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Human Molecular Genetics, 2004, Vol. 13, No. 8 839-849
DOI: 10.1093/hmg/ddh104
Human Molecular Genetics, Vol. 13, No. 8 © Oxford University Press 2004; all rights reserved

Gene-specific timing and epigenetic memory in oocyte imprinting

Diana Lucifero1, Mellissa R.W. Mann2, Marisa S. Bartolomei2 and Jacquetta M. Trasler1,*

1McGill University, Montreal Children's Hospital Research Institute and Departments of Pediatrics, Human Genetics, and Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada H3H 1P3 and 2Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

Received December 15, 2003; Accepted February 18, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Imprinted genes are differentially marked during germ cell development to allow for their eventual parent-of-origin specific expression. A subset of imprinted genes becomes methylated during oocyte growth in both mouse and human. However the timing and mechanisms of methylation acquisition are unknown. Here, we examined the methylation of the Snrpn, Igf2r, Peg1 and Peg3 differentially methylated regions in postnatal growing mouse oocytes. Our findings indicate that methylation was acquired asynchronously at these different genes. Further analysis of Snrpn DMR1 revealed that parental alleles retain an epigenetic memory of their origin as the two alleles were recognized in a parental-specific manner in the absence of DNA methylation. In addition, we show that methylation acquisition was probably related to oocyte diameter and coincided with the accumulation of Dnmt3a, Dnmt3b and Dnmt3L transcripts. Methylation of the repetitive retroviral-like intracisternal A particle also occurred during this same window of oocyte growth. These findings contribute to our understanding of the epigenetic mechanisms underlying imprint acquisition during female germ cell development and have implications for the practice of assisted reproductive technologies.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
DNA methylation is an epigenetic regulator of gene expression and acts as an important molecular mark underlying the parental-specific expression of genes subject to genomic imprinting (1). Imprinted genes account for the requirement of both maternal and paternal genomes in normal development and play significant roles in regulating embryo growth, placental function and neurobehavioral processes (2,3). Aberrant expression of several imprinted genes has been linked to the development of human diseases, including Beckwith–Wiedemann, Prader–Willi and Angelman syndromes (reviewed in 4).

To allow for the differential expression of imprinted genes, some epigenetic marking must distinguish the alleles inherited from the maternal versus the paternal genomes. To date DNA methylation remains the most widely investigated epigenetic modification associated with this differential marking of imprinted alleles. A number of genes regulated by imprinting contain differential methylation regions (DMRs) inherited from the gametes (5,6). In addition, DNA methylation is both a heritable and reversible epigenetic modification that is stably propagated after DNA replication and influences gene expression and chromatin condensation via the binding of factors such as methyl CpG binding proteins that subsequently recruit other gene silencing factors including histone deacetylases (reviewed in 7).

DNA methyltransferases (DNMTs) are a family of de novo and maintenance methylating enzymes responsible for the addition of a methyl group to the 5-position of cytosine within CpG dinucleotides (reviewed in 8). Dnmt3L mouse gene targeting studies demonstrate a critical role for this enzyme in the establishment of maternal methylation imprints in the female germ line (9,10). In addition, embryos derived via the transplantation of Dnmt3a–/–, Dnmt3b+/– ovaries have completely unmethylated Igf2r, Peg1, Peg3 and Snrpn DMRs (10). Mice deficient for DNA methyltransferases Dnmt1, Dnmt1o or Dnmt3L display both loss of allele-specific methylation and expression of imprinted genes implying at the very least a role for methylation in imprint maintenance (9–12).

The erasure, establishment and maintenance of imprints are dynamic processes that must be correctly reprogrammed with every reproductive cycle. From mouse studies, erasure occurs around the time that primordial germ cells enter the gonad (13–17). Imprint establishment occurs during gametogenesis, when maternal and paternal genomes are physically separated, and the timing of acquisition of genomic imprints is significantly different between the two germ lines. In the male germ line, methylation acquisition on H19, a maternally expressed gene, is initiated in prenatal prospermatogonia and complete postnatally by the pachytene stage of meiosis (18–20). Round spermatid microinjection experiments have confirmed that paternal imprints are complete by the haploid phase of spermatogenesis (21). In the female, elegant nuclear transplantation experiments in the mouse have suggested that imprint acquisition occurs in the postnatal growth phase of oogenesis (22,23). In complementary DNA methylation experiments, we have shown that the DMR1 methylation imprint on the paternally expressed bicistronic gene Snurf–Snrpn (24), hereinafter referred to as Snrpn, is acquired during oocyte growth and that establishment of the maternal methylation imprint on the gametic DMRs within Igf2r (25), Peg1 (26) and Peg3 (27), is complete in metaphase II (MII) oocytes (5). In agreement with our mouse data, a recent study on human oocytes indicated that SNRPN is methylated in late stage germinal vesicle (GV), metaphase I (MI) and MII oocytes (6). However, a thorough understanding of the timing and mechanisms underlying maternal methylation imprint establishment is lacking.

