Journal: Journal of Cell Science

Article Title: Oocyte-dependent activation of MTOR in cumulus cells controls the development and survival of cumulus–oocyte complexes

doi: 10.1242/jcs.182642

Figure Lengend Snippet: Suppression of Ddit4l mRNA expression in cumulus cells by oocytes, GDF9 and GDF9–BMP15 heterodimer. (A) qRT-PCR analysis of Ddit4l mRNA expression in cumulus cells of normal wild-type mouse COCs, oocytectomized cumulus cells (OOX) and oocytectomized cumulus cells co-incubated with F1 mouse fully grown oocytes (OOX+WT) that were cultured for 20 h. (B) qRT-PCR analysis of Ddit4l mRNA expression in cumulus cells of normal wild-type mouse COCs, oocytectomized cumulus cells and oocytectomized cumulus cells co-incubated with wild-type, Bmp15−/− and double-mutant mouse fully grown oocytes (designated as OOX+WT, OOX+Bmp15−/− and OOX+DM, respectively) that had been cultured for 20 h. (C) qRT-PCR analysis of Ddit4l mRNA expression in cumulus cells of normal wild-type mouse COCs, oocytectomized cumulus cells and oocytectomized cumulus cells treated with 100 ng/ml or 500 ng/ml recombinant mouse GDF9 (designated as G100 and G500, respectively) and cultured for 20 h. (D) qRT-PCR analysis of Ddit4l mRNA expression in cumulus cells of normal wild-type mouse COCs, oocytectomized cumulus cells and oocytectomized cumulus cells treated with increasing doses (0.35, 1, 3.5 ng/ml) of recombinant mouse GDF9–BMP15 heterodimer (designated as G:B) and cultured for 20 h. Data are presented as the mean±s.e.m. of fold changes relative to those of the COC group (n=3). Bars marked with different letters are significantly different, P<0.05 (one-way ANOVA followed by Tukey's HSD test).

Article Snippet: To test the effects of GDF9 and GDF9–BMP15 heterodimer on the expression of Ddit4l mRNA and phosphorylation of MTOR, RPS6KB1 and EIF4EBP1 protein in cumulus cells, oocytectomized cumulus cells were treated with 100- or 500-ng/ml recombinant mouse GDF9 (catalog no. 739-G9-010, R&D Systems) and/or various doses (0, 0.35, 1, 3.5 ng/ml) of recombinant mouse GDF9–BMP15 heterodimer (a gift from Prof. Martin M. Matzuk, Baylor College of Medicine, TX) and cultured for 20 h. To test the effect of inhibitors for SMAD 2 and/or SMAD3 on the expression of Ddit4l mRNA in cumulus cells of COCs, COCs were cultured in medium supplemented with or without 10 μM SB431542 or 20 μM SIS3 (EMD Biosciences) for 20 h. To test the effect of the same SMAD inhibitors on GDF9-controlled expression of Ddit4l mRNA, oocytectomized COCs cells were first treated with 10 μM SB431542 or 20 μM SIS3 for 1 h, and then treated together with 500 ng/ml GDF9 for another 19 h. The doses of SB431542 and SIS3 were chosen based on our previous studies ( Diaz et al., 2007b ; Su et al., 2010 ).

Techniques: Expressing, Quantitative RT-PCR, Incubation, Cell Culture, Mutagenesis, Recombinant

Journal: Journal of Cell Science

Article Title: Oocyte-dependent activation of MTOR in cumulus cells controls the development and survival of cumulus–oocyte complexes

doi: 10.1242/jcs.182642

Figure Lengend Snippet: Effects of SMAD2 and/or SMAD3 inhibitors on Ddit4l mRNA expression in cumulus cells. (A) qRT-PCR analysis of Ddit4l mRNA expression in cumulus cells of normal wild-type mouse COCs that had been treated with DMSO (designated as the ‘COC’ group), 10 μM SB431542 (COC+SB431542) or 20 μM SIS3 (COCs+SIS3) cultured for 20 h. (B) qRT-PCR analysis of Ddit4l mRNA expression in normal wild-type oocytectomized cumulus cells that had been treated with DMSO (the ‘OOX’ group), 500 ng/ml recombinant mouse GDF9 (OOX+G) or 500 ng/ml recombinant GDF9 in combination with 10 μM SB431542 (OOX+G+SB431542) or 20 μM SIS3 (OOX+G+SIS3) and cultured for 20 h. Data are presented as fold changes relative to control group (COC in A and OOX in B) and are shown as mean±s.e.m. (n=3). Bars marked with different letters are significantly different, P<0.05 (one-way ANOVA followed by Tukey's HSD test).

