Mol. Hum. Reprod. Advance Access originally published online on April 11, 2006
Molecular Human Reproduction 2006 12(6):407-411; doi:10.1093/molehr/gal040
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Genetic imprinting during impaired spermatogenesis
1Department of Urology and Pediatric Urology, 2Institute of Veterinary Anatomy, Histology and Embryology and 3Institute of Pathology, University of Giessen, Giessen Germany
4 To whom correspondence should be addressed at: Klinik und Poliklinik für Urologie und Kinderurologie, Rudolf-Buchheim-Strasse 7, 35385 Giessen, Germany. E-mail: klaus.steger{at}chiru.med.uni-giessen.de
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Abstract |
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Disorders in genetic imprinting are discussed as potential genetic risk in assisted reproduction technology (ART), where most of the natural selection mechanisms are bypassed. As currently only limited information about genomic imprinting in disruptive spermatogenesis is available, we analysed the imprinting state of the paternally methylated gene H19 in various germ cell populations derived from seminiferous tubules exhibiting impaired spermatogenesis. Different germ cell types were isolated by laser microdissection from human testicular paraffin sections. Although the methylation state of the maternally imprinted gene SNRPN was investigated by methylation-specific PCR (M-PCR) to establish the isolation method, methylation of H19 was analysed by a single-strand conformation-based method. Contamination by somatic Sertoli cells was excluded because of Sertoli cell-specific vimentin immunohistochemistry before germ cell laser microdissection. We demonstrate correct genetic imprints for H19 even in spermatogonia selected from seminiferous tubules exhibiting spermatogenic arrest at the level of spermatogonia, providing no evidence for incorrect genomic imprinting in spermatozoa from infertile men used for ICSI.
Key words: genetic imprinting/H19/impaired spermatogenesis/methylation-specific PCR/single-strand conformation polymorphism
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Introduction |
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Although most autosomal genes exhibit an equivalent expression between their maternal and paternal alleles, imprinted genes are known to express only one of them in a parent-of-origin-dependent manner (Reik and Walter, 2001a
,b). This is due to allele-specific differential DNA methylation of CpG dinucleotides within differentially methylated regions (DMRs) (Barlow, 1995). One of the best characterized DMR is localized on chromosome 11p15 and includes the reciprocal expressed genes IGF2 and H19 (Zhang and Tycko, 1992; Giannoukakis et al., 1993). H19 encodes for an untranslated mRNA that is transcribed exclusively from the unmethylated maternal allele but not from the methylated paternal allele (Figure 1).
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DNA methylation is a heritable epigenetic modification that is maintained during proliferation and differentiation processes. In the course of gametogenesis, these imprints are erased and re-established in a sex-specific manner because of the activity of DNA methyltransferases (DNMTs) (reviewed in Kierszenbaum, 2002). DNA methylation, in addition, may represent the most important epigenetic information that is transmitted from sperm to oocyte, as in haploid male germ cells, chromatin organization is severely changed because of histone-to-protamine exchange (reviewed in Steger, 1999). Aberrant regulation of imprinted genes is known to be responsible for various growth and behavioural syndromes. Within the gene cluster on chromosome 11p15, genetic and epigenetic alterations result in the Beckwith–Wiedemann syndrome (BWS) (Maher and Reik, 2000; Reik and Murrell, 2000).
Since the first report of a successful ICSI pregnancy (Palermo et al., 1992), the technique has become the mainstay of IVF clinics and is now the method of choice for the treatment of male factor infertility. However, it has been suggested that ICSI, by cancelling the natural selection of aberrant spermatozoa, may cause irregular embryo cell divisions because of mechanical manipulation of the gametes (Hewitson et al., 1999) and might transmit genetically related infertility (Ma et al., 2000). In addition, the use of testicular spermatozoa for ICSI raised the question whether the genetic imprinting of the gametes’ genome has already finished (Tycko et al., 1997). Indeed, several clinical studies reported an unexpected high incidence of imprinting disorders in children conceived with assisted reproduction technology (ART) (Cox et al., 2002; De Baun et al., 2003; Gicquel et al., 2003; Maher et al., 2003; Orstavik et al., 2003).
