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Department of BioScience, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

	Abstract

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In mammals, some genes categorized as imprinted genes are exclusively expressed either from maternal or paternal allele. This parental-origin-specific gene expression is regulated by epigenetic modification of DNA methylation in differentially methylated region (DMR), which is independently imposed during oogenesis and spermatogenesis. It is known that methylation of DMR in the female germ line is established during oocyte growth phase. However, the cause of the progression of methylation on DMR, due to either aging of mice or growth-size of oocyte was unclear up to now. Here, we analyzed the methylation of DMR for each eight imprinted genes (Igf2r, Lit1, Zac1, Snrpn, Peg1/Mest, Impact, Meg1/Grb10, and H19) by bisulfite sequencing methylation assay, using oocytes from 10 dpp (days post partum), 15 dpp, 20 dpp, and adult mice. To find whether the size of oocytes is the cause of methylation, above oocytes were classified into seven groups (each oocyte diameter ranging from 40 to 75 µm with intervals of 5 µm). The results from juvenile mice oocytes showed that DMR methylation progressed according to oocyte growth each imprinted gene. More than 85% of DMR methylation was achieved for both Igf2r, Zac1 & Lit1 with oocyte size of reaching 55 µm and Snrpn, Peg1/Mest, Impact, and Meg1/Grb10 with oocyte size of reaching 60 µm. Preferential methylation of maternal allele was observed in Zac1 and Peg1/Mest of juvenile oocytes and in Snrpn of juvenile and adult oocytes. The oocyte size-dependent-methylation progressed equally for all three different-age juvenile mice. The size-dependent-methylation was also recognized in the growing oocytes collected from adult mice, although the progress is slightly slower than that of juvenile mice. From these results, we concluded that DNA methylation is established with oocyte size dependent manner, not with aging of mice.

	Introduction

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Genomic imprinting is a unique molecular mechanism for controlling parental-origin-specific gene expression, depending on the DNA methylation status of the differentially methylated region (DMR) of each imprinted gene. In the germ-line, parental-specific DNA methylation is erased during the differentiation of primordial germ cells, and is independently re-established during spermatogenesis and oogenesis (Delaval & Feil 2004). However, when and how DNA methylation is reprogrammed remain major issues in the elucidation of the imprinting mechanism in mammals.

From among more than 80 imprinted genes/transcripts identified to date, only distal chr.7 (Igf2, H19, etc), distal chr.9 (Rasgrf1) and distal chr.12 (Dlk1, Gtl2, etc) have been identified as genes, which are epigenetically modified during spermatogenesis to achieve parental-origin-specific gene expression (Tremblay et al. 1995; Pearsall et al. 1999; Takada et al. 2002). The majority of imprinted genes are thought to be epigenetically modified during oocyte growth period. The de novo methyltransferases DNA methyltransferase 3a (Dnmt3a) (Okano et al. 1998) and DNA (cytosine-5-)-methyltransferase 3-like (Dnmt3L) (Aapola et al. 2000), which are responsible for methylation in germ-line are expressed throughout the oocyte growth phase (unpublished observation). The maternal genome from growing oocytes with 60–69 µm of juvenile mice is first capable of supporting embryo development, if it contributes to mature oocytes and fertilizes in vitro (Kono et al. 1996; Bao et al. 2000). This indicates that the epigenetic modification progress is not initiated until oocytes enter the growth phase with follicular development. Imprinted gene expression analysis in reconstructed parthenotes containing sets of haploid genome, both from an ovulated metaphase of the second meiotic stage (MII) oocyte and growing oocytes suggested that the epigenetic modification of each imprinted gene is imposed during oocyte growth at a specific time (Obata & Kono 2002). Furthermore, analysis of DNA methylation in the DMR of several imprinted genes showed a relationship between DNA methylation and oocyte growth (Lucifero et al. 2004).

However, the question of whether the epigenetic modification of each imprinted gene is imposed due to aging of mice or the growth-size of oocyte has been an unresolved issue. To clarify this issue, the precise imposed time was specified by investigating the DNA methylation of the DMR of each imprinted gene in growing oocytes obtained both from juvenile and adult mice. The results revealed that the maternal epigenetic modification required for the parental-origin-specific gene expression is imposed due to the growth-size of oocyte.

