Skip to main content
NCBI home page
Human Reproduction (Oxford, England) logoLink to Human Reproduction (Oxford, England)
. 2012 Nov 15;28(2):519–530. doi: 10.1093/humrep/des395

Haploinsufficiency of the paternal-effect gene Dnmt3L results in transient DNA hypomethylation in progenitor cells of the male germline

KM Niles 1,2, JR Yeh 3,4, D Chan 2,5, M Landry 2, MC Nagano 3,4,6, JM Trasler 1,2,5,7,*
PMCID: PMC3695691  PMID: 23159436

Abstract

STUDY QUESTION

How does haploinsufficiency of the paternal-effect gene Dnmt3L affect DNA methylation establishment and stability in the male germline?

SUMMARY ANSWER

Reduced expression of DNMT3L in male germ cells, associated with haploinsufficiency of the paternal-effect gene Dnmt3L, results in abnormal hypomethylation of prenatal germline progenitor cells.

WHAT IS KNOWN ALREADY

The DNA methyltransferase regulator Dnmt3-Like (Dnmt3L) is a paternal-effect gene required for DNA methylation acquisition in male germline stem cells and their precursors. In males, DNMT3L deficiency causes meiotic abnormalities and infertility. While Dnmt3L heterozygous males are fertile, they have abnormalities in X chromosome compaction and postmeiotic gene expression and sire offspring with sex chromosome aneuploidy. It has been proposed that the paternal effects of Dnmt3L haploinsufficiency are due to epigenetic defects in early male germ cells. DNA methylation is an essential epigenetic modification essential for normal germ cell development. Since patterns of DNA methylation across the genome are initially acquired in prenatal male germ cells, perturbations in methylation could contribute to the epigenetic basis of the paternal effects in Dnmt3L+/− males.

STUDY DESIGN, SIZE, DURATION

This is a cross-sectional study of DNA methylation in Dnmt3L+/+ versus Dnmt3L+/− male germ cells collected from mice at 16.5 days post-coitum (dpc), Day 6 and Day 70 (n = 3 per genotype, each n represents a pool of 2–20 animals). Additionally, DNA methylation was compared in enriched populations of spermatogonial stem cells (SSC)/progenitor cells from Dnmt3L+/+ and Dnmt3L+/− males following ∼2 months in culture.

MATERIALS, SETTING, METHODS

DNA methylation at intergenic loci along chromosomes 9 and X was examined by quantitative analysis of DNA methylation by real-time polymerase chain reaction at the time of initial acquisition of epigenetic patterns in the prenatal male germline (16.5 dpc) and compared with patterns in early post-natal spermatogonia (Day 6) and in spermatozoa in mice. DNA methylation status at CpG-rich sites across the genome was assessed in spermatogonial precursors from Day 4 male mice using restriction landmark genomic scanning.

MAIN RESULTS AND THE ROLE OF CHANCE

At 16.5 dpc, 42% of intergenic loci examined along chromosome 9 and 10% of those along chromosome X were hypomethylated in Dnmt3L heterozygotes. By Day 6 and in spermatozoa, germ cell DNA methylation was similar in heterozygous and wild-type mice. DNA methylation stability of acquired patterns in wild-type and Dnmt3L+/− SSC/progenitor cell culture was analyzed at numerous loci across the genome in cells cultured in vitro and collected at passages 6–28. While the methylation of most loci was stable in culture over time, differences at ∼1% of sites were found between Dnmt3L+/− and Dnmt3L+/+ cultures.

LIMITATIONS, REASONS FOR CAUTION

Evaluation of DNA methylation in SSCs can only be performed after a period of culture limiting the investigation to changes observed during culture when compared with DNA methylation differences between genotypes that could be present at the beginning of culture establishment.

WIDER IMPLICATIONS OF THE FINDINGS

The DNA methylation defects described here in prenatal male germline progenitor cells and SSC culture are the earliest epigenetic perturbations yet identified for a mammalian paternal-effect gene and may influence downstream epigenetic events in germ cells at later stages of development. Together, the results provide evidence of a ‘window’ of susceptibility in prenatal male germ cell precursors for the induction of epimutations due to genetic perturbations and, potentially, in utero environmental exposures.

STUDY FUNDING/COMPETING INTEREST(S)

Canadian Institutes of Health Research (CIHR) provided funding for J.M.T. (MOP229913) and M.C.N. (MOP86532). The authors have no conflicts of interest to declare.

Keywords: epigenetics, DNA methylation, Dnmt3L, spermatogonial stem cells, paternal effect

Introduction

Mammalian sperm carry unique epigenetic patterns on histones and DNA that are predicted to play an important role in normal embryonic development (Eckhardt et al., 2006; Oakes et al., 2007a; Hammoud et al., 2009; Brykczynska et al., 2010). Abnormalities in sperm DNA methylation patterns are associated with decreased fertility in mice and humans and raise concern for the potential of transmission of epimutations to the offspring (Kobayashi et al., 2007, 2009; Marques et al., 2009). The (cytosine-5) DNA methyltransferases (DNMT) DNMT3a and DNMT3L are essential for the acquisition of DNA methylation patterns in male germ cells that occurs predominantly in the late gestation prenatal period when germline stem cells are mitotically arrested (Bourc'his and Bestor, 2004; Kaneda et al., 2004; Webster et al., 2005). DNMT3 enzymes are highly conserved between mouse and human (Goll and Bestor, 2005). In addition, the timing of DNA methylation in human fetal male germ cells, along with the expression of DNMT3A, is similar to that in the mouse (Galetzka et al., 2007). In mice homozygous for targeted mutations in the epigenetic regulator, DNMT3L, a DNMT with no intrinsic methylating activity that co-operates with the de novo methylation enzyme DNMT3a2 (Chedin et al., 2002; Suetake et al., 2006), DNA methylation defects have been detected in prospermatogonia, type A spermatogonia and premeiotic spermatocytes at imprinted genes, repeat DNA elements as well as non-promoter, intergenic sites on chromosomes 4, 9 and X (Bourc'his and Bestor, 2004; Webster et al., 2005; Hata et al., 2006; Kato et al., 2007; La Salle et al., 2007; Niles et al., 2011).

While complete absence of DNMT3a and DNMT3L in male germ cells is associated with infertility, recent evidence indicates that DNMT3 variants and the presence of lower than normal levels of DNMTs in developing male germ cells may also perturb spermatogenesis and impact on the F1 offspring. A recent study performed in Chinese men has identified an association between male infertility with azoospermia and single-nucleotide polymorphisms in the DNMT3L gene, suggesting that human fertility may be affected by the expression level of DNMT3L (Huang et al., 2012). In a study of offspring conceived using assisted reproduction, Kobayashi et al. (2009) identified abnormal methylation of imprinted genes in abortuses that matched defects found in the paternal sperm; in two such cases, the fathers carried sequence variations in DNMT3L (Kobayashi et al., 2009). In mouse, Dnmt3L, in particular, was identified as the first example of a paternal-effect gene (Chong et al., 2007). Paternal-effect genes, when mutated, result in a phenotype in the wild-type offspring of heterozygous males. In the initial studies, an increased incidence of XO monosomy was found in the wild-type offspring of Dnmt3L heterozygous males mated to wild-type females (Chong et al., 2007). The authors hypothesized that haploinsufficiency of Dnmt3L was associated with abnormal epigenetic marking of DNA in male germ cells. Follow-up studies that were undertaken to better understand the basis of these paternal effects showed that the XY bodies from Dnmt3L heterozygous males were significantly longer than those from wild-type males with a concomitant increase in XY bearing sperm, and that there was deregulated expression of X-linked and autosomal genes in meiotic and haploid germ cells (Zamudio et al., 2011). While this was suggestive of underlying epigenetic defects, DNA methylation at imprinted and repeat sequences was unaffected in the spermatozoa of the Dnmt3L heterozygous males (Chong et al., 2007).

