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 6 June 2007
doi: 10.1242/dev.006882

Development 134, 2593-2603 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow A corrigendum has been published
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 Development
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 Sugiura, K.
Right arrow Articles by Eppig, J. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sugiura, K.
Right arrow Articles by Eppig, J. J.

Oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells

Koji Sugiura1, You-Qiang Su1, Francisco J. Diaz1, Stephanie A. Pangas2, Shweta Sharma3, Karen Wigglesworth1, Marilyn J. O'Brien1, Martin M. Matzuk2,4,5, Shunichi Shimasaki3 and John J. Eppig1,*

1 The Jackson Laboratory, Bar Harbor, ME 04609, USA.
2 Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA.
3 Department of Reproductive Medicine, University of California San Diego School of Medicine, La Jolla, CA 92093-0633, USA.
4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
5 Departments Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.


Figure 1
View larger version (69K):
[in this window]
[in a new window]

  		Fig. 1. Expression of Pfkp and Ldha mRNA and glycolysis in Gdf9+/- Bmp15-/- (double mutant, DM) and Bmp15-/- cumulus cells. (A) Localization of Pfkp and Ldha mRNA was detected by in situ hybridization using 22-day-old eCG-primed, either WT or DM, mice. CC, cumulus cells; MG, mural granulosa cells; O, oocytes; TC, theca cells. (Top) Brightfield images. (Bottom) Darkfield images. Scale bar: 500 µm. (B) Relative mRNA levels of Pfkp and Ldha in cumulus cells of WT, DM, Bmp15-/- or Gdf9+/- mice were examined using real-time PCR. (C) Relative glycolytic activity in COCs of WT, DM, Bmp15-/- or Gdf9+/- mice was measured as their ability to metabolize [5-3H]-glucose to 3H2O. Mean±s.e.m. The values indicated by different letters (a and b) are significantly different (P<0.05).

 

Figure 2
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 2. Effect of oocytes in promoting expression of Pfkp and Ldha mRNA and glycolytic activity in WT OOX cumulus cells. OOX cumulus cells of WT mice were co-cultured with oocytes of WT, DM, Bmp15-/- or Gdf9+/- mice for 20 hours; (A) relative mRNA levels of Pfkp and Ldha, and (B) glycolytic activity in the OOX cumulus cells of WT mice were examined. (C) Expression levels of Pfkp and Ldha mRNA in freshly isolated cumulus cells (Fresh), COCs and OOX cumulus cells cultured without (OOX) or with (+Oocyte) oocytes for 20 hours were assessed by real-time PCR analysis. Mean±s.e.m. The values indicated by different letters (a, b and c) are significantly different (P<0.05). (D) OOX cumulus cells of WT mice were co-cultured with oocytes of WT or Bmp15-/- mice, and phosphorylation status of SMAD1/5/8 and SMAD2 in the OOX cumulus cells of WT mice was examined. ß-actin (ACTB) was used as a loading control. (E) Expression levels of Bmp15, Gdf9, Fgf8, Tgfb2 and Bmp6 mRNA were compared between WT and Bmp15-/- oocytes. N.D., not detected. Mean±s.e.m.

 

Figure 3
View larger version (38K):
[in this window]
[in a new window]

  		Fig. 3. Inability of recombinant BMP15 alone, or in combination with GDF9, to stimulate expression of Pfkp or Ldha mRNA in cumulus cells. Recombinant BMP15 suppressed FSH-induced Lhcgr mRNA expression in mural granulosa cells and promoted cumulus expansion, but did not promote expression of Pfkp and Ldha mRNA in OOX cumulus cells. (A) Mural granulosa cells were cultured with recombinant BMP15 or oocytes (2 oocytes/µl) in the presence of FSH, and Lhcgr mRNA expression in the mural granulosa cells was examined. (B) OOX cumulus cells were treated with recombinant BMP15 or oocytes (2 oocytes/µl) in the presence of EGF, and degree of cumulus expansion was examined. (C) Representative photographs of expanded OOX cumulus cells treated with oocytes (left) or recombinant BMP15 (right). Scale bar: 500 µm. (D) OOX cumulus cells were treated with recombinant BMP15 or oocytes (2 oocytes/µl) and expression levels of Pfkp and Ldha mRNA in OOX cumulus cells were examined. (E) OOX cumulus cells were treated with recombinant BMP15 (500 ng/ml), GDF9 (500 ng/ml) or both, or co-cultured with oocytes (2 oocytes/µl) and expression levels of Pfkp and Ldha mRNA in OOX cumulus cells were examined. Mean±s.e.m. The values indicated by different letters (a, b, c and d) are significantly different (P<0.05).

 

Figure 4
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 4. Inability of recombinant TGFB2 and GDF9 to affect Pfkp and Ldha mRNA levels in cumulus cells. Although recombinant TGFB2 and GDF9 promoted cumulus expansion and recombinant GDF9 suppressed Lhcgr mRNA expression in mural granulosa cells, they did not promote expression of Pfkp and Ldha mRNA in OOX cumulus cells. OOX cumulus cells were treated with recombinant TGFB2 (A,B), GDF9 (D,E) or oocytes (2 oocytes/µl) and (A,D) degree of cumulus expansion and (B,E) expression levels of Pfkp and Ldha mRNA in OOX cumulus cells were examined. (C) Mural granulosa cells were cultured with recombinant GDF9 or oocytes (2 oocytes/µl), and Lhcgr mRNA expression in the mural granulosa cells was examined. Mean±s.e.m. The values indicated by different letters (a, b and c) are significantly different (P<0.05).

 

Figure 5
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 5. Effect of blocking contact between oocytes and cumulus cells on the elevated Pfkp and Ldha mRNA expression in OOX cumulus cells cultured with oocytes in vitro. (A) Contact between OOX cumulus cells and oocytes was prevented with a glass capillary. (B) A representative photograph of the culture condition. Scale bar: 200 µm. (C) Relative mRNA levels of Pfkp and Ldha in cumulus cells cultured alone (None), with oocytes (+Oocyte), or with oocytes but with contact prevented with the glass capillary [+Oocyte (no contact)]. The glass capillary was present in all cultures, even when there were no oocytes (None) or contact was allowed (+Oocyte). Mean±s.e.m. The values indicated by different letters (a, b) are significantly different (P<0.05).

 

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

  		Fig. 6. Expression of Fgf8b mRNA in oocytes. (A) Fgf8 mRNA was detected in oocytes in ovaries of both 12-day-old (12 day) and 22-day-old eCG-primed (22 day eCG) mice by in situ hybridization. (Left) Brightfield images. (Right) Darkfield images. Scale bar: 500 µm. (B) Schematic diagram of eight different Fgf8 isoforms, Fgf8a-h [modified from MacArthur et al. (MacArthur et al., 1995aGo)]. Positions of PCR primers used are indicated in the expanded region. (C) Oocyte cDNA were amplified with PCR and the products were separated by agarose electrophoresis. A representative gel photograph after ethidium bromide staining is shown. Approximate sizes are shown on the y-axis (bp). The numbers in parentheses indicate expected size of PCR products (bp).

 

Figure 7
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 7. Requirement for FGF signals to promote the expression of Pfkp and Ldha mRNA in OOX cumulus cells. (A) Dose-response of FGF8. OOX cumulus cells were cultured with (black circles) or without (white circles) oocytes (0.1 oocytes/µl) and treated with recombinant FGF8B, and expression levels of Pfkp and Ldha mRNA in the OOX cumulus cells were examined. As a control group, Pfkp and Ldha mRNA expression in OOX cumulus cells co-cultured with oocytes (2 oocytes/µl) is shown (black bars). (B) Effect of SU5402. OOX cumulus cells were co-cultured with oocytes (0.5 oocytes/µl) in the presence of either DMSO or SU5402 for 24 hours, and relative mRNA levels of Pfkp and Ldha in OOX cumulus cells were examined. (C) Reversibility of the effect of SU5404. OOX cumulus cells were co-cultured with oocytes (0.5 oocytes/µl) in the presence of either DMSO or SU5402. After 24 hours of culture, the OOX cumulus cells were washed thoroughly and cultured in a fresh medium for an additional 20 hours with freshly isolated oocytes (0.5 oocytes/µl), and relative mRNA levels of Pfkp and Ldha in the OOX cumulus cells were examined. Mean±s.e.m. The values indicated by different letters (a, b, c and d) are significantly different (P<0.05).

 

Figure 8
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 8. Co-treatment of recombinant BMP15 and FGF8B promoted expression of Pfkp and Ldha mRNA and glycolytic activity in OOX cumulus cells. OOX cumulus cells were treated with recombinant FGF8B (0, 10 or 100 ng/ml) with or without either BMP15 (500 ng/ml) or GDF9 (500 ng/ml), or co-cultured with oocytes (2 oocytes/µl), and (A) expression levels of Pfkp and Ldha mRNA and (B) glycolytic activity in OOX cumulus cells were examined. Mean±s.e.m. The values indicated by different letters (a, b and c) are significantly different (P<0.05).

 




© The Company of Biologists Ltd 2007