Wilhelm bernhard workshop on the cell nucleus

TOPOGRAPHY OF RIBOSOMAL DNA (rDNA) DETECTABLE BY FISH IN EARLY MOUSE EMBRYOS

¹Korobova F.V., ²Noniashvili E.M., ³Schoefer C., ¹Romanova L.G., ³Wachtler F., ²Dyban A.P., and ¹Zatsepina O.V.

¹Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAN, Moscow, and ²Institute of Experimental Medicine RAMN, St.-Petersburg, Russia; ³Histologisch-Embryologishes Institut, University of Vienna, Vienna, Austria.
It is well known that in preimplantation mouse embryos active ribosomal RNA (rRNA) genes are associated with the surface of so called nucleolar precursor bodies (NPBs), the characteristic structures, which are thought to serve for assembly of the functional nucleolus in early mammalian development. However, the question whether the NPBs are associated with all rDNA repeats and whether the topography of rDNA fluctuates respectively to its transcription and replication status still remains open. We applied fluorescence in situ hybridization (FISH) with mouse rDNA probes labeled with digoxigenin in order to map rDNA repeats in spreads of one- and two-cell mouse embryos. Before use, some one-cell embryos were incubated with okadaic acid, a potent phosphatase inhibitor that induced premature chromosome condensation. G1-, S-, and G2-type prematurely condensed chromosomes were found in pronuclei of embryos of different age, which reflects the embryo progression through the first cell cycle. Our data show that (1) irrespective to the transcription and replication activity of rDNA, the NPBs are not equal in their ability to support recruitment of rDNA repeats; (2) a part of rRNA genes is not associated with NPBs, but is “freely” located within the nucleoplasm; (3) resumption of rDNA transcription in late two-cell embryos is accompanied by significant rDNA unraveling as compared with early (transcriptionally inert) two-cell embryos; (4) in one-cell embryos, i.e. when rRNA genes are not transcribed, rDNA replication apparently occurs during the entire S-period and is asynchronous between individual chromosomal nucleolus organizing regions (NORs); (5) in one-cell mouse embryos, NPBs are involved in general arrangement of chromosomes within the pronuclei and are probably associated with the chromosomal NORs via the centromeric heterochromatin.

The research was supported by the INTAS (grant 96-1638) and Russian Foundation of Basic Researches (grants 02-04-49373 and 01-04-49810).