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First published online 27 March 2007
doi: 10.1242/jcs.000968

Journal of Cell Science 120, 1330-1340 (2007)
Published by The Company of Biologists 2007
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Oocytes determine cumulus cell lineage in mouse ovarian follicles

Francisco J. Diaz, Karen Wigglesworth and John J. Eppig*

The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA


Figure 1
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  		Fig. 1. Model depicting follicular growth and cumulus expansion. Preantral follicles under the stimulation of gonadotropins and oocyte factors develop into antral follicles. The formation of the antrum separates the granulosa cells into mural and cumulus cells. Mural cells (green) express Lhcgr, Cyp11a1 and Cd34 transcripts more highly than cumulus cells (red), whereas Slc38a3, Amh and Ar transcripts are highly expressed in cumulus cells. At ovulation, the LH surge induces cumulus expansion with associated increases in expansion-related transcripts (Has2, Ptgs2, Ptx3 and Tnfaip6). Real-time PCR analysis of mural and cumulus marker transcripts in freshly isolated COCs and mural cells is also shown. Transcripts were normalized to Rpl19 mRNA. *Indicates significant difference by Student's t-test P<0.05.

 

Figure 2
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  		Fig. 2. Immunolocalization of pSMAD2 in preantral (A), transitional preantral-antral (B) and antral follicles before (C) and after (D) 8 hours hCG (5 IU) treatment in vivo. (E) No staining was observed when the primary antibody was pre-incubated with pSMAD2 phosphopeptide. Arrows indicate cumulus-oocyte complexes. Bars, 50 µm. (F) Western blot immunostained for pSMAD2, total SMAD2 and ACTB in freshly isolated COCs and mural cells from antral follicles.

 

Figure 3
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  		Fig. 3. Oocytes stimulate pSMAD2 in cumulus cells. Western blot immunostained for pSMAD2 and ACTB in COCs cultured in media alone or with SB431542 (10 µM) and in OOX cells cultured alone, OOX cells co-cultured with FGO and co-cultured OOX cells treated with SB431542 (10 µM) for 15 hours. Graphical representation of relative pixel intensity normalized to ACTB pixel intensity is also shown. a,bP<0.05, significant differences.

 

Figure 4
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  		Fig. 4. Specificity of inhibitors. (A) Western blot of lysates collected from COCs treated with media only, SB431542 (10 µM) or SIS3 (20 µM) for 4 hours and immunostained for pSMAD2, total SMAD2, pSMAD3, pSMAD1/5/8 and ACTB proteins. (B) Western blot immunostained for pSMAD2 and ACTB of COCs treated with increasing concentration of SB431542 (0.1 µM to 100 µM) for 4 hours.

 

Figure 5
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  		Fig. 5. Regulation of mural transcripts Lhcgr, Cyp11a1 and Cd34. Effect of SB431542 (10 µM) (A) and SIS3 (20 µM) (B) on levels of mural transcripts in mural cells cultured alone or with FGOs (2 oocytes/µl) for 12 hours. Transcripts were measured by real-time PCR and normalized to Rpl19 mRNA. abcP<0.05, significant differences.

 

Figure 6
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  		Fig. 6. (A) Effect of OOX and FSH (100 ng/ml) on levels of mural transcripts in cumulus cells treated for 12 hours. Effect of SB431542 (10 µM) (B) or SIS3 (20 µM) (C) alone or in combination with FSH (100 ng/ml) on levels of mural transcripts in COCs cultured for 15 hours. Transcripts were measured by real-time PCR and normalized to Rpl19 mRNA. abcP<0.05, significant differences.

 

Figure 7
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  		Fig. 7. Regulation of cumulus transcripts (Slc38a3, Amh, and Ar) by oocytes. (A) Effect of OOX and oocyte co-culture on cumulus transcript levels in COCs and OOX cumulus cells cultured in vitro for 12 hours. Effect of SB431542 (10 µM) and FSH (100 ng/ml) (B) or SIS3 (20 µM) (C) on cumulus transcript levels in COCs cultured for 15 hours. Transcripts were measured by real-time PCR and normalized to Rpl19 mRNA. abcP<0.05, significant differences.

 

Figure 8
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  		Fig. 8. Effect of SB431542 (10 µM) (A) and SIS3 (20 µM) (B) on expression of cumulus transcripts in mural granulosa cells cultured alone or with FGOs (2 oocytes/µl) for 15 hours. Transcripts were measured by real-time PCR and normalized to Rpl19 mRNA. abcP<0.05, significant differences.

 

Figure 9
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  		Fig. 9. Involvement of SMAD signaling in cumulus expansion. (A) Effect of medium only, SB431542 (10 µM) or SIS3 (20 µM) on cumulus expansion in COCs treated with EGF (10 ng/ml) for 15 hours. White arrows indicate cumulus cells attached to the culture dish in COC treated with SIS3. (B) Expansion of COCs treated with EGF (10 ng/ml) alone or with 1 µM SB431542 or 0.1 µM SB431542 for 15 hours. (C) Effect of SB431542 (10 µM) or SIS3 (20 µM) on expression of cumulus expansion transcripts in COCs treated with EGF (10 ng/ml) for 6, 8 and 10 hours. Transcripts were measured by real-time PCR and normalized to Rpl19 mRNA. abcP<0.05, significant differences.

 

Figure 10
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  		Fig. 10. (A) Western blot immunostained for pSMAD2, pMAPK14, pMAPK3/1 and ACTB in COCs treated with EGF (10 ng/ml) alone or together with SB431542 (10 µM) for 4 hours. Graphical representations of pSMAD2, MAPK14 and MAPK3/1 pixel intensity normalized to ACTB are shown below a representative western blot (n=3). (B) Western blot immunostained for pSMAD2, total SMAD2 and ACTB in COCs treated with EGF (10 ng/ml) for 0, 4, 8 and 12 hours. Graphical representations of pSMAD2, total SMAD2, MAPK14 and MAPK3/1 pixel intensity, normalized to ACTB, are shown below a representative western blot (n=4). Note that each image shown is of the same blot sequentially stained for the proteins indicated. abcP<0.05, significant differences.

 

Figure 11
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  		Fig. 11. Summary model depicting the opposing gradients of oocyte-stimulated SMAD2 and FSH signaling in the antral follicle that result in divergent expression of mural and cumulus transcripts. Cumulus cells (red) form under the influence of the oocyte secreted factors (OSPs), whereas mural cells (green) form under the influence of FSH. Granulosa cells located in between oocyte and FSH signals exhibit intermediate levels of mural and cumulus transcripts (light green or pink) depending on their proximity to the basal lamina or the oocyte.

 

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© The Company of Biologists Ltd 2007