Article Snippet: To test the effects of GDF9 and GDF9–BMP15 heterodimer on the expression of Ddit4l mRNA and phosphorylation of MTOR, RPS6KB1 and EIF4EBP1 protein in cumulus cells, oocytectomized cumulus cells were treated with 100- or 500-ng/ml recombinant mouse GDF9 (catalog no. 739-G9-010, R&D Systems) and/or various doses (0, 0.35, 1, 3.5 ng/ml) of recombinant mouse GDF9–BMP15 heterodimer (a gift from Prof. Martin M. Matzuk, Baylor College of Medicine, TX) and cultured for 20 h. To test the effect of inhibitors for SMAD 2 and/or SMAD3 on the expression of Ddit4l mRNA in cumulus cells of COCs, COCs were cultured in medium supplemented with or without 10 μM SB431542 or 20 μM SIS3 (EMD Biosciences) for 20 h. To test the effect of the same SMAD inhibitors on GDF9-controlled expression of Ddit4l mRNA, oocytectomized COCs cells were first treated with 10 μM SB431542 or 20 μM SIS3 for 1 h, and then treated together with 500 ng/ml GDF9 for another 19 h. The doses of SB431542 and SIS3 were chosen based on our previous studies ( Diaz et al., 2007b ; Su et al., 2010 ).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Recombinant, Control

Journal: Journal of Cell Science

Article Title: Oocyte-dependent activation of MTOR in cumulus cells controls the development and survival of cumulus–oocyte complexes

doi: 10.1242/jcs.182642

Figure Lengend Snippet: ODPFs promote the differential activation of MTOR signaling in cumulus cells. (A,B) Western blot analysis of p-MTOR, p-RPS6KB1, p-EIF4EBP1 and ACTB in cumulus cells of COCs, oocytectomized cumulus cells (OOX) and oocytectomized cumulus cells co-cultured with normal wild-type fully grown oocytes (designated as OOX+FGO), or were treated with 500 ng/ml recombinant mouse GDF9 (designated as OOX+GDF9) or 3.5 ng/ml recombinant mouse GDF9–BMP15 heterodimer (designated as OOX+G:B) for 20 h. Representative western blot images are shown in A, and quantification of the signal intensity of each band from the western blot images is shown in B. Data are presented as the relative fold changes of signal intensity in each group compared to that in the COC after normalization to the corresponding ACTB expression values and are shown as mean±s.e.m. (n=3). Bars marked with different letters are significantly different, P<0.05 (one-way ANOVA followed by Tukey's HSD test). (C) Immunostaining of p-RPS6 on ovarian sections. The magnified images of a large antral follicle are shown on the right-hand side of the whole ovarian image, with arrows, arrowheads and asterisks pointing to cumulus, peri-antral and mural granulosa cells, respectively. p-RPS6 was stained in red, and DNA was stained in blue (4′,6-Diamidino-2-phenylindole, DAPI). Scale bars: 100 μm.

Article Snippet: To test the effects of GDF9 and GDF9–BMP15 heterodimer on the expression of Ddit4l mRNA and phosphorylation of MTOR, RPS6KB1 and EIF4EBP1 protein in cumulus cells, oocytectomized cumulus cells were treated with 100- or 500-ng/ml recombinant mouse GDF9 (catalog no. 739-G9-010, R&D Systems) and/or various doses (0, 0.35, 1, 3.5 ng/ml) of recombinant mouse GDF9–BMP15 heterodimer (a gift from Prof. Martin M. Matzuk, Baylor College of Medicine, TX) and cultured for 20 h. To test the effect of inhibitors for SMAD 2 and/or SMAD3 on the expression of Ddit4l mRNA in cumulus cells of COCs, COCs were cultured in medium supplemented with or without 10 μM SB431542 or 20 μM SIS3 (EMD Biosciences) for 20 h. To test the effect of the same SMAD inhibitors on GDF9-controlled expression of Ddit4l mRNA, oocytectomized COCs cells were first treated with 10 μM SB431542 or 20 μM SIS3 for 1 h, and then treated together with 500 ng/ml GDF9 for another 19 h. The doses of SB431542 and SIS3 were chosen based on our previous studies ( Diaz et al., 2007b ; Su et al., 2010 ).