In this study, our first aim was to investigate and compare the methylation profiles of the DMRs of imprinted genes from different chromosomal regions (Snrpn, Igf2r, Peg1 and Peg3) at five timepoints spanning the window of postnatal oocyte growth. Using bisulfite sequencing analysis, our findings suggest that, although each of these imprinted genes acquired methylation during postnatal oogenesis, methylation acquisition at these DMRs was asynchronous and may be governed by distinct mechanisms. Allele-specific methylation analysis of the Snrpn DMR revealed that, in the absence of DNA methylation, the parental identity of the Snrpn alleles was maintained as the maternally inherited allele acquired methylation prior to the paternally inherited allele and, thus, may be distinguishable via another epigenetic mechanism. This observed phenomenon of epigenetic memory was conserved when oocytes derived via the reciprocal cross were analyzed. We also show that methylation acquisition at the Snrpn DMR was related to oocyte diameter, and that the accumulation of Dnmt3a, Dnmt3b and Dmnt3L transcripts coincided with this increase in oocyte diameter and the timing of methylation acquisition on several imprinted genes. Analysis of the 5' long terminal repeat (LTR) within non-imprinted intracisternal A particle (IAP) elements indicates that methylation of this repetitive retroviral-like sequence occurred during the same window of postnatal oocyte growth, and suggests a similarity between imprinted genes harboring maternal methylation and repeat elements, and their targeting by DNMTs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Gene-specific establishment of DMR methylation during oocyte growth
In our first experiment, we assayed the timing of methylation acquisition for four imprinted genes harboring maternal methylation. In addition to being located on different chromosomes, each of the four genes has a well-defined DMR for which gamete-specific differences in methylation have been shown (5). The methylation status of a number of CpG sites within the Snrpn, Igf2r, Peg1 and Peg3 DMRs in 1, 5, 10, 15 and 25 dpp (days postpartum) oocytes was determined using bisulfite sequencing analysis (Fig. 1A–E and Supplementary Material Table 1). Non-growing oocytes from primordial follicles isolated at 1 (Fig. 1A) and 5 (Fig. 1B) dpp showed very few methylated CpG sites for all genes examined. In 10 dpp early-growing oocytes (Fig. 1C), which varied greatly in diameter (20–70 µm), we observed an increase in the number of methylated strands (percentage of strands hypermethylated defined as >50% of CpGs methylated on a given strand) for Peg3 (38%), Igf2r (15%) and Snrpn (11%), but not for Peg1 (0%). The majority of oocytes were probably isolated from preantral follicles.



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  		Figure 1. Gene-specific establishment of maternal methylation imprints for the Snrpn, Igf2r, Peg1 and Peg3 DMRs during postnatal oocyte growth. Snrpn resides within a well-characterized imprinting cluster on central chromosome 7, while Igf2r, Peg1 and Peg3 are located proximally on chromosomes 17, 6 and 7, respectively. Oocytes were analyzed at (A) 1, (B) 5, (C) 10, (D) 15 and (E) 25 dpp. Each line represents an individual clone. A solid circle indicates a methylated CpG site, an open circle denotes an unmethylated CpG and a missing circle represents a CpG site where the sequencing data were ambiguous. Space between strands of the same sample indicates that two sets of oocytes were independently bisulfite treated and analyzed. H19 methylation served as control for somatic cell contamination (data not shown). For each sample, the bisulfite methylation data were analyzed by computing both the percentage of clones >50% methylated and the percentage methylated CpGs out of the total number of CpGs analyzed, and then subjected to statistical analysis using Fisher's exact test (Supplementary Material Table 1).

 
The majority of oocytes isolated at 15 dpp were roughly 70–80 µm in diameter; we suggest that these oocytes were derived from early antral follicles. A number of smaller oocytes probably from less mature preantral follicles were also collected at this stage. While regions within the DMRs of Snrpn (83%), Igf2r (63%) and Peg3 (66%) continued to acquire methylation, Peg1 remained hypomethylated (0%; Fig. 1D). Complete methylation of strands was observed for the first time at this stage in oogenesis with the exception of Peg1.

Germ cells isolated from 25 dpp follicles were composed of GV oocytes that were fully grown (70–80 µm) and meiotically competent. Strikingly, all Peg1 strands sequenced were hypermethylated (Fig. 1E), suggesting that this gene underwent rapid de novo methylation late in oocyte development. Snrpn and Igf2r were 91 and 96% methylated, respectively, although a few strands remained hypomethylated at this late stage of oocyte growth. Peg3, which appeared to be one of the first genes to acquire methylation, was undermethylated compared to the other genes, although not significantly different from its methylation status at 15 dpp (Supplementary Material). Snrpn, Igf2r and Peg3 continued to acquire methylation as these DMRs were densely methylated in MII oocytes (5). These results indicate that the establishment of methylation imprints proceeded in a gene-specific manner while oocytes were arrested at prophase I and transitioned from primordial to antral follicles.

