and paternally imprinted genes, non-imprinted genes, intergenic and intronic sequences, and from the inactive X-chromosome in XX germ cells . DNA methylation is also lost from many endogenous retroviral elements (ERVs), although relatively new ERVs are protected and retain relatively high levels of DNA methylation [1–6].
In addition to DNA demethylation, germline reprogramming involves extensive reorganization of histone modifications [7, 8]. At E8.5, dimethylated lysine 9 on histone 3 (H3K9me2) is exchanged for trimethylated lysine 27 on histone 3 (H3K27me3), which is maintained in the germ cell nucleus until at least E12.5 , although transient loss of H3K27me3 has been reported at E11.5 .
H3K27me3 is catalyzed by Polycomb Repressive Complex 2 (PRC2) and leads to epigenetic repression of target sequences. PRC2 is comprised of three core components: Enhancer of Zeste 1 or 2 (EZH1 and EZH2); Suppressor of Zeste 12 (SUZ12); and Embryonic Ectoderm Development (EED). While EZH2 is the primary catalytic component of PRC2 [10–12], EED is critical for physically binding H3K27me3 via five tandemly repeated WD motifs, thereby playing an essential role in PRC2 assembly [13, 14]. Inactivation of any one of the three core PRC2 protein subunits severely compromises the functional activity of the complex and results in the loss of H3K27me3. Complete loss of PRC2 function in mice is embryonic lethal [15–17], while low function (hypomorphic) mutations [18, 19] or tissue-specific deletion leads to growth and skeletal malformations , heart and immune cell defects  and increased susceptibility to tumorigenesis . Conditional deletion of Eed in the male germline from around birth results in complete male infertility demonstrating an essential role for PRC2 in male germline development . Although deletion of Ezh2 in growing oocytes is compatible with normal fertility, offspring are born underweight, indicating that PRC2 acts as a maternal factor in oocytes . However, PRC2 is required for germline development in Drosophila oocytes, where it regulates cell cycle progression during oocyte specification . Moreover, PRC2 is required in the Caenorhabditis elegans germline to regulate the balance between H3K27me3 patterning catalyzed by MES 2, 3 and 6 (C. elegans PRC2), and MES4 which catalyzes H3K36me3 . Despite these observations, the function of PRC2 in male and female in mammalian fetal germ cells remains unknown.
H3K27me3 is enriched at the promoter regions of many PRC2 target genes and plays an essential role in cell differentiation [27, 28]. In fetal germ cells, H3K27me3 is enriched at developmental genes [29–31] and is also enriched on nucleosomes that are retained at the promoters of developmental genes in mature sperm, indicating that PRC2 may regulate epigenetic information that is transmitted to offspring [32–34].
In this study, we identify a key period of transient PRC2 enrichment in gonadal germ cells as they undergo epigenetic reprogramming. Moreover, we demonstrate that PRC2 is required for significant transient enrichment of H3K27me3 near the nuclear lamina, specifically during the developmental period in which germline DNA methylation levels are at their lowest. We propose that PRC2 and H3K27me3 are required for epigenetic reprogramming in fetal germ cells and may provide a transient mechanism that protects certain sequences from aberrant expression during the period of reduced DNA methylation in the developing germline.
H3K27me3 is highly enriched in fetal germ cells undergoing epigenetic reprogramming
PRC2 is transiently expressed in fetal germ cells undergoing epigenetic reprogramming
Relative staining intensities for PRC2 proteins in E10.5–E15.5 germ cells
H3K27me3 is enriched near the nuclear lamina in fetal germ cells undergoing epigenetic reprogramming
To quantify the proportion of germ cells that underwent a change from staining throughout the nucleus (uniform nuclear localization; UNL) to staining located toward the nuclear lamina (peripheral nuclear localization; PNL), we assessed the proportions of cells that had UNL and PNL at E10.5, E11.5, E12.5 and E15.5 in male and female gonads based on confocal images. At E10.5, H3K27me3 staining conformed to the UNL pattern in 75/90% of XX and XY germ cells (Fig. 5b). However, at E11.5 this pattern was significantly different, with 85/95% cells with H3K27me3 staining in the PNL pattern in XX and XY germ cells, and this pattern was maintained in E12.5 XX and XY germ cells. However, by E13.5 H3K27me3 staining returned to the UNL pattern in around 40% of XY germ cells but not in XX germ cells. By E15.5, 90/94% of XX and XY germ cells contained UNL, rather than PNL localization (Fig. 5b).