During normal spermatogenesis, the erasure of methylation marks of the maternally imprinted gene SNRPN (Manning et al., 2001) and the resetting of the paternally imprinted gene H19 (Kerjean et al., 2000; Marques et al., 2004) have been reported to be completed before germ cells enter meiosis. However, most of the testicular biopsies from infertile men exhibit mixed atrophy showing seminiferous tubules with at least qualitatively normal spermatogenesis in direct vicinity to tubules with spermatogenic arrest at the level of round spermatids, spermatocytes, and spermatogonia, and tubules with Sertoli cell-only (SCO) characteristics. As most of these biopsies contain some small areas with at least qualitatively normal spermatogenesis which allow testicular sperm extraction (TESE) to be carried out (Silber et al., 1995), in future, therapeutic testicular biopsies will play an increasing role for the treatment of male factor infertility. Therefore, in this study, we analysed the methylation state of the paternally imprinted gene H19 in different germ cell types derived from seminiferous tubules exhibiting impaired spermatogenesis.
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Materials and methods |
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Isolation of testicular cell types
Testicular samples were fixed in Bouin’s solution and embedded in paraffin. Sections of 6 µm were mounted on membrane slides (P.A.L.M. Microlaser Technologies, Bernried, Germany) which were pretreated with poly-L-lysine and UV. All following steps were performed under sterile conditions and at room temperature. After deparaffinization, slides were washed in washing buffer (0.1 M Tris, 0.1 M NaCl, pH 7.4) and pre-incubated in 5% bovine serum albumin (BSA) in washing buffer. Immunohistochemistry was performed with a mouse monoclonal antibody against vimentin (ready-to-use solution, clone V9, Dako, Hamburg, Germany) which is expressed in cells of mesodermal origin, e.g. Sertoli cells (Figure 2). After washing in washing buffer, slides were incubated with a biotinylated secondary antibody goat-anti mouse (Dako) in a 1:100 dilution in 5% BSA, rinsed and incubated with the Vectastain ABC reagent (Vector Laboratories, Burlingame, CA, USA). Colour development was performed with the VECTOR®NovaREDTM Substrate Kit (Vector Laboratories). Nuclei were counterstained with haematoxylin. After immunohistochemistry, slides were used for laser microdissection followed by laser pressure catapulting (LPC) with the MicroBeam system (P.A.L.M. Microlaser Technologies) to isolate different testicular cell types. The isolation of round spermatids is shown in Figure 2.
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We analysed spermatogonia from tubules with spermatogenic arrest at the level of spermatogonia, namely four probes from three patients each containing approximately 200 cells, and spermatocytes from tubules with spermatogenic arrest at the level of spermatocytes, namely 11 probes from six patients each containing 50–100 cells. Microdissected cells were catapulted in the cap of a 500-µl reaction tube. The cap contained 2 µl of mineral oil to achieve a better attachment of the catapulted cells. After closing the cap, the cells were spun down and ready for DNA extraction.
DNA extraction
DNA was extracted with the QIAamp Micro Kit (Qiagen, Hilden, Germany) for microdissected cells using carrier RNA, according to the manufacturer’s instructions. DNA was eluted with 20 µl of 10 mM Tris, pH 7.5.
