Epigenetic reprogramming in the porcine germ line
© Hyldig et al; licensee BioMed Central Ltd. 2011
Received: 2 August 2010
Accepted: 25 February 2011
Published: 25 February 2011
Epigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next generation. In addition to DNA demethylation, PGC are subject to a major reprogramming of histone marks, and many of these changes are concurrent with a cell cycle arrest in the G2 phase. There is limited information on how well conserved these events are in mammals. Here we report on the dynamic reprogramming of DNA methylation at CpGs of imprinted loci and DNA repeats, and the global changes in H3K27me3 and H3K9me2 in the developing germ line of the domestic pig.
Our results show loss of DNA methylation in PGC colonizing the genital ridges. Analysis of IGF2-H19 regulatory region showed a gradual demethylation between E22-E42. In contrast, DMR2 of IGF2R was already demethylated in male PGC by E22. In females, IGF2R demethylation was delayed until E29-31, and was de novo methylated by E42. DNA repeats were gradually demethylated from E25 to E29-31, and became de novo methylated by E42. Analysis of histone marks showed strong H3K27me3 staining in migratory PGC between E15 and E21. In contrast, H3K9me2 signal was low in PGC by E15 and completely erased by E21. Cell cycle analysis of gonadal PGC (E22-31) showed a typical pattern of cycling cells, however, migrating PGC (E17) showed an increased proportion of cells in G2.
Our study demonstrates that epigenetic reprogramming occurs in pig migratory and gonadal PGC, and establishes the window of time for the occurrence of these events. Reprogramming of histone H3K9me2 and H3K27me3 detected between E15-E21 precedes the dynamic DNA demethylation at imprinted loci and DNA repeats between E22-E42. Our findings demonstrate that major epigenetic reprogramming in the pig germ line follows the overall dynamics shown in mice, suggesting that epigenetic reprogramming of germ cells is conserved in mammals. A better understanding of the sequential reprogramming of PGC in the pig will facilitate the derivation of embryonic germ cells in this species.
Primordial germ cells derived from the epiblast of pre-gastrulating embryos are the founder population of the future gametes. A unique attribute of PGC is the acquisition of totipotency, which is required for the generation of a new organism. Extensive epigenetic reprogramming of PGC underlies the capacity of these cells for acquiring totipotency [1, 2]. Genome-wide DNA demethylation in mouse PGC results in the complete erasure of methylation marks in single-copy and imprinted genes, and a moderate reduction in retrotransposons and other repetitive elements [3–5]. This demethylation is a unique reprogramming event, most of which is restricted to a short window of time between E10.5-13.5 in the mouse, and is critical for erasing epigenetic memory and preventing the transmission of epimutations to the next generation [3, 4, 6]. Just before these major DNA demethylation events, changes in histone marks contribute to the establishment of a distinctive chromatin signature in PGC . Reduction in H3K9me2 is followed by an increase in H3K27me3 levels in migrating mouse PGC between E7.75 and E8.75, at a time when these cells undergo G2 arrest and transcriptional quiescence [3, 7]. When the PGC reach the genital ridges they undergo major conformational changes including loss of linker histone H1 and replacement of nucleosomal histones . Together, these dynamic events define a critical period for the epigenetic reprogramming of the mouse germ line.
Most of our knowledge in mammalian germ line development originates from studies in mice. A recent study demonstrated that mouse and rat embryonic germ (EG) cells share common ground state properties, suggesting that the molecular circuitry of pluripotency is conserved in rodents . Very little is known about the sequence of events during PGC development in other species , and studying these events in non-rodents is important for establishing the conserved mechanisms of PGC development in mammals.
The pig is a good model for studying mammalian development, due to the developmental and physiological similarities with most other mammals, including humans. Furthermore, the pig is also excellent for modelling human disease, and therefore great effort has been devoted to develop efficient genetic modification technologies in this species . Pig EG cell lines derived from gonadal PGC of E28-35 embryos have been used to generate transgenic animals . In the pig, migratory PGC can be identified in the dorsal mesentery of the hindgut in E18-20 and the colonisation of the genital ridges occurs around E23-24 . However, the events characterizing the epigenetic reprogramming of pig PGC remain largely unexplored. A recent report showed demethylation of the differentially methylated domain of IGF2-H19 gene cluster and centromeric repeats between E24-E28 followed by de novo methylation in male PGC by E30-E31, demonstrating that major DNA demethylation occurs in the pig germ line shortly after colonizing the gonadal ridges . There is also evidence that the imprinted gene PEG10 is biallelically expressed in EG cells derived from E27 embryos, indicating that demethylation has occurred . In the present study we extended these initial observations by investigating the methylation reprogramming of imprinted genes, retrotransposons and genome-wide histone modifications in migratory and gonadal PGC. We show that imprinted gene demethylation occurs asynchronously in pig PGC, with IGF2-H19 demethylation not beginning before E22, and IGF2R demethylation already starting in male PGC at this time point. We also show that SINE repeats undergo moderate progressive demethylation between E22-E31. Finally, we show that migratory pig PGC undergo reprogramming of H3K27me3 and H3K9me2 concurrent with a G2 arrest.
