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Review |
* Institute of Pathology, University of Bonn,
Bonn, Germany; and the Department of Urology
and Pediatric Urology, University of Giessen, Giessen, Germany.
| Correspondence to: Dr Klaus Steger, Klinik und Poliklinik für Urologie und Kinderurologie, Universitätsklinikum Giessen und Marburg GmbH, Standort Giessen, Justus-Liebig-Universität Giessen, Rudolf-Buchheim-Strasse 7, 35383 Giessen, Germany (e-mail: klaus.steger{at}chiru.med.uni-giessen.de). |
| Received for publication October 24, 2006; accepted for publication January 29, 2007. |
The total of the genetic information that is maintained in the nucleotide sequence of DNA in an organism is known as the genome. Although each cell of an organism contains exactly the same genome, only a specific subset of genes will be active at a certain time point in a particular cell. Whether a specific gene is active or silent is regulated by the epigenome, representing the total of the chromatin (DNA and histones) modifications that index the genetic information. The study of processes that are involved in establishing these chromatin modifications is called epigenetics. Epigenetic processes include 1) methylation of DNA at deoxycytosine residues within CpG dinucleotides located within gene promoters (CpG islands); 2) modification of core histones, such as acetylation, methylation, phosphorylation, and ubiquitinylation at specific amino acid residues within the amino terminus; and 3) RNA-associated silencing of gene expression by noncoding RNAs. All of these processes participate in the regulation of gene activity without affecting the genetic code. Although these processes are heritable, they are potentially reversible and therefore open up opportunities for epigenetic therapy (Figure 1).
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Epigenetic Processes
DNA Methylation—
Methylation of deoxycytosine residues in CpG dinucleotides requires the
activity of DNA methyltransferases (DNMTs), namely the maintenance DNMT1
(Bestor et al, 1988) or de novo DNMTs 3a and 3b
(Okano et al, 1998), which
catalyze the transfer of a methyl group from the S-adenosyl-methionine to the
5' position of cytosine in DNA. DNA methylation, in general, correlates
with gene activity in that hypomethylation is associated with transcriptional
activation, whereas hypermethylation is associated with transcriptional
repression. While hypermethylation has been suggested to be responsible not
for initiating but for maintaining gene silencing similar to the role of CpG
island hypermethylation on the inactive X chromosome
(Clark and Melki, 2002), its
repressive effect is believed to result from selective recognition of 5-methyl
CpG dinucleotides by 5-methyl-CpG–binding domain proteins that are known
to be involved in reading of DNA methylation marks
(Wade, 2001).
Specific information on DNA methylation in the testis. Recently analysis of 2600 gene loci revealed that the methylation status of testicular DNA is highly distinct from somatic tissues, exhibiting 8-fold the number of hypomethylated regions located within nonrepetitive sequences (Oakes et al, 2007). Most studies on DNA methylation in male germ cells, however, focus on ejaculated spermatozoa (see "Epigenetic Risks in ART"), tumor cells (see "The Formation of Testicular Cancer"), and expression of DNMTs that are responsible for DNA methylation. Detailed expression patterns are available for DNMTs 1, 3a, 3b, and 3-like (3L) (La Salle et al, 2004; Galetzka et al, 2007). In mice, expression of DNMT1 is down-regulated between days 14.5 and 15.5 of gestation and up-regulated around birth, suggesting a role in the maintenance of methylation patterns in proliferating spermatogonia. By contrast, expression of DNMTs 3a and 3b peaks around week 21 of gestation, suggesting a role in de novo methylation in mitotically arrested spermatogonia. This is consistent with the observation that Dnmt1–/– mice exhibit abnormal expression of imprinted genes (Li et al, 1992), whereas inactivation of DNMTs 3a and 3b blocks de novo methylation in early embryos (Okano et al, 1999). In addition, it has been suggested that DNMT3a generates a mark that is important for entrance of male germ cells into meiosis (Yaman and Grandjean, 2006). In the adult testis, a 5.2-kb Dnmt1 mRNA and its corresponding protein were demonstrated in spermatogonia, early spermatocytes, and round spermatids, whereas a 6.2-kb Dnmt1 mRNA was present in pachytene spermatocytes (Benoit and Trasler, 1994; Numata et al, 1994). Subsequently it was demonstrated that the Dnmt1 gene is regulated through alternative splicing, resulting in the production of 2 protein products, a somatic isoform that is also present in spermatogonia, early spermatocytes, and round spermatids and an oocyte-specific isoform (Mertineit et al, 1998). The pachytene spermatocyte–specific transcript is expressed at high levels in the testis but is not translated. Recently this expression pattern was confirmed in man. Interestingly, in infertile men revealing round spermatid maturation arrest, only pachytene spermatocytes exhibited positive signals for the DNMT1 protein, while the formerly positive round spermatids remained immunonegative (Omisanjo et al, 2007).
