Human Molecular Genetics, 2000, Vol. 9, No. 14 2183-2187
© 2000 Oxford University Press
Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis
1Laboratoire de Biologie de la Reproduction, 2Laboratoire d’Histologie Embryologie Cytogénétique and 3Laboratoire de Biochimie et Génétique Moléculaire, Université Paris V, Hôpital Cochin-Port-Royal, Pavillon Cassini, 123 Boulevard de Port-Royal, 75014 Paris, France and 4INSERM U257, Institut Cochin de Génétique Moléculaire (ICGM), Centre Hospitalier Universitaire-Cochin, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France
Received 25 May 2000; Revised and Accepted 12 July 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Parental-specific epigenetic modifications are imprinted on a subset of genes in the mammalian genome during germ cell maturation. However, the precise timing of their establishment remains to be determined. Methylation of CpG dinucleotides has been shown to be a part of the parental imprint. We have examined how the methylation pattern characteristic of the paternal allele in germ cells are established during human spermatogenesis. Two representative imprinted genes, H19 and MEST/PEG1, were studied. The experiments were performed using the bisulphite sequencing method on microdissected individual cells at different stages of male germ cell differentiation. We show that both genes are unmethylated in fetal spermatogonia, suggesting that all pre-existing methylation imprints are already erased by this stage. The MEST/PEG1 gene remains unmethylated at all subsequent post-pubertal stages of spermatogenesis, including mature spermatozoa. The methylation of H19 typical of the paternal allele first appears in a subset of adult spermatogonia and then is maintained in spermatocytes, spermatids and mature spermatozoa. Our results suggest that the methylation imprint inherited from the parents is first erased in the male germ line at an early fetal stage. The paternal-specific imprint is re-established only later, during spermatogonial differentiation in the adult testis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Normal mammalian development requires the presence of both a maternal and a paternal genome. This requirement is likely to be due to the existence of imprinted genes in the genome that are expressed from only one parental allele. Differential methylation of cytosine residues in CpG dinucleotides in critical regions of imprinted genes has been shown to be a part of the epigenetic imprint that differentiates the paternal and maternal alleles (1). Since methylation of DNA has been associated with the modulation of transcription, this mechanism could account for the silencing of one allele (2). Differentially methylated regions (DMRs) on the two parental alleles have been identified in the vicinity of many imprinted genes (3). Deletion of these DMRs can revert imprinting (4,5). Methylation is a heritable epigenetic modification which is erased and reset during male and female gametogenesis when differential marking of imprinted genes is hypothesized to occur. In addition, methylation is the only known epigenetic signal which can be retained by the paternal genome throughout male cell maturation, whereas chromatin organization is changed when histones are replaced by protamines in spermatids and mature spermatozoa (6).
The methylation pattern of several imprinted genes has been determined. The maternally expressed H19 gene is one of the best characterized examples. The transcriptionally active maternal allele is unmethylated whereas the inactive paternal allele is methylated (7,8). The DMR in the mouse H19 locus extends over 2 kb at 
4 kb upstream of the promoter of the gene (9–11). A similar pattern of methylation is observed for the human H19 gene which also exhibits maternal monoallelic expression (12). The oppositely imprinted mouse Mest/Peg1 and human MEST/PEG1 genes exhibit the same features, i.e. DMR unmethylated on the active paternal allele spanning the promoter, the first exon and part of the first intron (13–17).
The exact timing of the erasure of the old and establishment of the new methylation pattern remains unknown. In the present study, we have investigated this process in the human male germ line. We focussed our analysis on a subset of CpGs located at the 5' end of DMRs of the oppositely imprinted H19 and MEST/PEG1 genes. We made use of the bisulphite genomic sequencing technique on isolated microdissected cells to examine the methylation of these two regions at different stages of spermatogenesis (18,19). We report that both DMRs are already entirely unmethylated in fetal spermatogonia. Although the MEST/PEG1 remains unmethylated at all subsequent stages of adult germ cells, methylation of the H19 DMR can be detected starting from the adult spermatogonial stage. These findings suggest that the previous methylation imprint of these two representative genes is erased at fetal stages of spermatogenesis and that the specific paternal methylation pattern is established before meiosis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In a pilot experiment we have analysed the methylation of DMRs of the two oppositely imprinted genes, MEST/PEG1 and H19 in human adult somatic cells. DNA extracted from pools of lymphocytes was treated with bisulphite and the regions of interest were amplified by semi-nested PCR. The obtained fragments were subcloned. Typically 10 individual clones were sequenced for each amplification. As shown in Figure 1 the analysed fragments contained respectively 22 CpGs for MEST/PEG1 (Fig. 1A) and 19 CpGs for H19 (Fig. 1B) within the DMRs thought to be critical for the regulation of each of these two genes (10–17). Both methylated and unmethylated populations of clones were found. These populations are likely to represent the differentially methylated parental alleles.
