The human ␦ epithelial sodium channel (␦ENaC) subunit is related to the ␣-, ␤-, and ␥ENaC subunits that control salt homeostasis. ␦ENaC forms an amiloride-sensitive Na ؉ channel with the ␤ and ␥ subunits. However, the in vivo function of ␦ENaC is not known. To gain insight into the function of ␦ENaC, a yeast two-hybrid screen of a human brain cDNA library was carried out using the C-and N-terminal domains of ␦ENaC. A novel ␦ENaC-interacting protein called Murr1 (mouse U2af1-rs1 region) was isolated in the C-terminal domain screen. Murr1 is a 21-kDa protein mutated in Bedlington terriers suffering from copper toxicosis. The interaction of Murr1 and ␦ENaC was confirmed by glutathione Stransferase pulldown assay and coimmunoprecipitation. To test the functional significance of the interaction, Murr1 was coexpressed with ␦␤␥ENaC in Xenopus oocytes. Murr1 inhibited amiloride-sensitive sodium current in a dose-dependent manner. In addition, deletion of the last 59 amino acids of ␦ENaC abolished the inhibition. Murr1 also bound to the ␤-and ␥ENaC subunits and inhibited ␣␤␥ENaC sodium current. Therefore, these results suggest that Murr1 is a novel regulator of ENaC.

The aims were to investigate whether oocyte-secreted growth factors from a high (i.e. rat) and low (i.e. sheep) ovulation rate species could stimulate 3 H-thymidine incorporation in granulosa cells (GC) from antral follicles from the same or across species. Denuded oocytes (DO) were co-incubated with GC with or without specific antibodies to growth differentiating factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15). Co-incubations of DO-GC from the same or across species significantly increased thymidine incorporation in GC with increasing numbers of DO. GDF9 immuno-neutralisation reduced thymidine incorporation in rat GC co-incubated with either rat or ovine DO and in ovine GC co-incubated with ovine or rat DO. BMP15 immuno-neutralisation only reduced thymidine incorporation when ovine DO were co-incubated with either ovine or rat GC. Western blotting of oocytes co-incubated with GC identified GDF9 and BMP15 proteins for sheep and GDF9 protein for rats in oocyte lysates and incubation media. With respect to rat BMP15, a promature protein was identified in the oocyte lysate but not in media. Expression levels of GDF9 relative to BMP15 mRNA in DO co-incubated with GC were highly correlated (R 2 Z0.99) within both species. However, the expression ratios were markedly different for the rat and sheep (4.3 vs 1.0 respectively). We conclude that during follicular development, rat oocytes secrete little, if any, BMP15 and that GDF9 without BMP15 can stimulate proliferation of rat and ovine GC. In contrast, ovine oocytes secrete both BMP15 and GDF9, and both were found to stimulate proliferation in ovine and rat GC.

Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are secreted by the mammalian oocyte and are essential for ovarian follicular development, ovulation, and fertility. However, the secreted forms of the BMP15 and GDF9 proteins and the nature of cooperative molecular interactions between BMP15 and GDF9 previously reported have not been fully characterized. In this study, we found that recombinant mouse BMP15 and GDF9 are secreted as cleaved mature and proregion proteins, with BMP15 also secreted as uncleaved promature protein. Noncovalent interactions were identified between the mature and proregion proteins of each growth factor. Moreover, GDF9 mature protein was found to coimmunoprecipitate with the BMP15 proregion, suggestive of a heteromeric association between BMP15 and GDF9. Mouse GDF9 was found to exist mostly as a dimer of mature protein, in both the presence and absence of BMP15. In contrast, BMP15 formed mostly multimers of proregion and mature protein when combined with GDF9, providing further evidence for heteromeric interaction. Mouse BMP15 was found to act cooperatively with GDF9 in a rat granulosa cell thymidine incorporation bioassay and to signal through the BMPR2 and ACVR1B/TGFBR1/ACVR1C receptor-mediated pathways. Immunoneutralization experiments using GDF9 mature protein antibody indicated that these cooperative interactions are species specific. Additionally, immunoneutralization with proregion antibodies highlighted the involvement of the BMP15 proregion in BMP15/GDF9 cooperative interactions. Taken together, these findings support a novel hypothesis where the extracellular cooperative interactions of recombinant mouse BMP15 and GDF9 are multimeric, involving the proregion of BMP15, and may well be species specific.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted factors known to be involved in regulating the proliferation and differentiation of granulosa cells during follicular growth. The aims of this study were to determine the signalling pathways used by recombinant forms of murine and ovine GDF9 and BMP15 in combination (GDF9CBMP15) and the molecular complexes formed by combinations of these factors. Differences in the molecular forms of combinations of murine and ovine GDF9CBMP15 were observed by western blot analysis. Ovine GDF9CBMP15-stimulated 3 H-thymidine uptake was completely blocked by SMAD2/3 and nuclear factor-kB pathway inhibitors and partially blocked by a p38-mitogen-activated protein kinase (MAPK) inhibitor. Thymidine uptake by murine GDF9CBMP15 was reduced by the SMAD2/3 and extracellular signal-regulated kinase-MAPK pathway inhibitors and increased after addition of a c-Jun N-terminal kinase inhibitor. Stimulation of 3 H-thymidine uptake by GDF9CBMP15 from either species was not affected by the SMAD1/5/8 pathway inhibitor. In conclusion, both murine and ovine GDF9CBMP15-stimulated thymidine incorporation in rat granulosa cells was dependent on the SMAD2/3 signalling pathway but not the SMAD1/5/8 pathway. Divergence in the non-SMAD signalling pathways used by murine and ovine GDF9CBMP15 was also evident and may be due to the differences observed in the molecular complexes formed by these factors. These results are consistent with the hypothesis that the disparate cooperative functions of GDF9 and BMP15 in different species are mediated by divergent non-SMAD signalling pathways.