	Results

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DMR methylation progress in the growing oocytes from juvenile mice

The cause of the progression of methylation on DMR of imprinted genes (Fig. 1, Tables 1, 2), due to either the aging of mice or growth-size of oocyte was determined by using growing oocytes (Fig. 2). The oocytes with sizes of 40–60 µm (n = 2075), 40–70 µm (n = 2192), 40–75 µm (n = 4403), and 40–75 µm (n = 2520) for 10-, 15- and 20-day-old, and adult mice, respectively, were classified into following groups: (1) diameter of 40–45 µm (primary follicle stage 1 (PF1)), (2) 45–50 µm (primary follicle stage 2 (PF2)), (3) 50–55 µm (secondary follicle stage 1 (SF1)), (4) 55–60 µm (secondary follicle stage 2 (SF2)), (5) 60–65 µm (antral follicle stage 1 (AF1)), (6) 65–70 µm (antral follicle stage 2 (AF2)), and (7) 70–75 µm. Further, to gain a better understanding of the DMR methylation process, we analyzed the DMR methylation status of eight imprinted genes in six groups of oocytes from PF1 to AF2.

View larger version (31K): [in this window] [in a new window]   Figure 1  The relative position of mouse maternally imprinting Igf2r (GENBANK accession no. L06446), Lit1 (GENBANK accession no. AP001295), Zac1 (GENBANK accession no. Aj308559), Snrpn (GENBANK accession no. AF081460), Peg1/Mest (GENBANK accession no. AF017994), Impact (GENBANK accession no. AF232228) and Meg1/Grb10 (GENBANK accession no. AL663087) DMRs. Exons are depicted as solid boxes and are numbered. Horizontal arrowheads indicate the transcriptional orientation of imprinted genes. Open boxes represent differentially methylated regions (DMRs). Amplified regions enlarged below each gene and each CpG dinucleotide is represented with a circle.

View larger version (19K): [in this window] [in a new window]   Figure 2  Distributions of oocytes obtained from ovaries of 10-, 15- and 20-day-old and adult (8-week-old) mice.

View larger version (101K): [in this window] [in a new window]   Figure 3  Bisulfite sequencing analysis of DMR in maternally imprinted Igf2r, Zac1, Lit1, Impact, Snrpn, Peg1/Mest and Meg1/Grb10 in the growing oocytes from 10-, 15- and 20-day-old juvenile mice. Growing oocytes were classified into seven groups as follows: (1) PF1, diameter of 40–45 µm; (2) PF2, 45–50 µm; (3) SF1, 50–55 µm; (4) SF2, 55–60 µm; (5) AF1, 60–65 µm; (6) AF2, 65–70 µm; and (7) 70–75 µm (not analyzed). Each line of circles represents an individual sequence molecule, with each circle corresponding to a separate CpG dinucleotide. Closed and open circles indicate methylated and unmethylated CpGs. DNA polymorphisms found between C57BL/6 and DBA/2 in methylation analysis region of the Snrpn, Zac1 and Peg1/Mest, with maternal B6 allele sequences and paternal DBA sequences are identified as line of circles against the background of red region and the background of blue region, respectively.

It has been reported that the DMR for the Snrpn was methylated in the maternal allele prior to methylation in the paternal allele (Lucifero et al. 2004). It is uncertain that such asynchronous DNA methylation is a unique phenomenon, which occurs only in the Snrpn. Therefore, we performed an allele-specific methylation analysis for the Snrpn, Zac1, and Peg1/Mest genes using single nucleotide polymorphisms between C57BL/6 and DBA/2 mouse strains (Figs. 3, 4). Parental-origin-specific preferential methylation was clearly detected in the Snrpn of SF1/15-day and SF1/20-day oocytes and PF1, SF1, SF2 oocytes from adult mice. Further tendency of the preferential methylation was also observed in Peg1/Mest of SF2/10-day and SF2/15-day oocytes, and Zac1 of SF2/15-day and SF2/20-day oocytes.

