ИСТИНА

Nucleolar precursor bodies (NPBs) are numerous intranuclear entities, which appear in one-cell (zygotic) embryos in mammals of all species shortly after fertilization. NPBs are strikingly different from normal nucleoli in ultrastructure and synthetic activity. It is commonly accepted that NLBs "nucleate" the assembly of normal nucleoli at later stages of embryonic development, when the resumption of rRNA synthesis occurs. However, functions of NPBs in zygotic embryos, when rRNA genes are silent, remain unknown. In the current study, we address this issue by treatment of mouse zygotic embryos with okadaic acid (OA), an inhibitor of serine/threonine phosphoprotein phosphatases 1and 2A. OA is known to suppress the activity of maturation promoting factor and to induce premature condensation of chromosomes (PCC), while NPBs remain unaffected (Dyban et al., 1993). Chromosomal nucleolus organizing regions (NORs) were detected by fluorescence in situ hybridization (FISH) with anti-rDNA digoxigenin-labeled probes as described by Romanova et al. (2006). Before examining with an epifluorescence and a laser scanning confocal microscopes, embryo spreads were counterstained with a chromatin-binding dye DAPI. Our results for the first time show that (1) at all zygotic sub-stages (early, 19-21 h of post-hCG; middle, 22-26 h; late, 27-30 h), NOR-chromosomes are associated with NLBs. (2) The number of NLB-associated NORs correlates with the NPB size and increases from the early to late zygotes. (3) Beside the NOR-chromosomes, NPBs also interact with nonNOR-chromosomes that was particularly evident at the late zygote, when in each pronucleus all chromosomes become associated with the single NPB. (4) Chromosomal associations with NLBs are intermediated by their centromeric regions but not by the NORs themselves. We concluded that in both parental pronuclei and through the whole zygote, NLBs serve for general arrangement of chromatin/chromosomes, including the NOR-bearing ones.