Here, to explain the meiotic abnormalities reported by Zamudio et al. (2011), we postulated that the germ cells of Dnmt3L heterozygous males might have detectable DNA methylation abnormalities only early in development, in germline progenitor and stem cells either at the time of initial acquisition of DNA methylation in prenatal prospermatogonia or in post-natal spermatogonia. We compared DNA methylation patterns at loci known to acquire methylation in the prenatal period, in pure populations of 16.5 dpc prenatal germ cells and Day 6 post-natal germ cells from Dnmt3L heterozygote and wild-type males. In addition, spermatogonial stem cells (SSCs) were derived from Dnmt3L heterozygote and wild-type mice and cultured for 3–7 months in order to generate large numbers of germ cells for more extensive DNA methylation studies.

Materials and Methods

Mice

CD-1 mice were purchased from Charles River Canada Inc. (St-Constant, Quebec, Canada). Dnmt3Ltm1Bes mice were a gift from Timothy Bestor and Deborah Bourc'his (Bourc'his and Bestor, 2004). GOF/deltaPE-Oct4/GFP transgenic mice, which express GFP driven by the proximal promoter of Oct4, were a gift from Hans Schöler (Yoshimizu et al., 1999). Dnmt3L+/− females were crossed with GOF/deltaPE-Oct4/GFP males to obtain Dnmt3L+/−; GFP+ mice. Noon of the day on which the vaginal plug was identified was considered 0.5 dpc, while the day of birth was designated post-partum Day 0. All procedures were carried out in accordance with the Canadian Council on Animal Care and approved by the McGill University Animal Care Committee.

Isolation of male germ cells by flow cytometry

Dnmt3L+/+; GFP+ and Dnmt3L+/−; GFP+ paired testes were collected from male embryos at 16.5 dpc and male pups at Day 6 resulting from timed-pregnancies between Dnmt3L+/−; GFP+ females and males. Decapsulated testes were digested in 0.25% trypsin–EDTA (Gibco-BRL/Invitrogen) for at least 10 min at 37°C, dispersed and digested for at least another 10 min with DNase. The resulting cell suspension was washed twice and resuspended in sterile phosphate-buffered solution and DNase. GFP-positive prenatal prospermatogonia and undifferentiated (Day 6) post-natal spermatogonia were collected by a MoFlow cell sorter (Cytomation Inc., Ft Collins, CO, USA). No difference in isolation efficiency was noted between the genotypes. Germ cells obtained using FACS from the testes of groups of 2–20 animals were then pooled to a total of at least 50 000 germ cells needed for each of the three separate quantitative analyses of DNA methylation by real-time polymerase chain reaction (PCR) (qAMP) carried out across chromosomes 9 and X.

SSC culture

Immunomagnetic cell sorting and cell culture

A single-cell suspension from Dnmt3L+/+ and Dnmt3L+/− paired testes at Day 4 was prepared using a two-step enzymatic digestion as previously described (Ogawa et al., 1997; Nagano, 2004). The resulting cell suspension was filtered through a 40 μm cell strainer membrane to eliminate undigested fragments. Single-parameter (Thy-1.2) immunomagnetic sorting was used to produce a cell population enriched in SSCs as previously described (Ebata et al., 2005). Thy-1.2 is the single marker shown to give the highest efficiency of SSC enrichment (4- to 30-fold) as determined by SSC testicular transplantation assays (Kubota et al., 2004a; Oatley and Brinster, 2008). Briefly, single cells were incubated with biotinylated Thy-1.2 antibodies (BD Pharmingen) and subsequently reacted with streptavidin-coated M280 Dynabeads (Invitrogen). Thy-1.2-positive (Thy-1+) cells were isolated using a magnetic cell sorter to give an SSC-enriched population. Thus, the Thy-1+ cells were the source of the cells for the cultures and to refer to these cells or their derivatives in culture, we use the term SSC-enriched cells or SSCs throughout the text.

SSC-enriched cells were then cultured on a feeder layer of STO (SIM mouse embryo-derived thioguanine and ouabain-resistant) embryonic fibroblasts in a serum-free medium with 40 ng/ml recombinant human GDNF (R&D Systems), 300 ng/ml recombinant rat GFRA1 (R&D Systems) and 1 ng/ml FGF2 (Invitrogen), as described previously (Kubota et al., 2004b; Yeh et al., 2007). A medium change was performed every 3–4 days. Cultures were digested with 0.25% trypsin–EDTA and subcultured at a 1:1–1:3 dilution on Day 6 or 7 of culture. All cultures were maintained at 37°C in a humidified atmosphere of 5% CO2. SSC cultures were examined morphologically two or more times a week at the time of medium changes and passaging of the SSCs. Initially, the SSC-enriched cultures established from the Thy1+ cells contain SSCs and their daughter cells along with testicular somatic cells. The testicular somatic cells are known to disappear during the culture under the serum-free conditions that we used (Kubota et al., 2004b; Kubota and Brinster, 2008). SSCs were collected at passages 6–7, 8–9, 11–12 or 27–28 by gentle pipetting to mechanically separate stem/progenitor spermatogonia from the feeder layer (Yeh et al., 2011). Gentle pipetting is known to generate a >90% purity of germ cells (Ryu et al., 2005; Oatley et al., 2006) and we confirmed this in our hands (Yeh et al., 2011). Throughout culture, as monitored by M. Nagano, the morphology of the SSCs was similar to that in previously published photomicrographs from our laboratory (Yeh et al., 2007; Ebata et al., 2011; Yeh et al., 2011, 2012) as well as the morphology of SSCs in long-term (6 month) culture (Kubota et al., 2004b).

Purity of the isolated stem/progenitor spermatogonia was assessed with several approaches. First, we compared the restriction landmark genomic scanning (RLGS) methylation profiles of SSCs with those of 95% pure Type A spermatogonia generated in a previous study (Oakes et al., 2007b). The Type A spermatogonia were isolated using bovine serum albumin gradients from hundreds of testes from Day 8 mice; the isolated cells were used to prepare RLGS profiles and were not submitted to culture. The RLGS DNA methylation profiles of the SSCs collected at passages 6–28 in this study matched completely with respect to the presence and intensity of loci on the gels with those of Type A spermatogonia (data not shown). The observation that cultured SSCs and in vivo generated Type A spermatogonia had identical profiles provides strong evidence that our SSC-enriched profiles were >90% pure and that few DNA methylation changes occur in culture. Next, we compared DNA methylation RLGS profiles from stem/progenitor cells and STO feeder cells. As can be seen in Supplementary data, Fig. S1 (when compared with Fig. 4), the RLGS profiles from germ cells and the somatic STO feeder cells differ markedly. We also compared our SSC RLGS profiles with others in the laboratory where we had determined purity based on the absence of methylation at maternally methylated imprinted genes such as Snrpn; maternally methylated genes show 0% methylation in pure populations of germ cells. We only evaluated RLGS profiles that matched profiles of pure SSCs. Additionally, the maternally methylated imprinted locus U2af1-rs1 was evaluated in both STO feeder cells and stem/progenitor spermatogonia on the RLGS profiles.

Figure 4.

Figure 4

Open in a new tab

RLGS analysis of Dnmt3L+/+ SSC cultures at passages 6–7, 11–12 and 27–28 demonstrating no changes in DNA methylation.

