Postnatal epigenetic reprogramming in the germline of a marsupial, the tammar wallaby

Abstract

Background: Epigenetic reprogramming is essential to restore totipotency and to reset genomic imprints during mammalian germ cell development and gamete formation. The dynamic DNA methylation change at DMRs (differentially methylated regions) within imprinted domains and of retrotransposons is characteristic of this process. Both marsupials and eutherian mammals have genomic imprinting but these two subgroups have been evolving separately for up to 160 million years. Marsupials have a unique reproductive strategy and deliver tiny, altricial young that complete their development within their mother's pouch. Germ cell proliferation in the genital ridge continues after birth in the tammar wallaby (Macropus eugenii), and it is only after 25 days postpartum that female germ cells begin to enter meiosis and male germ cells begin to enter mitotic arrest. At least two marsupial imprinted loci (PEG10 and H19) also have DMRs. To investigate the evolution of epigenetic reprogramming in the marsupial germline, here we collected germ cells from male pouch young of the tammar wallaby and analysed the methylation status of PEG10 and H19 DMR, an LTR (long terminal repeat) and a non-LTR retrotransposons.

Results: Demethylation of the H19 DMR was almost completed by 14 days postpartum and de-novo methylation started from 34 days postpartum. These stages correspond to 14 days after the completion of primordial germ cell migration into genital ridge (demethylation) and 9 days after the first detection of mitotic arrest (re-methylation) in the male germ cells. Interestingly, the PEG10 DMR was already unmethylated at 7 days postpartum, suggesting that the timing of epigenetic reprogramming is not the same at all genomic loci. Retrotransposon methylation was not completely removed after the demethylation event in the germ cells, similar to the situation in the mouse.

Conclusions: Thus, despite the postnatal occurrence of epigenetic reprogramming and the persistence of genome-wide undermethylation for 20 days in the postnatal tammar, the relative timing and mechanism of germ cell reprogramming are conserved between marsupials and eutherians. We suggest that the basic mechanism of epigenetic reprogramming had already been established before the marsupial-eutherian split and has been faithfully maintained for at least 160 million years and may reflect the timing of the onset of mitotic arrest in the male germline.

Figure 1

Evaluation of the purity of the presumed germ cells isolated by FACS. (A) Single cell suspension of the D28 tammar pouch young testes labeled by the DDX4/VASA antibody. The white arrows indicate some of presumed germ cells showing the stronger fluorescence. (B) A result of FACS showing that the presumed germ cells with the stronger fluorescence are separable from the rest of cell populations in which some low level fluorescence was detectable. (C) The content of the presumed germ cell fraction after FACS showing the strong fluorescence in the most abundant cells. (D) Left: Illustration of the virtual DNA methylation pattern of the PEG10 DMR (a known maternally methylated DMR) in somatic cells and gonocytes (which should not be methylated in male germline). The white and black circles represent unmethylated and methylated CpG sites, respectively. Right: Result of COBRA (combined bisulphite and restriction analysis) at the PEG10 DMR. U and C bands are the uncut and cut bands indicating the amount of unmethylated and methylated alleles, respectively. Lane M, size marker. Lane 1, COBRA using the genomic DNA extracted from kidney (control somatic tissue). Lane 2, COBRA using the genomic DNA extracted from the presumed germ cells collected by FACS. Bottom: The vertical bar represent a single CpG site in the SGCE-PEG10 CpG island and the location of CpG site used for COBRA is indicated by the arrow head.

Figure 2

DNA methylation status of the H19 DMR, KERV-1 LTR and LINE1 5′ region in male pouch young germ cells at 1, 5, 10 and 14 days postpartum. (A) Single cell suspension of the D5 tammar pouch young gonads labeled by the SSEA1 antibody. The white arrows indicate some of presumed germ cells labeled very clearly. (B) The black and white circles represent methylated and unmethylated CpG sites, respectively. The KERV-1 and LINE1 data only show the number of methylated CpG in each sequence as CpG sites are not always conserved among clones because of the heterogeneous amplification of these repetitive sequences. No data for ‘n.d.’. (C) The vertical axis for the graph of H19 DMR methylation represents the percentage of methylated CpG sites based on the data shown in Figure 2B. In the graphs of retrotransposon methylation, the vertical axes represent total number of methylated CpG sites in each sequence instead of percentage. Blue horizontal bars indicate average of the data.

Figure 3

DNA methylation status of the H19 DMR, KERV-1 LTR and LINE1 5′ region in male pouch young germ cells at 20, 28, 32, 33 and 34 days postpartum and in adult testis. (A) The black and white circles represent methylated and unmethylated CpG sites, respectively. The KERV-1 and LINE1 data only show the number of methylated CpG in each sequence as CpG sites are not always conserved among clones because of the heterogeneous amplification of these repetitive sequences. No data for ‘n.d.’. (B) The vertical axis for the graph of H19 DMR methylation represents the percentage of methylated CpG sites based on the data shown in Figure 3A. In the graphs of retrotransposon methylation, the vertical axes represent total number of methylated CpG sites in each sequence instead of percentage. Blue horizontal bars indicate average of the data.

Figure 4

DNA methylation analysis by COBRA for the PEG10 and H19 DMRs, KERV-1 LTR and LINE1 5′ region in male pouch young germ and somatic cells at 7, 9, 12 and 14 days postpartum and in adult sperm. The gel pictures show the cut and uncut bands after digestion by the restriction enzymes indicated in each panel. The fluorescence positive and negative samples represent positively sorted presumed germ cells and negatively sorted somatic control cells. Adult sperm samples were labeled as ‘Sp’. The vertical axes of bar graphs represent ratio of the intensity of the cut bands reflecting the methylation level of each sample. In C and D, the regions and bands subjected to cut/uncut intensity calculation were labeled by U for uncut and C for cut, respectively. (A) PEG10 DMR, (B) H19 DMR, (C) KERV-1 LTR, (D) LINE1 5′ region.

Figure 5

Predicted DNA methylation dynamics during epigenetic reprogramming in the male germline of the tammar wallaby and corresponding stages in the mouse male germline. The vertical axes represent relative DNA methylation level and the horizontal axes represent days postpartum. In the top graph, the predicted methylation dynamics of the H19 and PEG10 DMRs were represented by the blue and broken grey lines, respectively. Demethylation of the H19 DMR was completed by 14 days postpartum and de-novo methylation started from 34 days postpartum. Retrotransposon methylation was not completely removed after the demethylation event in the germ cells, similar to the situation in the mouse. The corresponding stages in the mouse male germline were illustrated in the bottom. Thus, despite the occurrence of epigenetic reprogramming postnatally and the persistence of genome-wide undermethylation for 20 days in the postnatal tammar, the relative timing of germ cell reprogramming was conserved between marsupials and eutherians.