Allele-specific methylation on Snrpn
To gain a more in-depth understanding of the timing and mechanisms underlying maternal methylation imprint establishment, Snrpn was subjected to further analysis. Allele-specific methylation dynamics of the Snrpn DMR1 were assayed in postnatal F1 mouse oocytes by bisulfite sequencing, using strain-specific polymorphisms to differentiate the parental alleles (Fig. 2 and Supplementary Material Table 2). Methylation analysis of the Snrpn DMR1 in 1 dpp oocytes revealed both alleles to be essentially devoid of methylation, suggesting an equivalence of the parental alleles after methylation erasure (Supplementary Material Fig. 1). In 10 dpp oocytes (based on three independently collected sets of at least 500 oocytes each, from different litters), the maternal allele was substantially more methylated (67% strands hypermethylated) than the paternally derived allele (0%), suggesting an initial targeting of methylation to the maternally inherited Snrpn allele (Fig. 2A). Oocytes at 15 dpp (from two independently collected sets of at least 500 oocytes each, from different litters) continued to acquire methylation allele-specifically with the Snrpn maternal allele substantially more methylated (88%) than the paternal allele (22%; Fig. 2B). As observed in the non-allele-specific methylation analysis of Snrpn 15 dpp oocytes (Fig. 1), 15 dpp was the first timepoint where all 16 CpGs analyzed were methylated within a given strand. In these F1 samples, complete methylation occurred exclusively on the maternally inherited allele (Fig. 2B). Although some paternal strands were highly methylated, complete methylation of paternally inherited strands probably occurred later in oocyte growth.



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  		Figure 2. Allele-specific methylation status of Snrpn DMR1 in growing oocytes that were isolated at (A) 10 dpp, and (B) 15 dpp from CD1xB6(CAST7) (left) and B6(CAST7)xCD1 (right) F1 females. DNA isolation and bisulfite sequencing analysis were performed on four 10 dpp and three 15 dpp sets of F1 oocytes (roughly 500 each). Details as described in Figure 1. The maternally inherited allele appeared to acquire methylation prior to the paternal allele, suggesting that another epigenetic mark continues to distinguish Snrpn parental alleles in the absence of methylation in growing oocytes. See Supplementary Material Figure 1 for allele-specific methylation profile of Snrpn in 1 dpp oocytes and Supplementary Material Table 2 for statistical analysis of 10 and 15 dpp data.

 
To differentiate between strain-specific effects versus parental allele directed acquisition, DNA methylation was assayed in 10 and 15 dpp oocytes isolated from F1 pups derived via the reciprocal cross. A similar pattern of methylation establishment was observed with the maternal allele hypermethylated compared with the paternal allele (Fig. 2A and B, right). In 10 dpp oocytes, maternal and paternal strands exhibited 60 and 0% methylation, respectively. In 15 dpp oocytes, the maternal allele was 100% methylated while the paternal allele was 82% methylated. In contrast to expectation from the analysis of 1 dpp oocytes (Supplementary Material Fig. 1), the parental Snrpn alleles were not equivalent but retained their identity in the absence of a DNA methylation mark within the area of DMR1 examined.

Imprint establishment is related to oocyte diameter
Having established that maternal methylation imprints were conferred in a gene- and allele-specific manner, we next assessed whether the acquisition of Snrpn methylation was related to oocyte diameter. We isolated oocytes from 15 dpp females and grouped them according to diameter, with one pool containing oocytes that ranged between 20 and 50 µm and a second pool containing oocytes that were 60–80 µm. Bisulfite sequencing analysis of Snrpn showed that 15 dpp oocytes of small diameter were devoid of methylation (0%) while strands from larger diameter 15 dpp oocytes were hypermethylated (94%; Fig. 3A and Supplementary Material Table 3). Our observations that maternal methylation imprint establishment was related to oocyte diameter suggests that the accumulation of some enzyme(s) or factor(s) may be necessary for methylation imprint establishment.



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  		Figure 3. Snrpn DMR1 methylation profile of 15 dpp oocytes of different diameters. Methylation status of Snrpn DMR1 in (A) 20–50 µm and (B) 60–80 µm 15 dpp oocytes. Snrpn methylation acquisition appeared to be related to oocyte diameter. Details as described in Figure 1. See Supplementary Material Table 3 for statistical analysis.