DNA methylation analysis

Restriction landmark genomic scanning

Genomic DNA was isolated from Dnmt3L+/+ SSC cultures collected at passages 6–7, 11–12 and 27–28 as well as Dnmt3L+/+ and Dnmt3L+/− SSC cultures at passages 8–9 using proteinase K followed by phenol extraction. RLGS was performed as described previously (Okazaki et al., 1995). Briefly, two-dimensional profiles were created by digesting genomic DNA with methylation-sensitive NotI followed by radioactive end-labeling. Gels were exposed to a phosphorimager screen (Kodak) and were analyzed using the ImageQuant v5.1 software (GE Healthcare).

qAMP

DNA methylation analysis of germ cells was performed as described previously (Oakes et al., 2006). In previous studies (reviewed in Oakes et al., 2009), including those on chromosome 9 (Niles et al., 2011), DNA methylation levels were similar when measured by qAMP or bisulfite sequencing. Briefly, isolated DNA was digested with no enzyme (sham), a methylation-sensitive restriction enzyme (HhaI) or a methylation-dependent restriction enzyme (McrBC). Primers specific to regions at regular intervals spanning chromosomes 9 and X have been previously described (Oakes et al., 2007b; Niles et al., 2011). Additional intercalating regions on chromosome X were selected using the UCSC Genome Browser and PCR primers flanking the cut sites were designed (Supplementary data, Table S1). Real-time PCR was performed on digested templates using QuantiTect™ SYBR® Green PCR kit (Qiagen) according to the manufacturer's suggestions for the use of the Mx3000P PCR machine (Stratagene). The change in cycle threshold values relative to the sham-digested template was utilized to determine the percent methylation for the amplified region. Results are expressed as an average of the percent methylation obtained from each enzymatic digestion unless otherwise noted. Sites that had a DNA methylation difference of >10% when considering the standard error of the mean were identified to be different between the groups.

Bisulfite sequencing

DNA was isolated from Day 6 FACS purified germ cells from Dnmt3L+/+ and Dnmt3L+/− males. Bisulfite treatment was carried out using the EpiTect Bisulfite Kit according to the manufacturer's recommendations (Qiagen). Nested primers were designed for the Dnmt3L promoter active in embryonic stem cells (ESCs), prospermatogonia and SSCs as previously described (Hu et al., 2008): outside forward 5′-ATTTTAATGTGTGAG GTTTAGAGTTTTT-3′, outside reverse 5′-ACCTAAAAATCTCACAAAATTTCAAC-3′, inside forward 5′-GTTTTGAGTTTTATAGAATTTTATAATTTTT3′ and inside reverse 5′-AAA AACTATCAACATCAAAACTAAAAC-3′. Two rounds of PCR were completed. Each 23 µl PCR reaction contained 1 µl bisulfite converted DNA, 0.5 µl of each primer (10 µM), 12.5 µl PCR Mastermix (Promega Corporation) and 8.5 µl nuclease-free water. The following PCR conditions were used: 2 min at 94°C, 1 min at 53°C, 1 min at 72°C for two cycles followed by 30 s at 94°C, 1 min at 53°C, 1 min at 72°C for 40 cycles and 1 cycle of 10 min at 72°C. The second round of PCR was carried out in the same manner with 5 µl PCR product from the first round of PCR. PCR products were separated on a 2% agarose gel and extracted using the Qiagen MiniElute Gel Extraction Kit (Qiagen). PCR products were subcloned using the TOPO TA Cloning Kit (Invitrogen) and plasmid DNA was purified with the QIAprep Spin Miniprep Kit (Qiagen). Ten to 20 clones were selected per region and sent for sequencing at the McGill Genome Quebec Innovation Centre. Figures were created with the assistance of QUantification tool for Methylation Analysis (QUMA) (http://quma.cdb.riken.jp/) (Kumaki et al., 2008).

Statistical analysis

Data were analyzed with the aid of SigmaPlot computer program (SigmaPlot v11.0, Systat Software Inc., San Jose, CA, USA). The DNA methylation data obtained using qAMP were analyzed using the Mann–Whitney rank-sum test or t-test as indicated in the text and figure legends. In all cases, a P-value of ≤0.05 was considered significant. Data are presented as ±SEM. Data comparing percent methylation changes from 16.5 dpc to Day 6 in Dnmt3L+/− and Dnmt3L−/− germ cells were evaluated using a linear regression analysis. Only sites that obtained >60% methylation during normal germ cell development were used for this analysis.

Results

Transient DNA methylation differences between Dnmt3L+/+ and Dnmt3L+/− male germ cells

Evidence of increased sex chromosome aneuploidy in offspring sired by Dnmt3L+/− males (Chong et al., 2007; Zamudio et al., 2011) suggests the possibility of abnormal epigenetic modifications in the germline of Dnmt3L haploinsufficient males. However, in previous studies, we examined numerous sites across the genome at post-natal days 6–10 and found no significant DNA methylation differences in Dnmt3L+/− compared with Dnmt3L+/+ males (La Salle et al., 2007). Similarly, bisulfite sequencing of LINE-1 and IAP repeat regions and COBRA analysis of the imprint control regions or differentially methylated regions (DMRs) of H19 and Gtl-2 in sperm from Dnmt3L+/− when compared with wild-type males, revealed no noticeable methylation defects (Chong et al., 2007). Since the majority of DNA methylation acquisition occurs prior to the presence of type A spermatogonia (Oakes et al., 2007b), in the current study, we compared DNA methylation in both pre- and post-natal germ cells from Dnmt3L+/+ and Dnmt3L+/− mice. DNA methylation was examined in germ cells at numerous intergenic, non-promoter sites along chromosomes 9 and X that had previously been reported to become methylated in the male germline (Oakes et al., 2007b; Niles et al., 2011). As in previous studies, we measured DNA methylation using qAMP; this technique allows DNA methylation levels to be measured at multiple CpGs within regions of 100–150 bp in size and has been shown to correlate well with bisulfite sequencing results (Oakes et al., 2006). Intergenic sites were chosen that were spaced at ∼2.5 kb intervals and were not proximal (>10 kb away) to CpG islands or the transcriptional start site of a known gene and were in non-repetitive regions.

At 16.5 dpc, in germ cells from Dnmt3L+/− mice, 20 of 48 sites showed decreased DNA methylation on chromosome 9 (Fig. 1A), resulting in an overall significant loss of DNA methylation across the chromosome of 15.0% when compared with methylation levels in germ cells of Dnmt3L+/+ mice (P < 0.001) (Fig. 1D). For chromosome X at 16.5 dpc, while 6 of 58 sites showed lower DNA methylation levels in Dnmt3L+/− when compared with Dnmt3L+/+ mice (Fig. 2A), overall DNA methylation averaged along the chromosome was not significantly affected (Fig. 2D). By post-natal day 6, only one difference between genotypes was detected on chromosome 9 with none evident on chromosome X (Figs 1B and 2B) supporting our previously published data (La Salle et al., 2007). Finally, at Day 70, three minor differences were noted on chromosome 9 and none were noted on chromosome X (Figs 1C and 2C). The changes noted at both Day 6 and Day 70 did not significantly affect the overall DNA methylation averaged at sites along chromosome 9. Similarly, examination of DNA methylation at 31 intergenic sites across chromosome 4 in three sets of isolated germ cells (only one set was examined previously in La Salle et al., 2007) demonstrated no significant differences between genotypes at Day 6 and only a single difference at Day 70; overall DNA methylation levels along chromosome 4 were similar for Dnmt3L+/+ and Dnmt3L+/− germ cells (Supplementary data, Fig. S2A and B). Together, these data indicate that while DNA methylation appears unaltered in post-natal germ cells, it is significantly affected in Dnmt3L+/− male germ cells at 16.5 dpc on chromosomes 9 and visibly affected on chromosome X.

Figure 1.

Figure 1

Open in a new tab

Quantitative analysis of DNA methylation by real-time polymerase chain reaction (qAMP) examination of DNA methylation at sites separated by 2.5 Mb along chromosome 9 in Dnmt3L+/+ and Dnmt3L+/− male germ cells at (A) 16.5 dpc, (B) Day 6 and (C) Day 70. Error bars indicate standard error (n = 3). (D) Average DNA methylation for chromosome 9 at 16.5 dpc, Day 6 and Day 70 in Dnmt3L+/− and Dnmt3L+/+ male germ cells. Error bars indicate standard errors of the mean (n = 48). *P < 0.001 when comparing either two groups or two time points. (E) Linear regression analysis of percent change of DNA methylation in Dnmt3L−/− and Dnmt3L+/− male germ cells from 16.5 dpc and Day 6 (n = 20, P < 0.001).