DMR methylation progress in the growing oocytes from adult mice

Gene-specific DMR methylation progressed with oocyte size of juvenile mice. However, this finding did not include oocytes developed in the adult ovaries. The process of gene-specific DMR methylation in oocytes from adult ovaries was investigated by the DMR methylation status of the Zac1, Igf2r, Snrpn, Peg1/Mest, and Meg1/Grb10 in the growing oocytes collected from mature females (Fig. 4, Table 3). In PF2 oocytes from adults, the Igf2r, Snrpn, Peg1/Mest, and Meg1/Grb10 DMRs were not methylated, and the Zac1 DMR was only slightly methylated (5%). This pattern was similar to the oocytes observed in the juvenile mice. In SF1 oocytes, the rate of CpG-site methylation of the Zac1 DMR increased to 26.5%, and initial Igf2r DMR methylation (13.8%) was also observed with slight delay compared to the corresponding DMR methylation in juvenile mouse oocytes. However, the Snrpn DMR was not methylated in SF1 oocytes from adult mice, although approximately half of the Snrpn DMRs were methylated in the oocytes from juvenile mice. In SF2 oocytes, the Zac1 (66.7%) and Igf2r DMRs (36.3%) were methylated, and the Snrpn (32.0%), Peg1/Mest (20.1%), and Meg1/Grb10 DMRs (22.8%) began to show methylation at this stage as well. The Zac1 and Snrpn DMRs were almost fully methylated in oocytes at all stages of growth up to the AF1 stage. The Igf2r (71.7%), Peg1/Mest (40.2%) and Meg1/Grb10 DMRs (58.5%) were methylated in the AF1 oocytes, which were fully methylated until the oocytes developed to the AF2 stage. Thus, DNA methylation in the growing oocytes of adult ovaries also took place due to the size of oocyte, as it did in the oocytes obtained from the juvenile mice ovary. However, the progress of gene-specific DMR methylation in oocytes from adults tended to be slower than in those from juvenile mice.

View larger version (59K): [in this window] [in a new window]   Figure 4  Methylation analysis of DMR in maternally imprinted Igf2r, Zac1, Snrpn, Peg1/Mest and Meg1/Grb10 in the growing oocytes from adult (8-week-old) mice. For further information, see the legend to Fig. 3.

	Discussion

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Regulation of maternal imprinting as dependent upon oocyte size

The initiation of the methylation of CpG sites in DMRs for growing oocytes occurs when oocytes reach 40 µm, and methylation is completed when oocytes reach 65 µm. The timing of methylation was found to progress independently in each imprinted gene. For example, the DMR methylation of the Snrpn was initiated when the oocytes reached 40–45 µm, but there was still no sign of the initiation of methylation in the case of the Peg1/Mest. The order of the acquisition of methylation among maternally imprinting genes is thought to take place with proportion to essentiality of the gene for development. For example, Snrpn, Igf2r, and Lit1 are all methylated relatively early, and these genes are crucial to both development and growth, since a deficiency in any of these genes in mice is lethal (Lau et al. 1994; Wang et al. 1994; Yan et al. 1997; Zhang et al. 1997; Yang et al. 1998). However, Peg1/Mest, which was found to belong to a late-methylation group, is associated with maternal behavior and Peg1/Mest-mutant mice are already known to survive (Lefebvre et al. 1998).

Preferential maternal-allele-specific methylation in the DMR of the Snrpn was a unique case among oocytes derived from juvenile and adult mice, in which the DMR methylation clearly occurred in the maternal allele prior to the occurrence in the paternal allele. A similar preferential methylation was also observed in the DMRs of the Zac1 and Peg1/Mest in the juvenile mouse oocytes, but not in the adult oocytes. These results suggest that a secure memory mechanism is underlying the preferential methylation in the DMR of the Snrpn; however, it is still unknown how the maternal allele preferential methylation is induced (discussed in the later).

Gene expression analysis by RT-PCR and immunostaining showed that Dnmt3a and Dnmt3L are expressed and are located in the nuclei throughout the various stages of oocyte growth. Using Dnmt3a-conditional mutant mice, Kaneda et al. (2004) demonstrated that offspring from Dnmt3a-conditional mutant females died in utero, due to lack of both methylation and allele-specific expression at all imprinted loci modified in the maternal germ line. These findings, together with those using Dnmt3L-knockout mice suggest that both Dnmt3a and Dnmt3L are required for the methylation in most of the imprinted loci in the course of maternal methylation (Bourc’his et al. 2001; Hata et al. 2002). The results obtained clarify that the mechanisms required for oocyte size-dependent methylation is not responsible for the expression and localization of the de novo methylases Dnmt3a and Dnmt3L. Therefore, other regulatory factors could be proposed for the regulation of oocyte size-dependent methylation of the DMR of each imprinted gene.

Another mechanism involving the chromosome architecture is likely to be a fundamental importance for nuclear structure and function. In the nuclei of human blood cells, small and gene-dense chromosomes 17, 19 and 20 were shown to be preferentially located at a nuclear-interior position. In contrast, small and gene-poor 18 and Y chromosomes were found to be peripherally attached to the nuclear envelope. Furthermore, it has been observed that both smaller and larger chromosome territories are localized at the periphery and at the center of the nucleus (Boyle et al. 2001; Cremer et al. 2001). Growing oocytes at prophase of meiosis I are known to express genes actively (Bachvarova 1985). From these findings, it appears that the nuclear localization of chromatin, affecting the binding of methylase to the DMRs of DNA leads to the oocyte size-specific methylation for each imprinted gene.

