Epigenetics in Male Germ Cells

We provide an overview of epigenetic mechanisms comprising DNA methylation, histone modification, and RNA-associated silencing of gene expression by noncoding RNAs and subsequently focus attention on germ cell—specific epigenetic processes including histone to protamine exchange in haploid spermatids, genomic imprinting, and reprogramming of epigenetic information after fertilization. Finally, aberrant epigenetic reprogramming and its association with testicular cancer and possible risks for assisted reproductive technology (ART) techniques are discussed. 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). . Genetic regulation of gene activity occurs at the transcriptional level through binding of transcription factors to the promoter and at the translational level through binding of protein repressors to the 3′ untranslated region and the poly-A tail of the transcript. Alternative splicing of the transcript results in the generation of various isoforms with different functions. Mutations within the genetic code are heritable and nonreversible. Epigenetic regulation of gene activity involves DNA methylation, core histone modification, and RNA interference. Although epigenetic effects are heritable, they do not affect the genetic code and are potentially reversible. This article provides an overview of the above mentioned epigenetic processes and subsequently focuses attention on germ cell—specific epigenetic processes including 1) histone to protamine exchange in haploid spermatids that is known to be associated with core histone acetylation, 2) genomic imprinting causing specific genes to be expressed monoallelically in a parent of origin—dependent manner, and 3) epigenetic reprogramming after fertilization. Finally, aberrant epigenetic reprogramming and its association with testicular cancer and possible risks for ART techniques will be discussed. Although × chromosome inactivation represents one of the most dramatic examples of long-term gene silencing and monoallelic gene expression, it is beyond the scope of this review (see a recent review by Heard [2005]). 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 × 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). 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