 
Dnmt3 enzyme profiles during oocyte growth
As a first step in determining which DNMT is mediating the methylation of these imprinted DMRs during oogenesis, we assayed expression of Dnmt3a, Dnmt3b and Dnmt3L during postnatal oocyte growth using RT–PCR. Our results indicate that all three Dnmts were expressed during oocyte growth (Fig. 4). Dnmt3L transcripts appeared to be particularly abundant in growing oocytes and were differentially expressed in 20–50 versus 60–80 µm 15 dpp oocytes (Fig. 4, bottom right panel). The de novo methyltransferases, Dnmt3a and Dnmt3b, were expressed at lower levels, but with a similar expression profile to that seen for Dnmt3L where peak expression occurred in 15 dpp oocytes.



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  		Figure 4. Expression analysis of Dnmt3a, Dnmt3b and Dnmt3L during postnatal oogenesis. Oocytes were isolated at 5, 10, 15 and 25 dpp and from ovulated MII oocytes (C, minus reverse transcriptase control lane). Amplification of rabbit {alpha}-globin (R{alpha}G) was used as an internal control for RNA extraction and amplification efficiency. For Dnmt3a and Dnmt3b (top and middle panels) 150 oocytes were used per lane. For Dnmt3L, 25 oocytes were used per lane. Dnmt3a, Dnmt3b and Dnmt3L appeared to be expressed in growing oocytes as methylation was being acquired at maternally methylated DMRs. Dnmt3L transcripts appeared to be particularly abundant in growing oocytes. Bottom right panel, expression of Dnmt3L in 15 dpp oocytes (n=25) of different diameters. Oocytes were isolated at 15 dpp and grouped into two pools according to size: 20–50 µm (Sm for small) and 60–80 µm (Big). Dnmt3L expression was up-regulated as oocyte diameter increased.

 
IAP LTR methylation and expression in oocytes
To address the idea that methylation acquisition occurs postnatally in growing oocytes on a more global level, we assessed the methylation profile of non-imprinted IAPs during this same developmental window. IAP sequences are retrovirus-like transposable elements found in ~1000 copies in the mouse genome. Methylation of the 5'-LTR of IAPs is important for keeping these elements transcriptionally silent and inactive (28). Our results show that IAPs LTR methylation was also acquired during oocyte growth (Fig. 5A and Supplementary Material Table 4) with the percentage of hypermethylated strands in 1, 5, 10 and 15 dpp and MII oocytes at 12, 33, 82, 82 and 66%, respectively (15 dpp and MII methylation profiles not significantly different by Fisher's exact analysis, see Supplementary Material).



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  		Figure 5. IAP methylation and expression during postnatal oocyte growth. (A) Methylation status of the IAPs 5'-LTR in 1, 5, 10 and 15 dpp, and MII oocytes. Bisulfite results for 15 dpp and MII were obtained from two sets of oocytes. Details as described in Figure 1. See Supplementary Material Table 4 for statistical analysis. (B) Expression analysis of IAP type I transcripts in 5, 10, 15 and 25 dpp and MII oocytes (25 oocytes were used per lane) (C, control lane). IAP type I transcripts were expressed in 10, 15 and 25 dpp growing oocytes and appeared to be down-regulated in MII oocytes.

 
Using RT–PCR, we next determined whether IAP methylation during oocyte growth correlates with transcriptional silencing of IAP elements. IAP transcripts are divided into two subfamilies, type I and type II, which account for roughly 60 and 40% all IAPs, respectively (29). Our analysis, which investigated the expression profile of the more abundant subfamily, indicated that type I IAPs were present in 10, 15 and 25 dpp oocytes and were down-regulated in MII oocytes (Fig. 5B). Although this result may suggest that methylation resulted in IAP transcriptional down-regulation, it may also reflect the general phenomenon of decreased RNA synthesis in MII oocytes (30).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Methylation of imprinted genes occurred during oocyte growth
In this study, we show that a number of imprinted genes acquired their methylation imprint during postnatal oocyte growth and that, while some genes were methylated early in oocyte development, others were methylated in more mature fully grown oocytes. In our earlier study, we had examined Snrpn methylation at three timepoints during oocyte growth (1 and 10 dpp and in MII oocytes) (5). Here, by expanding our analysis to include additional timepoints as well as multiple DMRs within imprinted genes, we show that the acquisition of methylation imprints during oocyte development was a more generalized phenomenon. These findings are important in light of recent results indicating that human SNRPN is also methylated in GV oocytes (6). Several studies have suggested that there is an increased incidence of human imprinting disorders associated with alterations in the methylation of imprinted genes in children conceived by assisted reproductive technologies (ARTs) (31–35). We and others (36–39) have argued that it is thus critical to achieve a better understanding of the timing and mechanisms underlying imprint establishment and maintenance, as well as the conditions that may adversely affect imprints, such as ovulation protocols and culture conditions. The fact that maternal methylation imprints appear to be established during the later stages of oocyte development in human, as they are in mouse, provides support for the continued use of the mouse model in studying imprinting mechanisms.