Loss of DNMT3L, known to be present in precursors of spermatogonial stem cells, results in meiotic failure in spermatocytes, although these cells do not express DNMT3L (Bourc'his and Bestor, 2004). DNMT3L therefore represents an absolute requirement for male fertility; as in Dnmt3L–/– mice, pachytene spermatocytes exhibit homologous chromosomes that fail to align and form synaptonemal complexes, resulting in apoptosis of these cells (Webster et al, 2005). DNMT3L is thought to represent a methyltransferase, as it lacks enzymatic activity but is required for de novo methylation of imprinted genes in female (Bourc'his et al, 2001) and male germ cells (Bourc'his and Bestor, 2004). It has been demonstrated that expression of DNMT3L is regulated by 3 sex-specific promoters (Shovlin et al, 2007). Prospermatogonia encode a full-length protein that is involved in de novo methylation. Late pachytene spermatocytes activate a promoter located in intron 9 of the Dnmt3L gene. In haploid male germ cells, 3 truncated and noncoding transcripts can be observed. In contrast, in the ovary, an oocyte-specific promoter produces only 1 full-length transcript that is absent in Dnmt3L–/– female mice.
Practical approaches for analysis of DNA methylation. Practical approaches for analysis of DNA methylation can be divided roughly into 2 groups: those analyzing global methylation within the genome and those analyzing specific methylation within the promoter region of specific genes, namely the CpG islands, short regions of DNA (approximately 500 bp) with high CG content (>50%) that are present in 1% to 2% of gene promoters within the genome (Gardiner-Garden and Frommer, 1987). For global methylation analysis, the overall level of methyl cytosines within the genome is determined by applying either chromatographic methods or methyl-accepting capacity assays. For gene-specific methylation analysis, various methods are available. While former studies used methylation-sensitive restriction enzymes for DNA digestion followed by Southern detection or polymerase chain reaction (PCR) (Rein et al, 1998), at present the most common approach for analysis of DNA methylation is bisulfite genomic sequencing in combination with methylation-specific PCR. In brief, genomic DNA is subjected to sodium bisulfite treatment, which converts unmethylated cytosine residues into uracil residues but leaves methylated cytosines unaltered. Subsequent PCR amplification with specific primer pairs and sequencing provides the methylation status of every CpG dinucleotide in the amplified region (Figure 2). Semiquantitative data can be obtained with methylation-sensitive single-strand conformation analysis (SSCA), in which sodium bisulfite treatment of DNA is followed by PCR using primer pairs without a CpG site to avoid selective amplification of either methylated or unmethylated DNA and SSCA. Recently CpG island microarrays became available to identify unknown methylation hot spots or methylated CpG islands within the genome. In addition, there are numerous approaches to study the activity of DNMTs (Woo et al, 2005). For further information, refer to Fraga and Esteller (2002), Tollefsbol (2004), and Mager and Bartolomei (2005). Detailed protocols are available at http://www.epigenome-noe.net/ and http://www.protocol-online.org/.
