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First published online 28 November 2007
doi: 10.1242/dev.009068

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Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells

¹ The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA.
² Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA.
³ Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
⁴ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Venn diagram illustrating the number of unique transcripts whose steady-state level of expression is changed in mutant cumulus cells. Numbers indicate the number of transcripts (Unigene IDs) whose levels are changed significantly in pairwise comparisons. Overlapping areas of two or three circles represent the number of transcripts whose levels are commonly changed in the corresponding two- or three-comparison tests.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Real-time RT-PCR analysis of transcripts selected from microarray expression profiles. (A-J) Five categories of transcripts were selected for real-time RT-PCR analysis: transcripts that were most dramatically up-(A,F), or down-(B,G) regulated in mutant cumulus cells; transcripts that were specifically involved in certain metabolic pathways, e.g. sterol biosynthesis (C,H), bile acid biosynthesis (D,I), and glycolysis, purine metabolism, pyrimidine metabolism, pentose phosphate, and fructose and mannose metabolism (E,J). Black bars indicate expression levels detected by microarray, white bars indicate levels detected by real-time RT-PCR. Four sets of cumulus cell samples from each genotype were used for real-time PCR analysis and data are presented as mean ± s.e.m. of fold changes. *P<0.05; DM vs WT, or Bmp15-/- vs WT.

View larger version (39K): [in this window] [in a new window]   Fig. 3. The most highly affected pathways and functions in mutant cumulus cells. The 744 transcripts that were commonly changed in all three pairwise comparisons of cumulus transcriptomes in WT, Bmp15-/-, and DM mice were uploaded into the IPA platform, and canonical pathways and molecular functions analyses were carried out using Ingenuity Pathways Knowledge Base as reference dataset. (A) All canonical pathways identified that were significantly affected. (B) The 10 most affected molecular functions. The orange vertical line crossing all the bars in A and B indicates the threshold of significance (P=0.05), and bars above this line have a P-value of less than 0.05. (C) GenMAPP display of transcripts encoding enzymes required for cholesterol biosynthesis pathway. The list of all the genes on the array was uploaded into GenMAPP, and significantly downregulated transcripts were defined by the criteria of FC (fold change) <-1 and Fs P<0.01 in all three pairwise comparison analyses, and are shown in orange boxes. Upregulated transcripts are defined by the criteria of FC>1 and Fs P<0.01 in all three pairwise comparison analyses. No transcripts are identified to be upregulated by these criteria. Dotted boxes indicate that the transcripts in these boxes are represented by >1 probe set. The FC of each transcript is listed on the right side of the corresponding box. Only the FCDM vs WT of each transcript is shown here owing to space limitation. Minor modification of the original MAPP in GenMAPP was made here in order to cover most of the key enzymatic steps in this pathway, such as steps for producing FF-MAS and T-MAS. Panel C, by Michael Lieberman and Ned Mantei (2004), is reproduced from GenMAPP.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Effect of WT oocytes on expression of selected transcripts in WT OOX cumulus cells. WT COCs, OOX cumulus cells, and OOX cumulus cells + oocytes [two fully grown oocytes (FGO)/µl] were cultured for 20 hours, and expression of selected transcripts was detected by real-time RT-PCR using Rpl19 mRNA as internal control. (A) Transcripts upregulated in cumulus cells of both mutants. (B) Transcripts downregulated in cumulus cells of both mutants. (C) Transcripts encoding enzymes for cholesterol biosynthesis. (D) Transcripts involved in bile acid biosynthesis pathway. (E) Transcripts involved in other metabolic pathways: glycolysis, purine metabolism, pyrimidine metabolism, pentose phosphate, fructose and mannose metabolism, and inositol metabolism. Experiments were repeated three times. Data are presented as mean of the relative fold change in mRNA expression compared with COC group (control) ± s.e.m. Bars indicated with different letters are significantly different, P<0.05.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Reduction of cholesterol synthesis in mutant COCs and WT OOX cumulus cells. Cholesterol synthesis was measured as incorporation of [14C]acetate into cholesterol during culture. (A) A representative TLC image comparing radiolabeled cholesterol (indicated by arrow) in WT (lane1), Bmp15-/- (lane 2) and DM (lane 3) COCs. (B) Quantitative comparison of [14C]cholesterol levels in WT and mutant COCs. (C) A representative TLC image showing relative [14C]cholesterol (indicated by arrow) levels in WT cumulus cells of COCs (lane1), OOX cumulus cells (lane 2) and OOX cumulus cells + oocytes (lane 3). (D) Quantitative comparison of [14C]cholesterol levels in WT cumulus cells of COCs, OOX cumulus cells and OOX cumulus cells + oocytes. All experiments were repeated at least three times independently. Data are presented as mean of relative fold change compared with control (WT in panels B, CC/COC in panels D) ± s.e.m. Bars indicated with different letters are significantly different, P<0.05.

View larger version (93K): [in this window] [in a new window]   Fig. 6. Comparison of expression of transcripts encoding enzymes required for cholesterol biosynthesis in oocytes and cumulus cells. (A) Comparison of mRNA levels of transcripts encoding enzymes required for cholesterol biosynthesis in oocytes and cumulus cells, relative to levels of Rpl19 mRNA expressed by those cell types. Experiment was repeated three times independently. Data are presented as mean of the fold change of the mRNA levels relative to levels in oocytes (given a value of 1) ± s.e.m. *P<0.05, compared with levels in oocytes. (B) In situ hybridization of transcripts encoding enzymes required for cholesterol biosynthesis. The four images in each row show localization of transcripts indicated on the left side. In each row, the first two panels (from left) are low magnification, bright- and dark-field images of a 22-day-old eCG-primed ovary; the last two panels are high magnification, bright- and dark-field images of large antral follicles from the same ovary. Cumulus cells and oocyte in each follicle are indicated by an arrowhead and an arrow, respectively. Scale bars: 200 µm.

View larger version (33K): [in this window] [in a new window]   Fig. 7. Comparison of levels of cholesterol synthesized in oocytes and/or cumulus cells under various experimental conditions. (A) Comparison of levels of cholesterol synthesized in WT cumulus-cell-enclosed oocytes (CEOs) and denuded oocytes (DOs). The left panel is a representative TLC image showing levels of [14C]cholesterol (indicated by arrow) production in CEO and DOs. The right panel is the quantitative comparison of [14C]cholesterol levels in CEOs and DOs. (B) Comparison of levels of cholesterol synthesized in oocytes of WT, DM cumulus-oocyte complexes (COCs), and DM COCs that were co-cultured with WT fully grown oocytes (FGOs). The left panel is a representative TLC image showing levels of [14C]cholesterol (indicated by arrow) produced by oocytes in each group. The right panel is the quantitative comparison of [14C]cholesterol levels in oocytes of each group. (C) Comparison of levels of cholesterol synthesized in cumulus cells of WT, DM COCs, and DM COCs that were co-cultured with WT FGOs. The left panel is a representative TLC image showing levels of [14C]cholesterol (indicated by arrow) produced by cumulus in each group. The right panel is the quantitative comparison of [14C]cholesterol levels produced by cumulus cells in each group. All experiments were repeated four times. Data are presented as mean of the relative fold change in [14C]cholesterol levels (levels in controls, i.e., CEO in A, WT in B and C,=1) ± s.e.m. Bars indicated with different letters are significantly different (P<0.05) using ANOVA and Tukey's HSD test. The asterisk indicates significant difference by Student's t-test (P<0.05).