Gene-specific establishment of maternal methylation imprints
We first investigated the timing of methylation acquisition of four imprinted genes with a maternal methylation imprint, Snrpn, Igf2r, Peg1 and Peg3, in postnatal growing oocytes. Our use of bisulfite sequencing analysis, a very sensitive and powerful assay for investigating the methylation status of multiple CpGs within a region of interest, enabled five imprinted genes to be examined simultaneously in the same sets of oocytes, permitting the methylation of each gene to be analyzed in relation to the others. In each set of oocytes, H19 DMR methylation was examined first and the lack of methylated strands verified that there was no somatic contamination in the oocyte preparations (data not shown). Our findings indicate that methylation on the Snrpn, Igf2r, Peg1 and Peg3 DMRs was acquired asynchronously in a gene-specific manner while oocytes were arrested at prophase I and transitioned from primordial to antral follicles (summarized in Fig. 6).



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  		Figure 6. Schematic depicting the gene- and parental allele-specific dynamics underlying methylation acquisition during postnatal oogenesis. The top panel roughly depicts the methylation profiles of IAPs, Peg3, Igf2r, Snrpn and Peg1 in growing oocytes. Our findings indicate that non-imprinted IAPs (purple line) began acquiring methylation in non-growing and early-growing oocytes. Methylation was initiated on the Peg3, Igf2r (gray lines) and Snrpn (red and blue lines) DMRs in early-and mid growing oocytes (in descending order according to their percentage strands with >50% mCpGs), while Peg1 (gray line) remained hypomethylated in comparison to the other genes and acquired methylation late in oocyte development. Our results for Snrpn suggest a non-equivalence of the parental alleles in the absence of DNA methylation in growing oocytes, as the maternally inherited allele (red line) acquired methylation prior to the paternally derived allele (blue line). The bottom panel depicts the various stages of oocytes that were investigated.

 
An independent study inferred that maternal imprint acquisition occurred asynchronously based on expression of imprinted genes in parthenogenetic embryos derived from manipulated primordial to fully grown oocytes (23). However, these studies did not determine the mechanistic basis (i.e. DNA methylation or chromatin structure) of the asynchronous imprinting. Imprint establishment was suggested by Obata and Kono (23) to occur in primordial to primary follicle stages oocytes (5–15 dpp) for Snrpn, Znf127 and Ndn, in secondary follicle stage oocytes (10–20 dpp) for Peg3, Igf2r and p57kip2, in tertiary to early antral follicle stage oocytes (15–20 dpp) for Peg1 and in antral follicle stage oocytes for Impact (23). This suggested sequence of imprint establishment (23) closely resembled our results here, where Snrpn methylation was initiated in 10 dpp oocytes, Peg3 and Igf2r had similar methylation imprint profiles and Peg1 acquired methylation at a late stage in oocyte growth.

Each of the genes we investigated is found in different chromosomal regions: Snrpn is found within a well-characterized imprinting cluster that spans a large region of central chromosome 7, while Igf2r, Peg1 and Peg3 are located proximally on chromosomes 17, 6 and 7, respectively (www.mgu.har.mrc.ac.uk/imprinting/all_impmaps.html). We suggest that the gene-specific establishment of imprints may be explained by the fact that these genes are either present in distinct chromosomal regions or are within different surrounding chromatin environments.

Epigenetic memory in oocyte imprinting
Our non-allele-specific methylation analysis of the DMRs of each imprinted gene in 1 dpp oocytes, where the strands sequenced were devoid of methylation, suggests an equivalence of the parental alleles at this time. Further analysis of Snrpn revealed that parental alleles retained an epigenetic memory of their parental origin as the two alleles continued to be recognized in a parental-specific manner (summarized in Fig. 6). Our allele-specific analysis of imprint acquisition on Snrpn in oocytes indicates that the methylation imprint was initially established in preantral early growing oocytes on the maternally inherited allele. In contrast, the paternally inherited allele becomes methylated in more mature oocytes derived from antral follicles. Thus, our finding that the maternal and paternal alleles of Snrpn are differentially marked during oocyte growth indicates that the parental alleles were not equivalent, and retained their identity in the absence of DMR1 methylation.

Our findings for Snrpn are reminiscent of the sequence of methylation acquisition on the H19 DMR during spermatogenesis. Both parental alleles of H19 are unmethylated in 13.5 dpc (days postcoitum) prospermatogonia. Methylation is initiated and complete on the paternally inherited allele in 15.5 dpc prospermatogonia while methylation on the maternal allele is postponed until 18.5 dpc and is not completed until the end of meiosis I (19). Similar to Snrpn in the female germ line, this differential pattern of allele-specific methylation establishment on H19 indicates that some epigenetic modification in the male germ line distinguishes the parental alleles in the absence of DNA methylation.