EZH1/2 is required for H3K27me3 enrichment near the nuclear lamina in fetal germ cells undergoing epigenetic reprogramming
Our examination of EED, EZH2 and SUZ12 protein expression revealed that all three core PRC2 proteins were present in XX and XY germ cells between E11.5 and E12.5, coincident with the enrichment of H3K27me3 near the nuclear lamina (Figs. 2, 3, 4; Table 1). To determine whether PRC2 function was required for H3K27me3 enrichment near the nuclear lamina during this period of germ cell development, we blocked EZH1/2 function using the highly specific EZH1/2 inhibitor GSK126 . Initially, we validated the ability of GSK126 to block enrichment of H3K27me3 in germ cells. E11.5 whole gonad–mesonephros complexes were isolated from developing XX and XY embryos and cultured for 48 h on an organ culture membrane in medium containing increasing doses of GSK126 or vehicle control (DMSO). Flow cytometric analysis of GSK126 and control-treated gonads confirmed that GSK126 dose-dependently reduced H3K27me3 levels in germ cells in a 48-h period (Additional file 1: Fig. S2A). GSK126 was very well tolerated in these cultures, with even the highest dose having no effect on germ cell viability (Additional file 1: Fig. S2B). Moreover, we observed no impact on proliferation of either germ cells or somatic cells after 48-h treatment from E11.5 (Additional file 1: Fig. S2C, males shown). Similarly, in E12.5 fetal gonads cultured with 10 μm GSK126 for 72 h, germ cells entered mitotic arrest normally and somatic cell proliferation was normal, both in the Sertoli cell compartment and in the interstitial compartment (Additional file 1: Fig. S2D). This provided high confidence that GSK126 was well tolerated in fetal gonad cultures and demonstrated highly efficient, GSK126-dependent, depletion of H3K27me3 in germ cells within the 48- and 72-h culture periods.
With the exception of DNA methylation, reprogramming of epigenetic information in the developing germline is poorly understood. Here, we demonstrate that the epigenetic modifier complex PRC2, which catalyzes H3K27 methylation, is transiently expressed in germ cells soon after they enter the gonad and undergo the final phase of DNA demethylation . Coincident with increased PRC2 protein localization to the nucleus of germ cells, we observed substantial enrichment of H3K27me3 near the nuclear lamina in the gonadal germ cell population. This peripheral nuclear enrichment of H3K27me3 occurred during E11.5–E13.5, coinciding with the final phase of DNA demethylation and the lowest levels of DNA methylation in germ cells. By E15.5, H3K27me3 was again uniformly localized throughout nucleus in XX and XY germ cells. These findings demonstrate significantly three-dimensional reorganization of chromatin in the germ cell nucleus that is likely to be important for epigenetic reprogramming. In addition, we provide evidence that H3K27me3 enrichment near the nuclear lamina in E11.5–E13.5 germ cells is dependent on EZH1/2 activity, which comprise the essential catalytic proteins that mediate PRC2 activity. Combined, these data indicate that H3K27me3 mediates global reorganization of chromatin toward the nuclear lamina during the critical period during which germ cell DNA methylation levels are at their lowest. However, despite depletion of 80% of the H3K27me3 in male fetal germ cells, development of these cells was not altered within the 72-h culture window, and neither genes that mark male germline differentiation, nor known H3K27me3 targets, were altered.
