title: Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation.
abstract: Oocytes regulate follicle growth by secreting paracrine growth factors that act on neighbouring granulosa cells (GCs). Those factors identified to date are mainly members of the transforming growth factor-beta (TGFbeta) superfamily, but little is known about which specific receptor/signalling system(s) they employ. This study was conducted to determine the requisite pathways utilised by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded mouse oocytes are co-cultured with mural GCs. Oocytes, growth differentiation factor-9 (GDF9), TGFbeta1 and activin-A all promoted GC DNA synthesis, but bone-morphogenetic protein 6 (BMP6) did not. Subsequently, we tested the capacity of various TGFbeta superfamily receptor ectodomains (ECD) to neutralise oocyte- or specific growth factor-stimulated GC proliferation. The BMP type-II receptor (BMPR-II) ECD antagonised oocyte and GDF9 bioactivity dose-dependently, but had no or minimal effect on TGFbeta1 and activin-A bioactivity, demonstrating its specificity. The TGFbetaR-II, activinR-IIA and activinR-IIB ECDs all failed to neutralise oocyte- or GDF9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective native ligands. An activin receptor-like kinase (ALK) 4/5/7 inhibitor, SB431542, also antagonised both oocyte and GDF9 bioactivity in a dose-dependent manner. Consistent with these findings, oocytes, GDF9 and TGFbeta1 all activated SMAD2/3 reporter constructs in transfected GC, and led to phosphorylation of SMAD2 proteins in treated cells. Surprisingly, oocytes did not activate the SMAD1/5/8 pathway in transfected GCs although exogenous BMP6 did. This study indicates that oocyte paracrine factors primarily utilise a similar signalling pathway first identified for GDF9 that employs an unusual combination of TGFbeta superfamily receptors, the BMPR-II and a SMAD2/3 stimulatory ALK (4, 5 or 7), for transmitting their mitogenic actions in GC. This cell-signalling pathway may also have relevance in the hypothalamic-pituitary axis and in germ-somatic cell interactions in the testis.
authors.affiliation: Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, University of Adelaide, Australia. robert.gilchrist@adelaide.edu.au
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gilchrist
authors.forename: Lesley J
authors.initials: LJ
authors.lastname: Ritter
authors.forename: Samu
authors.initials: S
authors.lastname: Myllymaa
authors.forename: Noora
authors.initials: N
authors.lastname: Kaivo-Oja
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Dragovic
authors.forename: Theresa E
authors.initials: TE
authors.lastname: Hickey
authors.forename: Olli
authors.initials: O
authors.lastname: Ritvos
authors.forename: David G
authors.initials: DG
authors.lastname: Mottershead
mesh_terms: D028341:Activins / Q000378:metabolism; D000818:Animals; D055430:Bone Morphogenetic Protein 15; D055418:Bone Morphogenetic Protein 6; D052006:Bone Morphogenetic Protein Receptors, Type II / Q000235:genetics; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006107:Granulosa Cells / Q000166:cytology* / Q000187:drug effects; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D051379:Mice; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D019899:Paracrine Communication / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: C525902:Bmp15 protein, mouse; C525817:Bmp6 protein, mouse; D055430:Bone Morphogenetic Protein 15; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins; C525898:Gdf9 protein, mouse; D055429:Growth Differentiation Factor 9; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D051899:Smad2 Protein; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D028341:Activins; D004247:DNA; D052006:Bone Morphogenetic Protein Receptors, Type II