Strain-specific differences in mouse oocytes can affect epigenetic inheritance (40,41) and could explain the parental allele-specific methylation differences reported for H19 (19). Here, we analyzed Snrpn DMR1 methylation in reciprocal cross F1 oocytes to determine whether our allele-specific observations were dependent on the direction of the cross used to generate our oocytes. Our findings that the maternal allele was also hypermethylated when compared with the paternal allele in reciprocal cross oocytes imply that this differential establishment of maternal imprints was not strain-specific. However, it should be noted that, although the order of methylation was the same, the degree to which alleles were methylated appeared to be significantly different between the crosses. While these observations may reflect the fact that the number of oocytes collected with larger diameters may have varied between pools, they also suggest possible strain-specific differences in the DNMT content and activity of oocyte cytoplasms.

Together our results implicate the existence of another epigenetic mark in the two germ lines. We propose that differential chromatin structure directs the establishment of gametic imprints in a gene-specific manner and marks the parental alleles of imprinted genes during germ cell development. While methylation imprints are erased in PGCs, other epigenetic modifications may not be removed. The remaining epigenetic modifications may direct DNA methylation exclusively to the Snrpn maternal DMR1 in oocytes before 10 dpp and to the paternal H19 DMR in prospermatogonia prior to 18.5 dpc.

Differential modifications on the histone tails of imprinted parental alleles have been described. Allele-specific acetylation and methylation profiles of specific histone residues have been reported for Igf2, H19, Igf2r, Snrpn and U2af1-rs1 (42–45). We propose that chromatin modifications may mark the alleles of imprinted genes during early germ cell development and initially target DNA methylation to one parental allele. Collecting the number of germ cells needed to carry out the allele-specific chromatin immunoprecipitation assays or downscaling and adapting such assays to allow analysis of chromatin modifications in germ cells remains a significant challenge to the testing of this hypothesis.

Methylation acquisition at Snrpn DMR1 correlated with an increase in oocyte diameter and the accumulation of Dnmt3a, Dnmt3b and Dnmt3L transcripts
During postnatal female germ cell development, the oocyte grows greatly in volume and accumulates a variety of factors necessary for both meiotic maturation and early embryo development (30). We concluded from our results on oocytes from 1, 5, 10, 15 and 25 dpp mice that there is a progressive stage-related increase in methylation of the imprinted genes examined. Since the oocytes were selected at different ages, it is also possible that there is an age effect. Experiments to test an age effect would be challenging and require the isolation of oocytes of different sizes from the adult ovary. However, our results on Snrpn methylation in oocytes of different sizes isolated from the ovaries of 15 dpp mice support our conclusion of stage-specific methylation. Our observations that maternal methylation imprint establishment was related to oocyte diameter suggests that the accumulation of some enzyme(s) or regulatory molecule(s) necessary for imprint establishment is the limiting factor in methylation imprint establishment. We therefore examined the expression of the most recently characterized DNMT enzymes, DNMT3a, DNMT3b and DNMT3L, in growing oocytes.

DNMT3a and DNMT3b are de novo methyltransferases (46), and their involvement in establishing methylation imprints has been suggested by the observation that Igf2r, Peg1, Peg3 and Snrpn DMRs are unmethylated in embryos derived via the transplantation of Dnmt3a–/–, Dnmt3b+/– ovaries (10). Dnmt3L mouse knockout studies demonstrate an essential role for this enzyme in the establishment of maternal methylation imprints in the female germ line (9,10). As Dnmt3L does not share any of the conserved DNMT catalytic motifs responsible for enzymatic activity, it is postulated to be a regulator of maternal imprint establishment. Our results indicate that all three enzymes are expressed during oocyte growth with similar expression profiles (Fig. 4). Dnmt3L transcripts appear to be particularly abundant in growing oocytes, further supporting an important role for this DNMT in the establishment of methylation imprints. Expression of Dnmt3L in growing oocytes, but not primary oocytes, was also reported by Bourc'his et al. (9). It has been shown that DNMT3L stimulates methylation by DNMT3a (but not DNMT3b) at the Snrpn and Igf2r DMRs in vitro (47). In growing oocytes, DNMT3L may serve to recruit de novo methyltransferases such as DNMT3a to the DMRs of imprinted genes and direct methylation at these sites. While a number of candidate factors have been postulated to play a role in maternal imprint establishment, there remains the possibility that other proteins such as methyl-binding and chromatin remodeling proteins or some yet to be characterized DNMT may also be involved.

IAP methylation and expression in oocytes and implications for the repeat-like nature of imprinted gene DMRs
Having shown that DNA methylation is established at the DMRs of several imprinted genes in growing oocytes, we were interested in determining whether a non-imprinted class of sequences that is known to be methylated acquires methylation within the same developmental window. The great majority of CpGs present in the mammalian genome are contained within repetitive DNA elements. We chose to analyze the methylation status of the 5'-LTR of IAP retroviral repeat sequences. Several studies have investigated the methylation status of IAP elements in germ cells and embryos as a means of gaining insights into the methylation reprogramming events that occur during development. Previous studies have suggested that IAPs are hypomethylated in non-growing primary oocytes (48,49) and hypermethylated in fully grown oocytes (48,50) and, like imprinted genes, appear to resist demethylation in the zygote and preimplantation embryo (50). We now show using bisulfite sequencing that multiple CpG sites within the 5'-LTR of IAP elements were relatively undermethylated in 1 dpp oocytes, and acquired methylation during postnatal oocyte growth in a manner comparable to imprinted genes.