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Compared with acetylation, signaling by methylation is more complex, as lysines can be mono-, di-, or trimethylated, arginines can be mono- or dimethylated, and both site specificity and the number of methyl groups modulate the epigenetic signal. Methylation of lysine residues is mediated by Su(var)3–9, enhancer-of-zeste, trithorax protein (SET) domains that can recruit other proteins through their chromodomains. Chromodomains themselves are linked to transcriptional repression through the formation of heterochromatin or heterochromatin-like complexes and might ultimately signal epigenetic gene expression (Bottomley, 2004). Histone demethylation is performed by either Jumonji C domain–containing histone demethylase that is specific for H3K36 (Klose et al, 2006; Tsukada et al, 2006) or lysine-specific demethylase that is specific for mono- and dimethylated H3K4 (Forneris et al, 2005, 2006).
Processes of histone modification and DNA methylation often involve dynamic interactions that can either reinforce or inhibit epigenetic changes. It has been demonstrated that the N-terminal domain of DNMT1 is able to bind to HDACs and can suppress gene transcription through facilitation of histone deacetylase activity (Roundtree et al, 2000). Furthermore, there is evidence that cooperation between DNA methylation and histone modification is involved in the regulation of imprinted gene allele–specific expression: 1) several imprinting loci, such as the insulin-like growth factor 2 (IGF2)-H19 domain (Grandjean et al, 2001) and the SNRPN domain (Gregory et al, 2001), display acetylation of particular histone amino acid residues exclusively on the expressed allele and 2) DNMT complexes associate with HDACs (Fuks et al, 2000, 2005). Recently silencing of the melanoma antigen–encoding (MAGE) gene family was reported to require both promoter demethylation and histone deacetylation (Wischnewski et al, 2006). Both epigenetic silencers and activators for gene transcription are polycomb group genes representing part of multitask protein complexes including HDAC and histone methyltransferase activities (Valk-Lingbeek et al, 2004; Grimaud et al, 2006).
Specific information on histone modifications in the testis. Hyperacetylation of histone H4 has been reported to be associated with histone to protamine exchange in haploid spermatids (Meistrich et al, 1992; Hazzouri et al, 2000; Sonnack et al, 2002). Recently it was demonstrated that the double bromodomain-containing protein BRDT, which has been localized in haploid spermatids, binds hyperacetylated histone H4 before accumulating in condensed chromatin (Govin et al, 2006). Therefore, it has been suggested that BRDT is involved in organizing the spermatozoon's genome by mediating a general histone acetylation–induced chromatin compaction and maintaining a differential histone acetylation of specific regions. Misregulation of histone H4 acetylation is associated with impaired spermatogenesis. In infertile men exhibiting Sertoli cell only (SCO) syndrome, Sertoli cell nuclei displayed an increase in histone H4 acetylation (Faure et al, 2003). In mice, treatment with the HDAC inhibitor trichostatin A (TSA) resulted in reversible infertility due to meiotic arrest at the level of pachytene spermatocytes (Fenic et al, 2004).
Practical approaches for analysis of histone modifications. Chromatin immunoprecipitation (ChIP) has emerged as a powerful tool for the study of interactions between DNA and chromatin-associated proteins, such as transcription factors and chemically modified core histones, across a defined DNA domain (Das et al, 2004). Two approaches can be distinguished by their methods of chromatin preparation: nChIP using native chromatin prepared by micrococcal nuclease digestion of nuclei (Hebbes et al, 1988) and xChIP using cross-linked chromatin prepared by adding formaldehyde to cells and exposing cells to ultraviolet irradiation (Solomon et al, 1988). Although ChIP at present is most accurate when there is an abundance of material (>107 cells) and only 1 cell type in the sample, it facilitates a precise and complex analysis of particular loci or even of whole genomes when combined with microarray technology (ChIP on chip) (Buck and Lieb, 2004) (Figure 2). Recently a protocol was reported that allows the analysis of as few as 100 cells using carrier chromatin ChIP in combination with PCR (O'Neill et al, 2006; Turner et al, 2006). For further information, refer to Tollefsbol (2004) and Mager and Bartolomei (2005). Detailed protocols are available at http://www.epigenome-noe.net/.
