spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online November 7, 2008
doi: 10.1242/10.1242/dev.023747

Development 135, 3935-3945 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bui, H.-T.
Right arrow Articles by Wakayama, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bui, H.-T.
Right arrow Articles by Wakayama, T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

The cytoplasm of mouse germinal vesicle stage oocytes can enhance somatic cell nuclear reprogramming

Hong-Thuy Bui*,{ddagger}, Sayaka Wakayama, Satoshi Kishigami{dagger}, Jin-Hoi Kim*, Nguyen Van Thuan* and Teruhiko Wakayama{dagger},{ddagger}

RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Kobe 650-0047, Japan.


Figure 1
View larger version (32K):
[in this window]
[in a new window]

  		Fig. 1. Schema of the experiments. (A) Injection of mouse cumulus cell nuclei (black) into GV nucleus, GV cytoplasm, GV intact or GV enucleated oocytes. (B) Cumulus cells were permeabilized with streptolysin O (SLO, 40 minutes), washed and incubated for 45 minutes in HEPES-CZB medium containing an ATP-generating system with or without (control) GV oocyte cytoplasmic lysate. Intact and treated cumulus cells were examined for the intensity of histone H3 methylation at lysine 9 (Me-H3-K9). GV nuclei were removed from the GV oocyte lysate before use (a). Scale bar: 35-40 µm. (b,c) Hoechst-stained GV nucleus at high magnification. Scale bar: 20 µm. The lower figure indicated fibroblasts treated as above, their membrane resealed and cultured for 1, 2, 3 or 4 weeks. These cells were then collected to examine Oct4 and nuclear lamin A (LMNA) using RT-PCR. (C) Cumulus cells were treated with GV oocyte cytoplasmic lysate and transferred into enucleated MII oocytes. The oocytes were activated and cultured until the blastocyst stage to examine Oct4 and Cdx2 immunoreactivities. Some embryos were transferred to pseudopregnant surrogate mothers to obtain cloned pups.

 

Figure 2
View larger version (50K):
[in this window]
[in a new window]

  		Fig. 2. Expression of histone H3 acetylation at lysine 14 (Ac-H3-K14) in somatic nuclei injected into the nucleus or cytoplasm of mouse GV oocytes. (A) Immediately after somatic injection into both the nucleus and cytoplasm. (B) (a-f) 2 hours after somatic injection into the nucleus and the cytoplasm. (g-l) 7 hours after somatic injection into the nucleus and the cytoplasm. (C) Control oocyte non-treated dbcAMP at 7 hours. Scale bars: 20 µm. Arrows indicate injected cumulus cell nuclei.

 

Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 3. Expression of HDAC and histone acetylation in mouse oocytes. (A) Expression of HDAC activity in nuclei, cytoplasm and oocytes at the GV stage. The positive and negative controls were crude nuclear extract from MCF4 cells and were used according to the procedure outlined in the Cyclex HDAC assay kit. (B) Expression of HDAC activity in oocytes during maturation at various meiotic stages: GV (0 hours), GVBD (3 hours), MI (7 hours) and MII (17 hours). (C) Deacetylation of histone H3-K9 in somatic nuclei transferred into enucleated GV oocytes (a-h) or in control intact GV oocytes (i-l). Oocytes were cultured at various times to obtain the equivalent meiotic stages: GV-like stage (0 hours); GVBD (3 hours); MI-like stage (7 hours) and MII-like stage (17 hours). Scale bars: 20 µm.

 

Figure 4
View larger version (57K):
[in this window]
[in a new window]

  		Fig. 4. Demethylation of histone H3-K9 in somatic nuclei after being injected into intact mouse GV oocytes and enucleated GV oocytes. (A,B) Oocytes were examined after culture for 0, 1, 3 and 7 hours. Methylation of H3-K9 is shown in green and microtubules are stained for β-tubulin in red. (B) After nuclear transfer in enucleated GV oocytes, somatic chromosome formed structures similar to fully grown GV chromosome after 1 hour (m) and similar to late GV chromosome after 2 hours (n). OC, oocyte chromosome; SC, somatic chromosome. Arrow indicates an intact cumulus cell before injection. Scale bars: 20 µm.