The similarity in de novo methylation acquisition suggests a likeness in the nature and regulation of IAPs and imprinted genes, as has been suggested by others (50). It has been shown that the short tandem repeat elements within the DMRs of some imprinted genes are important for imprinting (51). DNMTs and other components of the complex that drive methylation imprints may recognize these repeats as the signal directing methylation. From our results, methylation appears to be targeted to IAPs earlier during oocyte growth than the imprinted genes we examined. We speculate that the degree to which imprinted genes are repeat-like may be one of the factors involved in determining the timing of methylation imprint acquisition.

DNA methylation has been proposed as an IAP transcriptional silencing mechanism (48). Embryos completely deficient in the major maintenance methyltransferase Dnmt1 show a 50- to 100-fold increase in the expression of all classes of IAP transcripts (48). Because our IAP expression analysis was limited to type I transcripts whereas our bisulfite methylation analysis detected the majority of IAPs with LTRs, a direct correlation between IAP methylation and expression levels may not be appropriate. However, data from our analyses of growing oocytes, showing active transcription of type I IAP elements that were probably methylated, suggest that methylation may not be sufficient to repress IAP transcription and that other epigenetic mechanisms are probably required for silencing.

Significance
Maternal imprinting defects have been reported for a number of clinical syndromes. In one syndrome, thought to represent a global disorder of imprinting in the female germ line, biparental hydatidiform moles develop as a result of a maternal imprinting establishment defect (52,53). Moreover, several reports have linked ARTs to an increased incidence of rare imprinting diseases (reviewed in 36,37). Methylation analysis of affected ART-conceived children has revealed a consistent defect in the methylation status of the maternally inherited allele at the SNRPN (Angelman syndrome) and KCNQ1OT1 (Beckwith–Wiedemann syndrome) DMRs (31–35). It has been postulated that genetic defects (52,53) and techniques used in human ARTs (36,37) may perturb maternal imprint establishment and/or maintenance. Our data and those of others (23) indicate that some maternal imprints are established relatively late in oogenesis. Since imprint acquisition during oocyte growth may be vulnerable to ARTs, such as in vitro maturation of oocytes and ovarian hyperstimulation, epigenetic evaluation of oocyte manipulations should be included in prospective studies on ARTs and imprinting disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Oocyte collections and mice
Oocytes were isolated from dissociated ovaries at 1, 5, 10, 15 and 25 dpp while MII oocytes were collected from 7- to 8-week-old females as described (5,54) and were washed free of somatic cells and stored at –80°C until ready for use. Briefly, ovaries of 1, 5, 10 and 15 dpp mice were dissected in PBS, pH 7.2, and transferred to conical tubes containing 2 ml of 3 mg/ml polyvinylpyrrolidone (Sigma) prepared in PBS, 2 mg/ml collagenase (Sigma), 0.025% trypsin (Gibco BRL) and 0.02 mg/ml DNase (Sigma). The contents were shaken at 250 rpm in a 37°C incubator for 3–10 min depending on the age of the ovary and subsequently diluted by half with Hepes-buffered MEM, pH 7.2 (Gibco BRL), modified as described (55). The dissociation of oocyte–cumulus cell complexes was carried out by vigorously drawing the MEM-H/PBS solution in and out of a micropipette. GV oocytes were isolated by puncturing ovarian follicles of 25 dpp mice using a needle. MEM-H used for the isolation of 25 dpp oocytes was supplemented with 50 µg/ml dibutyryl cyclic AMP to prevent GV breakdown. MII oocytes were collected from 7- to 8-week-old females that were superovulated by injection of 7.5 IU of pregnant mares' serum gonadotropin (Sigma), followed 44–48 h later by 5 IU of human chorionic gonadotropin (Sigma). Twenty hours post-hCG, MII oocytes were recovered from the oviducts and the cumulus cells were dispersed with 1 mg/ml hyaluronidase (Roche Diagnostics). For all stages collected, oocytes were picked up using a mouth-controlled drawn-out glass pipette and washed free of somatic cells by transfer through three dishes of MEM-H. Only cumulus-free, non-fragmented and ‘healthy’ looking oocytes were chosen for analysis. Somatic cell contamination was eliminated as a source of methylated strands, as the H19 DMR, that is paternally methylated, was completely unmethylated for each sample (data not shown).