Techniques: Activation Assay, Western Blot, Cell Culture, Recombinant, Expressing, Immunostaining, Staining

Journal: Reproduction (Cambridge, England)

Article Title: REGULATION OF AMH BY OOCYTE SPECIFIC GROWTH FACTORS IN HUMAN PRIMARY CUMULUS CELLS

doi: 10.1530/REP-17-0421

Figure Lengend Snippet: Cumulus cells were treated for 48 hours with vehicle (C), GDF9 (2.5, 10ng/ml), BMP15 (2.5, 10ng/ml), or the combination of GDF9 and BMP15 (0.1, 0.6, 2.5, 5, or 10ng/ml of each). Amh mRNA levels were determined by qPCR and expressed as relative to Rpl19. Columns represent the mean ± SEM, Columns with different letters differ significantly a–b P<0.05, a–c P<.001 vs controls, one-way ANOVA, Bonferroni test, n=15.

Article Snippet: Cumulus cells were treated with human recombinant GDF9 (R&D Systems), BMP15 (R&D Systems), and/or FSH (Serono) with or without specific inhibitors of intracellular signaling; H89, GF109203X (EMD Biosciences-Calbiochem), MK2206, LDN-193189 (Selleck Chemicals), U0126 (Millipore), or SB431542 (Tocris).

Techniques:

Journal: Reproduction (Cambridge, England)

Article Title: REGULATION OF AMH BY OOCYTE SPECIFIC GROWTH FACTORS IN HUMAN PRIMARY CUMULUS CELLS

doi: 10.1530/REP-17-0421

Figure Lengend Snippet: A) Cumulus cells from three different patients (Patient 1, 2, and 3) were treated with 5 ng/ml of both GDF9 and BMP15 (GB) for 48 hours. AMH β-actin (ACTB) protein levels were determined by Western blotting. On the left, the average (± SEM) of the relative optical density units of AMH to ACTB is shown (**P< 0.001, t-test, n=3). B) Cumulus cells from Patient 4 were treated with 2.5, 5, or 20 ng/ml of both GDF9 and BMP15. AMH protein levels were determined as in A. Expression of ACTB is shown as a loading control.

Article Snippet: Cumulus cells were treated with human recombinant GDF9 (R&D Systems), BMP15 (R&D Systems), and/or FSH (Serono) with or without specific inhibitors of intracellular signaling; H89, GF109203X (EMD Biosciences-Calbiochem), MK2206, LDN-193189 (Selleck Chemicals), U0126 (Millipore), or SB431542 (Tocris).

Techniques: Western Blot, Expressing, Control

Journal: Reproduction (Cambridge, England)

Article Title: REGULATION OF AMH BY OOCYTE SPECIFIC GROWTH FACTORS IN HUMAN PRIMARY CUMULUS CELLS

doi: 10.1530/REP-17-0421

Figure Lengend Snippet: Cumulus cells were treated for 48 hours with GDF9 and BMP15 (G+B; 5 ng/ml of each) in the presence or absence of FSH (50 ng/ml). Amh mRNA levels were determined by qPCR and expressed relative to Rpl19. Columns with different letters differ significantly a–b P<0.001, a–c P<0.05, b–c P<0.01, one-way ANOVA, Bonferroni test, n=11.

Article Snippet: Cumulus cells were treated with human recombinant GDF9 (R&D Systems), BMP15 (R&D Systems), and/or FSH (Serono) with or without specific inhibitors of intracellular signaling; H89, GF109203X (EMD Biosciences-Calbiochem), MK2206, LDN-193189 (Selleck Chemicals), U0126 (Millipore), or SB431542 (Tocris).

Techniques:

Journal: Reproduction (Cambridge, England)

Article Title: REGULATION OF AMH BY OOCYTE SPECIFIC GROWTH FACTORS IN HUMAN PRIMARY CUMULUS CELLS

doi: 10.1530/REP-17-0421

Figure Lengend Snippet: Cumulus cells were treated for 1 hour with the following specific SB431542 (SMAD2/3; 10 μM), or LDN-193189 (SMAD1/5/8; 100 nM) followed by a 48-hour co-treatment with GDF9 and BMP15 (5 ng/ml of each). Amh mRNA levels were determined 48 h after by qPCR and expressed relative to Rpl19. Columns with different letters differ significantly a–b P<0.001, one-way ANOVA, Bonferroni test, n=9.

Article Snippet: Cumulus cells were treated with human recombinant GDF9 (R&D Systems), BMP15 (R&D Systems), and/or FSH (Serono) with or without specific inhibitors of intracellular signaling; H89, GF109203X (EMD Biosciences-Calbiochem), MK2206, LDN-193189 (Selleck Chemicals), U0126 (Millipore), or SB431542 (Tocris).

Techniques: