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First published online 1 November 2005
doi: 10.1242/jcs.02644

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Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins

Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville Road, Woodville, South Australia, 5011, Australia

View larger version (90K): [in a new window]   Fig. 1. Representative images of confocal laser scanning microscopy of DNA fragmentation in cumulus cells, as detected by TUNEL (green label). All cell nuclei are also stained with propidium iodide (red). Positive control DNAse 1-treated OOX showed very strong apoptotic staining (99%) (A), negative control did not reveal any apoptotic signals (0%) indicating specific labelling (B), expanded COC after culture with low apoptotic labelling (9%) (C), compared with OOX with higher apoptotic labelling (35%) (D). Light micrograph of an unexpanded intact COC before culture (E) and a denuded oocyte (F), generated by the mechanical removal of cumulus cells from a COC.

View larger version (14K): [in a new window]   Fig. 2. A dose response of oocyte-secreted factors on cumulus cell apoptosis in the absence (A) or presence (B) of FSH. Oocytectomized complexes (OOX) were cultured with increasing numbers of denuded oocytes (DO) and at the maximum dose were effective at reducing apoptosis to the control COC levels. FSH also significantly reduced apoptosis in COCs and OOXs. Points represent average percentage of apoptotic cumulus cells (mean ±s.e.m.). Values from points with different labels; a,b,cdiffer significantly (P<0.001).

View larger version (33K): [in a new window]   Fig. 3. Pattern of apoptosis within cumulus complexes in relation to proximity to oocyte-secreted factor origin. Diameters of unexpanded COCs and OOXs were measured after culture without FSH, using confocal microscopy and divided into three layers: inner, middle and outer layers. Each layer representing 33% of the radius (A). The incidence of apoptosis was lowest closest to the oocyte, regardless of whether the oocyte was inside the complex [(C), COC inner layer] or oocytes were on the outside of the complex [(D), OOX outer layer], and apoptosis increased with increasing distance from the oocyte (B). Both C and D images are exaggerated examples to illustrate pictorially the morphogenic gradient of apoptosis through the cumulus cell layers. *Significantly different (two-way ANOVA; P=0.026).

View larger version (25K): [in a new window]   Fig. 4. Dose response of the putative oocyte-secreted factors; GDF-9, BMP-6, BMP-15 on cumulus cell apoptosis. OOX were cultured with increasing concentrations of GDF-9 (0-175 ng/ml), BMP-6 (0-100 ng/ml), and BMP-15 (0-20% v/v), either in the absence (A,C,E) or presence (B,D,F) of FSH. Cumulus cell apoptosis was unaffected by GDF-9 and attenuated in a dose-dependent manner by BMP-6, but more notably by BMP-15. FSH independently attenuated apoptosis regardless of treatment or complex type. Points represent average percentage of apoptotic cumulus cells (mean ±s.e.m.). Values from points with different labels; a,b,cdiffer significantly (A,B; P<0.001). Asterisks represent significant difference (P<0.001) relative to the control (OOX) for that factor (C-F).

View larger version (41K): [in a new window]   Fig. 5. Effect of denuded oocytes (DO), GDF-9 and BMP-15 on OOX expression of Bcl-2 and Bax proteins as examined by western blot analysis. Groups of 35 OOX were loaded in each lane after the following treatments: lane 1, 10% v/v 293H (control conditioned medium); lane 2, control (OOX alone); lane 3, 132 ng/ml GDF-9; lane 4, 10% v/v BMP-15; lane 5, 0.7 DO/µl. Band intensities were quantified by densitometry and are expressed relative to the 293H control, from three replicate experiments (mean ±s.e.m.). Bars with different superscripts within a group (a,bBcl-2, y,zBax) are significantly different (P<0.001).

View larger version (17K): [in a new window]   Fig. 6. Protection of cumulus cells from staurosporine-induced apoptosis by denuded oocytes (DO), BMP-6 and BMP-15. OOX alone or co-cultured with 35 DO, 10 ng/ml BMP-6, or 10% v/v BMP-15, were exposed to either 0.1 µM or 1.0 µM staurosporine (STS) in the last 6 hours of incubation. Oocytes, BMP-6 and BMP-15 all prevented staurosporine-induced cumulus cell apoptosis. Asterisks represent OOX means significantly different (P<0.001) relative to the OOX control.

View larger version (19K): [in a new window]   Fig. 7. Effect of BMP antagonists on cumulus cell apoptosis. OOX were cultured with 10% v/v BMP-15 in the presence of increasing doses of follistatin (0-100 µg/ml) (A), and OOX were cultured with 10 ng/ml BMP-6 in the absence or presence of a high neutralizing dose of 20 µg/ml of a BMP-6 neutralizing antibody (NAb) (B). Suppression of cumulus cell apoptosis by BMP-15 was antagonized by follistatin. The NAb effectively antagonized the anti-apoptotic effects of BMP-6. Points and bars represent average percentage of apoptotic cumulus cells (mean ±s.e.m.). Values from points with different labels; a,b,cdiffer significantly (P<0.001).

View larger version (22K): [in a new window]   Fig. 8. Role of BMP-15 and BMP-6 in the anti-apoptotic actions of oocytes on cumulus cells. OOX co-cultured with denuded oocyte (25 DOs) were treated with 50 µg/ml follistatin, 20 µg/ml BMP-6 NAb, or a combination of the two (A). Both follistatin and the BMP-6 NAb were effective at partially antagonizing the anti-apoptotic effects of oocytes, however neither completely restored apoptosis to OOX levels, either alone or combined. Co-culturing OOX with DO or treatment with BMP-15 alone or BMP-6 alone decreased cumulus cell apoptosis (B). Combined treatment of OOXs with BMP-6 and BMP-15 did not further decrease apoptosis levels beyond that of BMP-15 alone, suggesting no additive effect of these two BMPs. Bars represent average percentage of apoptotic cumulus cells (mean ±s.e.m.). Values from bars with different labels; a,b,cdiffer significantly (P<0.001).

View larger version (24K): [in a new window]   Fig. 9. Effect of BMP-7 and its antagonist gremlin on cumulus cell apoptosis. OOXs were cultured with 10% v/v BMP-15 in the presence of increasing doses of gremlin (0-40 µg/ml) (A). OOX were also co-cultured with 100 ng/ml BMP-7 and/or 10% BMP-15 in the presence or absence of 2 µg/ml gremlin (B). Gremlin did not antagonize the suppressive effect of BMP-15 on cumulus cell apoptosis, whereas it did that of BMP-7. Bars and points represent average percentage of apoptotic cumulus cells (mean ±s.e.m.). Values from bars with different labels; a,b,cdiffer significantly (P<0.001).

View larger version (49K): [in a new window]   Fig. 10. Proposed model by which the paracrine network of BMP/GDF-9 growth factors and their binding proteins interact to regulate apoptosis in the COC microenvironment. Oocyte-secreted BMP-15 and BMP-6 signal through the cumulus cell receptor BMPR-II to actively prevent cumulus cell death. Oocyte-secreted GDF-9, also acting through BMPR-II but using a different type-I receptor to the BMPs, does not prevent cumulus cell apoptosis but induces cumulus cell gremlin expression. GDF-9-stimulated gremlin expression may in turn block theca and granulosa cell-derived BMPs from competing with BMP-15, BMP-6 and GDF-9 for BMPR-II binding. In addition, follistatin produced by mural granulosa cells and present in follicular fluid, may limit the anti-apoptotic effects of oocyte BMP-15 specifically to the COC microenvironment.