A popular approach to intervene directly in the chromatin remodeling process and to monitor changes in gene activity is to inhibit HDACs by HDAC inhibitors such as TSA, leading to increasing acetylation of the gene promoter and changes in gene activity, if the gene is regulated by histone (de)acetylation (Liu et al, 2006). Recently Heltweg et al (2005) presented a nonradioactive HDAC assay in which substrates with known and unknown HDAC activity are treated with an HDAC inhibitor. The HDAC activity is calculated relative to the conversion value.
RNA-Associated Silencing— Since analysis of the eukaryotic transcriptome revealed a large number of noncoding RNAs (ncRNAs) containing a high density of stop codons and lacking any extensive open reading frames, it has become clear that RNA plays a profound and complex role in the regulation of gene expression (Costa, 2005). ncRNAs are 20 to 22 nucleotides in length and include micro RNAs that are generated from hairpin-shaped precursors and small interfering RNAs (siRNAs), which are generated from double-stranded RNAs (dsRNAs) (Figure 4). Regulation of gene expression occurs at either the transcriptional level (eg, via control of chromatin organization) or the posttranscriptional level (eg, via control of mRNA stability and protein synthesis). It happens through RNA-associated silencing that requires the presence of Argonaute family proteins and is operable through RNA interference (RNAi) (Kawasaki et al, 2005). RNAi constitutes a powerful natural and valuable tool for the dynamic silencing of specific gene expression in basic research with therapeutic potential (Barik, 2005; Cejka et al, 2006).
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Practical approaches for analysis of RNA-associated silencing. During the last decades loss of function studies were traditionally performed on knockout mice to obtain mutant embryonic cells. While this remains a laborious method, RNAi has evolved to be a powerful novel technique for highly specific post-translational gene silencing in which siRNAs work as sequence-specific RNAi mediators. Gene function was eliminated by applying RNAi approaches in mouse embryonic stem cells to direct cell differentiation (Lykke-Andersen, 2006). In the RNAi pathway, silencing of gene expression occurs in response to the introduction of dsRNAs into the cell of interest (Figure 4). For further information, refer to Duxbury and Whang (2004), Sandy et al (2005), and Lykke-Andersen (2006). Detailed protocols are available at http://www.protocol-online.org/.
Germ Cell–Specific Epigenetic Processes
Histone to Protamine Exchange in Haploid Spermatids—
During spermatogenesis, restructuring of the nuclear chromatin is
accompanied by a gradual replacement of part of the somatic histones by
testis-specific subtypes, such as H1t, H1t2, HILS1, H2A.X, TH2B, H3.3A, and
H3.3B (Kimmins and Sassone-Corsi,
2005). Subsequently both somatic and testis-specific histones are
replaced by protamines. Protamine-DNA interactions are followed by chromatin
condensation, resulting in a total stop of gene expression in haploid
spermatids (Steger, 1999,
2001). Protamines also
undergo several chemical alterations including phosphorylation,
dephosphorylation (Marushige and Marushige, 1997), and disulfide bond
formation (Calvin and Bedford,
1971) (Figure 5).
In man, histone to protamine exchange is only about 85% complete
(Tanphaichitr et al, 1978).
This dual nucleohistone/nucleoprotamine structure may correspond to a specific
differential arrangement of chromatin regions according to potential functions
(Gardiner-Garden et al, 1998;
Wykes and Krawetz, 2003). The
presence of genomic islands that maintain a somatic-like chromatin structure
despite the global transition into the nucleoprotamine structure may
correspond to an essential mark for the establishment of adequate epigenetic
information in the offspring. Gatewood et al
(1987,
1990) demonstrated
sequence-specific packaging of DNA in nucleohistone and nucleoprotamine
components, suggesting that DNA linked to nucleohistone may represent sites of
active transcription. There is now evidence to suggest that the paternal
genome may be involved in starting early gene expression in the zygote.