A previous study reported reorganization of germ cell chromatin soon after germ cells enter the gonad . This led to the proposal that H3K27me3 is removed and replaced in gonadal germ cells to ensure that epigenetic information is reset in the fetal germline . However, another study concluded that H3K27me3 is maintained at relatively constant levels in gonadal germ cells, but fluctuates with cell cycle progression . In the present study, we used flow cytometry to quantify H3K27me3 levels per cell and directly assess the variation in H3K27me3 levels in gonadal germ cells at E11.5, E13.5 and E15.5. In addition, we examined H3K27me3 in sections of gonads using both standard confocal imaging and super-resolution imaging at E10.5, E11.5, E12.5, E13.5 and E15.5. Using flow cytometry, we detected high levels of H3K27me3 in E11.5 germ cells, which moderately declined in E13.5 and E15.5 germ cells. H3K27me3 levels in germ cells were fourfold to sevenfold higher than those detected in the gonadal somatic cells at all stages analyzed. Consistent with this, confocal and super-resolution imaging revealed higher levels of H3K27me3 in germ cells than in somatic cells at all stages analyzed. Using these combined approaches, we found no evidence of extensive loss of H3K27me3 in either XX or XY germ cell after they entered the gonad. This is consistent with the conclusions of Kagiwada et al. , who also observed relatively constant levels of H3K27me3 in gonadal germ cells, although levels fluctuated with cell cycle. It remains possible that H3K27me3 is removed and replaced in a very short temporal window. However, based on our findings we conclude that widespread loss of H3K27me3 is unlikely to occur as part of epigenetic reprogramming in mouse germ cells in the few days after they enter the developing gonads.
Rather than loss of H3K27me3 from germ cells at E11.5, we observed substantial transient enrichment of H3K27me3 near the germ cell nuclear lamina. This persisted until E13.5, but by E15.5 H3K27me3 was again localized throughout the germ cell nucleus in both XX and XY germ cells. In male germ cells, the redistribution of H3K27me3 back through the nucleus at E15.5 coincided with the initiation of de novo DNA methylation in mitotically arrested male germ cells. However, H3K27me3 was also distributed throughout the nucleus by the time female germ cells exit the cell cycle and have entered meiotic prophase by E15.5 .
The redistribution of H3K27me3 toward the nuclear lamina in E11.5–E13.5 male and female germ cells is both striking and intriguing. One possibility is that H3K27me3 regulates specific gene expression programs in the male and female germlines during this time. However, our analysis of global gene expression patterns in which H3K27me3 was depleted by 80% revealed no differences in gene expression in the 72-h window analyzed. Markers of male germline development were expressed normally, and there was no evidence that known targets of PRC2/H3K27me3 were de-repressed. This was surprising as previous studies have identified genes that are enriched for H3K27me3 in male and female germ cells during this period, suggesting that PRC2-mediated H3K27me3 enrichment might substantially regulate sex-specific germ cell development [29–31]. One caveat for the current study is that around 20% of the global H3K27me3 levels remained in germ cells after drug treatment, and this may be sufficient to maintain normal regulation of gene expression. However, dSTORM imaging clearly demonstrated that the H3K27me3 that remained in GSK126-treated cells was no longer localized toward the nuclear lamina, demonstrating that at least this function was compromised in the drug-treated gonads. Moreover, as we observed similar enrichment of H3K27me3 near the nuclear lamina in both sexes, and inhibition of EZH1/2 and depletion of H3K27me3 affected this equally in both sexes, it is unlikely that the global reorganization of H3K27me3 we observed can explain the regulation of sex-specific programs. It is more likely that H3K27me3 mediates a repressive activity that affects sequences common to both male and female germ cells.
Based on the current data, the most plausible explanation for the enrichment of H3K27me3 near the nuclear periphery is that H3K27me3-enriched chromatin is relocalized to the nuclear lamina in order to achieve appropriate transcriptional repression. Although it remains possible that H3K27me3 is removed from the center of the nucleus between E10.5 and E11.5 and re-established in the center of the nucleus between E13.5 and E15.5, we consider this unlikely for two reasons. Firstly, blocking EZH1/2 which catalyzes H3K27me3 prevented enrichment of H3K27me3 at the germ cell nuclear periphery in gonads cultured from E11.5 for 24 or 48 h. Secondly, studies in other cell systems provide substantial evidence that localization of chromatin to the nuclear periphery is associated with transcriptional repression [45, 46].