Bisulphite treatment
Sodium bisulphite (SBS) treatment was performed to convert unmethylated cytosines to uracil and leaving methylated cytosines unchanged. Extracted DNA was denatured by the addition of 2.2 µl of freshly prepared 3 M NaOH and incubation at 37°C for 15 min and 95°C for 3 min. After denaturation, the samples were directly put on ice. After the addition of 200 µl of SBS solution (667 µl of 40 mM hydroquinone, 10 ml of 2.8 M SBS, 400 µl of 10 M NaOH), the samples were overlayed with mineral oil and incubated overnight at 55°C in the dark. DNA was purified using the QiaEXII system (Qiagen). Elution was done two times with 25 µl of 10 mM Tris, pH 7.5, for 10 min at 50°C. The purified DNA was desulphonated by the addition of 5.5 µl of 3 M NaOH and incubation at 37°C for 15 min. Fifty-five microlitres of 6 M ammonium acetate, pH 7, and 220 µl of pure EtOH were added for DNA precipitation. Precipitated DNA was washed with 100 µl of EtOH 70%. After centrifugation and drying, the DNA was eluted in 10 µl of 10 mM Tris, pH 7.5.
PCR amplification of bisulphite-treated DNA
Amplification of the SNRPN region was performed separately for each allele applying heminested highly sensitive PCR with the primers already published (Manning et al., 2001). PCR reactions were performed in a 30-µl volume containing the DNA suspended in 5 µl of 10 mM Tris, pH 7.5, PCR buffer gold (Applied Biosystems, Lincoln, NE, USA), 2 mM MgCl2, 0.33 mM of each dNTP, 0.5 µM of the corresponding primer and 0.3 µl (1.5 U) of Amplitaq Gold polymerase (Applied Biosystems). After activation of the polymerase at 95°C for 10 min, DNA was amplified in 30 cycles for 30 s at 95, 62 and 72°C followed by a final extension at 72°C for 10 min. PCR results were analysed by electrophoresis in a 2% agarose gel by staining the DNA with SYBR®Green (Sigma, Munich, Germany).
Amplification of the H19 region was done with the following primers: H19for (corresponding to nucleotides 6099–6121, GenBank accession no. AF087017 [GenBank] ) 5'-GTATAGTATATGGGTATTTTTGG-3' and H19rev (nucleotides 6326–6303) 5'-CTATAAATATCCTATTCCCAAATA-3'.
These primers allow the amplification of both alleles, the methylated and the unmethylated alleles by spanning a region with 18 differentially methylated CpGs.
DNA was amplified in a 30-µl volume containing 10 µl of the extracted and bisulphite-treated DNA, PCR buffer gold, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.66 µM of each primer and 0.5 µl (2.5 U) of Amplitaq Gold polymerase. After activation of the polymerase at 95°C for 10 min, DNA was amplified in 40 cycles for 45 s at 95, 56 and 72°C followed by a final extension at 72°C for 10 min.
Single-strand conformation analysis
The alleles of the H19 amplicons were analysed on the basis of single-strand conformation polymorphism (SSCP). The single-strand conformation analysis (SSCA) gel was poured between the clean plates of the Minigel-Twin system (Biorad, Munich, Germany) containing the following components: 9% acrylamide (29:1 arcrylamide : bisacrylamide), x1 TBE buffer (89 mM Tris-borate, 2 mM EDTA, pH 8.0), 0.07% APS and 0.0014% TEMED. The gel was polymerized for 2 h at room temperature and was kept at 4°C overnight before starting the run. Four microlitres of the PCR reaction was added to 4 µl of loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue and 0.05% xylene cyanol) and denatured at 95°C for 5 min and directly transferred on ice. After loading the gel, the run was performed for 8 h at 4°C and a constant voltage of 50 V. After the run, the bands were silver stained using the GelCode®SilverSNAP® Stain Kit II (Pierce, Rockford, IL, USA).
Construction of paternal and maternal controls for SSCA
To compare the resulting bands after the analysis of the microdissected cell types in SSCA, we needed to produce controls, which show only the methylated or unmethylated pattern (Figure 4A and B). For the methylated paternal control, we used ejaculated spermatozoa which were laser microdissected (about 100 spermatozoa per extraction) after the ejaculate of fertile men was streaked out on a membrane slide. The unmethylated pattern of H19 can be found in oocytes. Owing to restrictions in the use of oocytes for research, we generated an artificial maternal control. First, we amplified bisulphite-treated DNA derived from somatic tissue (prostate) with the H19-specific primers. The resulting amplicons of 227 bp length display a mixture of the methylated and the unmethylated fragments. Because of the differences in sequence which were introduced by bisulphite treatment, we were able to make use of a polymorphism in a TaqI restriction site. This site was only present in the originally methylated paternal amplicons. By restriction with TaqI, the paternal amplicons were cut into two smaller fragments. The remaining maternal fragment (227 bp) was gel extracted with QiaExII and used for SSCA.
