Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 1. Individual effects of BMP15 or GDF9 on preantral follicle growth in vitro. Follicles were mechanically isolated and cultured individually in 96-well plates for 72 h in a range of concentrations of BMP15 (a,b) or GDF9 (c,d) and with 10ng/ml FSH (b,d). Variation in follicle size and morphology exposed to different treatments are shown (e). Values are means SEM of 4-6 cultures. Comparisons between treatment groups were made within each time point by ANOVA and Bonferroni multiple comparisons test where *P .05 and **P .01 vs 0ng/ml.

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: In Vitro, Isolation, Cell Culture

Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 2. Combined effects of BMP15 and GDF9 on preantral follicle growth in vitro. Follicles were mechanically isolated and cultured individually in 96-well plates in BMP15 (250ng/ml), GDF9 (250ng/ml) or equal amounts (250ng/ml) of both and overall size relative to control was assessed at each time point (a). The co-operative effect of both ligands on follicle growth was assessed further using a fixed concentration of GDF9 (250ng/ml) and a range of concentrations of BMP15 (0-250ng/ml) (b). Values are means SEM (n 8 (a) and n 5 (b) cultures). Comparisons between treatment groups were made within each time point by ANOVA and Bonferroni multiple comparisons test where *P .05 and **P .01 vs control.

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: In Vitro, Isolation, Cell Culture, Control, Concentration Assay

Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 3. Effect of BMP15 and/or GDF9 on GC proliferation and apoptosis in cultured preantral follicles. Follicles were fixed after 24 or 48 h culture with BMP15 (250ng/ml), GDF9 (250ng/ml) or equal amounts (250ng/ml) of both. Sections of follicles were stained for Ki67 (green) or cleaved caspase-3 (red) and counterstained with DAPI (blue), examples at 24h are shown (a). Positive staining in the GC compartment was quantified for Ki67 and cleaved caspase-3 at 24h (b) and 48h (c). Values are means SEM (n 4-6 follicles/group). *P .05, **P .01 vs control (Cntrl). Scale bar represents 50 m.

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: Cell Culture, Staining, Control

Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 4. Candidate gene expression in cultured preantral follicles exposed to BMP15 and/or GDF9. Preantral follicles were cultured for 24 h in BMP15 (250ng/ml), GDF9 (250ng/ml) or equal amounts (250ng/ml) of both. Samples from each group consist of four pooled follicles that were snap frozen and analyzed by qRT-PCR. Relative gene expression was determined by normalization against Atp5b as described in Materials and Methods and expressed as fold changes relative to untreated control samples (Cntrl). Values are means SEM (n 4 ovaries/cultures). Groups with different letters are statistically different (P .05).

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: Gene Expression, Cell Culture, Quantitative RT-PCR, Control

Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 6. Effect of dorsomorphin (DSM) on preantral follicle growth in the presence of BMP15. Follicles were cultured for 72 h in the presence of BMP15 (250ng/ml) or BMP15 (250ng/ml) DSM (1.0 M) and follicle size was assessed in relation to controls at each time point (a). In a separate experiment follicles were cultured for 48h in the presence of both BMP15 and GDF9 (250ng/ml each) and follicle size was assessed in relation to those cultured with both ligands and DSM (1.0 M) (b). Values are means SEM (n 4 cultures each). *P .05, **P .01 vs Control.

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: Cell Culture, Control

Journal: Endocrinology

Article Title: Investigations of TGF-β signaling in preantral follicles of female mice reveal differential roles for bone morphogenetic protein 15.

doi: 10.1210/en.2012-2251

Figure Lengend Snippet: Figure 7. Effect of SB505124 on preantral follicle growth in vitro. Follicles were mechanically isolated and cultured individually in 96-well plates for 72 h to test the effects of SB505124 on follicle growth in the presence or absence of GDF9 (a), BMP15 (b) or the combination of BMP15 GDF9 (c). All values shown are means SEM (n 6, 4 and 6 cultures for a, b and c respectively). Comparisons between treatment groups were made within each time point by ANOVA and Bonferroni multiple comparisons test. Groups with different letters are statistically different (P .05).

Article Snippet: Follicles were exposed to a range of concentrations of recombinant mouse (rm) GDF9 or recombinant human (rh) BMP15 (R&D Systems Inc., Abingdon, UK).

Techniques: In Vitro, Isolation, Cell Culture

Journal: Endocrinology

Article Title: Heparan sulfate proteoglycans regulate responses to oocyte paracrine signals in ovarian follicle morphogenesis.

doi: 10.1210/en.2012-1181

Figure Lengend Snippet: FIG. 1. Inhibition of cumulus expansion by heparin is restored by GDF9 treatment. Cumulus oocyte complexes were cultured under in vitro maturation conditions (MEM 2% fetal calf serum, 10 mIU/ml FSH, 10 ng/ml Egf) without or with exogenous heparin (300 g/ml) or heparin Gdf9 (500 ng/ml). A–C, Representative images of control (A), heparin-treated (B), and heparin GDF9 (C)-treated COC after 12 h culture. CEI was scored by a trained observer blinded to treatments. Mean SEM of CEI was from three independent experiments (D). Values indicated by different characters are significantly different P 0.001 by one-way ANOVA and Tukey’s post hoc test.