Figure 2.

Figure 2

Open in a new tab

qAMP examination of DNA methylation at sites separated by 2.5 Mb along chromosome X in Dnmt3L+/+ and Dnmt3L+/− male germ cells at (A) 16.5dpc, (B) Day 6 and (C) Day 70. Error bars indicate standard error (n = 3). (D) Average DNA methylation for chromosome X at 16.5 dpc, Day 6 and Day 70 in Dnmt3L+/− and Dnmt3L+/+ male germ cells. Error bars indicate standard errors of the mean (n = 48).

DNA methylation is delayed in the Dnmt3L+/− male germline

We previously reported that DNA methylation at 24 sites along chromosome 9 was very low in 16.5 dpc germ cells from Dnmt3L-null (Dnmt3L−/−) mice when compared with wild-type mice (19 versus 71%), but that significant catch-up occurred by Day 6 after birth, such that average germ cell DNA methylation was 61% for Dnmt3L−/−mice and 82% for wild-type mice (Niles et al., 2011). The results indicated a delay in germ cell methylation associated with complete DNMT3L deficiency and we proposed that the catch-up might be due to prolonged exposure of later germ cells to DNMT3a. Here, we examined whether a similar delay might also be evident in Dnmt3L haploinsufficient germ cells. A comparison of DNA methylation between 16.5 dpc and Day 6 on chromosome 9 in Dnmt3L+/− germ cells revealed a significant change in DNA methylation of 28.7% between the two time points (P < 0.001) compared with a significant 13.4% change in Dnmt3L+/+ germ cells (P < 0.012) (Fig. 1D). At Day 6, DNA methylation was no longer significantly different between Dnmt3L+/+ and Dnmt3L+/− germ cells.

In order to determine whether particular sites were consistently affected by this delay in DNA methylation, 24 chromosome 9 sites examined in both the current and previous studies (Niles et al., 2011) were compared in Dnmt3L+/+, Dnmt3L+/− and Dnmt3L−/− germ cells at 16.5 dpc (Supplementary data, Fig. S3A). At all sites examined, DNA methylation levels in Dnmt3L+/− were intermediary between the severely delayed Dnmt3L−/− germ cells and the wild-type controls. Additionally, Dnmt3L−/− germ cells at Day 6 showed similar levels of DNA methylation as Dnmt3L+/− germ cells at 16.5 dpc, suggesting a potential Dnmt3L dose-dependent effect (Supplementary data, Fig. S3B). Finally, linear regression analysis found a significant negative correlation (R = − 0.904, n = 20, P < 0.001) between the percent change between 16.5 dpc and day 6 in Dnmt3L−/− and Dnmt3L+/− germ cells (Fig. 1E); the results indicate that those sites that have a large DNA methylation change in Dnmt3L−/− germ cells have a minimal change in haploinsufficient germ cells because they may have already attained the appropriate level of DNA methylation, i.e. are sites where catch-up can occur despite complete DNMT3L deficiency. Together the results support the concept of a delay in DNA methylation dependent on the amount of DNMT3L present in early germ cells.

Although chromosome X also showed some site-specific DNA methylation differences at 16.5 dpc in Dnmt3L+/− male germ cells (Fig. 2A), at no time point was there a significant chromosome-wide effect on methylation along the X chromosome (Fig. 2D) as had been found for chromosome 9 (Fig. 1D). However, when comparing results from the current and previous (La Salle et al., 2007) studies, as had been noted for chromosome 9 (Supplementary data, Fig. S3B), chromosome X Dnmt3L−/− germ cells at Day 6 also showed similar levels of DNA methylation as in Dnmt3L+/− germ cells at 16.5 dpc (Supplementary data, Fig. S4), suggesting the potential for a delay of DNA methylation at specific sites on the X chromosome as well.

The delay in DNA methylation does not appear to depend on methylation of the Dnmt3L promoter

Expression of Dnmt3L in the epiblast of early post-implantation embryos as well as ES cells is controlled by DNA methylation of its own promoter mediated by DNMT3a, DNMT3b and DNMT3L (Hu et al., 2008). Dnmt3L is known to be expressed in a highly controlled manner in prenatal male germ cells with the peak at ∼15.5 dpc and low levels of expression in undifferentiated Type A and Type A spermatogonia shortly after birth (La Salle et al., 2007). Whether the down-regulation of Dnmt3L expression in post-natal germ cells is accompanied by promoter DNA methylation is not known. In order to determine if Dnmt3L expression might be controlled by DNA methylation in male germ cells and whether promoter methylation differs in haploinsufficient germ cells, bisulfite sequencing of the Dnmt3L promoter, that is active in ESCs, prospermatogonia and SSCs (Jia et al., 2007; Hu et al., 2008; O'Doherty et al., 2011), was performed on DNA from Dnmt3L+/+ and Dnmt3L+/− germ cells FACS-purified from Day 6, a time following the major peak of expression (Fig. 3). The Dnmt3L promoter showed similar low levels of methylation of 5.7 and 10.8%, respectively, in Dnmt3L+/+ and Dnmt3L+/− germ cells.

Figure 3.

Figure 3

Open in a new tab

DNA methylation analysis of the Dnmt3L promoter using bisulfite sequencing. Open and dark circles indicate an unmethylated and methylated CpG, respectively.

DNA methylation is stable in long-term SSC cultures

Results from the FACS-purified germ cells indicated that there were DNA methylation defects in prenatal germ cells of Dnmt3L+/− mice that were no longer detectable in post-natal germ cells. However, our quantitative analysis of DNA methylation was limited due to the small numbers of germ cells available. Thus, subtle defects in DNA methylation might be present in post-natal spermatogonia that could not be detected by the site-specific approach we used. In order to test for methylation defects in Dnmt3L+/− germ cells at a larger number of loci across the genome as well as to test if Dnmt3L haploinsufficient germ cells were more susceptible to losing methylation in culture, we took advantage of a recently developed SSC culture technique (Kubota et al., 2004b; Yeh et al., 2011). SSC cultures allow the expansion of SSC and non-stem progenitors, thereby providing the means to examine not only large numbers of germ cells but also to test the long-term epigenetic stability of a stem/progenitor cell population. DNA methylation at some imprinted and repeat loci has been shown to be stable during maintenance of SSC cultures (Kanatsu-Shinohara et al., 2005; Lee et al., 2009), but the loci analyzed were limited. To expand examination of DNA methylation in stem/progenitor cells to a larger number of loci across the genome, DNA was first extracted from wild-type SSC cultures collected over 7 months at passages 6–7, 11–12 and 27–28 and analyzed using RLGS, a technique that allows visualization of nearly 3000 differentially methylated NotI sites in the genome (Oakes et al., 2007b). With this technique, complete loss or gain as well as decreases or increases in methylation can be detected by phosphorimager analysis of the gels. Detailed analysis of multiple samples per passage number showed no evidence of increases or decreases in methylation over the course of long-term culture (Fig. 4). Our RLGS results on a large number of sites of different types (CpG island, repeat, intergenic) across the genome indicate that DNA methylation is remarkably stable in wild-type SSCs for at least 5–6 months (i.e. from passage 6 to passage 28) in culture.

