Immunoneutralization of growth differentiation factor 9 reveals it partially accounts for mouse oocyte mitogenic activity.

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)beta 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 TGFbeta1 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 microg/ml) decreased the mitogenic activity of oocytes but only by approximately 45% at the maximum dose of mAb. Just 5 microg/ml of mAb-GDF9-53 neutralized 90% of recombinant mGDF9 mitogenic activity, but only 15% of oocyte activity. Unlike mAb-GDF9-53, a TGFbeta pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGF beta 1-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.

cDNA cloning, expression studies and chromosome mapping of human type I serine/threonine kinase receptor ALK7 (ACVR1C).

Transforming growth factor-beta (TGF-beta) superfamily related growth factors signal by binding to transmembrane type I and type II receptor serine/threonine kinases (RSTK), which phosphorylate intracellular Smad transcription factors in response to ligand binding. Here we describe the cloning of the human type I RSTK activin receptor-like kinase 7 (ALK7), an orthologue of the previously identified rat ALK7. Nodal, a TGF-beta member expressed during embryonic development and implicated in developmental events like mesoderm formation and left-right axis specification, was recently shown to signal through ALK7. We found ALK7 mRNA to be most abundantly expressed in human brain, pancreas and colon. A cDNA encoding the open reading frame of ALK7 was obtained from a human brain cDNA library. Furthermore, a P1 artificial chromosome (PAC) clone containing the human ALK7 gene was isolated and fluorescent in situ hybridization (FISH) on metaphase chromosomes identified the gene locus as chromosome 2q24.1-->q3. To test the functionality of the ALK7 signaling, we generated recombinant adenoviruses containing a constitutively active form of ALK7 (Ad-caALK7), which is capable of activating downstream targets in a ligand independent manner. Infection with Ad-caALK7 of MIN6 insulinoma cells, in which ALK7 has previously been shown to be endogenously expressed, led to a marked increase in the phosphorylation of Smad2, a signaling molecule also used by TGF-betas and activins.