Indeed, it is highly plausible that transcriptional repression plays an essential role in maintaining genomic integrity during epigenetic reprogramming. DNA methylation is at its lowest level soon after germ cells enter the gonad. This is considered to involve a period during which germ cells may be vulnerable to aberrant expression of sequences such as ERVs that would otherwise contain DNA methylation and be repressed [2, 6, 47–49]. Previous studies have demonstrated enrichment of H3K27me3 at ERVs including young LTR and LINE elements, and at intergenic regions, introns and ICRs in male and female gonadal germ cells undergoing epigenetic reprogramming [6, 31]. Although repression of ERVs is in part achieved by SETDB1 through its catalysis of H3K9me3 in E13.5 fetal germ cells, this activity does not account for the repression of all ERVs, indicating that other mechanisms also repress ERVs during this period . Moreover, recent work has demonstrated a role for H3K27me3 in ERV repression in embryonic stem cells in which DNA methylation levels are reduced by treatment with 5-azacytidine and vitamin C .
Interestingly, although H3K27me3 was enriched near the nuclear lamina in germ cells undergoing reprogramming, EZH2, EED and SUZ12 staining was consistently detected throughout the nucleus. Yet blocking EZH2 activity prevented enrichment of the remaining H3K27me3 near the nuclear lamina. This implies that although PRC2 is required for catalyzing H3K27me3 in the nucleus at this stage, the complex is not relocated toward the nuclear lamina with H3K27me3-enriched chromatin. One possible explanation is that relocalization of H3K27me3 may depend on an additional mechanism(s) in the germ cell nucleus.
In this study, we demonstrate PRC2-dependent enrichment of H3K27me3 near the nuclear lamina when DNA methylation is at its lowest level in germ cells, but no change in expression of protein coding genes, with the caveat that EZH1/2 was blocked and H3K27me3 depleted in a limited 72-h window. However, EZH1/2-dependent reorganization of H3K27me3 in both sexes suggests that H3K27me3 plays roles other than repressing coding genes during this developmental period. One possibility may be that PRC2 and H3K27me3 play a transient role in repressing ERVs during epigenetic reprogramming of the germline through a mechanism that involves their relocalization to the nuclear periphery and sequestration into repressive chromatin structures. However, further work is required to determine whether this is the case.
We have identified substantial nuclear reorganization of the repressive histone modification H3K27me3 in germ cells as they undergo epigenetic reprogramming and reach their lowest DNA methylation levels. This occurs during a transient period of PRC2 enrichment in germ cells of both sexes and is dependent on EZH2 activity, the enzyme that catalyzes H3K27 methylation. Determining the repressive mechanisms that regulate germline epigenetic state during germline reprogramming is essential for understanding how altered epigenetic function in the germline contributes to inherited disease. It is therefore imperative that further work seeks to understand the mechanisms that regulate epigenetic and genetic integrity in the germline prior to fertilization.
Mouse strains, animal housing, breeding and ethics
Mice were housed at Monash Medical Centre Animal Facility using a 12-h light–dark cycle. Food and water were available ad libitum, and room temperature was 21–23 °C with controlled humidity. With the exception of breeding pairs and neonatal mice (up to three weeks old), males and females were weaned at 21 days and kept in cages of up to five individuals. All animal work was undertaken in accordance with Monash University Animal Ethics Committee (AEC) approvals. Temporal and spatial profiling of PRC2 components and H3K27me3 was carried out on embryos collected from Oct4 [Pou5f1]-eGFP on 129svJ background males crossed with Swiss females (obtained from Monash Animal Services). Females were checked for vaginal plugs daily, and detection of a plug was noted as E0.5. Embryos E12.5 or older were sexed by the presence (male) or absence (female) of testis cords in gonads. Embryos younger than E12.5 were sexed using PCR as described .