Results & Discussion
OCT4 expression identifies the early pig germ line
These results show that OCT4 is expressed in migratory and early gonadal PGC and can be used as a reliable marker of pig PGC between E17-E31. Furthermore, it is expressed in the majority of putative germ cells at E42. We therefore used OCT4 staining followed by FACS sorting to obtain purified PGC at different developmental stages.
Reprogramming of gender specific methylation imprints at CTCF3 in IGF2-H19gene cluster is initiated after germ cell arrival to the genital ridges
Reprogramming of gender specific imprints of the IGF2Rgene is initiated in porcine germ cells prior to arrival in the genital ridges
We next examined the methylation status of the DMR2 located in intron 2, which is maternally methylated in mice , human , cattle  and sheep . Our analysis from bisulfite converted brain DNA showed that this region is differentially methylated (Figure 3), suggesting that this region plays a role in imprinting control of the pig IGF2R. We used this fragment to investigate the dynamic methylation reprogramming in purified PGC from porcine embryos of different developmental stages. In mice, DMR2 demethylation of Igf2r begins as early as E9.5 in migratory PGC , indicating that a gonadal environment is not needed to initiate DNA demethylation. We found that only male porcine PGC from E22 embryos show low levels of methylation with only 11.36% methylated CpGs. Gender specific differences were not observed in the methylation level of this gene in migratory mouse PGC . Importantly, although at this developmental stage the gonadal primordium has the characteristics of an indifferent gonad , SRY and its downstream target SOX9 are expressed in the migratory path of pig PGC between E21-E23 [39, 40], indicating that at the molecular level sexual dimorphism has already been established. Thus, demethylation of IGF2R in male PGC provides evidence supporting sex specific differences in the germ cells at this stage. The levels of methylation remained low in mature pig sperm (Figure 3), in agreement with Igf2r methylation reported in mice  and sheep sperm .
Interestingly, early gonadal PGC from female E22 and E25 embryos showed approximately 50% methylation, indicating that demethylation had not yet initiated. In PGC from female E29-31 embryos this DMR2 was almost completely demethylated, and by E42 the methylation level reached 63%, indicating de novo methylation by this stage (Figure 3). Since the same E42 samples were used to analyse the methylation status of H19, which is almost completely unmethylated in PGC (Figure 2), we think it is unlikely that the samples were contaminated with somatic cells. In mice the Igf2r DMR2 remains unmethylated in female germ cells until after birth, where de novo DNA methylation is acquired during oocyte growth [43, 44]. The precocious de novo methylation observed in female pig PGC suggests that acquisition of DNA methylation in the Igf2r is controlled differently in the two species. In line with our observations, a recent report showed that sheep oocytes derived from small preantral follicles possess a monoallelic pattern of methylation , indicating that precocious IGF2R methylation also occurs in sheep.
Together, our results demonstrate that imprinted DMR2 of IGF2R in the pig undergoes methylation reprogramming, with a precocious onset of demethylation in male migratory PGC, and early de novo methylation initiated in female germ cells before birth.
Short Interspersed Nuclear Elements are partially demethylated in the developing germ line
The overall reduction in methylation of SINE repeats is lower compared to the reported demethylation of centromeric repeats, which show extensive and gender specific demethylation in PGC at similar stages . This suggests that the different genomic contexts of interspersed versus centromeric repeats can impact on the demethylation machinery in PGC.
Cell cycle distribution and dynamics of histone modifications in porcine PGC
Next, we examined the DNA content of FACS sorted PGC to determine their cell cycle stage. The earliest stage of PGC that we were able to isolate was from E17 embryos, which showed a great proportion of cells in G2 (44%). This distribution resembles the patterns reported for murine PGC at about E9.75, a time point just following the G2 arrest observed between E7.5-E9 in the PGC population . In contrast, the porcine PGC from E22, E25 and E29-31 show nearly identical distribution displaying a clear G1 peak, a small broad S phase and a minor G2 peak (15-21%) (Figure 5Y). This cell cycle distribution resembles that of mouse somatic cells , and that of the somatic fraction of the porcine cell suspension used for sorting in this study (data not shown). These results show that the dynamic changes in H3K27me3 and H3K9me2 in pig PGC correspond overall with the pattern described for mouse migratory PGC . It is interesting however, that we observe these dynamic changes occurring over a longer period of about 6 days, which is more than three times the interval required in mice. The protraction of this process is likely due to the slower development in the pig.
