Postnatal epigenetic reprogramming in the germline of a marsupial, the tammar wallaby
© Suzuki et al.; licensee BioMed Central Ltd. 2013
Received: 11 January 2013
Accepted: 8 May 2013
Published: 3 June 2013
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.
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.
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.
Genome-wide dynamic changes of epigenetic states during mammalian germ cell development, called epigenetic reprogramming, are essential to restore totipotency and to renew parental imprinting in the male and female germ cells [1–4]. In mice, loss of DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2) followed by the gain of H3K27me3 are the first gross epigenetic changes observed in migrating primordial germ cells (PGCs) between E7.5 and E9.5 [5, 6]. Then, the second wave of DNA demethylation which is associated with the erasure of parental imprinting, promoter methylation of germline genes and with the reduction of retrotransposon methylation takes place around E11.5, just after PGCs have entered into the genital ridges [7–10]. From E14.5, de-novo DNA methylation dependent on the actions of the DNMT3 family re-establishes paternal imprints and methylation of retrotransposons in G1-arrested male germ cells, known as prospermatogonia or male gonocytes [11–18].
In higher vertebrates, genomic imprinting has been identified in eutherian and marsupial mammals [19–24]. However, of the 16 or so eutherian imprinted genes examined so far in marsupials, only six are imprinted [23–35]. Furthermore, there are only two DMRs, associated with PEG10 and H19, that have been discovered so far, in marsupials, both in the tammar wallaby [24, 30]. The tammar H19 DMR was identified as a germline DMR because it was fully methylated in adult testes . However, the precise timing of epigenetic reprogramming in the developing germ cells of marsupials has never been established. Eutherians and marsupials 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 . In the tammar, most PGCs complete their migration to the genital ridges just before birth . Post-migratory PGCs continue to proliferate after birth, and it is only after 25 days postpartum that female germ cells begin to enter meiosis while male germ cells enter into G1-phase mitotic arrest [39, 40]. To compare the evolution of epigenetic reprogramming between this distantly related mammal and the mouse, we analysed the methylation dynamics of the H19 DMR, which is the only paternal DMR discovered in marsupials so far, an LTR and a non-LTR retrotransposons in the male germline of the tammar wallaby during the postnatal proliferation and early mitotic arrest stages.
Results and discussion
Isolation of germ cells from the tammar pouch young testes
DNA demethylation in the germline of tammar male pouch young
De-novo DNA methylation in the germline of tammar male pouch young
We next determined when de-novo methylation took place at the H19 DMR and the retrotransposons. At 20, 28, 32 and 33 days postpartum, the H19 DMR was still nearly fully unmethylated, suggesting that the undermethylated states observed at 14 days postpartum had persisted at least until these stages (Figure 3A). At the same time, these data demonstrate that the effect of somatic cell contamination during germ cell separation to the results of methylation analyses was negligible, so we assume the faint cut bands in the PEG10 DMR COBRA in Figures 1 and 4 may not be a simple reflection of somatic cell contamination. Also the methylation level of both LTR and non-LTR retrotransposons at 20 and 28 days postpartum was similar to that at 14 days postpartum (Figures 2B and C and 3A and B). On the other hand, we detected de-novo DNA methylation of the H19 DMR in three different animals at 34 days postpartum, indicating that 34 days postpartum is the critical stage for the acquisition of de-novo methylation and that it occurs rapidly. The increase of methylation in the retrotransposons was detected at 32 days postpartum, two days earlier than de-novo methylation of the H19 DMR (Figure 3A, B). It is possible that the methylation machinery responds more quickly to the retrotransposons retaining some degree of methylation than the fully unmethylated H19 DMR. Alternatively, the methylation machinery might be slightly differently recruited to the H19 DMR and to the retrotransposons. The G1-phase entry into mitotic arrest begins only after 25 days postpartum in the tammar male germline and is not complete until after day 50. Considering that germ cell development in the tammar wallaby takes much longer than in mouse and occurs postpartum, the relative timing and pattern of de-novo DNA methylation in the male germ cell development as well as the timing of demethylation is remarkably similar in both species. In mouse male germ cells undergoing mitotic arrest, NANOS2 maintains their arrested state and induces male-type germ cell differentiation including the expression of DNMT3L, an essential factor for the establishment of paternal imprinting and retrotransposon methylation . The orthologue of NANOS2 is found in the tammar genome (Hickford and Renfree, unpublished). Although the precise molecular pathway between NANOS2 and DNMT3L expression is still largely unknown, the similar relative timing of de-novo DNA methylation in the male germline of tammar and mouse, which starts shortly after the entry into mitotic arrest in both species, suggests that the molecular basis connecting these events has been conserved between marsupials and eutherians. The orthologues of the factors essential for paternal imprinting establishment in the mouse germline, such as DNMT3A, DNMT3L and BORIS/CTCFL, are also present in marsupials [48, 49]. These orthologues most likely play the same critical role to establish the methylation imprint in the marsupial H19 DMR, which occurs at a similar relative time in the male germ cell development as in that of the mouse.
