Constitutive heterochromatin during mouse oogenesis: the pattern of histone H3 modifications and localization of HP1alpha and HP1beta proteins.

Centromeres are the fragments of DNA that are responsible for proper chromosome segregation. They consist of centromeric chromatin surrounded by blocks of pericentric heterochromatin, playing an important role in centromere function. In somatic cells, the pericentric domains have a specific pattern of epigenetic modifications of core histones and contain specific pericentric proteins. These features are probably more important for the centromere function than the sequence of the centromeric DNA itself. In somatic cells, the HP1alpha and HP1beta proteins are indispensable for constitutive heterochromatin formation and maintenance. We have analyzed the localization of these proteins in the primordial, growing, fully-grown, and maturing mouse oocytes. Additionally, we have analyzed post-translational modifications of histone H3, which can influence HP1alpha and HP1beta association with the heterochromatin. We showed that the regions of constitutive heterochromatin have a distinct pattern of histone H3 acetylation and di-, and trimethylation of its lysine 9. We demonstrated that HP1beta protein was present in pericentric chromatin domains in primordial oocytes, growing (transcriptionally active) oocytes, and in fully-grown oocytes, and was released to the cytoplasm after germinal vesicle breakdown. In contrast, the HP1alpha was never detected in primordial oocytes, was first detected in pericentric heterochromatin in growing oocytes, dissociated from pericentric heterochromatin in fully-grown oocytes, and it was never detected in maturing oocytes. The presence of HP1alpha and HP1beta proteins on the heterochromatin of transcriptionally active oocytes and their absence in transcriptionally silent oocytes suggest that they are necessary for the repression of RNA synthesis in heterochromatin domains of transcribing oocytes.

Accumulation and dynamics of proteins of the MCM family during mouse oogenesis and the first embryonic cell cycle.

We describe the localization of three proteins of the minichromosome maintenance (MCM) family, Mcm2, -6 and -7 in mouse ovarian oocytes. We showed that Mcm proteins are stored in two forms: soluble and insoluble. Soluble Mcm2, -6 and -7 were uniformly distributed in the nuclei of ovarian oocytes. Insoluble Mcm2 and Mcm7 (but not Mcm6) were detected in the nuclei of resting, growing and fully-grown transcribing oocytes. In transcriptionally inactive fully-grown oocytes, Mcm2 underwent redistribution and Mcm7 disappeared. A similar effect was observed when transcription in growing oocytes was inhibited with alpha-amanitin. We postulate that in mouse oogenesis, the insoluble Mcm proteins are engaged in processes related to regulation of transcription and/or chromatin organization. In oocytes preparing for meiotic maturation, aggregates of the insoluble form of Mcm2 fragmented, dispersed and ultimately disappeared from the nuclei. Numerous Mcm2-positive deposits were observed in the cytoplasm of maturing oocytes. In the one-cell embryo, insoluble Mcm2 appeared in the G1 nucleus, persisted in the S phase and was undetectable in the G2 nucleus. Such behavior of Mcm2 supports its involvement in chromatin licensing in the first embryonic cell cycle.