Critical Period of Nonpromoter DNA Methylation Acquisition during Prenatal Male Germ Cell Development

Abstract

The prenatal period of germ cell development is a key time of epigenetic programming in the male, a window of development that has been shown to be influenced by maternal factors such as dietary methyl donor supply. DNA methylation occurring outside of promoter regions differs significantly between sperm and somatic tissues and has recently been linked with the regulation of gene expression during development as well as successful germline development. We examined DNA methylation at nonpromoter, intergenic sequences in purified prenatal and postnatal germ cells isolated from wildtype mice and mice deficient in the DNA methyltransferase cofactor DNMT3L. Erasure of the parental DNA methylation pattern occurred by 13.5 days post coitum (dpc) with the exception of approximately 8% of loci demonstrating incomplete erasure. For most loci, DNA methylation acquisition occurred between embryonic day 13.5 to 16.5 indicating that the key phase of epigenetic pattern establishment for intergenic sequences in male germ cells occurs prior to birth. In DNMT3L-deficient germ cells at 16.5 dpc, average DNA methylation levels were low, about 30% of wildtype levels; however, by postnatal day 6, about half of the DNMT3L deficiency-specific hypomethylated loci had acquired normal methylation levels. Those loci normally methylated earliest in the prenatal period were the least affected in the DNMT3L-deficient mice, suggesting that some loci may be more susceptible than others to perturbations occurring prenatally. These results indicate that the critical period of DNA methylation programming of nonpromoter, intergenic sequences occurs in male germline progenitor cells in the prenatal period, a time when external perturbations of epigenetic patterns could result in diminished fertility.

DNA methylation in spermatogenesis and male infertility

“…However, in males, DNMT3L was initially detected at 12.5 days after fertilization of the egg, and the levels peaked at 15.5 days and remained upregulated until birth (77)(78)(79). However, the expression of DNMT3L was significantly 79 following birth and was found to be low in mature germ cells (36)(37)(38), suggesting an association between DNMT3L and DNA methylation. In addition, the inactivation of DNMT3L was shown to cause mitotic delays, chromosome synapsis errors and the ceasing of spermatogenesis at the zygotene stage of mitosis, thus inhibiting the maturation of germ cells (46)(47)(48).…”

“…They are considered to differ mechanistically due to inherent differences in their catalytic domains, and it has been suggested that DNMT3a is distributive while DNMT3b is processive (31)(32)(33)(34)(35)(36). The third significant member in the DNMT3 family is DNMT3L, which is considered to be required for the establishment of maternal imprints in oocytes (37)(38)(39)(40)(41), and has also been shown to be expressed during spermatogenesis (37,(42)(43)(44). SAM, as the methyl donor for DNMTs, is formed through the addition of adenosine triphosphate to methionine, which is catalyzed by the methionine adenosyltransferase enzyme.…”

Dynamics of 5-hydroxymethylcytosine during mouse spermatogenesis

“…In this line, one of the main contributions of our study is that we have identified this small but dynamic proportion. It is believed that demethylation and remethylation of repeats is mostly completed before birth 9,[11][12][13] . In the present study, we show that the 5hmC status in repeats keep changing during spermatogenesis even after birth.…”

“…In mice, the majority of methylation in male germ cells is completed before type A SG (SG-A) are formed at 6 days post-partum (dpp), while those at a small number of sites continues to occur until the formation of pachytene SC (pacSC) 10 . Imprinted genes, repetitive sequences and non-promoter intergenic regions complete DNA methylation before birth 9,[11][12][13] . However, little is known about whether and how global DNA methylation changes in germ cells after birth.…”

DNMT3L promotes quiescence in postnatal spermatogonial progenitor cells

“…DNMT3L itself does not possess enzymatic activity but acts as a processive catalyst and cooperates with other DNA methyltransferases (DNMTs) via its C-terminus to promote the de novo methylation of DNA sequences, including retrotransposons, gene bodies and the regulatory sequences of imprinted genes (Bourc'his et al, 2001;Chedin et al, 2002;Hata et al, 2002;Bourc'his and Bestor, 2004;Suetake et al, 2004;Kato et al, 2007;Holz-Schietinger and Reich, 2010;Niles et al, 2011;Smallwood et al, 2011;Van Emburgh and Robertson, 2011;Arand et al, 2012;Kobayashi et al, 2012;Ichiyanagi et al, 2013). In addition, DNMT3L contains a plant homeodomain (PHD)-like domain that can recruit histone modifiers, such as histone deacetylase 1 (HDAC1), or bind to the Nterminus of histone H3 when the lysine 4 of histone H3 is unmethylated (Aapola et al, 2002;Deplus et al, 2002;Jia et al, 2007;Ooi et al, 2007).…”