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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
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