Gonad collection, dissociation, fixation, antibody staining and flow cytometry are as previously described . Gonad samples stained with rabbit IgG control antibody were used as negative controls to set flow cytometry gates for H3K27me3 intensity, and mesonephros samples were used as a germ cell negative control to gate for eGFP. Cell proliferation was assessed by dosing cultured gonads with 20 μm 5-ethynyl-2 deoxyuridine (EdU) for the final 2 h of culture prior to tissue collection, dissociation and fixation. Cell cycle analysis was carried out as described  with germ cells identified by their expression of mouse vasa homologue (MVH) and somatic cells identified by anti-Mullerian hormone (AMH) staining. Cells were stained with 20 μg/ml propidium iodide, allowing quantitation of cellular DNA content. Proliferation was measured by gating EdU-positive cells against propidium iodide to identify cells actively in S-phase. Cells in G1 and G2/M were identified by their DNA content and the absence of S-phase activity. All flow cytometry was performed on a FACS Canto instrument and data analyzed in FlowJo and GraphPad Prism. For all analyses, 3–9 biological replicates were analyzed and statistical significance determined using one-way ANOVA with Tukey’s multiple comparison or t test as appropriate. P values <0.05 were considered significant.
Tissue fixation and embedding
Gonads were fixed in 4% paraformaldehyde (PFA) in PBS for IF and confocal imaging, or 2% PFA plus 0.02% glutaraldehyde (GA) in PBS (dSTORM super-resolution microscopy) for 20–90 min according to embryo age (Additional file 1: Table S1). Samples were then washed twice in PBS and left in 30% sucrose in PBS overnight at 4 °C. Samples were then placed in disposable cryostat molds (Sakura Finetek, 4565) filled with OCT (Sakura Finetek, 4583) and frozen in dry ice. Blocks were and stored at −80 °C.
Eight-micron sections were cut from OCT-embedded gonads fixed in 4% PFA, mounted on SuperFrost Plus slides and dried for 5 min before immersing in 1× PBS. Sections were then permeabilized by incubation in 1% Triton × 100 (Sigma, T8787) in PBS for 10 min at room temperature (RT). Slides were washed three times for 5 min each in PBS. Sections were blocked in PBS containing 5% BSA (Sigma, A9647) and 10% donkey serum (Sigma, D9663) and incubated for 45 min at RT. Blocking solution was replaced by PBS containing 1%BSA and appropriately diluted primary antibodies (Additional file 1: Table S2) and incubated for 1 h at RT. Slides were washed three times for 5 min in PBS and secondary antibodies diluted in 1% BSA in 1 x PBS according to antibody dilutions outlined in Additional file 1: Table S3. Secondary antibody incubation was carried out in a dark box for 1 h at RT. Slides were washed three times in PBS (5 min each wash) and mounted in ProLong Gold ® containing DAPI (Life Technologies, P36931) and left in a dark box over night to dry. For control slides, only a secondary antibody was applied. Confocal images were taken as single optical sections using a Nikon ® C1 inverted confocal microscope. All pictures were taken at 80×, using a 40× oil immersion lens. Nuclear localization of H3K27me3 was assessed in 3–6 biological replicates per embryonic stage. Each germ cell was visually assessed for UNL or PNL staining based on the presence of staining throughout the nucleus or only around the nuclear periphery, respectively. Cell counts were recorded using ImageJ Plugin Cell Counter. Data were expressed as the percentage of cells with UNL and PNL staining and analyzed for statistically significant differences between stages using one-way ANOVA with Tukey’s multiple comparison test or differences between treatments using Student’s t test with P ≤ 0.05 considered significant. H3K27me3 mean intensity was measured in three biological replicates for each culture period (E11.5 +48 h male and females, >119 cells analyzed; E11.5 +24 h males, >68 cells analyzed). ImageJ ROI manager was used to calculate the mean intensity for H3K27me3, and unpaired t tests were used to statistically analyze treatments groups, with P ≤ 0.05 considered significant.
dSTORM super-resolution imaging
Two-micron sections were cut from OCT-embedded gonads fixed in 2% PFA and 0.02% GA (Additional file 1: Table S1) and mounted on poly-l-lysine (Sigma) coated #1.5 Coverslips (Grale HDS) enclosed within 8-well sticky chambers (Ibidi, 80828) and stained.
