Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells.
ABSTRACT The acquisition of genomic methylation in the male germ line is initiated prenatally in diploid gonocytes, while DNA methylation in the female germ line is initiated postnatally in growing oocytes. We compared the temporal expression patterns of the DNA methyltransferases, DNMT1, DNMT3a, DNMT3b, and DNMT3l in the male and female germ lines. DNMT1 expression was examined by immunocytochemistry and Northerns with an emphasis on the prenatal period. In the female, there is a gradual down-regulation of DNMT1 protein in prenatal meiotic prophase I oocytes that is not associated with the production of an untranslated transcript, as it is in the male; these results suggest that the mechanism of meiotic down-regulation differs between the sexes. In the male, DNMT1 is unlikely to play a role in the prenatal acquisition of germ line methylation patterns since it is down-regulated in gonocytes between 14.5 and 18.5 days of gestation and is absent at the time of initiation of DNA methylation. To search for candidate DNMTs that could be involved in establishing methylation patterns in both germ lines, real-time RT-PCR was used to simultaneously study the expression profiles of the three DNMT3 enzymes in developing testes and ovaries; DNMT1 expression was included as a control. Expression profiles of DNMT3a and DNMT3l provide support for an interaction of the two enzymes during prenatal germ cell development and de novo methylation in the male. DNMT3l is the predominant DNMT3 enzyme expressed at high levels in the postnatal female germ line at the time of acquisition of DNA methylation patterns. DNMT1 and DNMT3b expression levels peak concomitantly, shortly after birth in the male, consistent with a role in the maintenance of methylation patterns in proliferating spermatogonia. Together, the results provide clues to specific roles for the different DNMT family members in de novo and maintenance methylation in the developing testis and ovary.
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ABSTRACT: DNA methylation is a well-characterized epigenetic modification involved in gene regulation and transposon silencing in mammals. It mainly occurs on cytosines at CpG sites but methylation at non-CpG sites is frequently observed in embryonic stem cells, induced pluriotent stem cells, oocytes and the brain. The biological significance of non-CpG methylation is unknown. Here, we show that non-CpG methylation is also present in male germ cells, within and around B1 retrotransposon sequences interspersed in the mouse genome. It accumulates in mitotically arrested fetal prospermatogonia and reaches the highest level by birth in a Dnmt3l-dependent manner. The preferential site of non-CpG methylation is CpA, especially CpApG and CpApC. Although CpApG (and CpTpG) sites contain cytosines at symmetrical positions, hairpin-bisulfite sequencing reveals that they are hemimethylated, suggesting the absence of a template-dependent copying mechanism. Indeed, the level of non-CpG methylation decreases after the resumption of mitosis in the neonatal period, whereas that of CpG methylation does not. The cells eventually lose non-CpG methylation by the time they become spermatogonia. Our results show that non-CpG methylation accumulates in non-replicating, arrested cells but is not maintained in mitotically dividing cells during male germ-cell development.Nucleic Acids Research 11/2012; · 8.03 Impact Factor
Article: Signaling through the TGF Beta-Activin Receptors ALK4/5/7 Regulates Testis Formation and Male Germ Cell Development.[show abstract] [hide abstract]
ABSTRACT: The developing testis provides an environment that nurtures germ cell development, ultimately ensuring spermatogenesis and fertility. Impacts on this environment are considered to underlie aberrant germ cell development and formation of germ cell tumour precursors. The signaling events involved in testis formation and male fetal germ cell development remain largely unknown. Analysis of knockout mice lacking single Tgfβ family members has indicated that Tgfβ's are not required for sex determination. However, due to functional redundancy, it is possible that additional functions for these ligands in gonad development remain to be discovered. Using FACS purified gonadal cells, in this study we show that the genes encoding Activin's, TGFβ's, Nodal and their respective receptors, are expressed in sex and cell type specific patterns suggesting particular roles in testis and germ cell development. Inhibition of signaling through the receptors ALK4, ALK5 and ALK7, and ALK5 alone, demonstrated that TGFβ signaling is required for testis cord formation during the critical testis-determining period. We also show that signaling through the Activin/NODAL receptors, ALK4 and ALK7 is required for promoting differentiation of male germ cells and their entry into mitotic arrest. Finally, our data demonstrate that Nodal is specifically expressed in male germ cells and expression of the key pluripotency gene, Nanog was significantly reduced when signaling through ALK4/5/7 was blocked. Our strategy of inhibiting multiple Activin/NODAL/TGFβ receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFβ and Activin signaling during testis formation and male germ cell development.PLoS ONE 01/2013; 8(1):e54606. · 4.09 Impact Factor
Article: Spata22, a novel vertebrate-specific gene, is required for meiotic progress in mouse germ cells.[show abstract] [hide abstract]
ABSTRACT: The N-ethyl-N-nitrosourea-induced repro42 mutation, identified by a forward genetics strategy, causes both male and female infertility, with no other apparent phenotypes. Positional cloning led to the discovery of a nonsense mutation in Spata22, a hitherto uncharacterized gene conserved among bony vertebrates. Expression of both transcript and protein is restricted predominantly to germ cells of both sexes. Germ cells of repro42 mutant mice express Spata22 transcript, but not SPATA22 protein. Gametogenesis is profoundly affected by the mutation, and germ cells in repro42 mutant mice do not progress beyond early meiotic prophase, with subsequent germ cell loss in both males and females. The Spata22 gene is essential for one or more key events of early meiotic prophase, as homologous chromosomes of mutant germ cells do not achieve normal synapsis or repair meiotic DNA double-strand breaks. The repro42 mutation thus identifies a novel mammalian germ cell-specific gene required for meiotic progression.Biology of Reproduction 01/2012; 86(2):45. · 4.01 Impact Factor