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First published online 22 August 2006
doi: 10.1242/jcs.03105

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Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation

¹ Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, University of Adelaide, Australia
² Programme for Developmental and Reproductive Biology, Biomedicum Helsinki, and Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Finland

View larger version (28K): [in a new window]   Fig. 1. Oocyte stimulation of granulosa cell DNA synthesis - comparison with GDF9 and BMP6. (A) Mural GCs were cultured with increasing numbers of oocytes per well or increasing doses of recombinant BMP6 or GDF9 and assessed for [3H]thymidine incorporation after 24 hours. Points are means ± s.e.m. from triplicate wells and are representative of three replicate experiments. (B) Mural GCs were cultured alone or together with DOs (125 DOs/125-µl well) for 6 hours, RNA was extracted, reverse transcribed and subjected to real-time RT-PCR for Ccnd2. *mRNA levels significantly increased with oocyte co-culture. (C) Mural GCs were cultured alone, treated with BMP6 (50 ng/ml) or co-cultured with 16 oocytes per well, each with and without a neutralising dose (20 µg/ml) of a BMP6 monoclonal neutralising antibody (NAb) or a control IgG (20 µg/ml). Columns represent means ± s.e.m. from triplicate wells, representative of three replicate experiments. BMP6 NAb had no significant effect (P>0.05) on oocyte-stimulated GC DNA synthesis.

View larger version (20K): [in a new window]   Fig. 2. Interactions between GDF9 and TGFß1 or oocyte-stimulated granulosa cell DNA synthesis. Mural GCs were cultured with increasing doses of mouse GDF9 either alone or in the presence of DOs (16 DOs/125-µl well) or human TGFß1 (0.5 ng/ml). After 24 hours of culture the labelled thymidine incorporated into cells was counted. Points are means ± s.e.m. from at least triplicate wells from five replicate experiments. The stimulatory effects of TGFß1 and oocytes were additive to the stimulatory actions of GDF9 at low doses of GDF9 [means with an asterisk are significantly different to GDF9 alone at that dose (2-way ANOVA; P<0.01)], whereas this additive effect was lost at higher doses of GDF9 (>8 ng/ml for DOs; >15 ng/ml for TGF-ß1; dose GDF9 X treatment interaction, P<0.001).

View larger version (22K): [in a new window]   Fig. 3. Specificity and potency of the BMP receptor-II ectodomain at neutralising oocyte- and GDF9-stimulated mural GC DNA synthesis. (A) Mural GCs were cultured with DOs (12 DOs/125-µl well), mouse GDF9 (40 ng/ml), human activin A (20 ng/ml), and human TGFß1 (0.5 ng/ml), in either the absence or the presence of a maximum neutralising dose of BMP receptor-II ectodomain (BMPR-II ECD; 2 µg/ml). Bars represent means ± s.e.m. from at least triplicate wells and are representative of three replicate experiments. Asterisks represent significant neutralisation (P<0.01) caused by the ECD. Control is mural GC alone or with mitogen. (B) Mural GCs were cultured either with or without DOs (12 DOs/125-µl well; ) or 40 ng/ml mouse GDF9 (), in either the absence or the presence of increasing doses of BMPR-II ECD. Points are means ± s.e.m. from at least triplicate wells, expressed as a fraction of the control (mitogen with no ECD), and are representative of three replicate experiments. An asterisk represents the lowest dose of BMPR-II ECD that is significantly less than the 100% control (P<0.001).

View larger version (46K): [in a new window]   Fig. 4. Specificity of oocyte-secreted factors for BMPR-II within the superfamily of TGFß type-II receptors. Mural GC thymidine incorporation was stimulated with either (A) human TGFß1 (0.5 ng/ml), (B) mouse GDF9 (40 ng/ml), (C) human activin A (10 ng/ml) or (D) 12 oocytes/well. Each mitogen was also treated separately with four different soluble receptor ectodomains; BMPR-II, TGFßR-II, ActR-IIA and ActR-IIB, each at 2 µg/ml. Bars are means ± s.e.m from triplicate wells, expressed as a fraction of the positive control (mitogen with no ECD), and are representative of four replicate experiments. An asterisk represents significant antagonism relative to the positive control (P<0.05).

View larger version (20K): [in a new window]   Fig. 5. Expression of BMP receptor-II mRNA in cumulus cells and BMP receptor-II ectodomain (BMPR-II ECD) neutralisation of oocyte-stimulated cumulus cell DNA synthesis. (A) Mural granulosa cells and cumulus-oocyte complexes were collected from antral follicles, cumulus cells were generated by denuding cumulus-oocyte complexes, RNA was extracted, reverse transcribed and subjected to real-time RT-PCR. There was no significant difference between cell types in the level of expression of BMPR-II mRNA. (B) Oocytectomised complexes (OOX) were generated by microsurgically removing the oocyte contents from cumulus-oocyte complexes. OOX were cultured for 24 hours either alone, together with 30 DOs/well, or with oocytes and 2 µg/ml of BMPR-II ECD. Columns are means ± s.e.m. from three replicate experiments. Columns with different superscript letters are significantly different (P<0.001).

