Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 11, 11

Epigenetic Reprogramming in the Porcine Germ Line

Affiliations

Epigenetic Reprogramming in the Porcine Germ Line

Sara M W Hyldig et al. BMC Dev Biol.

Erratum in

  • BMC Dev Biol. 2011;11. doi:10.1186/1471-213X-11-42

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figures

Figure 1
Figure 1
Identification of PGC by immunostaining. The top panel shows transversal sections of porcine embryos in the area where the PGC are found; hind gut of E17 (Figure 1A), genital ridges or primitive gonads of E22 (Figure 1B), E25 (Figure 1C), E31 (Figure 1D) and E42 (Figure 1E). Arrows indicate the PGC containing tissue (hind gut, genital ridges or gonads). Arrowhead depicts the mesonephric connection. The bottom panel shows double fluorescence immunostaining of the OCT4 and SSEA-1 in transversal sections of porcine embryos of the ages E17 (Figure 1F), E22 (Figure 1G), E25 (Figure 1H), E31 (Figure 1I) and E42 (Figure 1J). 5-7 PGC containing sections of one embryo of each stage were stained. Scale bars = 10 µm
Figure 2
Figure 2
Methylation dynamics of the IGF2-H19 gene cluster. Methylation of the CpG regulatory box CTCF3 region for IGF2-H19 gene cluster was investigated by bisulfite sequencing. A DNA pool from germ cells of 6-8 embryos of each gender in the stages E22, E25, E29-31 and E42 was bisulphite converted and used for the analysis after one PCR reaction and subsequent transformation and cloning. The position of the CTCF3 is indicated on the schematic representation of the gene cluster and the sequence of the investigated fragment after bisulfite mutagenesis is showed below. Empty and filled circles indicate unmethylated and methylated CpGs, respectively. 12-18 clones were analysed from each group. Each horizontal line represents one clone. Percent methylation mean ± SEM for each group is indicated below.
Figure 3
Figure 3
Methylation dynamics of the IGF2R gene. The two CpG islands of the IGF2R gene are known from other species as Differentially Methylated Region 1 (DMR1) and 2 (DMR2). A fragment of these regions was investigated for methylation of CpGs (See Additional file 2). The positions of the DMRs are indicated on the schematic representation of the gene and the sequences of the investigated fragments after bisulfite mutagenesis are showed above and below, respectively. DNA from liver and heart of an E45 embryo and a pool of DNA from ten E31 brains were analysed for DMR1. A DNA pool from germ cells of six-eight embryos of each gender in the stages E22, E25, E29-31 and E42 was used for the analysis of DMR2. Furthermore, DNA pools from a sperm sample and from ten E31 brains were included. The DNA was bisulphite converted and used for the analysis after one PCR reaction and subsequent transformation and cloning. Empty and filled circles indicate unmethylated and methylated CpGs, respectively. 11-15 clones were analysed from each group. Each horizontal line represents one clone. Percent methylation mean ± SEM for each group is indicated below.
Figure 4
Figure 4
Methylation dynamics of short interspersed repeats. Short Interspersed Nuclear Elements (SINE) were investigated for their methylation level in the porcine germ line. A DNA pool from germ cells of 13-16 embryos of the stages E22, E25, E29-31 and E42 was bisulphite converted and used for the analysis after one PCR reaction and subsequent transformation and cloning. 11-24 clones were analysed from each group. Due to high mutagenic rate in this type of elements, single clones are not identical regarding number and position of CpGs. The mean methylation level was calculated as suggested by Yang et al. [52] and results shown in the diagram. The sequence of an example of the investigated fragments after bisulfite mutagenesis is shown. Bars on the columns indicate SEM. E: embryonic stage.
Figure 5
Figure 5
Cell cycle distribution and H3K27 trimethylation and H3K9 dimethylation in porcine PGC. Reprogramming of histone modifications H3K27me3 and H3K9me2 was investigated by immunohistochemistry in paraffin sections of porcine E15 (n = 1, Figure 5A-D, M-P), E17 (n = 1, Figure 5E-H, Q-T) and E21 (n = 1, Figure 5I-L, U-X) embryos. Micrographs show the histone modifications in green (Figure 5A, E, I, M, Q, U). PGC are identified by OCT4 expression in red and counterstained with Hoechst for DNA stain in blue. Arrowheads mark PGC. Figure 5Y shows the cell cycle distribution after FACS analysis of PGC during development (n = 13-24 for each stage). Arrowheads denote the G1 and G2 peaks. Scale bars = 10 μm.
Figure 6
Figure 6
Diagramatic representation of the dynamic events during reprogramming of the germ cells in the mouse and the pig. Schematic overview of the events studied in the current report compared with the same events in mouse PGC. Erasure of Igf2/H19 imprints occurs in gonadal PGC of both species. Male pig migratory PGC lose IGF2R imprints before reaching the gonads, in contrast to the findings in mice [37], where demethylation occurs at the same time in male and female PGC after entering the gonad. Remodeling of repetitive sequences follows a similar dynamic in mice and pig PGC, with partial demethylation followed by remethylation after arrival to the genital ridges. The major changes in H3K9me2 and H3K27me3 occur in migratory PGC prior to their arrival to the genital ridge and are concurrent with the G2 arrest. The timelines for embryonic age are aligned according to the time points of PGC specification and arrival in the genital ridges for both species. Coloured boxes on the left hand side show the level of each epigenetic mark in somatic cells. Coloured lines depict presence of the indicated epigenetic marks at respective time points, and the lack of colour reflects the absence of the marks.

Similar articles

See all similar articles

Cited by 10 articles

See all "Cited by" articles

References

    1. Surani MA, Hayashi K, Hajkova P. Genetic and epigenetic regulators of pluripotency. Cell. 2007;128:747–62. doi: 10.1016/j.cell.2007.02.010. - DOI - PubMed
    1. Saitou M, Yamaji M. Germ cell specification in mice: signaling, transcription regulation, and epigenetic consequences. Reproduction. pp. 931–42. - DOI - PubMed
    1. Seki Y, Hayashi K, Itoh K, Mizugaki M, Saitou M, Matsui Y. Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice. Dev Biol. 2005;278:440–58. doi: 10.1016/j.ydbio.2004.11.025. - DOI - PubMed
    1. Hajkova P, Erhardt S, Lane N, Haaf T, El-Maarri O, Reik W, Walter J, Surani MA. Epigenetic reprogramming in mouse primordial germ cells. Mech Dev. 2002;117:15–23. doi: 10.1016/S0925-4773(02)00181-8. - DOI - PubMed
    1. Popp C, Dean W, Feng S, Cokus SJ, Andrews S, Pellegrini M, Jacobsen SE, Reik W. Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency. Nature. pp. 1101–5. - DOI - PMC - PubMed

MeSH terms

LinkOut - more resources

Feedback