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Histone to protamine exchange is associated with core histone acetylation, as acetyl groups turn the basic state of histones into a neutral one which, as a consequence, decreases the affinity of histones for DNA and allows protamines to interact with DNA. In rat (Meistrich et al, 1992), mouse (Hazzouri et al, 2000), and man (Sonnack et al, 2002), hyperacetylated histones have been demonstrated in mitotically active spermatogonia and in elongating spermatids, where histone to protamine exchange takes place. Interestingly, impaired spermatogenesis has been reported to be associated with both precocious (in pachytene spermatocytes) and reduced (in round spermatids) hyperacetylation of histone H4, possibly followed by incorrect histone to protamine exchange resulting in round spermatid maturation arrest (Sonnack et al, 2002). Whether this represents a cause or a consequence of impaired spermatogenesis remains to be elucidated.
Following fertilization, male and female pronuclei exhibit functional differences, although they reside within the same zygotic cytoplasm. In mice, the male pronucleus has been demonstrated to exhibit a volume 1.65-fold greater and transcriptional activity twofold higher than that of the female pronucleus. Both volume and transcriptional activity were approximately equal after the first mitosis of the embryo (Liu et al, 2005). In addition, sperm constitutive heterochromatin was found to be enriched for nucleosomes that carry specific histone modifications that are transmitted to the oocyte. This suggests an epigenetic mechanism for inheritance of chromosomal architecture (van der Heijden et al, 2006). Authors evaluated the dynamics of sperm chromatin remodeling in the zygote directly after gamete fusion and found histone H4 acetylated at lysines 8 and 12 prior to full decondensation of the sperm nucleus. This suggests that these marks are transmitted by the spermatozoon.
Genomic Imprinting— Genomic imprinting describes an epigenetic process by which sex-specific marks, so-called imprints, on certain chromosomal regions, so-called imprinted genes, are established in a parent of origin–dependent manner and thus expressed monoallelically. This functional asymmetry of parental genomes was discovered through nuclear transplantation experiments demonstrating that parthenogenetic mouse embryos die by day 10 of gestation (Barton et al, 1984; McGrath and Solter, 1984). Data from Kono et al (2004) suggest that paternal imprinting is obligatory for descendants to prevent parthenogenesis. Researchers created the first viable parthenogenetic mouse using a reconstructed oocyte with 2 haploid sets of the maternal genome derived from a fully grown oocyte and a nongrowing oocyte. The mouse developed to adulthood and was able to produce offspring. This development was possible by appropriate expression of the IGF2 and H19 genes, as the genome from the nongrowing oocyte was obtained from mutant mice carrying a deletion within the H19 gene that results in the absence of maternal-specific methylation.
Only a few imprinted genes have been found to acquire a methylation mark in the male germ line, and only 3 of these genes have been studied in detail, namely IGF2/H19 (Davis et al, 1999, 2000; Ueda et al, 2000), Rasgrf1 (Li et al, 2004), and Gtl2 (Li et al, 2004). IGF2/H19 is a reciprocally imprinted locus exhibiting paternal IGF2 and maternal H19 expression. Therefore, the noncoding RNA gene H19 exhibits a paternal imprinting pattern with hypermethylation on the silent paternal allele and hypomethylation on the expressed maternal allele (Figure 6). At present, there is no evidence that H19 has a function other than acting as a regulatory decoy for IGF2. A 2-kb differentially methylated domain (DMD) located –2 to –4 kb relative to the H19 transcriptional start site on the paternal allele acts as the imprinting center, the deletion of which results in loss of imprinting of both the IGF2 and H19 genes (Arney, 2003; Smith et al, 2004).
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In humans, 2 imprinting clusters have been extensively described: one on human chromosome 11p15 is linked to the pathogenesis of Beckwith-Wiedemann syndrome (BWS) and another on 15q11–13 is linked to Angelman syndrome (AS)/Prader-Willi syndrome (PWS) (Falls et al, 1999) (Figure 7). See further information in "Epigenetic Risks in ART."
