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BOR - Papers in Press, published online ahead of print May 5, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028852
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BIOLOGY OF REPRODUCTION 71, 732–739 (2004)
DOI: 10.1095/biolreprod.104.028852
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Immunoneutralization of Growth Differentiation Factor 9 Reveals It Partially Accounts for Mouse Oocyte Mitogenic Activity1

R.B. Gilchrist2,3, L.J. Ritter3, M. Cranfield4, L.A. Jeffery5, F. Amato3, S.J. Scott3, S. Myllymaa5, N. Kaivo-Oja5, H. Lankinen6, D.G. Mottershead5, N.P. Groome4, and O. Ritvos5

Research Centre for Reproductive Health,3 Department of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, University of Adelaide, Woodville, SA 5011, Australia School of Biological and Molecular Sciences,4 Oxford Brookes University, Headington, Oxford OX3 0BP, United Kingdom Programme for Developmental and Reproductive Biology,5 Biomedicum Helsinki and Department of Bacteriology Immunology, Peptide and Protein Laboratory,6 Department of Virology, Haartman Institute, University of Helsinki, 00014 Helsinki, Finland

Paracrine factors secreted by oocytes play a pivotal role in promoting early ovarian follicle growth and in defining a morphogenic gradient in antral follicles, yet the exact identities of these oocyte factors remain unknown. This study was conducted to determine the extent to which the mitogenic activity of mouse oocytes can be attributed to growth differentiation factor 9 (GDF9). To do this, specific anti-human GDF9 monoclonal antibodies were generated. Based on epitope mapping and bioassays, a GDF9 neutralizing antibody, mAb-GDF9-53, was characterized with very low cross-reactivity with related transforming growth factor (TGF)ß superfamily members, including BMP15 (also called GDF9B). Pep-SPOT epitope mapping showed that mAb-GDF9-53 recognizes a short 4-aa sequence, and three-dimensional peptide modeling suggested that this binding motif lies at the C-terminal fingertip of mGDF9. As predicted by sequence alignments and modeling, the antibody detected recombinant GDF9, but not BMP15 in a Western blot and GDF9 protein in oocyte extract and oocyte-conditioned medium. In a mouse mural granulosa cell (MGC) bioassay, mAb-GDF9-53 completely abolished the mitogenic effects of GDF9, but had no effect on TGFß1 or activin A-stimulated MGC proliferation. An unrelated IgG at the same dose had no effect on GDF9 activity. This GDF9 neutralizing antibody was then tested in an established oocyte-secreted mitogen bioassay, where denuded oocytes cocultured with granulosa cells promote cell proliferation in a dose-dependent manner. The mAb-GDF9-53 dose dependently (0–160 µg/ml) decreased the mitogenic activity of oocytes but only by ~45% at the maximum dose of mAb. Just 5 µg/ml of mAb-GDF9-53 neutralized 90% of recombinant mGDF9 mitogenic activity, but only 15% of oocyte activity. Unlike mAb-GDF9-53, a TGFß pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGFß1-induced DNA synthesis. This study has characterized a specific GDF9 neutralizing antibody. Our data provide the first direct evidence that the endogenous GDF9 protein is an important oocyte-secreted mitogen, but also show that GDF9 accounts for only part of total oocyte bioactivity.

1 The Adelaide component of this work was supported by Project Grant 207761 from the National Health and Medical Research Council (NHMRC) of Australia. R.B.G. is the recipient of the FTT Fricker Medical Research Associateship from the University of Adelaide. The European contribution was partly funded by QLK6-2000-00338-OVAGE grant from the European Commission to O.R. and N.P.G. The work of L.J., S.M., N.K-O, D.G.M, and O.R. was supported by grants from the Academy of Finland, the Finnish National Technology Agency, the Juselius Foundation, the Novo Nordisk Foundation, and the Helsinki University Central Hospital Funds. L.A.J. and N.K.-O. are recipients of Ph.D. studentships from the GSBM and HBGS graduate schools at the University of Helsinki.

2 Correspondence. FAX: 61 08 8222 7521; robert.gilchrist{at}adelaide.edu.au




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