DNA methylation in SSC cultures is affected by Dnmt3L haploinsufficiency

In order to determine whether Dnmt3L haploinsufficiency affects DNA methylation in stem/progenitor cells, Dnmt3L+/+ and Dnmt3L+/− cells were collected from cultures at passages 8–9 and DNA methylation was examined with RLGS. Attempts to establish cultures of Dnmt3L−/− stem/progenitor cells were unsuccessful. There were no methylation differences noted when comparing the gels from independently derived lines of Dnmt3L+/+ cells. In contrast, changes were found at 12 sites, 8 were hypomethylated and 4 were hypermethylated in Dnmt3L+/− compared with Dnmt3L+/+ cultures (Table I, Fig. 5). The identities of 2 of the 12 loci are known and correspond to the genes Pfn2 and the maternally methylated imprinted locus U2af1-rs1. Dnmt3L+/+ stem/progenitor cells demonstrated no methylation at the U2af1-rs1 site as would be expected for a maternally methylated imprinted locus in the male germline. It was, however, fully methylated in Dnmt3L+/− stem/progenitor cells. Thus, DNA methylation patterns in cultured stem/progenitor spermatogonia were altered by Dnmt3L haploinsufficiency even at an early passage number.

Table I.

Summary of changes seen on RLGS in Dnmt3L haploinsufficient SSC cultures.

RLGS ID Location+/− 500 bp Locus Genic location Dnmt3L+/+ Dnmt3LI+/− Dnmt3LK+/− Dnmt3LL+/− Type of change
2C31 chr3:57976407–57977407 Pfn2 3′ 1 1 1 0.5 Hyper
3E7 chr11:22923940–22924940 U2af1-rs1 5′ 4 0 0 0.5 Hyper
1D2 2 3 3 3 Hypo
1D3 2 3 3 3 Hypo
1D4 2 3 3 3 Hypo
Novel 0 2 2 2 Hypo
2D35 3 0.5 0.5 0.5 Hyper
2E57 0 1 1 1 Hypo
2F29 2 4 3 3 Hypo
2F40 0 2 0 2 Hypo
3C5 0 1 0.5 0.5 Hypo
3D24 4 2 1 0.5 Hyper
Open in a new tab

Intensities of spots are scored for each Dnmt3L genotype from 0 to 4 representing 100–0% DNA methylation, respectively.

Figure 5.

Figure 5

Open in a new tab

RLGS analysis of Dnmt3L+/+ (control) and Dnmt3L+/− SSC cultures at passages 8–9. White arrows indicate sites of hypermethylation compared with control and black arrows indicate sites of hypomethylation compared with control.

Discussion

In this paper, we examined DNA methylation in Dnmt3L+/− males at pre- and post-natal time points in vivo as well as utilized the in vitro SSC culture model to enable a more detailed examination of DNA methylation patterns. Our data clearly show that DNA methylation defects were present transiently in Dnmt3L heterozygotes in prospermatogonia at the time of initial prenatal acquisition of DNA methylation patterns (16.5 dpc); in contrast, by 6 days after birth, DNA methylation levels in spermatogonia of Dnmt3L heterozygotes were similar to those of wild-type mice. While DNA methylation patterns at numerous different types of loci across the genome were stable in cultured stem/progenitor spermatogonia from wild-type mice, they were altered in Dnmt3L heterozygotes. The results provide evidence of the earliest epigenetic defect yet described in a paternal-effect gene. Such early DNA methylation defects may be responsible for downstream effects such as the sex chromosome and meiotic gene expression abnormalities previously reported in the germ cells of Dnmt3L heterozygotes (Chong et al., 2007; Zamudio et al., 2011). In addition, the results provide evidence of a ‘window’ of susceptibility in prenatal male germ cell precursors for the induction of epimutations due to genetic perturbations (e.g. Dnmt3L haploinsufficiency) or in utero environmental exposures.

As a potential explanation for the decreased levels of DNA methylation in the 16.5 dpc Dnmt3L+/− germ cells, we examined methylation of the Dnmt3L promoter that is active in stem cells and prospermatogonia. Methylation of the Dnmt3L promoter was similarly low in both the Dnmt3L+/− and Dnmt3L+/+ germ cells. These results indicate that post-natal down-regulation of Dnmt3L expression is regulated by factors other than promoter DNA methylation in male germ cells and suggest that Dnmt3L promoter methylation is unlikely to be an explanation for differences in DNA methylation dynamics between Dnmt3L+/+ and Dnmt3L+/− mice.

With the in vivo approach based on FACS isolation of germ cells, by Day 6, all the sites on both chromosomes 9 and X that had decreased DNA methylation at 16.5 dpc in Dnmt3L+/− germ cells attained the same level of DNA methylation as in the Dnmt3L+/+ germ cells. The results suggest that by Day 6, a sufficient period of time had passed to allow DNA methylation acquisition to be completed in the Dnmt3L+/− germ cells and indicate the capacity of the DNA methylation machinery to complete the DNA methylation necessary prior to meiosis. In addition, most sites examined on chromosomes 9 and X had no significant DNA methylation differences between Dnmt3L+/+ and Dnmt3L+/− germ cells at Day 70. The exception to this was three sites on chromosome 9 that had small DNA methylation differences between the wild-type and the heterozygote mice. It is difficult to determine whether these changes are sufficient to have a biological effect on male germ cell development as the specific amount and localization of tolerable DNA methylation perturbation needed to affect fertility is unknown. However, in light of the findings of Chong et al. (2007) and Zamudio et al. (2011), it is likely that the prenatal DNA methylation defects may contribute significantly to the meiotic and sex chromosome defects reported in the Dnmt3L+/− mice.

While Dnmt3L haploinsufficient germ cells appear to eventually reach expected levels of DNA methylation prior to meiosis, the evidence of increased sex chromosome aneuploidy in offspring of Dnmt3L+/− sires (Chong et al., 2007) suggests that the DNA methylation delay may play a role in preventing successful germ cell development and reproduction. We postulate that early DNA methylation defects may interfere with the timing of other epigenetic events in the male germline. In particular, covalent modifications of the histones such as methylation, phosphorylation and acetylation are also important in determining the epigenetic control of the genome. One such modification, histone 3 lysine 4 (H3K4) methylation, undergoes dynamic changes throughout spermatogenesis (Godmann et al., 2007). Similar to the absence of DNMT3L in males, the absence of MEISETZ, a H3K4 methyltransferase, results in infertility, indicating the importance of H3K4 methylation in male germ cell development (Hayashi et al., 2005).

Both H3K4 and H3K9 methylation have been linked to DNA methylation. Numerous studies have demonstrated an inverse relationship between DNA methylation and H3K4 methylation (Fournier et al., 2002; Vu et al., 2004; Delaval et al., 2007). H3K4 di-methylation was found to be decreased within the DMRs of the paternally methylated imprinted genes H19, Rasgrf1 and Gtl2 as well as the repetitive element LINE 1 compared with maternally methylated imprinted genes and somatic genes in pre- and post-meiotic male germ cells (Delaval et al., 2007). Imprinting control regions in intergenic and intragenic regions located away from CpG islands also have the presence of overlapping H3K4 trimethylation and H3K9 trimethylation on the active and repressed alleles, respectively (Dindot et al., 2009). These findings indicate that the inverse relationship between H3K4 methylation and DNA methylation may be important in determining the localization of epigenetic modifications during male germline development. Recently, it was determined that DNMT3L can bind to the N-terminal tail of Histone (H) 3 only if the lysine 4 was unmethylated and that the H3 tail is required for de novo DNA methylation (Hu et al., 2009). Mono-, di- or tri-methylation of the lysine 4 prohibits DNMT3L binding (Ooi et al., 2007). Subsequently, it was shown that DNMT3a also binds H3 tails that are unmodified at K4 (Otani et al., 2009). A positive correlation has also been found between H3K9me3 and DNA methylation (Meissner et al., 2008) and, in a related finding, Zhang et al. (2010) have demonstrated that chromatin substrates with the H3K9me3 modification were methylated efficiently by DNMT3a and DNMT3a/DNMT3L complexes. Together the findings point toward an explanation for the inverse relationship between DNA and H3K4 methylation, the positive relationship between DNA and H3K9 methylation, as well as a potential mechanism directing localization and memory for site-specific epigenetic modifications.