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In order to be able to analyse the methylation status of each gene in a single cell, we have used the down-scaled modification of the bisulfite sequencing technique (19). Individual spermatozoa were included in agarose beads and all subsequent reactions were performed in the same tube. The relative frequency of successful amplification after bisulfite treatment was approximately the same for MEST/PEG1 and H19: 4 of 23 and 3 of 23, respectively. As expected, every sequenced clone obtained from mature spermatozoa was unmethylated at the MEST/PEG1 locus and methylated at the H19 locus.
We then examined the methylation status of each region in round and elongated spermatids identified on the basis of their morphology (Fig. 2A). The cells were micromanipulated with a glass needle, grouped into pools of three, included in agarose beads and subjected to bisulphite analysis. The methylation pattern of both MEST/PEG1 and H19 was found to be identical to that found in mature spermatozoa, indicating that the methylation imprint of these two genes is already established at this stage of germ cell differentiation. Therefore, we have extended our analysis to earlier stages.
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Fetal spermatogonia, adult spermatogonia and spermatocytes I were microdissected with a glass needle from cryostat sections of a 24-week-old fetal testis specimen and adult testicular biopsies (Fig. 2B). The different cell types were identified on the basis of their morphology and location within the tubules. As a control, fetal Sertoli cells and fetal testicular fibroblasts were also microdissected. In these two somatic cell types, both the methylated and the unmethylated allele populations were found for each gene confirming that there was no bias for amplification of one of the alleles (20). Using the same conditions, we found that fetal spermatogonia carried only unmethylated alleles of H19 and MEST/PEG1 genes. This result shows that the initial methylated imprint inherited from the parents has already been erased by this early stage. As expected, the MEST/PEG1 gene conserved the unmethylated status in adult spermatogonia and spermatocytes I. In contrast, unmethylated and methylated alleles of the H19 gene were detected in different cells at the adult spermatogonial stage. Therefore, the methylation of this critical region of the gene, typical of the paternal allele, is acquired in adult spermatogonia. Because it was not possible to identify subclasses of spermatogonia on the basis of morphological criteria, the timing of methylation imprint could not be determined with more precision. However, it is clear that de novo methylation of H19 is complete before cells enter meiosis, since only methylated alleles were detected in the spermatocytes I.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The results presented here show that the original methylation imprint on the MEST/PEG1 and H19 DMRs is erased before the fetal spermatogonial stage in the human male germ line. The MEST/PEG1 gene remains unmethylated at all subsequent stages including mature spermatozoa. In contrast, methylation at the H19 locus is acquired at the adult spermatogonial stage. These cells represent the last stage of differentiation before the meiotic division. Therefore, the establishment of the methylation imprint in the male germ line can be divided into two distinct phases: (i) erasure of pre-existing parental methylation marks; and (ii) establishment of the paternal methylation imprint in a broad sense of the term, which is independent of the allele actually methylated. Other authors also observed the loss of allele-specific methylation imprints in both the male and female primordial germ cells (PGCs) of the mouse (21,22). Expression of imprinted genes in the germ line, such as H19, become biallelic at day 11.5 of mouse embryonic development (E11.5) (23,24), also suggesting that pre-existing imprints are erased by this stage.
However, asynchronous replication—a general characteristic of imprinted genes—is conserved in primordial germ cells and can still be seen in primitive type-A spermatogonial stem cells isolated from 6-day-old mice (25). Interestingly, we found that the DMRs of the H19 and the MEST/PEG1 genes are unmethylated in fetal spermatogonia in humans, but also during the equivalent stages in mice (A. Kerjean and A. Pàldi, unpublished data) suggesting that asynchronous replication occurs even in the absence of parent-specific methylation. This dissociation between the parent-specific replication timing and methylation suggests that cells are still able to discriminate parental alleles even in the absence of methylation marks. The asynchrony between the two alleles disappears before spermatogonia enter meiosis (25), at the same stage when the DMR of H19 becomes fully methylated, suggesting that the paternal epigenotype is acquired at this stage.
Other studies also support the view that the spermatogonial stage is crucial in the establishment of the new parental imprint. The de novo methylation observed in this study during human spermatogenesis is consistent with the transient localization of the mouse DNMT1 protein in the nuclei of spermatogonia and leptotene/zygotene spermatocyte stages (26,27). In the case of the RSV-Ig-myc transgene, Chaillet et al. (28) found that the paternally transmitted transgene was undermethylated at the primitive spermatogonial stage and became methylated later during spermatogenesis. Finally, Shamanski et al. (29) have shown that the paternal methylation imprint is largely or entirely established by the spermatid stage.