 

Figure 5
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 5. Chromosome morphology and histone modifications. (A) The morphologies of mouse oocytes and injected donor nuclei are classified according to GV oocyte maturation status. Histone H3 methylation at lysine 9 (Me-H3-K9), phosphorylation at serine 10 (P-H3-S10) and serine 28 (P-H3-S28) of oocyte and somatic cell nuclei injected into intact and enucleated GV oocytes were examined simultaneously up to 17 hours after injection. (B) Expression of methylation H3-K9 in somatic nuclei injected into the cytoplasm (a-c) and nucleus (d-f), then cultured for 3 hours with dbcAMP. Control oocyte (g-i) was cultured same time without dbcAMP. Arrows indicate injected cumulus nuclei. (C) About 10% of enucleated GV oocytes extruded an extremely large first polar body at 17 hours after cumulus cell injection (c). Scale bar:20 µm.

 

Figure 6
View larger version (69K):
[in this window]
[in a new window]

  		Fig. 6. Effects of mouse GV oocyte cytoplasmic lysates on somatic nuclei and cloned embryos. (A) Intensity of histone H3-K9 methylation in cumulus cell nuclei; Me-H3-K9 is shown in green and nuclear membranes are stained by lamin B in red. (b) Intact cumulus; (e) permeabilized cumulus without cytoplasmic lysates (control) and (h) permeabilized cumulus with cytoplasmic lysates. (B) Intact and cumulus cell nuclei treated with GV cytoplasmic lysates were transferred to enucleated MII oocytes, then the oocytes were activated and cultured to the blastocyst stage. ICSI-generated embryos were used as controls. The intensity of H3-K9 methylation of zygote and blastocyst stages is shown in fertilized embryos (b,h); in cloned embryos with intact cumulus cells (d,j); and in cloned embryos with cumulus cells treated with GV oocyte cytoplasmic lysate (f,l). Arrows indicate the nuclei at metaphase or anaphase/telophase stages with high levels of Me-H3-K9. Scale bars: 20 µm. (C) Quantification of methylated histone H3-K9 in pronuclear, two-cell and blastocyst stage embryos (ICM, inner cell mass; TE, trophectoderm). Each column represents the normalized mean value of these intensities per developmental stage, except for blastocysts, and distinguishes between ICM and TE. The data are presented as the mean±s.e.m.

 

Figure 7
View larger version (35K):
[in this window]
[in a new window]

  		Fig. 7. Expression of Oct4, a pluripotency marker, and repression of nuclear lamin A (LMNA), a tissue-specific marker, in cells treated with GV cytoplasmic lysate and cultured in DMEM or ES medium. B6D2F1 strain mouse fibroblasts were treated with GV cytoplasmic lysate and cultured for 0, 1, 2, 3 or 4 weeks in DMEM with 10% FBS (DMEM medium) or in medium designed for embryonic stem cell culture (ES medium). (A) PCR analysis of Oct4 and LMNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalization control. Expression of Oct4 and repression of LMNA is clearly shown after 4 weeks of culture in both DMEM and ES medium. (B,C) Quantitative RT-PCR analysis of Oct4 (B) and LMNA (C). SC, somatic cell; ES, embryonic stem cell. Asterisks indicate the significant differences between expression of Oct4 and repression of LMNA at 4 weeks of culture.

 

Figure 8
View larger version (60K):
[in this window]
[in a new window]

  		Fig. 8. Production of high-quality cloned embryos and offspring. (A) Expression of Oct4 and Cdx2 in expanded mouse blastocysts derived from normally fertilized embryos, from embryos produced by nuclear transfer with intact cumulus and from embryos produced by nuclear transfer with cumulus cells treated with GV oocyte cytoplasmic lysates. The classification is based on the numbers of ICM blastomeres expressing immunoreactivity for Oct4. Type I, more than 15 positive cells; type II, 10-15 positive cells; type III, fewer than 10 positive cells. Oct4 is stained green and Cdx2 is red. Scale bar: 20 µm. (B) Proportions of blastocysts classified into these three types. (C) Cloned mice were born from enucleated MII oocytes injected with cumulus nuclei treated with cytoplasmic lysates of GV oocytes.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008