Demethylation and de-novo methylation in the male germline of a marsupial occurs over a prolonged period postpartum. Despite the occurrence of epigenetic reprogramming postnatally and the persistence of genome-wide undermethylation for 20 days in the postnatal tammar, the relative timing and mechanism of germ cell reprogramming was 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 that it is tightly correlated with the onset of mitotic arrest in the male tammar wallaby.
Animals and tissue collection
Tammar wallabies (Macropus eugenii) of Kangaroo Island origin were maintained in our breeding colony in grassy, outdoor enclosures. Lucerne cubes, grass and water were provided ad libitum and supplemented with fresh vegetables. Gonads or testes were collected from pouch young aged between 1 and 34 days postpartum. The pouch young age was determined by plotting head length against growth curves for the tammar . Experimental procedures conformed to Australian National Health and Medical Research Council (2004) guidelines and were approved by the Animal Experimentation Ethics Committees of the University of Melbourne.
Preparation of single cell suspension
Gonads or testes were torn using a needle in 0.25% Trypsin/EDTA (Invitrogen) and were incubated for 10 min at 37°C. The gonadal/testicular cells were dissociated by 30 pipetting strokes with 1 mL plastic tips followed by 10 strokes with 200 μL plastic tips. The cell samples were passed through 40 μm cell strainer (BD Biosciences).
Germ cell labeling
The cells were fixed in 4% PFA/PBS for 20 min at room temperature and then permeabilised in 0.1% Triton X-100/PBS for 15 min at room temperature. The primary antibody reactions were performed in 0.1% BSA and 0.05% Tween 20/PBS containing the SSEA1 antibody (1/30 of total reaction volume, MC-480; Developmental Studies Hybridoma Bank at the University of Iowa) or the DDX4/VASA antibody (1/300 of total reaction volume, ab13840; Abcam) for 30 min at room temperature. The cells were washed in 0.1% Tween 20/PBS and were labeled by the secondary antibodies (Invitrogen) in the same solution as the primary antibody reaction. The labeled single cell suspension samples were passed through 40 μm cell strainer (BD Biosciences) before fluorescence activated cell sorting, FACS (MoFlo Cell Sorter, Beckman Coulter and FACS Aria III, BD Biosciences).
DNA methylation analyses
Genomic DNA was extracted from the germ cells collected by FACS using a Wizard Genomic DNA Purification Kit (Promega). Purified genomic DNA was treated with a sodium bisulphite solution as described previously [60, 61]. After the bisulphite treatment for the genomic DNA, 30 to 38 cycles of PCR with the genomic DNA templates corresponding to 100 to 5,000 cells were carried out using the following primer pairs.
PEG10 DMR Forward: 5′- CCTCCCATTAACTTTAAAATCACC -3′
PEG10 DMR Reverse: 5′- ATTGTAGTAATGGGGTAGGTTATG -3′
H19 DMR Forward: 5′- GAATGGGTTAGATGAGGGTAGTATAG -3′
H19 DMR Reverse: 5′- TATCAAACACCAAAACCACAAATAA -3′
H19 COBRA Forward: 5′- TTATTTTGGAGAAAATTTGAAGATAAGTAG -3′
H19 COBRA Reverse: 5′- TATCCTAAAACATCAAAACCTAAATTAAAC -3′
KERV-1 LTR Forward: 5′- TAAACTCAATTCCATATAAACAATCTC -3′
KERV-1 LTR Reverse: 5′- TTTTTGTTTTGTAAGGGTTTTTTAG -3′
LINE1 Forward: 5′- GGAGATTTTTGTTTTAGAGAGATTTGTAAA -3′
LINE1 Reverse: 5′- TATAAAAACACCCCACTCCCCTCTC -3′
The PCR products for COBRA (combined bisulphite and restriction analysis) were digested with 1 to 10 units of MluCI, AciI, TaqI (New England Biolabs) or HinfI (TaKaRa) restriction enzymes for 2-3 h at 37°C or 65°C for TaqI. The intensity of the cut and uncut bands was quantified by ATTO CS Analyzer 3 software (ATTO). The PCR products for H19 DMR and retrotransposons were cloned, and the clones were sequenced. The sequence data were analysed by QUMA (quantification tool for methylation analysis; http://quma.cdb.riken.jp) .
We thank Alison Bradfield and Scott Brownlees for assistance with the animals, Helen Clark and Bonnie Dopheide for technical assistance and Drs. Hongshi Yu and Danielle Hickford for help in collecting tissue. Fluorescence activated cell sorting was operated by Dr. Matt Burton at Murdoch Children’s Research Institute, Royal Children's Hospital and Susumu Ito at Research Center for Human and Environmental Sciences, Shinshu University.
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