1. The enucleolation of mammalian oocytes and one-cell stage embryos J. Fulka, Jr. Institute of Animal Science Prague, CZ fulka@vuzv.cz (……breaking the old developmental biology dogma?)

3. Nucleolus in differentiated cells: plurifunctional nucleolus (biosynthesis and processing of RNA components of ribo-protein complexes, involved in cell cycle regulation, differentiation, development, etc. Nucleolus in fully grown oocytes and early embryos: ?????? – commonly accepted “store of material(s) from which nucleoli are formed in developing embryos”

4. fibrillar, granular and dense fibrillar components dense fibrillar Nucleolus Precursor Body NPB

5. GV MI A-TI MII PNs CCA -/PCC - CCA+/PCC+ CCA-/PCC- CCA+/PCC+ CCA-/PCC- CCA: chromosome condensation activity; PCC: premature chromosome condensation

6. Enucleolation

7. 1.6 ng of proteins

8. Anti – triMe H4K20 DAPI 99% of NPB material removed by enucleolation

9. CTR ENU

10. Development - YES Development – NO (2-cell stage arrest) ICSI of control and enucleolated oocytes confirms classical theory

11. Blastocysts 2-cell stage ENU – 1 cell CTR – 1 cell SC or ESC nuclear transfer

12. Nucleolus re-injection No development GV MI MII IVF No NCL offspring NCLs + development compromised – WHY?

13. Enucleolation of one cell stage mouse embryos MPN

14. Enucleolation of one cell stage mouse embryos EARLY LATE no development ?

15. Enucleolation of late one cell stage mouse embryos

17. Is NPB indeed the storage site of material(s) from which nucleoli are formed in developing embryos? ….. and if not, it is good for WHAT?

18. RESEARCH ARTICLE The maternal nucleolus plays a key role in centromere satellite maintenance during the oocyte to embryo transition Helena Fulka1,* and Alena Langerova2 © 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1694-1704 doi:10.1242/dev.105940

19. REC CO-FISH Control – Major SatellitesEnucleolated – Major Satellites Disrupted MajS, MajS bridging

20. REC Control – Minor SatellitesEnucleolated – Minor Satellites Disrupted MinS, MinS bridging

21. Nucleolus precursor body makeover Unlike somatic cells, the nucleus of the oocyte and very early embryo contains a morphologically distinct nucleolus called the nucleolus precursor body (NPB). Although this enigmatic structure has been shown to be essential for normal mammalian development, its precise function remains unclear. In this issue, Helena Fulka and Alena Langerova now demonstrate (p. 1694) a crucial role for the NPB in regulating major and minor satellite DNA sequences and chromosome dynamics in the mouse. Absence of the NPB during the first embryonic cell cycle causes a significant reduction in satellite DNA sequences, and the authors also observe extensive chromosome bridging of these sequences during the first embryonic mitosis. The authors further demonstrate that the NPB is unlikely to be involved in ribosomal gene activation and processing as previously believed, since this process can still occur in NPB-depleted early embryos. This study uncovers an interesting and novel role for the NPB in early embryogenesis.

22. Cytoplast without membrane components …. and what else?

23. Cytoplast with membrane components

24. Nucleus transfer, PCC

25. Decondensation NT 1h PCC 16h CHXM Cytoplast: membrane (-), NPB (-) Cytoplast: membrane (+), NPB (+)

26. Special thanks! Helena Fulka Sugako Ogushi Hirohisa Kyogoku Alena Langerova