Article Snippet: In the indicated experiments, IVM cultures used either the above control conditions, or included heparin (300 g/ml; Sigma catalog no. H3149) or heparin GDF9 (R&D Systems, Minneapolis, MN; catalog no. 739-G9, 500 ng/ml).

Techniques: Inhibition, Cell Culture, In Vitro, Control

Journal: Endocrinology

Article Title: Heparan sulfate proteoglycans regulate responses to oocyte paracrine signals in ovarian follicle morphogenesis.

doi: 10.1210/en.2012-1181

Figure Lengend Snippet: FIG. 2. Heparin blocks cumulus-specific gene expression and signal transduction mediated by GDF9. A–D, Expression levels of cumulus-specific Has2 (A) and Tnfaip6 (B) or mural granulosa cell specific; Cyp11a1 (C) and Lhcgr (D) transcripts were evaluated after 12 h culture in no treatment (control), heparin (300 g/ml), or heparin GDF9 (500 ng/ml). Expression levels are normalized to rpL19 mRNA loading control assayed in parallel and expressed relative to expression in a pool of whole-ovary calibrator RNA sample. Values indicated by asterisks are significantly different (P 0.05) by using one-way ANOVA and Tukey’s post hoc test. E, Phosphorylated SMAD2 abundance in extracts of COC after 6 h culture in the indicated treatments. Hep, Heparin. Equivalent protein content was loaded in each lane, and -actin was evaluated to confirm equal sample loading. F, Venn diagram illustrating the number of gene transcripts identified in microarray analysis with significantly up- or down-regulated after heparin or SB431542 treatment compared with controls. Numbers indicate the number of transcripts with significantly up- or down-regulated expression level in pairwise comparisons against controls. Overlapping areas represent the number of transcripts whose levels are commonly altered by both treatments. Genes with significantly altered steady-state expression were defined by P 0.05 after ANOVA with step-up adjustment and greater than 2-fold change in expression level (n 4 independent experiments). Mwt, Molecular weight.

Article Snippet: In the indicated experiments, IVM cultures used either the above control conditions, or included heparin (300 g/ml; Sigma catalog no. H3149) or heparin GDF9 (R&D Systems, Minneapolis, MN; catalog no. 739-G9, 500 ng/ml).

Techniques: Gene Expression, Transduction, Expressing, Control, Microarray, Molecular Weight

Journal: Endocrinology

Article Title: Heparan sulfate proteoglycans regulate responses to oocyte paracrine signals in ovarian follicle morphogenesis.

doi: 10.1210/en.2012-1181

Figure Lengend Snippet: FIG. 3. Mouse and human GDF9 bind to heparin with high affinity. IMAC-purified murine (upper panel) or human (lower panel) recombinant GDF9 condition media were incubated with heparin sepharose then eluted in stepwise increasing NaCl concentrations as described in Materials and Methods. Samples were separated on reducing SDS-PAGE gel and GDF9 was detected by Western blot (described in Supplemental Methods) probing with mAb-53 directed against the mature peptide. M, Molecular weight marker (sizes in kilodaltons indicated on the left). SM, Starting material; FT, flowthrough. Arrowheads represent GDF9 (bands at 60 kDa are the intact promature protein; bands at 17 kDa are the processed mature monomer). The lower molecular band is likely a degradation product.

Article Snippet: In the indicated experiments, IVM cultures used either the above control conditions, or included heparin (300 g/ml; Sigma catalog no. H3149) or heparin GDF9 (R&D Systems, Minneapolis, MN; catalog no. 739-G9, 500 ng/ml).

Techniques: Purification, Recombinant, Incubation, SDS Page, Western Blot, Molecular Weight, Marker

Journal: Endocrinology

Article Title: Heparan sulfate proteoglycans regulate responses to oocyte paracrine signals in ovarian follicle morphogenesis.

doi: 10.1210/en.2012-1181

Figure Lengend Snippet: FIG. 6. GDF9 regulates proteoglycan gene expression in COC. Expression of proteoglycans, Glypican 1, Syndecan 4, and Betaglycan, was evaluated in COC after 12 h IVM culture [MEM EGF (10 ng/ml) and FSH (10 mIU/ml)] either with no treatment (control) or treated with heparin (300 g/ml) or heparin GDF9 (500 ng/ml). Expression levels are normalized to rpL19 mRNA loading control assayed in parallel and presented as expression relative to a calibrator sample of whole-ovary RNA. Values are mean SEM of three independent experiments. Those with different characters are significantly different (P 0.05). #, P 0.0549, one-way ANOVA and Tukey’s post hoc test.

Article Snippet: In the indicated experiments, IVM cultures used either the above control conditions, or included heparin (300 g/ml; Sigma catalog no. H3149) or heparin GDF9 (R&D Systems, Minneapolis, MN; catalog no. 739-G9, 500 ng/ml).

Techniques: Gene Expression, Expressing, Control