Ovary

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

Abstract
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 identity of these oocyte factors remains 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 characterised with very low cross-reactivity with related TGF- superfamily members, including BMP15 (also called GDF9B). Pep-SPOT epitope mapping showed that mAb-GDF9-53 recognizes a short 4-aa sequence, and 3D-peptide modelling suggested that this binding motif lies at the C-terminal fingertip of mGDF9. As predicted by sequence alignments and modelling, 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 TGF-1 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 co-cultured with granulosa cells promote cell proliferation in a dose-dependent manner. 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 TGF--pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGF-1-induced DNA synthesis. This study has characterised 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 shows that GDF9 accounts for only part of total oocyte bioactivity.

Key words: Ovary • Follicle • Granulosa cells • Growth factors • Ovum

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