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

Abstract

Communication between oocytes and their companion somatic cells promotes the healthy development of ovarian follicles, which is crucial for producing oocytes that can be fertilized and are competent to support embryogenesis. However, how oocyte-derived signaling regulates these essential processes remains largely undefined. Here, we demonstrate that oocyte-derived paracrine factors, particularly GDF9 and GDF9-BMP15 heterodimer, promote the development and survival of cumulus-cell-oocyte complexes (COCs), partly by suppressing the expression of Ddit4l, a negative regulator of MTOR, and enabling the activation of MTOR signaling in cumulus cells. Cumulus cells expressed less Ddit4l mRNA and protein than mural granulosa cells, which is in striking contrast to the expression of phosphorylated RPS6 (a major downstream effector of MTOR). Knockdown of Ddit4l activated MTOR signaling in cumulus cells, whereas inhibition of MTOR in COCs compromised oocyte developmental competence and cumulus cell survival, with the latter likely to be attributable to specific changes in a subset of transcripts in the transcriptome of COCs. Therefore, oocyte suppression of Ddit4l expression allows for MTOR activation in cumulus cells, and this oocyte-dependent activation of MTOR signaling in cumulus cells controls the development and survival of COCs.

Keywords: Apoptosis; Cumulus cells; DDIT4L; Female infertility; MTOR; Oocyte.

Fig. 1.

Upregulation of Ddit4l expression in mutant cumulus cells. (A) Measurements of the steady-state levels of Ddit4l mRNA in wild-type (WT), double-mutant (DM) and Bmp15^−/− cumulus cells by using microarray analysis (left bar graph) and quantitative real-time RT-PCR (qRT-PCR, right bar graph) analyses. Data are presented as mean±s.e.m. of fold changes relative to the wild-type group (n=3). Bars marked with different letters are statistically different, P<0.05 (one-way ANOVA followed by Tukey's HSD test). (B) Immunohistochemical detection of DDIT4L protein in the large antral follicles of wild-type and double-mutant ovaries. Arrows and asterisks indicate cumulus and mural granulosa cells, respectively. Scale bars: 100 μm. (C) Quantification of cumulus cells (CC) stained positively by a DDIT4L-specific antibody as shown in B. DDIT4L-positive and non-positive cumulus cells were counted, and the percentage of the positively staining cells relative to the total number of cumulus cells in the same follicle was calculated. A total of six large antral follicles per genotype was counted. *P<0.05 (t-test) compared with wild type.

Fig. 2.

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).

Fig. 3.

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).

Fig. 4.

Differential expression of Ddit4l in granulosa cells. Bright- (A) and dark- (B) field micrographs of ovarian sections after in situ hybridization analysis for Ddit4l mRNA. The magnified images of a large antral follicle are shown on the right-hand side of the whole ovarian images, with arrows and asterisks indicating cumulus and the mural granulosa cells, respectively. (C) qRT-PCR analysis of the levels of Ddit4l mRNA in isolated cumulus cells (CC) and mural granulosa cells (MGC). Data are presented as mean±s.e.m. (n=3) of fold changes relative to the CC group. *P<0.05 (t-test), compared with the CC group. (D) Micrographs of ovarian sections after immunohistochemical staining of DDIT4L protein. The magnified image of a large antral follicle is shown on the right-hand side of the whole ovarian image, with the arrow and asterisk pointing to cumulus and mural granulosa cells, respectively. Scale bars: 100 μm.

Fig. 5.

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.

Fig. 6.

Knockdown of Ddit4l in cumulus cells induces the activation of MTOR signaling. (A) qRT-PCR analysis of Ddit4l mRNA expression in cumulus cells before and after culture in monolayer for 24 h. Data are presented as the relative fold changes of Ddit4l mRNA in the cultured group (designated as 24 h) compared with the non-cultured group (designated as 0 h) and are shown as mean±s.e.m. (n=3). *P<0.05 (t-test), compared with the 0-h group. (B) qRT-PCR analysis of Ddit4l mRNA expression in monolayer-cultured cumulus cells after transfection with or without control siRNA or siRNA against Ddit4l. Data are presented as the relative fold changes of Ddit4l mRNA in the transfected group compared with that of the non-transfected group (designated as Control) 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,D) Western blot analysis of the expression of p-MTOR, p-RPS6KB1, p-EIF4EBP1 and ACTB in monolayer-cultured cumulus cells after transfection with or without control siRNA or Ddit4l siRNA. Representative western blot images are shown in C, and the quantitative analysis of the signal intensity of each band in the western blot images is shown in D. Data are presented as the relative fold changes of signal intensity in siRNA-transfected groups compared with those of the non-transfected groups (Control) after normalization with 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).

Fig. 7.

Inhibition of MTOR signaling in COCs compromises oocyte developmental competence and cumulus cell survival. (A) Western blot analysis of p-RPS6KB1, p-EIF4EBP1 and ACTB in COCs that had been treated for 14 h with various doses (i.e. 0, 1, 5 and 10 μM) of Torin1. One set of representative images is shown, and similar results were obtained in three independent experiments. (B) The effect of initially treating COCs for 24 h with various doses (i.e. 0, 1, 5 and 10 μM) of Torin1 on subsequent oocyte fertilization (left panel; percentage of two-cell-stage embryo calculated from the total number of MII- oocytes) and preimplantation development (right panel; percentage of blastocysts calculated from the total number of two-cell-stage embryos). Data 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,D) Analysis of apoptosis by TUNEL staining in COCs after being treated for 14 h with various doses (i.e. 0, 1, 5 and 10 μM) of Torin1. The quantification of TUNEL-staining-positive cells in COCs is shown in C. Data are presented as mean±s.e.m. (n=3) of the fold changes in the number of apoptotic cells in the treated groups relative to those in the Control group, which was treated only with DMSO. Bars marked with different letters are significantly different, P<0.05 (one-way ANOVA followed by Tukey's HSD test). Representative confocal microscope images of TUNEL-stained COCs are shown in D. Apoptotic cells are marked in magenta, and DNA is stained in blue (4′,6-Diamidino-2-phenylindole, DAPI). Scale bars: 100 μm.

Fig. 8.

Comparison of transcripts changed by MTOR inhibition in vitro and hCG administration in vivo, and qRT-PCR validation of a subset of transcripts identified by microarray analysis to be changed after MTOR inhibition. (A) Venn diagrams showing the transcripts that were commonly up- (left panel) and downregulated (right panel) through MTOR inhibition in vitro and hCG administration in vivo. (B) qRT-PCR (qPCR) validation of transcripts that were most dramatically upregulated by Torin1 treatment in COCs. (C) qRT-PCR validation of transcripts that were either most dramatically downregulated or that encode important proteins essential for cumulus cell development and function. (D) qRT-PCR validation of transcripts encoding EGF-like growth factors. (E) qRT-PCR validation of transcripts essential for cumulus expansion. (F) qRT-PCR validation of transcripts that encode enzymes for cholesterol biosynthesis. Gray bars indicate expression detected by using a microarray; black bars indicate levels detected by using qRT-PCR. Four sets of COC samples were used for qRT-PCR analysis, and data are presented as the mean±s.e.m. of fold changes relative to levels in the DMSO-treated (Control) group. The changes in the expression of all the transcripts examined were statistically significant (P<0.05; one-way ANOVA followed by Tukey's HSD test).