Chromosome Research 1994, 2, 147-152 DNA methylation changes during mouse spermatogenesis Jesüs del Mazo, Giorgio Prantera, Miguel Torres & Marina Ferraro Received 5 July 1993, received in revised form 9 November 1993; Accepted for publication by H. C. Macgregor 23 November 1993 Genomic imprinting in mammals is thought to be mediated by differences in the methylation level of cytosine residues in the genome. These differences in DNA methylation are thought to be generated during the development of the germ line. To characterize the profile of global methylation of the mouse genome during male gametogenesis, we have quantified the relative level of methylation in individual cells during meiosis and spermatogenesis. A decrease in the level of DNA methylation is observed from meiotic cells to elongated spermatids. The erasure of the somatic pattern of methylation during spermatogenesis sug- gests the existence of a subsequent mechanism gen- erating the parental specific methylation patterns leading to genomic imprinting of specific alleles. Key words: gametogenesis, imprinting, in situ nick- translation, methylation, mouse, Mus musculus, sperma- togenesis Introduction In mammals, genomic imprinting of maternal and paternal chromosomes is a mechanism that generates differential expression of aUeles in relation to their parental origin. This phenomenon is thought to play a crucial role in the embryonic development of mam- mals (Solter 1988). Alteration of the genomic imprinting pattern has also been identified as a possible cause of genetic disorders, including some human diseases (Reik 1989, Clarke 1990, Hall 1990). The molecular basis for this epigenetic inheritance has not been clearly deli- neated. However, any proposed mechanism should meet the following requisites: it should ensure the inheritance of the imprinting pattern through subse- quent rounds of DNA replication, and its reversal when passing through the female or male gamete (Sapienza et al. 1989). Methylation of DNA at the cytosine residues is an epigenetic phenomenon, which possesses the requisites of heritability and reversibility (Razin et al. 1984). Moreover, changes in DNA methylation have been also associated with the regulation of gene expression (Boyes & Bird 1991), and clinical manifestation of some human diseases (Oberlé et al. 1991). For these reasons, DNA methylation has been proposed as a possible mechanism to imprint genes, chromosome regions, or specific chromosomes (Swain et al. 1987, Prantera & Ferraro 1990, Grant et al. 1992). Although we cannot exclude fertilization as the mo- ment when male and female pronuclei are differentially imprinted, the most accepted hypothesis is that parental imprinting takes place, or at least begins, during the development of germ cells. Thus, changes in the methylation pattern should occur during gameto- genesis. In fact, different methylation patterns in ga- mete cells versus somatic tissues have been described for specific genes (Groudine & Conkin 1985, Driscoll & Migeon 1990) or DNA sequences (Sandford et al. 1984). In this work, we have applied a sensitive technique that shows changes in the global methylation levels of DNA in individual cells during mouse spermatogen- esis. We have quantified the changes in DNA methyla- tion for different cell types during the progression of spermatogenesis. Our results suggest a model for the participation of methylation in the generation of par- ental imprinting. Jesüs del Mazo (corresponding author) and Miguel Torres are at the Centro de Investigaciones Biolögicas (C.S.I.C.), Veläzquez 144, 28006- Madrid, Spain. Tel: ( + 34) 1 5644562 ext 4324; Fax: ( + 34) 15627518. Giorgio Prantera is at The Departimento di Agrobiologia e Agrochimica, Universitd della Tuscia, Viterbo, Italy. Marina Ferraro is at the Departimento di Genetica e Biologia Molecolare, Universitä La Sapienza, Roma, Italy. © 1994 Rapid Communications of Oxford Ltd Chromosome Research Vol 2 1994 147