The enucleolation of mammalian oocytes and one-cell stage

embryos

J. Fulka, Jr.Institute of Animal Science

Prague, CZfulka@vuzv.cz

(……breaking the old developmental biology dogma?)

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”

fibrillar, granular anddense fibrillar components

dense fibrillar

Nucleolus Precursor Body NPB

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

Enucleolation

1.6 ng of proteins

Anti – triMe H4K20

DAPI

99% of NPB material removed by enucleolation

CTR

ENU

Development - YES

Development – NO(2-cell stage arrest)

ICSI of control and enucleolated oocytes confirms classical theory

Blastocysts 2-cell stage

ENU – 1 cell CTR – 1 cell

SC or ESC nuclear transfer

Nucleolus re-injection

No development

GV MI MII IVF No NCL

offspring

NCLs +

development compromised – WHY?

Enucleolation of one cell stage mouse embryos

MPN

Enucleolation of one cell stage mouse embryos

EARLY LATE

no development ?

Enucleolation of late one cell stage mouse embryos

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?

RESEARCH ARTICLEThe maternal nucleolus plays a key role in centromere satellitemaintenance during the oocyte to embryo transitionHelena Fulka1,* and Alena Langerova2

© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1694-1704 doi:10.1242/dev.105940

REC

REC

CO-FISH

Control – Major Satellites Enucleolated – Major Satellites

Disrupted MajS, MajS bridging

REC

Control – Minor Satellites Enucleolated – Minor Satellites

Disrupted MinS, MinS bridging

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 isunlikely to be involved in ribosomal gene activation and processing aspreviously believed, since this process can still occur in NPB-depletedearly embryos. This study uncovers an interesting and novel role for theNPB in early embryogenesis.

Cytoplast without membrane components

…. and what else?

Cytoplast with membrane components

Nucleus transfer, PCC

Decondensation

NT 1h PCC

16h CHXM

Cytoplast: membrane (-), NPB (-)

Cytoplast: membrane (+), NPB (+)

Special thanks!

Helena FulkaSugako Ogushi

Hirohisa KyogokuAlena Langerova