Davis et al. (30) showed that the sequences proximal to the promoter of the mouse H19 gene are differentially methylated in diploid, mitotic spermatogonia. The reason for this discrepancy is not completely clear but may be due to the purity of the germ cell population studied. Davis et al. (30) used fractionation cell procedure producing only 80–90% pure cell populations. In contrast, our analysis was done on individually identified cells microdissected under a microscope.
In conclusion, establishment of the male-specific methylation imprint of the H19 DMR occurs before proliferating spermatogonia enter the meiotic division. The change in DNA methylation could reflect cellular differences at this stage of spermatogenesis and could be associated with genome reorganization during the transition from proliferation (self-renewing pool of stem cells) to differentiation (meiotic prophase cells). It is possible therefore, that the acquisition of the typical paternal methylation profile at the H19 and MEST/PEG1 genes is a part of a genome-wide reorganization of the sperm genome and may represent a critical step of spermatogenesis itself.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Isolation of spermatogenic cells
Ejaculates from three normal men were used in this study as a source of spermatozoa and spermatids. Spermatozoa were selected by centrifugation on a two-layer PureSperm gradient (90 and 45%) by standard procedure (JCD, Paris, France) (31). Enrichment for spermatids from ejaculates was achieved by four repetitive ‘swim-up’ steps (0.1 ml of the non-migrating semen cell suspension stratified on the bottom of 0.5 ml of human tubal fluid-serum 7.5% for 30 min) (HTF medium; Irvine Scientific, Santa Ana, CA). The cell fraction containing the majority of immature germ cells was then centrifuged on a two-layer PureSperm. The different types of spermatids were identified by simple microscopic observation (Fig. 2A).
Fetal spermatogonia, spermatogonia and spermatocytes I were isolated from frozen testicular biopsies (two adult testes, one 24 week fetal testis) with normal apparent morphology of spermatogenesis. Cryostat sections were prepared and stained with haematoxylin and eosin (12.5 µm sections) (Fig. 2B).
Spermatozoa and spermatids were micromanipulated using an intracytoplasmic sperm injection micropipette. Microdissection was performed on cryostat sections using glass microneedles (70 µm tip diameter) controlled by a Narishige micromanipulator (NT-88; Narishige, Tokyo, Japan). To eliminate the risk of amplification of contaminating exogenous DNA, tools and tubes were treated with ultraviolet light (254 nm for 30 min). Individual cells were picked up into a sperm injection pipette or glass microneedle under microscopic observation and deposited into a 10 µl drop of molten 2% low-melting point agarose. The agarose/cell drop was immediately solidified on ice and the agarose beads were collected into a 0.2 ml PCR tube. Control tubes containing all reagents but no cells were systematically included.
Bisulphite treatment of agarose beads
Bisulphite treatment of peripheral blood lymphocytes was performed using standard procedures (32). The bisulphite protocol on agarose beads was performed using a slight modification of a published procedure (19). Briefly, the drop containing a defined number of cells (1–3 cells) was laid over 200 µl of lysis solution (0.5 M EDTA pH 8, 1% L-sarcosine; 2 mg/ml proteinase K). After overnight incubation at 50°C, the proteinase K was inactivated in 40 µg/ml PMSF. Denaturation of the DNA strands was achieved by adding 60 µl of freshly prepared sodium hydroxide (0.3 M NaOH) for 30 min. Aliquots (170 µl) of bisulphite solution (4 M sodium bisulphite, 125 mM hydroquinone, pH 5.0) were added to each reaction tube. The reaction mixtures were overlaid with mineral oil and incubated in the dark for at least 4 h at 50°C. Desulphonation was achieved in 0.2 M NaOH for 15 min. The reactions were neutralized with 0.2 vol of 1 M hydrochloric acid. Finally, beads were washed in 1x TE. PCR reactions were performed directly on the beads.
PCR amplifications, cloning and sequencing of bisulphite-treated DNA
PCR was performed in 10 mM GeneAmp PCR Buffer II, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.5 µM of each primer and 1.25 U of AmpliTaq Gold DNA polymerase (Perkin Elmer, Norwalk, CT). Two rounds of amplification were done using the following conditions: 94°C for 10 min followed by 40 cycles of 94°C for 45 s, 61°C for 45 s, 72°C for 1 min and a 10 min extension step at 72°C. To generate the product of MEST/PEG1, amplification with primer pair F609–R898 was followed by semi-nested amplification with F609–R827 corresponding to 22 CpGs (Fig. 1A). The sequences of the primers, with the nucleotide position of the first base indicated in parentheses (GenBank accession no. Y10620, nucleotides 609–898) are as follows: F609, 5'-tygttgttggttagttttgtayggtt-3', where Y is either C or T; R898, 5'-aaaaataacaccccctcctcaaat-3'; R827, 5'-cccaaaaacaaccccaactc-3'. To generate the product of H19, amplification with primer pair F6005–R6326 was followed by semi-nested amplification with F6115–R6326 corresponding to 19 CpGs (Fig. 1B). The sequences of the primers (GenBank accession no. AF087017, nucleotides 6005–6326) are as follows: F6005, 5'-aggtgttttagttttatggatgatgg-3'; R6326, 5'-tcctataaatatcctattcccaaataacc-3'; F6115, 5'-tgtatagtatatgggtatttttggaggttt-3'. The amplified DNA was cloned into the CloneAmp pAMP1 system (Life Technologies, Gaithersburg, MD) and sequenced using an ABI Prism 377 DNA Sequencer (Perkin Elmer).
