DNA methylation and demethylation events during meiotic prophase in the mouse testis.

Abstract

The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein 1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated; in contrast, the methylation of Il-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during meiotic prophase in the mouse testis.Mammalian DNA contains only one modified base, 5-methylcytosine (47); 5-methylcytosine is found primarily in the dinucleotide sequence 5'-CpG-3' (11). Methylation changes in deoxycytosine residues of the dinucleotide sequence CpG in mammalian DNA have been implicated in the transcriptional activation of genes (for a review, see reference 6), changes in chromatin structure (18,24), and parental imprinting (39,43,45). Most of the genes whose methylation patterns have been examined so far in mammalian spermatozoa are those that are expressed after fertilization. For many genes there are clear differences between CpG methylation in spermatozoa and that in somatic tissues. Many genes in spermatozoa are more highly methylated at CpG sites than in the somatic tissue where they are expressed; when these genes become methylated during germ cell development remains to be determined (22,27,28,34,36,46,48). Studies in the chicken indicate that de novo methylation events occur somewhere in the transition between spermatogonia and spermatocytes (18). In contrast, several DNA satellite sequences are less methylated in spermatozoa than in various somatic DNAs (9,15,16,35,42,44); this undermethylation of satellite sequences occurs as early as primitive type A, type A, and type B spermatogonia (35).Little is known about the methylation patterns of genes that are expressed exclusively in the testis; that is, whether the methylation of testis-specific genes changes during germ cell development in the testis and how such methylation changes relate to the transcription of these genes. We have used DNA from the testes of prepubertal mice of different ages and enriched populations of male germ cells to monitor the location and nature of methylation changes for three testis-specific genes during spermatogenesis.The DNA methylation patterns for three postmeiotically expressed mouse genes encoding nuclear chromatin proteins, transition protein 1 (mTP1) (25), protamine 1 (mPl), and protamine 2 (mP2) (19,20), were examined. mTP1 (26, 51), mPl, and mP2 (23, 50) are expressed at similar times during spermatogenesis, and their gene products serve a similar function in the testis (for a review, see references 19 and 20). We have compared the DNA methylation ...

Testis-specific expression of a metallothionein I-driven transgene correlates with undermethylation of the locus in testicular DNA.

“…In the germinal epithelium the locus encoding transition protein 1 is demethylated prior to actual expression of the gene (56). Thus it is possible that the undermethylation ofthe transgene represents activation of such a mechanism as part of normal testis-specific gene regulation.…”

Adverse Effects of 5-Aza-2′-Deoxycytidine on Spermatogenesis Include Reduced Sperm Function and Selective Inhibition of de Novo DNA Methylation

“…DNA was extracted from purified spermatozoa using proteinase K followed by phenol extraction for Southern blot and restriction landmark genomic scanning (RLGS) analysis as described previously (Okazaki et al, 1995). Southern blots were done as described previously (Trasler et al, 1990) and visualized by autoradiography. Major and minor satellite probes were constructed by PCR amplification of genomic DNA using primers described previously (Lehnertz et al, 2003).…”

Recombinant Human DNA (Cytosine-5) Methyltransferase

“…In mammals, the patterns of methylation are inherited from both parental genomes but are erased and reconstructed (de novo methylation) in somatic cells following implantation (13,14). Such patterns are then copied and maintained by hemimethylation of the daughter strands during semiconservative DNA synthesis in S phase (maintenance methylation) (15, 16).…”