View larger version (29K): [in a new window]   Fig. 6. Activation of granulosa cell SMAD2 and SMAD3 transducer molecules by GDF9 and oocytes. (A) Mural GCs were transiently transfected with a SMAD3-responsive CAGA-luciferase plasmid, and subsequently left untreated, treated with 0.5 ng/ml TGFß1, or treated with increasing doses of GDF9 (or 293H: the negative v/v control of conditioned medium from untransfected 293H cells) for 48 hours, and then relative luciferase activity was measured from cell extracts. (B) Mural GCs were cultured for 90 minutes either alone (control), co-cultured with oocytes (500 DOs/1-ml well), with human activin A (50 ng/ml), or mouse GDF9 (40 ng/ml), then extracted granulosa cell proteins were subjected to 10% SDS-PAGE and western blotting using SMAD2 and phospho-SMAD2 antibodies. Mural GC SMAD2 was phosphorylated by treatment with mGDF9, oocytes and, to a lesser extent, activin A.

View larger version (20K): [in a new window]   Fig. 7. Oocyte-paracrine factors signal through the TGFß/activin intracellular pathway, and not through the BMP pathway. Mural GCs were transiently transfected during culture with either a SMAD3-responsive CAGA-luciferase plasmid (A) or a SMAD1-responsive BRE-luciferase reporter plasmid (B), then treated with various TGFß superfamily growth factors or were co-cultured with 60 oocytes/250-µl well. GDF9 was used at 40 ng/ml. TGFß1 (0.5 ng/ml) is a positive control for CAGA (and negative control for BRE), and BMP6 (50 ng/ml) is a positive control for BRE (and negative controls for CAGA). 293H is the GDF9 negative control: an equivalent v/v of conditioned medium from untransfected 293H cells. Columns represent means ± s.e.m. from four replicate experiments. Oocytes significantly (P<0.01) increased mural GC CAGA-luciferase, but not BRE-luciferase activity (P>0.05); asterisks indicate significantly different to control (P<0.01).

View larger version (24K): [in a new window]   Fig. 8. Inhibition of oocyte- and GDF9-stimulated SMAD3 activation using the ALK4/5/7 kinase inhibitor, SB431542. Mural GCs were transiently transfected during culture with either a SMAD3-responsive CAGA-luciferase or a SMAD1-responsive BRE-luciferase reporter plasmid. (A) CAGA-luciferase was stimulated by TGFß1, which in turn was dose-dependently antagonised by treatment with SB431542. The carrier, DMSO, at a v/v dose equivalent to 0.5 µM of SB431542, did not affect TGFß-induced mural GC CAGA-luciferase activity. (B) BMP6 (50 ng/ml)-stimulated BRE-luciferase activity was not affected by a maximum dose of 2 µM SB431542. (C) Treatment of mural GCs with 0.5 µM SB431542 completely antagonised GDF9-activation (60 ng/ml) and oocyte-activation (60 oocytes/well) of SMAD3-responsive CAGA luciferase activity; asterisk indicates significantly different to control (oocytes alone; P<0.05).

View larger version (18K): [in a new window]   Fig. 9. Inhibition of oocyte- and GDF9-stimulated CC and mural GC proliferation using the ALK 4/5/7 kinase inhibitor, SB431542. (A) Cumulus cells (OOX) were treated with GDF9 (250 ng/ml) or DOs (0.8/µl), with or without 4 µM SB431542 and cultured for 6 hours, and Ccnd2 mRNA levels were examined using real-time RT-PCR. Columns are means ± s.e.m. from six replicate experiments and columns with different superscripts are significantly different (P<0.05). (B) Mural GCs were cultured for 24 hours and thymidine incorporation was stimulated with either 20 ng/ml GDF9, 0.5 ng/ml TGFß1, 16 DOs/well or 50 ng/ml BMP6, all of which, in turn, were antagonised by SB431542 at 1.0 µM. An equivalent dose of the SB431542 carrier DMSO did not affect ligand-stimulated mural GC DNA synthesis. Columns are means ± s.e.m. from triplicate wells and are representative of three replicate experiments; asterisks indicate significantly different to control (mitogen alone; P<0.001). (C) Oocyte (14/well)- and GDF9 (20 ng/ml)-stimulated mural GC DNA synthesis were inhibited in a dose-dependent manner with increasing concentrations of SB431542. Points are means ± s.e.m. from triplicate wells and are representative of three replicate experiments. Asterisks represent the lowest dose of SB431542 that is significantly less than the positive control (P<0.05).