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Epigenetic Reprogramming— Epigenetic reprogramming represents a stepwise process whereby the somatic-like epigenetic pattern undergoes erasure and is transformed into the sex-specific pattern of mature germ cells. It takes place at 2 critical periods, namely gametogenesis, which is essential for genomic imprinting, and preimplantation, which is essential for embryo development (Reik et al, 2001; Rousseaux et al, 2004; Allegrucci et al, 2005). Exact timing of epigenetic reprogramming ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which the new parental imprints are established. Aberrant reprogramming before specification of the germ line will involve both somatic and germ cells and therefore may result in the inheritance of an epigenetic trait.
At fertilization, sperm and oocytes represent a different epigenetic organization in that 1) differentially methylated regions have been established at various imprinted loci, 2) the sperm genome is more methylated than the oocyte genome, and 3) chromatin has been compacted with protamines in sperm and with histones in oocytes. After fertilization, decondensation of the male pronucleus is followed by a rapid and active demethylation, as both male- and female-specific epigenetic programs are reset to allow the gametes to give rise to a zygote that is totipotent and thus able to develop into a new organism (Mayer et al, 2000; Oswald et al, 2000). Primordial germ cells (PGCs) possess a somatic-like methylation pattern that is erased before they start their migration into the gonadal ridge, namely between days 10.5 and 12.5 post coitum (Davis et al, 1999, 2000; Reik et al, 2001; Hajkova et al, 2002; Yamazaki et al, 2003; Lucifero et al, 2004b), making PGCs inadequate donors for reproductive cloning (Yamazaki et al, 2003). While erasure of the paternally inherited DNA is completed within 1 day, suggesting an active process, demethylation of maternally inherited DNA happens more slowly by a passive process. Following the entry of PGCs into the gonadal ridge, the embryonic genome-wide methylation pattern is re-established in a sex-specific manner by de novo methylation (Kafri et al, 1992; Davis et al, 2000; Obata and Kono, 2002). While repeated sequences, promoters of tissue-specific genes, and gene-coding regions are generally hypermethylated, most CpG sites in CpG islands are protected from methylation. However, some of them undergo differential allelic methylation (eg, CpG islands on the inactive X chromosome and of imprinted genes) (Yoder et al, 1997). While paternal methylation imprints are acquired early, maternal methylation imprints are obtained late during gametogenesis (Davis et al, 1999, 2000; Hajkova et al, 2002; Obata and Kono, 2002; Yamazaki et al, 2003; Lucifero et al, 2004b). In the female germ line, imprints are known to be re-established in growing oocytes, whereas the timing of methylation re-establishment in imprinted genes is less clear for the male germ line but is assumed to be established in gonocytes.
Interestingly, Li et al (2004) observed differences in both timing and extent of methylation within the DMD among different imprinted genes. While methylation on H19 and Rasgrf1 was erased at day 12.5 of gestation, that of Gtl2 was retained. De novo methylation occurred in parallel between days 12.5 and 17.5 of gestation but was incomplete until the mature sperm stage in the male. Different acquisition of paternal imprints have also been reported for H19 and SNRPN (Davis et al, 1999, 2000; Ueda et al, 2000; Lucifero et al, 2004b). The paternally imprinted gene H19 becomes methylated during spermatogenesis, with the paternally inherited alleles becoming methylated prior to the maternally inherited alleles, namely during fetal stages on the paternal allele and during perinatal stages on the maternal allele, continuing postnatally through the onset of meiosis. By contrast, the maternally imprinted gene SNRPN becomes methylated during oogenesis with the maternally inherited alleles becoming methylated prior to the paternally inherited alleles.
Aberrant Epigenetic Reprogramming
The Formation of Testicular Cancer—
Recent data suggest that cancer has a fundamentally common basis that is
grounded in a polyclonal epigenetic disruption of stem/progenitor cells, which
might be a crucial target for cancer risk assessment and chemoprevention
(Feinberg et al, 2006).