Accumulating evidence suggest that DNA methylation is closely linked to other epigenetic modifications. Evidence from oocytes indicated that an absence of maternal DNMT3L may lead to a decrease and loss of allele-specificity of H3K9 trimethylation, H4K20 trimethylation and H2A/H4R3 dimethylation in the embryo, suggesting a possible role for DNA methylation in guiding histone modifications (Henckel et al., 2009). In sperm, however, the H19-Igf2 DMR is methylated at different times depending on the parental origin of the chromosome, with the paternal chromosome completing methylation more rapidly. H3K4 trimethylation has been found to be increased on the maternal allele of H19-Igf2 when compared with the paternal allele, suggesting that H3K4 methylation may also guide DNA methylation localization (Lee et al., 2010). The results presented here suggest that the delay in DNA methylation in the prenatal Dnmt3L+/− male germ cells may contribute to downstream effects on fertility and reproduction. With increasing evidence of an interplay between DNA methylation and histone methylation, it would be worth examining whether alterations in other epigenetic modifications such as the methylation of H3K4 or H3K9 are found in prenatal Dnmt3L+/− male germ cells. Further research into the patterns and timing of histone modifications as well as their interrelationship with DNA methylation at specific sites at these critical stages will need to be performed.

SSC cultures may eventually provide a tool to better understand the timing and the relationship between the various epigenetic processes within the male germline. To date, one of the most widely used stem cell culture systems, ESCs have been shown, using RLGS, the technique also used in our studies, to have unstable DNA methylation patterns in culture, depending on the culture system, time in culture and passage number (Allegrucci et al., 2007). In order to utilize SSCs as a tool to examine the effects of DNMT3L haploinsufficiency, it was important to first determine the stability of DNA methylation in SSC cultures. Examination of DNA methylation using RLGS in wild-type SSCs maintained up to 28 passages (∼7 months) was unable to identify any differences between passages 6–7, 11–12 and 27–28. This supports previous data that showed stable DNA methylation at imprinted genes and repeat regions in SSC cultures maintained in a slightly different culture system (Kanatsu-Shinohara et al., 2005; Lee et al., 2009) and expands this evaluation to many sites of different types across the genome.

Our experiments used RLGS to examine DNA methylation at numerous sites in the genome in SSCs from Dnmt3L haploinsufficient mice. Dnmt3L+/− stem/progenitor spermatogonia collected at passages 8–9 demonstrated differences at 12 sites, 8 hypomethylated and 4 hypermethylated compared with Dnmt3L+/+ cells. The differences found may be due to changes that occur in vivo during the prenatal period prior to collection of the cells for culture or in vitro at sites that have delayed DNA methylation in Dnmt3L+/− germ cells and are still undergoing modifications during this time period. Alternatively, these differences may be due to increased susceptibility of Dnmt3L+/− SSCs to the effects of culture. The maternally methylated imprinted locus U2af1-rs1 demonstrated a change from no methylation in Dnmt3L+/+ stem/progenitor cells to 100% methylation in Dnmt3L+/− stem/progenitor cells. Previous work by Chong et al. determined that no DNA methylation changes occurred at imprinted loci between Dnmt3L+/+ and Dnmt3L+/− male germ cells, indicating that this perturbation likely occurred during the culture process in Dnmt3L+/− stem/progenitor cells. These results suggest that normal levels of DNMT3L may be necessary for the continued acquisition and/or the maintenance of DNA methylation during SSC culture and could imply that Dnmt3L+/− stem/progenitor cells are more susceptible to DNA methylation perturbation. Thus, normal levels of DNMT3L may be necessary for the continued acquisition and/or the maintenance of DNA methylation during SSC culture. This may be especially true in cases such as Dnmt3L haploinsufficiency where DNA methylation has already been perturbed in vivo prior to SSC collection and culture establishment. Thus, haploinsufficiency of Dnmt3L may not be compensated during culture for epigenetic errors already present in germ cells at the start of culture. The use of the SSC culture system, where large numbers of cells can be produced, is a promising approach to uncover epigenetic defects in male germ cells. Future experiments could be designed to modulate the expression of Dnmt3L in the SSC culture system in an attempt to correct underlying DNA methylation defects in Dnmt3L+/− male germ cells and DNA methylation could be examined at high resolution using next-generation sequencing technology.

DNA methylation is a critical process for male germ cell development and fertility. Additionally, it may be linked to other epigenetic processes that are also key elements of successful germ cell development. Our studies show that, not only is DNA methylation established in the prenatal period, but it can be delayed in Dnmt3L haplosinsufficient mice. The temporary lag in DNA methylation acquisition may have downstream consequences that thus far have not been examined. Further examination of this timing and interactions with other epigenetic modifications in in vivo and in vitro models will be crucial in understanding the epigenetic patterning that is essential for normal male germ cell development.

Supplementary data

Supplementary data are available at http://humrep.oxfordjournals.org/.

Authors' roles

K.M.N.: participation in study design, execution, analysis, manuscript drafting and critical discussion. J.R.Y.: study execution, manuscript drafting and critical discussion. D.C.: study execution. M.L.: study execution. M.C.N.: participation in study design, analysis, manuscript drafting and critical discussion. J.M.T.: participation in study design, analysis, manuscript drafting and critical discussion.

Funding

Canadian Institutes of Health Research (CIHR) provided funding for J.M.T. (MOP229913) and M.C.N. (MOP86532).

Conflict of interest

None declared.

Supplementary Material

Supplementary Data
supp_28_2_519__index.html (1.5KB, html)

Acknowledgements

We would like to thank Timothy Bestor and Déborah Bourc'his for the gift of the Dnmt3L mice and Hans Schöler for the gift of the GOF 18/delta PE-Oct4/GFP mice. We would like to thank Giles Hooker for his assistance with the statistical analysis and Martine Dupuis and Eric Massicotte at the Institut de Recherches Cliniques de Montréal for their expertise in flow cytometry. K.M.N. was a recipient of the CIHR Institute of Genetics Walter and Jessie Boyd & Charles Scriver MD/PhD Studentship Award. J.R.Y. was a recipient of a studentship grant from the Centre for the Study of Reproduction (CSR). D.C. was a recipient of Fonds de la recherche en santé du Québec (FRSQ) and CIHR Doctoral Research Awards. J.M.T. is a James McGill Professor of McGill University. All authors are members of the Research Institute of The McGill University Health Centre which is supported by the FRSQ.