Super-resolution images were recorded using a custom-built (Monash Micro Imaging) dSTORM instrument based on an Olympus IX-71 microscope equipped with Plan ApoChromat oil immersion 100× 1.4NA objective (Olympus part #N1480900), 1.6× magnification changer, Toptica 488 nm laser (200 mW), Gem 561 nm laser (500 mW), and Oxxius 637 nm laser (150 mW), suitable Olympus fluorescence filter cubes and Andor iXon Ultra 897 High Speed EMCCD camera with single-photon sensitivity for single-molecule detection. The final excitation steering mirror and beam expansion lenses were mounted on a translation stage for free adjustment of the total internal reflection fluorescence (TIRF) angle. The system was operated at a TIRF angle appropriate for the respective sample to concentrate excitation power and reduce background fluorescence. Samples were mounted on a manual x,y translation stage to minimize sample drift.
Alexa Fluor® 647 super-resolution imaging was performed in imaging buffer at pH8.0 containing 50 mM MEA and a glucose scavenging system (10% Glucose, 40 μg/ml catalase, 0.5 mg/ml glucose oxidase) in PBS. The sample was illuminated continuously with 70 mW 637 nm laser power at the appropriate TIRF angle. After an initial pumping period of <30 s to drive dyes into the dark state, single-molecule blinking time series were acquired for 10,000 frames at a camera EM gain of 50 and exposure time of 20 ms.
The acquired data were reconstructed to super-resolved images of 10-nm pixel size using the open-source software rapidSTORM version 3.3.1. Blinks with a local signal-to-noise ratio (SNR) <80 were discarded. Images were first color coded for temporal appearance of blinks to detect sample drift, then (if necessary) corrected for drift using the linear drift correction available in rapidSTORM and exported as 8-bit grayscale images.
Analysis of H3K27me3 staining in dSTORM super-resolution images
Radial histogram analysis was carried out for all representative super-resolution images for each embryonic time point and treatment group. This involved the use of an algorithm (macro was installed and analyzed on ImageJ) to measure staining intensity of H3K27me3, beginning at the center of the cell nucleus out to the nuclear periphery. H3K27me3 intensity was measured in ever-increasing concentric circles (radial diameter) expanding in at 0.1 μm increments from the center of the nucleus until the nuclear lamina was reached. Values on each radial diameter line were collated and averaged to provide values ± standard error of the mean across 3–6 biological replicates. Data were processed and graphed in Microsoft Excel.
All culture reagents were purchased from Life Technologies unless otherwise stated. Embryos were collected at E11.5 from Swiss females mated to 129T2svJ Oct4-eGFP transgenic males. Gonad plus mesonephros was cultured on 30-mm organotypic cell culture inserts (Merck Millipore; PICM03050) in 1200-μl culture media (250 μm sodium pyruvate, 15 mM Hepes, 1X nonessential amino acids (Life Technologies, 11140), 1 mg/ml N-acetylcysteine (Sigma, A9165), 55 μm β-mercaptoethanol (Life Technologies, 21985) and 10% FCS in DMEM/F12 with Glutamax (Life Technologies, 10565) containing either DMSO (vehicle control) or 10 μm GSK126 (EZH1/2 inhibitor; SelleckChem, S7061). Media preparations also contained 1× penicillin/streptomycin (Life Technologies, 15070). Gonads were randomly allocated to each culture treatment condition and cultured for 48 h in 37 °C/5% CO2 conditions. Culture media was refreshed daily. Gonads were processed for flow cytometry, FACS, IF and dSTORM super-resolution imaging.