Epigenetic chemotherapy with promising drugs that induce DNA demethylation and
partially reactivate silenced genes in cancer cells to restore their function,
such as 5-aza-2'-deoxycytidine and its oral counterpart zebularine
(Daskalakis et al, 2002;
Cheng et al, 2003) and the
histone deacetylase inhibitor TSA (Cameron
et al, 1999), are currently entering clinical trials and are
justified in patients who fail other treatment regiments. In general, 2
apparently contrasting phenomena coexist within cancer cells
(Jones and Baylin, 2002;
Esteller, 2005;
Fraga and Esteller, 2005;
Laird, 2005), namely a
profound loss of global 5-methylcytosine genomic content together with
discrete areas of dense hypermethylation. Changes of the methylation profile
are also accompanied by modulated expression profiles of DNMTs.
Global hypomethylation is primarily due to the loss of methylation from formerly hypermethylated repetitive sequences of the genome and has been linked to chromosomal instability, reactivation of oncogenes, and cancer testis genes such as MAGE-1 (De Smet et al, 1996). It is particularly severe in pericentromeric regions and predisposes to chromosomal instability, which results in translocations and loss of heterozygosity (Eden et al, 2003; Yeh et al, 2003). In contrast, hypermethylation occurs at CpG islands, most of which are unmethylated in normal cells and located in the promoters of certain tumor suppressor genes.
Testicular germ cell tumors (TGCTs) in adults possess a pluripotential nature and display protein histology from germ cells to embryonal and somatic cell differentiation. These properties make TGCTs a unique model for studying germ cell development and gametogenesis in respect to DNA reprogramming. TGCTs originate from a precursor lesion known as carcinoma in situ (Skakkebaek, 1972), or intratubular germ cell neoplasia unclassified (IGCNU) (Mostofi et al, 1987). IGCNU is thought to originate from a transformed fetal germ cell, gonocytes, or PGCs (Oosterhuis and Looijenga, 2005). Up until now little has been known about DNA methylation and histone modification in TGCTs. It has been shown that compared with nonseminomas, seminomas have demethylated DNA (Smiraglia et al, 2002). This finding is in line with the results of gene expression profiling and increased expression of DNMT3b in embryonal carcinoma compared with seminoma (Almstrup et al, 2005; Korkola et al, 2006). We recently detected by immunohistochemistry and in situ hybridization an increase of DNMT1 expression in nonseminomatous TGCTs, while the expression of HDAC was not differentially regulated between normal and neoplastic germ cells (Omisanjo et al, 2007). Gene expression analysis and subsequent immunohistochemical study revealed that a proposed aneuploidy-inducing centromere variant of histone H3, CENPA, is overexpressed in IGCNU and TGCTs (Biermann, in preparation).
The status of genomic imprinting of TGCTs has proven useful in identifying their cell of origin and in explaining their developmental potential. TGCTs comprise 3 entities (Oosterhuis and Looijenga, 2005):
Furthermore, in up to 80% of invasive TGCTs, relative overrepresentation of 12p sequences is due to 1 or more copies of isochromosome 12p (Atkin and Baker, 1982). However, the number of 12p sequences does not correlate with stage of disease and treatment resistance (Roelofs et al, 2000).
While normal cells are characterized by hypermethylated CpG islands resulting in silencing of associated tumor suppressor genes, transformation into cancer cells is associated with a release of the tight control of DNA methylation followed by a disruption of the well-balanced dynamic interplay between methylation and demethylation of these CpG islands.