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Results |
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In a pilot experiment, we isolated spermatocytes as well as round and elongated spermatids from testicular biopsies with normal spermatogenesis applying laser microdissection and LPC. For reasons of better morphological orientation and to avoid contamination with somatic Sertoli cells, we performed immunohistochemistry for the intermediate filament protein vimentin before LPC, which results in a Sertoli cell-specific signal within the seminiferous epithelium (Figure 2). Subsequent to LPC, we performed DNA extraction, SBS treatment and methylation-specific PCR (M-PCR) of the SNRPN region. M-PCR with the primers specific for the methylated maternal allele resulted in a 128-bp PCR product solely in the positive control, whereas all germ cell specimens were completely negative. By contrast, spermatocytes, round and elongated spermatids revealed a strong signal at 99 bp when primers specific for the unmethylated paternal allele were used. Summarized, all analysed germ cells exhibited the correct unmethylated imprinting state of the SNRPN region (Figure 3).
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Opposite to SNRPN, H19 is methylated on the paternal allele. To analyse the resetting process in different stages of testicular germ cell development, we amplified the H19 DMR with primer pairs that allow the amplification of both the primary methylated and secondary to SBS treatment unmethylated region. Two hundred and twenty-seven base pair fragments were obtained containing originally methylated (paternal) and originally unmethylated (maternal) amplicons. As SBS treatment before M-PCR transforms methylation differences into sequence differences, paternal amplicons contain a TaqI restriction site and can be cut into a 103-bp and a 124-bp fragment, whereas maternal amplicons cannot be restricted by TaqI. The different alleles could be detected by SSCA. Ejaculated spermatozoa isolated by LPC served as control for the paternal-specific methylation state. For maternal-specific imprinting, namely the absence of methylation at the H19 DMR, maternal amplicons were extracted from agarose gel and used as control in SSCA (Figure 4A and B).
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We analysed spermatogonia from tubules with spermatogenic arrest at the level of spermatogonia and spermatocytes from tubules with spermatogenic arrest at the level of spermatocytes. As shown in Figure 4C, both spermatogonia and spermatocytes displayed only two bands in SSCA resembling the paternal-specific methylation pattern that was, in addition, demonstrated in ejaculated spermatozoa (paternal control) but not in the unmethylated control corresponding to the maternal state. Somatic DNA from prostate revealed four bands resembling the single strands of the paternal and maternal alleles.
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Discussion |
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TESE in combination with ICSI, at present, is the method of choice for the treatment of severe male factor infertility. For several years, however, there has been intense discussion concerning the genetic risks of ART (Cox et al., 2002
; De Baun et al., 2003; Gicquel et al., 2003; Maher et al., 2003; Orstavik et al., 2003). Although ICSI by cancelling the natural selection of aberrant spermatozoa has been suggested to transmit genetically related infertility (Ma et al., 2000), the combination of ICSI with TESE, in addition, raised the question whether the genetic imprinting of the gametes’ genome has already been finished (Tycko et al., 1997). In this study, we focused on the question whether infertile patients with spermatogenic arrest at the level of spermatocytes and spermatogonia reveal delayed or even incorrect resetting of their imprinting marks. This is important in so far as therapeutic testicular biopsies play an increasing role for the treatment of male factor infertility, namely the extraction of spermatozoa subsequently used for ICSI. As our data demonstrate correct imprinting resetting of H19 even in spermatogonia isolated from seminiferous tubules with severe spermatogenic impairment, it is very unlikely that spermatozoa obtained from TESE do not exhibit the correct paternal-specific methylation of H19.