FACS and cell viability
Four to six cultured gonads were collected, pooled together and dissociated with 0.25% trypsin as previously described. The reaction was stopped by adding 500 μl of culture media containing 10% fetal calf serum, and cells were collected by centrifugation. Cells were resuspended in 300 μl culture media containing 10% fetal calf serum and propidium iodide added to a final concentration of 2 μg/ml. Cells were run on an Influx 2 cell sorter. Non-viable cells were quantified and excluded as a percentage of the whole single cell population based on propidium iodide staining. Oct4-eGFP positive (germ) and negative (somatic) cells separated based on eGFP fluorescence and collected in separate tubes. Cells were collected by centrifugation and snap frozen for RNA extraction.
RNA preparation and expression microarray analysis
RNA was extracted from FACS-purified cells (~20,000 cells per sample, n = 3 for vehicle and GSK126 treatments, respectively) using TRIzol reagent, treated with Turbo DNAse 1 (Ambion) according to the manufacturer’s instructions and resuspended in RNAase-free water. RNA quantity and quality were assessed using a Qubit instrument and a Bioanalyzer. Total RNA (100 ng/sample) was labeled using Agilent One Colour Low Input Quick Amp Labelling Kit v.6.6, and dye incorporation assessed using a NanoDrop ND-1000 Spectrophotometer. In total, 600 ng of Cy3-labeled cRNA (specific activity >6 pmol Cy3/μg) was fragmented at 60 °C according to the Agilent protocol and hybridized to Agilent SurePrint G3 Mouse Gene Expression 8 × 60K Arrays for 17 h at 67 °C according to manufacturer’s instructions. Arrays were washed and immediately scanned on an Agilent C, DNA microarray scanner using one-color scan settings for 8 × 60K arrays. The scanned images were analyzed with Feature Extraction Software 126.96.36.199 (Agilent) using default parameters (protocol GE1-1100_Jul11 and Grid: 028005_D_F_20120201) to obtain background subtracted and spatially detrended Processed Signal intensities. Data were analyzed using GeneSpring GX software using the proprietary analysis pathway. In addition, raw signal intensities for a selection of genes were independently normalized against validated reference genes for fetal germ cells using intensities across all probes for Sdha, Canx and MapK1 and the data expressed as normalized signal intensities.
anti-Mullerian hormone (also known as Mullerian inhibitory substance)
Embryonic Ectoderm Development
endogenous retroviral elements
Enhancer of Zeste 1
Enhancer of Zeste 2
fluorescence-activated cell sorting
dimethylated lysine 9 on histone 2
trimethylated lysine 9 on histone 3
trimethylated lysine 27 on histone 3
long interspersed nuclear elements
LINE subgroup 1
mouse vasa homologue (also known as DDX4)
peripheral nuclear localization
Polycomb Repressive Complex 2
SET domain, bifurcated 1
short interspersed elements
Suppressor of Zeste 12
uniform nuclear localization
PW conceived the project. PW, LP, JS and KH designed the experiments. LP, KH, KE and JS performed the experiments. LP, KE, KH, JS and PW analyzed the data. LP, JS and PW wrote the paper. All authors read and approved the final manuscript.
This work was supported by National Health and Medical Research Grants GNT1043939 and GNT1051223 awarded to PSW, funding from the Monash University Faculty of Medicine, Nursing and Health Sciences funding granted to PW and the Victorian Government’s Operational Infrastructure Support Program. LP was supported by an Australian Postgraduate Award. We thank Monash Animal Research Platform staff for assistance with mouse care, Camden Lo (Monash Micro Imaging) for imaging advice and Jodee Gould (Monash Health Translational Precinct Medical Genomics Facility) for assistance with micro-array studies.
The authors declare that they have no competing interests.
Availability of data and materials
The expression array datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. All other data generated or analyzed during this study are included in this published article and its supplementary information files.
Ethics approval and consent to participate
All experiments in involving were performed under ethical approval and in accordance with guidelines and regulations of Monash University Animal Ethics Committee.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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