Epigenetic Risks in ART— Several cases of AS (Cox et al, 2002; Orstavik et al, 2003; Horsthemke and Ludwig, 2005), PWS (Horsthemke and Ludwig, 2005), BWS (de Baun et al, 2003; Giquel et al, 2003; Maher et al, 2003; Halliday et al, 2004; Chang et al, 2005; Horsthemke and Ludwig, 2005), and retinoblastoma (Moll et al, 2003) with genomic imprinting disorders have been documented in children conceived via ART. Possible epigenetic risks linked to ART techniques may result from either the use of sperm with incomplete reprogramming or in vitro embryo procedures during a time window when epigenetic reprogramming occurs (De Rycke et al, 2002; Lucifero et al, 2004a; Niemitz and Feinberg, 2004; Horsthemke and Ludwig, 2005; Thompson and Williams, 2005; Horsthemke and Buiting, 2006).
With regard to the technique, it is unclear whether the problem is hormonal stimulation, gamete manipulation, or culture conditions. Superovulation has been suggested, which would lead to the maturation of epigenetically imperfect oocytes that would not have been ovulated without treatment or disturbing the process of DNA methylation in the oocyte (Chang et al, 2005; Horsthemke and Ludwig, 2005). It has been suggested that in vitro culture of gametes, zygotes, and embryos, representing a common feature of all ART techniques, results in an accumulation of epigenetic alterations leading to an enhancement in fetal growth and heavier newborn cattle, known as large offspring syndrome. By contrast, in mice and humans, there seems to be a reduction in birth weight (Sinclair et al, 2000; Young and Fairburn, 2000). Although low birth weight (Schieve et al, 2002), congenital malformations (Hansen et al, 2002), and imprinting disorders (Gosden et al, 2003) in some ART children can at least in part be attributed to epigenetic variation, it is possible that some subfertile couples have a genetic predisposition to epigenetic instability, which makes their offspring more susceptible to epigenetic changes—independently of whether or not the children are conceived by ART.
Testicular sperm extraction (TESE) in combination with intracytoplasmic sperm injection (ICSI) at present is the method of choice for the treatment of severe male factor infertility. Although it has been suggested that ICSI, by canceling the natural selection of aberrant spermatozoa, transmits genetically related infertility (Ma et al, 2000), the combination of ICSI with TESE also raises the question whether the genetic imprinting of the gametes' genome has already been finished (Tycko et al, 1997). During murine spermatogenesis, genomic imprinting of the H19 gene is completed within pachytene spermatocytes. Interestingly, the paternally inherited alleles become methylated prior to the maternally inherited alleles (Davis et al, 1999, 2000). However, insemination of oocytes with round spermatids is associated with poor embryo development that may be due to epigenetic abnormalities; the paternal genome in zygotes derived from immature spermatozoa has been demonstrated to be highly methylated, while the normal paternal genome is hypomethylated (Kishigami et al, 2006). During human spermatogenesis, erasure of methylation marks of the maternally imprinted gene SNRPN (Manning et al, 2001) and 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. Hartmann et al (2006) focused on the question whether infertile patients with spermatogenic arrest 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 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 will not exhibit the correct paternal-specific methylation of H19. However, the existence of sporadic hypomethylated CpG sites that have been described in ejaculated spermatozoa of oligozoospermic men could not be completely excluded (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 analyzed 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 analysis of the cells. In a very small proportion of spermatogonia- and spermatocyte-derived amplicons, Kerjean et al (2000) detected a nearly fully methylated allele that showed a single unmethylated CpG. This CpG may correspond to nucleotide 7966 (GenBank accession number AF125183), which is known to represent a C/T polymorphic site within the CTCF-binding site (Frevel et al, 1999). A tyrosine in position 7966 might be confused with an unmethylated CpG. This polymorphic site was also confirmed by Marques et al (2004), who analyzed the methylation pattern of H19 in ejaculated spermatozoa from normozoospermic and oligozoospermic patients. Although several reports exist on children with imprinting defects who were conceived by ART, it remains questionable whether there is a real risk of paternal transmission of imprinting defects or whether the main risk of epigenetic diseases after ART might be oocyte related.
Footnotes
Supported in part by German Research Foundation (Beutsche Forschungsgemeinschaft) grants BI1265/1-1 and STE892/8-1.
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