Recently, histone modifications have also been suggested as one of the possible factors in the regulation of the expression of imprinted genes. The expression of imprinted genes in the placenta, located on the distal region of chromosome 7 was occurred with both dimethylation at Lys9 of histone H3 and trimethylation at Lys27 of histone H3, but not with methylation of the DMR (Lewis et al. 2004; Umlauf et al. 2004). Further, the expression of the CDKN1C, a putative tumor suppressor was accompanied by loss of both CpG methylation and histone H3K9 at DMR of the LIT1 (Soejima et al. 2004). Lucifero et al. (2004) previously demonstrated the preferential methylation of the maternal DMR of the Snrpn during oocyte growth in juvenile mice. This preferential maternal methylation was also seen in the Zac1 and Peg1/Mest in this current result. Histone modification may provide a clue to the preferential methylation of the maternal DMR of imprinted genes during oocyte growth. CpG methylation of the Prader–Willi syndrome of imprinting-center was lost in the ES cells harboring a homozygous mutation of the Dnmt1, but normal levels of H3 Lys-9 methylation were maintained. The imprinted expression of Snrpn was also maintained in the ES cells (Xin et al. 2003). It has also been reported that in the nonexpressed maternal allele, H3K4, K14, and K18 in the DMR of the Snrpn were all methylated, but not acetylated (Gregory et al. 2001). Thus, histone modifications appear to function as imprinting mechanisms, and it suggests that they are also involved in preferential DMR methylation in the maternal allele of the Snrpn, Zac1 and Peg1/Mest. In any case, the significance of the preferential methylation of the maternal allele remains unknown at present.

In conclusion, the present study with use of a mouse model confirmed that maternal imprinting progresses in accordance with oocyte growth, not by aging or maturation. The oocytes completed maternal imprinted and acquired competence to support development before reaching to the full size. However, further studies will be needed to give a satisfactory explanation of the molecular mechanism underlying the oocyte size-dependent methylation of each imprinted gene.

	Experimental procedures

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Collection of oocytes and sperm

Oocytes were all derived from (C57BL/6 x DBA) F1 mice. The growing oocytes were collected from ovarian follicles of 10-, 15- and 20-day-old, and adult mice. These ovaries were dissected into small pieces using a 27G needle in M2 medium, containing 240 µM dbcAMP (Sigma) and treated with 1 mg/mL collagenase (Sigma) for 10 min, followed by 0.05% trypsin-0.53 mM EDTA (Gibco) for 10 min. After washing several times, the isolated growing oocytes were collected and then treated with 0.5% proteinase (Sigma) to remove zona pellucida. The denuded oocytes were classified into seven groups, based on the size of oocyte.

DNA isolation

DNA was isolated by the Proteinase K/SDS method, as described by Zuccotti & Monk (1995). In this case, 50–1000 oocytes were resuspended in 18 µL of lysis solution containing 2 µg E. coli tRNA, 1 mM SDS, and 280 µg/mL Proteinase K. The samples were incubated for 30–90 min at 37 °C, and were subsequently incubated for 15 min at 98 °C.

Bisulfite sequencing

The isolated DNA was treated with sodium bisulfite using a CpGenome modification kit (CHEMICON). The bisulfite-converted DNA was amplified by nested (seminested) PCR, using an Advantage cDNA PCR Kit (BD Biosciences) for Igf2r (Stoger et al. 1993), Lit1 (Yatsuki et al. 2002), Zac1 (Smith et al. 2002), Snrpn (Shemer et al. 1997), Peg1/Mest (Lefebvre et al. 1997), Impact (Okamura et al. 2000), Meg1/Grb10 (Arnaud et al. 2003) and H19 (Tremblay et al. 1995) (Fig. 1). The primers and PCR protocols for the amplification of all target genes are given in Tables 1 and 2. To purify the PCR products, the DNA fragments were separated by electrophoresis using 2% agarose gel. Then the bands were excised and purified with the Wizard SV gel and PCR Clean-Up System (Promega). Purified DNA was cloned into a pGEM T-Easy Vector (Promega). Plasmid DNA was isolated using a Flexi Prep Kit (Amersham Biosciences) and sequenced with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The bisulfite efficiency exceeded 98%.

	Acknowledgements

 
This work was supported by a grant from the Bio-oriented Technology Research Advancement Institution (BRAIN), Japan.

	Footnotes

 
Communicated by: Denise P. Barlow

^* Correspondence: E-mail: tomohiro{at}nodai.ac.jp

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Received: 6 October 2005
Accepted: 20 December 2005

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