For the Snrpn diameter specific methylation analysis, 15 dpp were collected as described above, measured and pooled into the 20–50 and 60–80 µm size groups using a stage micrometer. For the other methylation and expression experiments, all 1–5 dpp and 25 dpp oocytes collected were 10–20 and 70–80 µm in diameter, respectively, whereas our 10 and 15 dpp oocyte pools were more heterogeneous. At 10 dpp, the oocytes collected ranged from 20 to 70 µm in diameter with greater than 50% of oocytes being larger than 50 µm. At 15 dpp, roughly 90% of oocytes were greater than 60 µm and ranged in diameter from 20 to 80 µm.

Oocytes used in the non-allele specific experiments were obtained from CD-1 mice (Charles River Canada, St Constant, QC, Canada). C57BL/6J and Mus musculus castaneus were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). To facilitate the isolation of F1 hybrid mice for the allele-specific bisulfite analysis of Snrpn in oocytes, a strain of mice that served as the source of M. m. castaneus allele was used (56). Natural matings between CD1 and C57BL/6J(CAST-7) mice were used to produce females for the isolation of oocytes. Parental origin of the sequenced strands was determined using strain-specific single nucleotide polymorphisms for Snrpn and H19 (56). Experiments were performed in compliance with the guidelines set by the Canadian Council for Animal Care.

DNA isolation and bisulfite sequencing
DNA was isolated from 450 to 500 oocytes at 1, 5, 10, 15 and 25 dpp, digested using HindIII (Gibco BRL) and bisulfite treatment was carried out as previously described (5,57). At least two independent sets of oocytes from mice of different litters were collected and bisulfite treated separately; all six genes were amplified from each set of oocytes. Nested PCR amplification for Snrpn, Igf2r, Peg1, Peg3, H19 and non-nested PCR amplification for IAPs were carried out on each set of isolated oocytes as previously described (5,12). Clones containing the appropriate inserts were sequenced using an ABI 310 sequencer. Only sequences with >95% bisulfite conversion efficiency were used for analysis. Sequence differences between clones with similar CpG methylation profiles were verified to ensure unique clones were represented.

Primers
Primers specific for bisulfite-converted DNA for Snrpn, Igf2r, Peg1, Peg3, H19 and IAPs were as previously described (5,12). The regions analyzed for each of these genes are within CpG islands. We examined a total of 16 CpG sites in a 419 bp fragment of Snrpn (2151–2570 bp, AF081460), seven CpG sites in a 205 bp fragment of Igf2r (796–1001 bp, L06446), 23 CpG sites in a 562 bp fragment of Peg1 (1089–1651 bp, AF017994), 18 CpG sites in a 286 bp fragment of Peg3 (2770–3056 bp, AF105262), 16 CpG sites in a 422 bp fragment of H19 (1304–1726 bp, U19619) and nine CpG sites in a 212 bp fragment of IAPs (100–312 bp, M17551).

RT–PCR
Total RNA was extracted from roughly 500 each 5, 10, 15 and 25 dpp and MII oocytes using Trizol reagent (Invitrogen) supplemented with 10 µg of mussel glycogen (Boehringer Ingelheim) (58). Prior to RNA extraction 0.125 pg of rabbit globin mRNA (Sigma) was added per oocyte for each sample. Amplification of rabbit {alpha}-globin was used as an internal control for RNA extraction and amplification efficiency (59). Oocyte RNA pellets were dissolved in 50 µl of DEPC-treated water and 25 or 150 oocytes were used in the SuperScript One-Step RT–PCR System (Invitrogen) to analyze Dnmt3a, Dnmt3b, Dnmt3L and IAP (type I) expression levels. RT–PCR products were electrophoresed through 2% agarose gels. The amplification product sizes were as follows: Dnmt3a, 601 bp (671–1272 bp, NM_007872); Dnmt3b, 506 bp (556–1062 bp, AF068628); Dnmt3L, 533 bp (632–1165 bp, NM_019448); IAPs, 418 bp [1651–2069 bp (29), M17551]. See Supplementary Material for primer sequences. Primers were designed to span introns for the transcripts investigated. RT–PCR experiments were repeated on at least two samples of RNA from independently collected and extracted oocytes at each timepoint.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We wish to thank H. Clarke, T. Davis and T. Kelly for helpful suggestions and advice. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) to J.M.T. D.L. is the recipient of CIHR and Fonds de la recherche en santé du Québec (FRSQ) studentships. J.M.T. is a William Dawson Scholar of McGill University and a Scholar of the FRSQ. M.S.B. is an assistant investigator of the Howard Hughes Medical Institute. M.R.W.M. was supported by a grant from the Lalor Foundation.


   FOOTNOTES
 
* To whom correspondence should be addressed at: McGill University, Montreal Children's Hospital Research Institute, 4060 Ste. Catherine Street West, Montreal, Quebec, Canada H3Z 2Z3. Tel: +1 5144124400, ext. 25235; Fax: +1 5144124331; Email: jacquetta.trasler{at}mcgill.ca


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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