References

  1. Allegrucci C, Wu YZ, Thurston A, Denning CN, Priddle H, Mummery CL, Ward-van Oostwaard D, Andrews PW, Stojkovic M, Smith N, et al. Restriction landmark genome scanning identifies culture-induced DNA methylation instability in the human embryonic stem cell epigenome. Hum Mol Genet. 2007;16:1253–1268. doi: 10.1093/hmg/ddm074. [DOI] [PubMed] [Google Scholar]
  2. Bourc'his D, Bestor TH. Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature. 2004;431:96–99. doi: 10.1038/nature02886. [DOI] [PubMed] [Google Scholar]
  3. Brykczynska U, Hisano M, Erkek S, Ramos L, Oakeley EJ, Roloff TC, Beisel C, Schubeler D, Stadler MB, Peters AH. Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa. Nat Struct Mol Biol. 2010;17:679–687. doi: 10.1038/nsmb.1821. [DOI] [PubMed] [Google Scholar]
  4. Chedin F, Lieber MR, Hsieh CL. The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a. Proc Natl Acad Sci USA. 2002;99:16916–16921. doi: 10.1073/pnas.262443999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chong S, Vickaryous N, Ashe A, Zamudio N, Youngson N, Hemley S, Stopka T, Skoultchi A, Matthews J, Scott HS, et al. Modifiers of epigenetic reprogramming show paternal effects in the mouse. Nat Genet. 2007;39:614–622. doi: 10.1038/ng2031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Delaval K, Govin J, Cerqueira F, Rousseaux S, Khochbin S, Feil R. Differential histone modifications mark mouse imprinting control regions during spermatogenesis. EMBO J. 2007;26:720–729. doi: 10.1038/sj.emboj.7601513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dindot SV, Person R, Strivens M, Garcia R, Beaudet AL. Epigenetic profiling at mouse imprinted gene clusters reveals novel epigenetic and genetic features at differentially methylated regions. Genome Res. 2009;19:1374–1383. doi: 10.1101/gr.089185.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ebata KT, Zhang X, Nagano MC. Expression patterns of cell-surface molecules on male germ line stem cells during postnatal mouse development. Mol Reprod Dev. 2005;72:171–181. doi: 10.1002/mrd.20324. [DOI] [PubMed] [Google Scholar]
  9. Ebata KT, Yeh JR, Zhang X, Nagano MC. Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro. Exp Cell Res. 2011;317:1319–1329. doi: 10.1016/j.yexcr.2011.03.013. [DOI] [PubMed] [Google Scholar]
  10. Eckhardt F, Lewin J, Cortese R, Rakyan VK, Attwood J, Burger M, Burton J, Cox TV, Davies R, Down TA, et al. DNA methylation profiling of human chromosomes 6, 20 and 22. Nat Genet. 2006;38:1378–1385. doi: 10.1038/ng1909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fournier C, Goto Y, Ballestar E, Delaval K, Hever AM, Esteller M, Feil R. Allele-specific histone lysine methylation marks regulatory regions at imprinted mouse genes. EMBO J. 2002;21:6560–6570. doi: 10.1093/emboj/cdf655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Galetzka D, Weis E, Tralau T, Seidmann L, Haaf T. Sex-specific windows for high mRNA expression of DNA methyltransferases 1 and 3A and methyl-CpG-binding domain proteins 2 and 4 in human fetal gonads. Mol Reprod Dev. 2007;74:233–241. doi: 10.1002/mrd.20615. [DOI] [PubMed] [Google Scholar]
  13. Godmann M, Auger V, Ferraroni-Aguiar V, Di Sauro A, Sette C, Behr R, Kimmins S. Dynamic regulation of histone H3 methylation at lysine 4 in mammalian spermatogenesis. Biol Reprod. 2007;77:754–764. doi: 10.1095/biolreprod.107.062265. [DOI] [PubMed] [Google Scholar]
  14. Goll MG, Bestor TH. Eukaryotic cytosine methyltransferases. Annu Rev Biochem. 2005;74:481–514. doi: 10.1146/annurev.biochem.74.010904.153721. [DOI] [PubMed] [Google Scholar]
  15. Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR. Distinctive chromatin in human sperm packages genes for embryo development. Nature. 2009;460:473–478. doi: 10.1038/nature08162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hata K, Kusumi M, Yokomine T, Li E, Sasaki H. Meiotic and epigenetic aberrations in Dnmt3L-deficient male germ cells. Mol Reprod Dev. 2006;73:116–122. doi: 10.1002/mrd.20387. [DOI] [PubMed] [Google Scholar]
  17. Hayashi K, Yoshida K, Matsui Y. A histone H3 methyltransferase controls epigenetic events required for meiotic prophase. Nature. 2005;438:374–378. doi: 10.1038/nature04112. [DOI] [PubMed] [Google Scholar]
  18. Henckel A, Nakabayashi K, Sanz LA, Feil R, Hata K, Arnaud P. Histone methylation is mechanistically linked to DNA methylation at imprinting control regions in mammals. Hum Mol Genet. 2009;18:3375–3383. doi: 10.1093/hmg/ddp277. [DOI] [PubMed] [Google Scholar]
  19. Hu YG, Hirasawa R, Hu JL, Hata K, Li CL, Jin Y, Chen T, Li E, Rigolet M, Viegas-Pequignot E, et al. Regulation of DNA methylation activity through Dnmt3L promoter methylation by Dnmt3 enzymes in embryonic development. Hum Mol Genet. 2008;17:2654–2664. doi: 10.1093/hmg/ddn165. [DOI] [PubMed] [Google Scholar]
  20. Hu JL, Zhou BO, Zhang RR, Zhang KL, Zhou JQ, Xu GL. The N-terminus of histone H3 is required for de novo DNA methylation in chromatin. Proc Natl Acad Sci USA. 2009;106:22187–22192. doi: 10.1073/pnas.0905767106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Huang JX, Scott MB, Pu XY, Zhou-Cun A. Association between single-nucleotide polymorphisms of DNMT3L and infertility with azoospermia in Chinese men. Reprod Biomed Online. 2012;24:66–71. doi: 10.1016/j.rbmo.2011.09.004. [DOI] [PubMed] [Google Scholar]
  22. Jia D, Jurkowska RZ, Zhang X, Jeltsch A, Cheng X. Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature. 2007;449:248–251. doi: 10.1038/nature06146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kanatsu-Shinohara M, Ogonuki N, Iwano T, Lee J, Kazuki Y, Inoue K, Miki H, Takehashi M, Toyokuni S, Shinkai Y, et al. Genetic and epigenetic properties of mouse male germline stem cells during long-term culture. Development. 2005;132:4155–4163. doi: 10.1242/dev.02004. [DOI] [PubMed] [Google Scholar]
  24. Kaneda M, Okano M, Hata K, Sado T, Tsujimoto N, Li E, Sasaki H. Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature. 2004;429:900–903. doi: 10.1038/nature02633. [DOI] [PubMed] [Google Scholar]
  25. Kato Y, Kaneda M, Hata K, Kumaki K, Hisano M, Kohara Y, Okano M, Li E, Nozaki M, Sasaki H. Role of the Dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse. Hum Mol Genet. 2007;16:2272–2280. doi: 10.1093/hmg/ddm179. [DOI] [PubMed] [Google Scholar]
  26. Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C, Utsunomiya T, Sasaki H, Yaegashi N, Arima T. Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Hum Mol Genet. 2007;16:2542–2551. doi: 10.1093/hmg/ddm187. [DOI] [PubMed] [Google Scholar]
  27. Kobayashi H, Hiura H, John RM, Sato A, Otsu E, Kobayashi N, Suzuki R, Suzuki F, Hayashi C, Utsunomiya T, et al. DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm. Eur J Hum Genet. 2009;17:1582–1591. doi: 10.1038/ejhg.2009.68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kubota H, Brinster RL. Culture of rodent spermatogonial stem cells, male germline stem cells of the postnatal animal. Methods Cell Biol. 2008;86:59–84. doi: 10.1016/S0091-679X(08)00004-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kubota H, Avarbock MR, Brinster RL. Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod. 2004a;71:722–731. doi: 10.1095/biolreprod.104.029207. [DOI] [PubMed] [Google Scholar]