All the procedures involving animals have been approved by the School of Biosciences Ethics Review Committee (University of Nottingham, UK). Embryos were collected from British Landrace sows or Yorkshire X Landrace gilts artificially inseminated or mated 15 (n = 1), 17 (n = 14), 18 (n = 13), 21 (n = 1), 22 (n = 15), 25 (n = 14), 29 (n = 4), 31 (n = 11) and 42 (n = 18) days prior to embryo collections. Embryos were recovered from the pregnant uteri within between 30 min and 2 hrs of slaughter.
One embryo of each of the stages E15, E17, E21, E22, E25, E31 and E42 were fixed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C. Tissue was hereafter dehydrated through increasing ethanol concentrations to xylene and embedded in paraffin. Transversal sections of 4-5 μm thickness containing the PGC were collected on SuperFrost Plus microscope slides (Menzel, Braunschweig, Germany).
Tissue preparation for methylation analysis
Hindgut or genital ridges/early gonads were dissected from each embryo and roughly chopped before treatmentwith 0.1% collagenase/0.1% dispase for 11 minutes and subsequently 1 minute in 0.25% trypsin with EDTA at 37°C. Tissue was disintegrated by gentle pipetting after addition of Dulbecco's Modified Eagle Medium (DMEM) with 4-10% fetal bovine serum (FBS) and centrifuged 5 minutes at 600 × g. Cells were resuspended in FBS with 10% DMSO and stored in liquid nitrogen up to 10 weeks.
The putative IGF2R gene was identified by aligning the porcine partial coding sequence (Accession number AF339885) to the porcine genome (assembly version 8, Pre.Ensembl). The promoter region and exon 1 of the gene were deduced using the annotated IGF2R gene sequences of Bos Taurus (Accession number NM174352). The putative DMRs were identified by the freeware CpG Island Searcher .
DNA extraction, gender determination and bisulfite conversion
Genomic DNA was extracted from porcine embryo tissue using Blood and Tissue DNA extraction kit (Qiagen, Hilden, Germany). The amount of extracted DNA was quantified on a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and a maximum of 1 μg was used for bisulfite conversion. For gender determination we followed the protocol reported by . Primers used are presented in Table 1. For bisulfite mutagenesis DNA was converted with EZ DNA Methylation-Gold kit (Zymo Research, Orange, CA, USA) and eluted in 10 μl nuclease free water following manufacturer's instructions.
PCR amplification of bisulfite converted DNA
The bisulfite converted DNA was amplified by PCR. All primers, annealing temperatures and sizes of products are listed in Table 1. The PCR amplification consisted of a denaturing step of 5 min at 95°C followed by 50-52 cycles of 30 sec at 94°C, 30 sec at 57°C - 64°C and 1 min at 72°C. Finally, there was an extra elongation step of 15 min at 72°C. The amplified products were analysed by electrophoresis on 2% agarose gels. The amplified products were sequenced by direct sequencing after purification with Qiagen Gel Extraction kit (Qiagen, Hilden, Germany) or as individual clones after transformation using pGEM-T EasyVector System (Promega, Charbonniéres, France) in Escherichia coli DH5α. The obtained nucleotide sequences were analysed with the freeware Chromas Lite (Technelysium Pty Ltd). The methylation level of repeat sequences was calculated using the approach proposed by Yang et al. . The method is based on the assumption that the mutation rate for CpG → TpG is identical on the two strands. Briefly, the number of potential CpGs in the investigated sequence was identified for all positions where one or more of the clones had a methylated CpG (See Table 1 for approximate numbers of investigated CpGs). Unmethylated CpGs were then calculated as TpGs deducted the number of TpAs (representing TpG mutations on the opposite strand) in the potential CpG positions. The efficiency of the genomic DNA conversion was evaluated by the number of non-converted non-CpG cytosines and no clones carrying more than one of these were included in the analyses.
