title: Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins.
abstract: Paracrine factors secreted by the oocyte regulate a broad range of cumulus cell functions. Characteristically, cumulus cells have a low incidence of apoptosis and we proposed that this is due to oocyte-secreted factors acting in an anti-apoptotic manner. Bovine cumulus-oocyte complexes (COC) were aspirated from abattoir-derived ovaries and oocytectomized (OOX) by microsurgical removal of the oocyte. OOX were treated with doses of either denuded oocytes (DO) or various growth factors for 24 hours (+/- rFSH; 0.1 IU/ml). Proportions of apoptotic cumulus cells were assessed using TUNEL and laser confocal scanning microscopy followed by image analysis. Quantification of Bcl-2 and Bax proteins in OOX was undertaken by western analysis. Oocyte removal led to a significant increase in cumulus cell apoptosis compared with COC controls (35% versus 9% TUNEL positive, respectively; P<0.001). Levels of OOX apoptosis were significantly reversed (P<0.001) in a dose-dependent manner when co-cultured with oocytes. Furthermore, the anti-apoptotic effect of oocyte-secreted factors followed a gradient from the site of the oocyte(s). Growth differentiation factor 9 (GDF9) had no significant effect on cumulus cell apoptosis. By contrast, cumulus cell apoptosis was significantly (P<0.001) reduced by bone morphogenetic proteins (BMP) 15, 6 or 7. Accordingly, levels of anti-apoptotic Bcl-2 were high in OOX+DO and OOX+BMP15 and low with OOX+GDF9 or OOX alone, whereas the reverse was observed for pro-apoptotic Bax. DO, BMP15 and BMP6 were also able to protect cumulus cells from undergoing apoptosis induced by staurosporine. FSH partially prevented apoptosis in all treatment groups (P<0.001). Follistatin and a BMP6 neutralizing antibody, which antagonized the anti-apoptotic effects of BMP15 and BMP6, respectively, whether alone or combined, blocked approximately 50% of the anti-apoptotic actions of oocytes. These results are the first to demonstrate that oocyte-secreted factors, and particularly BMP15 and BMP6, maintain the low incidence of cumulus cell apoptosis by establishing a localized gradient of bone morphogenetic proteins.
authors.affiliation: 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.
authors.forename: Tamer S
authors.initials: TS
authors.lastname: Hussein
authors.forename: David A
authors.initials: DA
authors.lastname: Froiland
authors.forename: Fred
authors.initials: F
authors.lastname: Amato
authors.forename: Jeremy G
authors.initials: JG
authors.lastname: Thompson
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gilchrist
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D019485:Bone Morphogenetic Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D038681:Follistatin / Q000494:pharmacology; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D008954:Models, Biological; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019899:Paracrine Communication; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D019485:Bone Morphogenetic Proteins; D038681:Follistatin; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D005640:Follicle Stimulating Hormone; D019311:Staurosporine