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr A. Viellefond and A.M. Hagnéré for providing testicular biopsies; Drs P. Billuard, T. Bienvenue, A. Carrié, J. Chelly, M. Delpech, M. Fellous, C. Poirot and R. Zemni for useful suggestions. We thank F. Poirier for helpful comments on the manuscript.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +33 1 44 41 24 56; Fax: +33 1 44 41 24 62; Email: paldi@cochin.inserm.fr
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C. Allegrucci, A. Thurston, E. Lucas, and L. Young Epigenetics and the germline Reproduction, February 1, 2005; 129(2): 137 - 149. [Abstract] [Full Text] [PDF]
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 F. Weber, M. A. Aldred, C. D. Morrison, C. Plass, A. Frilling, C. E. Broelsch, K. A. Waite, and C. Eng Silencing of the Maternally Imprinted Tumor Suppressor ARHI Contributes to Follicular Thyroid Carcinogenesis J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1149 - 1155. [Abstract] [Full Text] [PDF]
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D. Lucifero, J.R. Chaillet, and J. M. Trasler Potential significance of genomic imprinting defects for reproduction and assisted reproductive technology Hum. Reprod. Update, January 1, 2004; 10(1): 3 - 18. [Abstract] [Full Text] [PDF]
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 A. B. Bowman, J. M. Levorse, R. S. Ingram, and S. M. Tilghman Functional Characterization of a Testis-Specific DNA Binding Activity at the H19/Igf2 Imprinting Control Region Mol. Cell. Biol., November 15, 2003; 23(22): 8345 - 8351. [Abstract] [Full Text] [PDF]
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E. Geuns, M. De Rycke, A. Van Steirteghem, and I. Liebaers Methylation imprints of the imprint control region of the SNRPN-gene in human gametes and preimplantation embryos Hum. Mol. Genet., November 15, 2003; 12(22): 2873 - 2879. [Abstract] [Full Text] [PDF]
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O. El-Maarri, M. Seoud, P. Coullin, U. Herbiniaux, J. Oldenburg, G. Rouleau, and R. Slim Maternal alleles acquiring paternal methylation patterns in biparental complete hydatidiform moles Hum. Mol. Genet., June 15, 2003; 12(12): 1405 - 1413. [Abstract] [Full Text] [PDF]
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M. De Rycke, I. Liebaers, and A. Van Steirteghem Epigenetic risks related to assisted reproductive technologies: Risk analysis and epigenetic inheritance Hum. Reprod., October 1, 2002; 17(10): 2487 - 2494. [Abstract] [Full Text] [PDF]
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T. Li, T. H. Vu, K.-O. Lee, Y. Yang, C. V. Nguyen, H. Q. Bui, Z.-L. Zeng, B. T. Nguyen, J.-F. Hu, S. K. Murphy, et al. An Imprinted PEG1/MEST Antisense Expressed Predominantly in Human Testis and in Mature Spermatozoa J. Biol. Chem., April 12, 2002; 277(16): 13518 - 13527. [Abstract] [Full Text] [PDF]
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B. K. Jones, J. Levorse, and S. M. Tilghman A human H19 transgene exhibits impaired paternal-specific imprint acquisition and maintenance in mice Hum. Mol. Genet., February 1, 2002; 11(4): 411 - 418. [Abstract] [Full Text] [PDF]
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 A. Kerjean, A. Vieillefond, N. Thiounn, M. Sibony, M. Jeanpierre, and P. Jouannet Bisulfite genomic sequencing of microdissected cells Nucleic Acids Res., November 1, 2001; 29(21): e106 - e106. [Abstract] [Full Text] [PDF]
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 D. T. Schneider, A. E. Schuster, M. K. Fritsch, J. Hu, T. Olson, S. Lauer, U. Gobel, and E. J. Perlman Multipoint Imprinting Analysis Indicates a Common Precursor Cell for Gonadal and Nongonadal Pediatric Germ Cell Tumors Cancer Res., October 1, 2001; 61(19): 7268 - 7276. [Abstract] [Full Text] [PDF]
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