Immunohistochemistry on PFA fixed, and paraffin embedded tissue
Sections were deparaffinated in xylene and rehydrated through descending concentrations of ethanol. The epitopes were demasked by 15 minutes microwave boiling of the slides in TE-buffer (0,01 M Tris, 0,001 M EDTA), pH 8.0 (AppliChem) or 0.01 M citrate buffer (pH 6.0) followed by 15 minutes cool down and 15 minutes wash in demineralised water. Tissue was permeabilised in 1% Triton X-100, blocked in 2% BSA/PBS prior to 1 hour incubation with primary antibodies; rabbit monoclonal anti-H3K27me3 (Upstate; 1:200), mouse monoclonal anti-H3K9me2 (Abcam, 1:200) and goat polyclonal anti-OCT3/4 (SantaCruz; 1:200). Negative controls were incubated in blocking buffer. After extended washes, the sections were incubated for 40 minutes with secondary antibodies; Alexa Fluor ® 594 conjugated donkey anti-goat IgG (Invitrogen; 1:250), Alexa Fluor ® 488 conjugated donkey anti-rabbit IgG (Invitrogen; 1:250) and Alexa Fluor ® 488 conjugated donkey anti-mouse IgG (Invitrogen; 1:250). For chromogenic detection the ABC technique was performed using the Vectastain Elite ABC kit (Vector Laboratories, Peterborough, U.K.) with DAB (Vector Laboratories, Peterborough, U.K.) as a substrate to visualise the positive cells. The sections were counterstained with haematoxylin and mounted using DPX mounting media (VWR International Ltd., Poole, U.K.). For immunofluorescence slides were mounted in Fluorescence Mounting Medium (DakoCytomation) and pictures of areas containing PGC were captured in 40× magnification with Leica DMRB fluorescence microscope through Leica DFC350FX camera.
Immunocytochemistry on ethanol fixed cell suspensions
Cell suspensions were thawed and added DMEM medium with 10% FBS. The cells were spun down and resuspended in medium twice to wash out DMSO before ice cold 99% ethanol was added dropwise to a final concentration of 70%. Cells were fixed at -20°C for 20 min. Before fixation, the suspension was filtered through a 30 μm nylon mesh (Miltenyi, Bergisch Gladbach, Germany) to ensure single cell suspension. Cells were washed twice in PBS with 0.1% Tween-20 and 1% BSA, permeabilised 30 min in 2% Triton X 100 with 0.1 mg/ml RNase A. The cells were resuspended in 5% BSA in PBS and incubated 1 hour 4°C to block unspecific antibody binding. Cells were incubated with goat anti-OCT3/4 antibody over night at 4°C (SantaCruz, 1:500 in blocking buffer), washed twice and incubated 1 hour RT with Phycoerythrin (PE)-conjugated donkey anti-goat IgG (AbCam, 1:100 in blocking buffer). Finally, the cells were washed three times before added 7-amino-actinomycin D (Invitrogen) to a final concentration of 4 μM. Cell suspensions were stored cold and in the dark until analysis. Negative controls were treated identically but incubated in blocking buffer instead of either the first or both antibodies. In addition, cells of the human embryonic kidney 293T cell line were used as negative cell samples while mouse embryonic stem cells were used as positive cell samples for adjustment of the flow cytometer.
Fluorescence-activated cell sorting (FACS) analysis
Cell suspensions were analysed on an Altra Flow Cytometer (Beckman Coulter, Brea, CA, USA). Signals for forward scatter, side scatter and fluorescence (PE for OCT4 and 7-AAD for DNA content) were collected for a minimum of 50000 cells in each group. Representative FACS plots are shown in additional file 3. Data were analyzed using WinMDI (http://facs.scripps.edu/software.html; authored by Dr. J. Trotter (The Scripps Research Institute, California, USA), with FSC/SSC and pulse width gating to exclude doublets. Cells were sorted on the basis of their OCT4 expression into a negative and a positive sample. The positive samples contained a minimum of 500 putative PGC. Cell cycle analysis was carried out using the freeware Cylchred (Dr. T. Hoy, Cardiff University, School of Medicine (Cardiff, UK) to give the proportion of cells in each phase of the cell cycle.
SMWH was supported by grants from K. Hoejgaards Foundation, N. & F.S. Jacobsens Foundation, C. & O. Brorsons travel grant for younger scientists, the joint Foundation between S. Chr. Soerensens & wifes Memory Foundation, the Association of Farmers associations of Jutland, The Foundation J. Skrikes Establishment, and G. J. Soerensens & wifes Foundation. DAC was supported by scholarship from CONACYT-Mexico. Part of this study was supported by grants from The University of Nottingham and the Royal Society to RA.
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