Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation.
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.
Gilchrist
Robert B
RB
Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, University of Adelaide, Australia. robert.gilchrist@adelaide.edu.au
Ritter
Lesley J
LJ
Myllymaa
Samu
S
Kaivo-Oja
Noora
N
Dragovic
Rebecca A
RA
Hickey
Theresa E
TE
Ritvos
Olli
O
Mottershead
David G
DG
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
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