Dynamic Nuclear Organization of the Genome During Fetal Male Germ Cell Development.

Abstract

In mice, male germ cells enter mitotic arrest at around 13.5 days postcoitum (dpc) and maintain G0/G1 cell cycle stage until after birth. During the mitotic arrest, male germ cells undergo epigenetic reorganization, which is essential for completion of male sex differentiation and reproductive function. We previously showed that when 15.5-19.5 dpc male germ cells are used as donors for nuclear transfer experiments, most of cloned two-cell embryos are developed to blastocysts (Yamazaki et al., 2003). These data indicate that the transition of male germ cells into a G0/G1 cell cycle state increase the reprogramming potential of the genome. Previously, constitutive heterochromatin was thought to be remarkably stable in composition and transcriptionally inert. However, several lines of recent evidence suggest that a global reorganization and spatial clustering of constitutive heterochromatin play a critical role in epigenetic plasticity during cellular differentiation. In this study, we investigated molecular links between the global organization of the genome and the epigenetic plasticity of male germ cells throughout fetal (12.5-19.5 dpc) and neonatal development (4 days postpartum). To reveal the global organization of the genome, constitutive heterochromatin was monitored by DAPI and by immunostaining of histone H3 trimethylated on lysine 9 (H3-triMetK9) and heterochromatin protein 1 (HP1α). The H3-triMetK9 and HP1α stainings were confined to small, discrete foci coincident with DAPI-bright, constitutive heterochromatin in male germ cells at 12.5-13.5 dpc. On the other hand, between 13.5-19.5 dpc, these three stainings showed a diffused, poorly defined regions, and the number of heterochromatin foci significantly decreased. These morphological changes occurred during the transition of male germ cells from mitotic stage into a G0/G1 cell cycle state (13.5-15.5 dpc), and remained constant during G0/G1 cell cycle stage. To assess these global changes at a local level, we compared histone methylation levels at major satellite repeats by chromatin immunoprecipitation analyses. We observed enrichment of H3-triMetK9 across major satellite repeats between 12.5-15.5 dpc, but then observed loss of H3-triMetK9 during 17.5-19.5 dpc. Also, we analyzed the expression profiles of histone methyltransferases (Suv39h1 and Suv39h2), and histone demethylase (Jmjd2b) with real-time RT-PCR. Expression of Suv39h1 and Suv39h2 was downregulated at 15.5 dpc, while the expression of Jmjd2b was upregulated at 17.5 dpc. A marked decrease of H3-triMetK9 levels at major satellite repeats at 17.5 dpc might be caused by preceding loss of the activity of the histone methyltransferases and also the increased activity of the histone demethylase. Taken together, these results showed that constitutive heterochromatin organization is drastically modified at global and local levels during prespermatogenesis. The decrease in epigenetic histone modifications characteristic of heterochromatin suggests that chromatin conformation in male germ cells are globally more open in G0/G1 cell cycle stage compared to mitotically active stage. These chromatin properties of G0/G1 male germ cells might reflect a functionally important hallmark of epigenetic plasticity. (Research was supported by NIH HD042772.)