Owing to mixed atrophy in testicular biopsies, the isolation of germ cells from testicular cell suspensions, as has been reported by Manning et al. (2001), for our question, represents an inappropriate technique, because isolated germ cells could no longer be assigned to specific seminiferous tubules exhibiting different forms of spermatogenic impairment. Kerjean et al. (2000) performed microdissection on cryosections followed by methylation analysis of testicular germ cells. According to our experience, however, the correct identification of specific germ cell types in cryosections is very difficult because of the poor morphology. In this study, we therefore decided to use paraffin material in combination with laser microdissection and LPC opening new possibilities in the histological identification and isolation of germ cells. The immunohistochemical staining of the disruptive somatic Sertoli cells with vimentin provides additional help.
To analyse major methylation differences within bisulphite-treated DNA, in this study, we used SSCA and demonstrated that spermatogonia and spermatocytes from seminiferous tubules with impaired spermatogenesis exhibit the same methylation pattern for H19 as ejaculated spermatozoa from normozoospermic men. In addition, we were able to differentiate between the maternally unmethylated and the paternally methylated alleles of this gene. These results clearly indicate that the main resetting of H19 is already finished in spermatogonia, even in disruptive spermatogenesis. However, we could not completely exclude the existence of sporadic hypomethylated CpG sites that have been described in ejaculated spermatozoa of oligozoospermic men (Marques et al., 2004).
Kerjean et al. (2000) reported that a small portion of amplicons derived from spermatogonia of normal testes displayed the unmethylated allele of H19. It may be possible that these amplicons result from a small fraction of spermatogonial stem cells, suggesting that the resetting of the analysed region takes place at an early spermatogonial stage. The fact that this unmethylated allele of H19 was not found in spermatogonia of impaired spermatogenesis might be explained by the different isolation and cell type-specific analysation of the cells. We isolated several cells of one cell type for a single DNA extraction and amplification. If only one cell of this isolation shows the unmethylated allele, this fact might be overlapped by the methylated alleles of the other cells. In a very small proportion of spermatogonia- and spermatocyte-derived amplicons, Kerjean et al. (2000) detected a nearly fully methylated allele that shows a single unmethylated CpG. To our understanding, this CpG corresponds to nucleotide 7966 (GenBank accession no. AF125183
[GenBank]
) that is known to represent a C/T polymorphic site (Frevel et al., 1999) within the CTCF binding site. A tyrosine in position 7966 might be confused with an unmethylated CpG. This polymorphic site was also confirmed by Marques et al. (2004) who analysed the methylation pattern of H19 in ejaculated spermatozoa from normozoospermic and oligozoospermic patients.
Although several reports exist on children with imprinting defects that have been conceived by ART, it remains questionable whether there is a real risk of paternal transmission of imprinting defects. The cases of Angelman syndrome after ICSI described by Cox et al. (2002) and Orstavik et al. (2003) are due to missing methylation on the maternal SNRPN imprinting control region. In the analysed cases of epigenetically caused Beckwith–Wiedemann syndrome (BWS) that occurred after ART, the methylation of the maternally imprinted KCNQ1 imprinting centre region (ICR) is defective (De Baun et al., 2003; Gicquel et al., 2003; Maher et al., 2003). This suggests that the main risk of epigenetic diseases after ART might be oocyte related. This is consistent with the finding that the only identified common factor of 12 BWS children conceived by different reproductive techniques is ovarian stimulation (Chang et al., 2005).
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Acknowledgements |
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Funding of this research program was provided by German Research Foundation (DFG) Research Training Group 533: Cell–Cell Interaction in Reproduction. Funding to pay the Open Access publication charges for this article was provided by the efficiency-oriented funds (LOM) of the Department of Urology and Pediatric Urology, Justus-Liebig-University of Giessen.
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Notes |
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Sonja Hartmann is a member of the DFG Research Training Group 533 Cell-Cell Interaction in Reproduction.
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Submitted on February 16, 2006; accepted on March 17, 2006.
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