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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
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 Related articles in JCS
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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bomar, J.
Right arrow Articles by Collas, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bomar, J.
Right arrow Articles by Collas, P.
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?

Differential regulation of maternal and paternal chromosome condensation in mitotic zygotes

Jacqueline Bomar1, Pedro Moreira1, John J. Balise1 and Philippe Collas2,*

1 Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA
2 Institute of Medical Biochemistry, University of Oslo, PO Box 1112 Blindern, Oslo 0317, Norway



View larger version (51K):

[in a new window]
  		Fig. 1. Distribution of AKAP95 in mouse somatic cells, gametes and embryos. (A) Immunofluorescence analysis of AKAP95 in mouse cumulus cells and in situ-prepared nuclear matrices after methanol fixation, using an affinity-purified anti-rat AKAP95 antibody. DNA was counterstained with Hoechst 33342. (B) HeLa cells and mouse cumulus cells were immunoblotted using anti-AKAP95 antibodies with or without a competitor AKAP95(387-692) peptide. (C) Immunoblotting analysis of AKAP95 in MII oocytes, sperm, DTT-treated decondensed sperm `halos' and PN embryos. Protamines were immunoblotted to demonstrate availability of chromatin sperm antigens on blots.

(D) Immunofluorescence distribution of AKAP95 in MII oocytes and in vitro cultured preimplantation embryos. Arrowheads point to MII chromosomes. Arrows point to a metaphase blastomere. (E) MII oocytes were activated with either 10 mM SrCl2, 10 µg/ml cycloheximide (CHX) or 10 mM SrCl2 together with 5 µg/ml actinomycin D. Activated oocytes were fixed at the PN stage and labeled using anti-AKAP95 antibodies. Insets, MII and SrCl2-activated oocytes were immunoblotted using anti-AKAP95 antibodies. DNA was stained with Hoechst 33342. FPN and MPN, female and male pronucleus, respectively. PB, polar body. Bars, 10 µm (A); 20 µm (D,E).

 


View larger version (33K):

[in a new window]
  		Fig. 2. RNA in situ hybridization analysis of AKAP95 message in MII oocytes. Oocytes were fixed and hybridized with a biotinylated probe against rat AKAP95 cDNA. Hybridization was detected with TRITC-conjugated avidin (red). DNA was counterstained with 0.2 µg/ml Hoechst 33342 (blue). Samples were also pre-treated with DNAse I (+DNAse) or 100 µg/ml RNAse A (+RNAse) prior to fixation. Note the restricted localization of AKAP95 mRNA around the meiotic spindle. PB, polar body. Dotted circles delineate the oocyte. Bar, 20 µm.

 


View larger version (25K):

[in a new window]
  		Fig. 3. Distribution of mCAP-D2 in mouse gametes. (A) Immunoblotting analysis of mCAP-D2 in indicated cell types using an affinity-purified antibody against hCAP-D2. (B) Immunofluorescence localization of mCAP-D2 in MII oocytes. Arrowhead points to MII chromosomes. DNA was stained with Hoechst 33342. PB, polar body. Bar, 20 µm.

 


View larger version (46K):

[in a new window]
  		Fig. 4. AKAP95 associates with maternal chromosomes at first mitosis, whereas mCAP-D2 is recruited to both chromosome complements. (A) Sperm were labeled with Hoechst 33342 (blue) before ICSI into oocytes. Resulting ICSI embryos were fixed at the PN stage, total DNA was labeled with PI (red) and AKAP95 localized by immunofluorescence (green). Merge image shows AKAP95 labeling restricted to the FPN (yellow). (B,C) Mitotic zygotes produced by ICSI of Hoechst-labeled sperm as in A were fixed and AKAP95 (B) and mCAP-D2 (C) were examined by immunofluorescence. (D) Mitotic embryos were also double-labeled with anti-AKAP95 mAb47 and anti-hCAP-D2 antibodies. `F' and `M', female and male chromatin, respectively; PB, polar body. Bars, 20 µm.

 


View larger version (74K):

[in a new window]
  		Fig. 5. AKAP95 is required for condensation of maternal chromosomes. (A) Zygotes produced by ICSI of Hoechst-labeled sperm were injected into the FPN or the MPN with 250 pg AKAP95(387-450). Embryos were fixed, counterstained with PI (red) and labeled using anti-AKAP95 polyclonal antibodies (green). (B-D) Injected embryos were cultured to mitosis, fixed and analyzed by DNA staining and AKAP95 immunofluorescence using the polyclonal antibody or mAb47 (insets in B and C only). (B) Mitosis after MPN injection. (C) Mitosis after FPN injection. (D) Mitosis after FPN injection with 250 pg AKAP95(387-692). `F' and `M', female and male chromatin, respectively; PB, polar body. Dotted circles delineate embryos. Bar, 20 µm.

 


View larger version (51K):

[in a new window]
  		Fig. 6. AKAP95 is required for mCAP-D2 targeting to maternal, but not paternal, chromatin. (A,B) Injection of AKAP95(387-450) in the FPN abolishes mCAP-D2 targeting to female chromatin. The FPN of zygotes produced as in Fig. 5A was injected with (A) 250 pg AKAP95(387-450) or (B) 250 pg AKAP95(387-692). Embryos progressed to mitosis and were examined by total DNA staining with PI (red) and mCAP-D2 immunofluorescence (green). (C,D) AKAP95(387-692) rescues condensation of maternal chromosomes. Mitotic embryos with decondensed female chromatin and condensed male chromosomes were cytoplasmically injected with (C) 5 ng AKAP95(387-692) or (D) 5 ng AKAP95(387-450) as a negative control. Embryos were fixed 2 hours later and analyzed by DNA labeling with PI and by immunofluorescence using the anti-AKAP95 polyclonal antibody. Arrowheads point to female chromatin. Color coding for C and D is shown. `F' and `M', female and male chromatin, respectively. Dotted circles delineate the embryos. Bar, 20 µm.

 


View larger version (20K):

[in a new window]
  		Fig. 7. Male chromosomes condense and segregate at mitosis in the absence of AKAP95. (A) The FPN of normal fertilized PN stage embryos was mechanically removed and (B) resulting embryos were allowed to enter mitosis and (C) cleave to the two-cell stage. Indicated stages were examined by DNA staining with PI (red) and immunofluorescence analysis of AKAP95 (green). Merged fluorescence images are shown. Dotted circles delineate embryos or blastomeres. PB, polar body. Bar, 20 µm.

 


View larger version (14K):

[in a new window]
  		Fig. 8. AKAP95 is required for cleavage beyond the two-cell stage. The nucleus of one blastomere of a two-cell stage embryo was injected with 250 pg AKAP95(387-450) in a buffer containing a 150 kDa FITC-dextran as a tracer (arrow), while the second blastomere was injected with unlabeled peptide buffer. Each nucleus of control two-cell stage embryos was also mock-injected with buffer (Mock-injected, right panel). Embryos were allowed to cleave to the 8-16-cell stage. Nuclei were labeled with Hoechst 33342. Arrowheads point to a metaphase plate in the cleaving buffer-injected blastomere (Mitosis). PB, polar body. Bar, 20 µm.

 

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 2002