  30. Kubota H, Avarbock MR, Brinster RL. Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA. 2004b;101:16489–16494. doi: 10.1073/pnas.0407063101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kumaki Y, Oda M, Okano M. QUMA: quantification tool for methylation analysis. Nucleic Acids Res. 2008;36:W170–W175. doi: 10.1093/nar/gkn294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. La Salle S, Oakes CC, Neaga OR, Bourc'his D, Bestor TH, Trasler JM. Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L. BMC Dev Biol. 2007;7:104–121. doi: 10.1186/1471-213X-7-104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lee J, Kanatsu-Shinohara M, Ogonuki N, Miki H, Inoue K, Morimoto T, Morimoto H, Ogura A, Shinohara T. Heritable imprinting defect caused by epigenetic abnormalities in mouse spermatogonial stem cells. Biol Reprod. 2009;80:518–527. doi: 10.1095/biolreprod.108.072330. [DOI] [PubMed] [Google Scholar]
  34. Lee DH, Singh P, Tsai SY, Oates N, Spalla A, Spalla C, Brown L, Rivas G, Larson G, Rauch TA, et al. CTCF-dependent chromatin bias constitutes transient epigenetic memory of the mother at the H19-Igf2 imprinting control region in prospermatogonia. PLoS Genet. 2010;6:e1001224. doi: 10.1371/journal.pgen.1001224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Marques CJ, Francisco T, Sousa S, Carvalho F, Barros A, Sousa M. Methylation defects of imprinted genes in human testicular spermatozoa. Fertil Steril. 2009;94:585–594. doi: 10.1016/j.fertnstert.2009.02.051. [DOI] [PubMed] [Google Scholar]
  36. Meissner A, Mikkelsen TS, Gu H, Wernig M, Hanna J, Sivachenko A, Zhang X, Bernstein BE, Nusbaum C, Jaffe DB, et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature. 2008;454:766–770. doi: 10.1038/nature07107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nagano M. Spermatogonial transplantation. In: Gardner DK, Lane MWA, editors. A Laboratory Guide to the Mammalian Embryo. Oxford: Oxford University Press; 2004. pp. 334–351. [Google Scholar]
  38. Niles KM, Chan D, La Salle S, Oakes CC, Trasler JM. Critical period of nonpromoter DNA methylation acquisition during prenatal male germ cell development. PLoS One. 2011;6:e24156. doi: 10.1371/journal.pone.0024156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Oakes CC, La Salle S, Robaire B, Trasler JM. Evaluation of a quantitative DNA methylation analysis technique using methylation-sensitive/dependent restriction enzymes and real-time PCR. Epigenetics. 2006;1:146–152. doi: 10.4161/epi.1.3.3392. [DOI] [PubMed] [Google Scholar]
  40. Oakes CC, La Salle S, Smiraglia DJ, Robaire B, Trasler JM. A unique configuration of genome-wide DNA methylation patterns in the testis. Proc Natl Acad Sci USA. 2007a;104:228–233. doi: 10.1073/pnas.0607521104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Oakes CC, La Salle S, Smiraglia DJ, Robaire B, Trasler JM. Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells. Dev Biol. 2007b;307:368–379. doi: 10.1016/j.ydbio.2007.05.002. [DOI] [PubMed] [Google Scholar]
  42. Oakes CC, La Salle S, Trasler JM, Robaire B. Restriction digestion and real-time PCR (qAMP) Methods Mol Biol. 2009;507:271–280. doi: 10.1007/978-1-59745-522-0_20. [DOI] [PubMed] [Google Scholar]
  43. Oatley JM, Brinster RL. Regulation of spermatogonial stem cell self-renewal in mammals. Annu Rev Cell Dev Biol. 2008;24:263–286. doi: 10.1146/annurev.cellbio.24.110707.175355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Oatley JM, Avarbock MR, Telaranta AI, Fearon DT, Brinster RL. Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc Natl Acad Sci USA. 2006;103:9524–9529. doi: 10.1073/pnas.0603332103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. O'Doherty AM, Rutledge CE, Sato S, Thakur A, Lees-Murdock DJ, Hata K, Walsh CP. DNA methylation plays an important role in promoter choice and protein production at the mouse Dnmt3L locus. Dev Biol. 2011;356:411–420. doi: 10.1016/j.ydbio.2011.05.665. [DOI] [PubMed] [Google Scholar]
  46. Ogawa T, Arechaga JM, Avarbock MR, Brinster RL. Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol. 1997;41:111–122. [PubMed] [Google Scholar]
  47. Okazaki Y, Okuizumi H, Sasaki N, Ohsumi T, Kuromitsu J, Hirota N, Muramatsu M, Hayashizaki Y. An expanded system of restriction landmark genomic scanning (RLGS Ver. 1.8) Electrophoresis. 1995;16:197–202. doi: 10.1002/elps.1150160134. [DOI] [PubMed] [Google Scholar]
  48. Ooi SK, Qiu C, Bernstein E, Li K, Jia D, Yang Z, Erdjument-Bromage H, Tempst P, Lin SP, Allis CD, et al. DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature. 2007;448:714–717. doi: 10.1038/nature05987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Otani J, Nankumo T, Arita K, Inamoto S, Ariyoshi M, Shirakawa M. Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX-DNMT3-DNMT3L domain. EMBO Rep. 2009;10:1235–1241. doi: 10.1038/embor.2009.218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Ryu BY, Kubota H, Avarbock MR, Brinster RL. Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. Proc Natl Acad Sci USA. 2005;102:14302–14307. doi: 10.1073/pnas.0506970102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Suetake I, Morimoto Y, Fuchikami T, Abe K, Tajima S. Stimulation effect of Dnmt3L on the DNA methylation activity of Dnmt3a2. J Biochem (Tokyo) 2006;140:553–559. doi: 10.1093/jb/mvj185. [DOI] [PubMed] [Google Scholar]
  52. Vu TH, Li T, Hoffman AR. Promoter-restricted histone code, not the differentially methylated DNA regions or antisense transcripts, marks the imprinting status of IGF2R in human and mouse. Hum Mol Genet. 2004;13:2233–2245. doi: 10.1093/hmg/ddh244. [DOI] [PubMed] [Google Scholar]
  53. Webster KE, O'Bryan MK, Fletcher S, Crewther PE, Aapola U, Craig J, Harrison DK, Aung H, Phutikanit N, Lyle R, et al. Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis. Proc Natl Acad Sci USA. 2005;102:4068–4073. doi: 10.1073/pnas.0500702102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yeh JR, Zhang X, Nagano MC. Establishment of a short-term in vitro assay for mouse spermatogonial stem cells. Biol Reprod. 2007;77:897–904. doi: 10.1095/biolreprod.107.063057. [DOI] [PubMed] [Google Scholar]
  55. Yeh JR, Zhang X, Nagano MC. Wnt5a is a cell-extrinsic factor that supports self-renewal of mouse spermatogonial stem cells. J Cell Sci. 2011;124:2357–2366. doi: 10.1242/jcs.080903. [DOI] [PubMed] [Google Scholar]
  56. Yeh JR, Zhang X, Nagano MC. Indirect effects of Wnt3a/beta-catenin signalling support mouse spermatogonial stem cells in vitro. PLoS One. 2012;7:e40002. doi: 10.1371/journal.pone.0040002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yoshimizu T, Sugiyama N, De Felice M, Yeom YI, Ohbo K, Masuko K, Obinata M, Abe K, Scholer HR, Matsui Y. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev Growth Differ. 1999;41:675–684. doi: 10.1046/j.1440-169x.1999.00474.x. [DOI] [PubMed] [Google Scholar]
  58. Zamudio NM, Scott HS, Wolski K, Lo CY, Law C, Leong D, Kinkel SA, Chong S, Jolley D, Smyth GK, et al. DNMT3L is a regulator of X chromosome compaction and post-meiotic gene transcription. PLoS One. 2011;6:e18276. doi: 10.1371/journal.pone.0018276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zhang Y, Jurkowska R, Soeroes S, Rajavelu A, Dhayalan A, Bock I, Rathert P, Brandt O, Reinhardt R, Fischle W, et al. Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail. Nucleic Acids Res. 2010;38:4246–4253. doi: 10.1093/nar/gkq147. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
supp_28_2_519__index.html (1.5KB, html)
supp_des395_des395supp.doc (27.5KB, doc)
supp_des395_des395supp_fig1.tif (6.9MB, tif)
supp_des395_des395supp_fig2.tif (243.2KB, tif)
supp_des395_des395supp_fig3.tif (195.3KB, tif)
supp_des395_des395supp_fig4.tif (119.3KB, tif)
supp_des395_des395supp_table1.doc (45.5KB, doc)

Articles from Human Reproduction (Oxford, England) are provided here courtesy of Oxford University Press

RESOURCES