The Molecular Forms of GDF9 In A Range of Mammalian Species

<p>Growth Differentiation Factor (GDF) 9 is a member of the transforming growth factor β (TGFβ) superfamily that is exclusively expressed within and secreted from, the oocyte. This protein has generated much interest as it has been found to play a major role in follicular growth and maturation in mammals, and may be involved in determining litter size. Like most TGFβ family members, it is synthesised as a pre-pro-mature protein and is cleaved at various stages to allow the biologically active mature form to bind its Type II receptor. The aim of this study was to improve our understanding of the different molecular forms of GDF9 that are present within ovarian follicles of a range of mammalian species that differ in litter size. To achieve this aim, Western blotting experiments were performed to illustrate the molecular forms that were present within, and secreted from, the oocytes of rats, pigs, sheep and red deer. The detection of bands that represented the different molecular forms of GDF9 was undertaken using a monoclonal antibody that targeted a conserved region in the mature form of ovine GDF9.  The predominant forms of GDF9 found within and secreted from the oocyte across the species were the promature and cleaved mature forms of GDF9. Densitometry analysis of the Western blots revealed that pig, sheep, and red deer had significantly more of the promature, than the mature, form within the oocyte. Conversely, there were no significant differences between the levels of promature and mature forms of GDF9 in the secreted media. Moreover, the levels of the specific molecular forms of GDF9 were not different between pigs, sheep and red deer. The levels of GDF9 in rat samples were low which may be due to a lower affinity of the monoclonal GDF9 antibody due to sequence differences between rat and ovine GDF9.  Interestingly, applying a crosslinking reagent to the oocyte lysate and conditioned media samples revealed the appearance of a high molecular size band. The appearance of this band, which was more prominent in the rat and pig, was concomitant with the disappearance of the mature GDF9 band. The differential levels of these presumptive GDF9 multimers in these two species that have large litters may suggest that rat and pig mature GDF9 binds other oocyte secreted factors more readily than GDF9 from mono-ovulatory species. Importantly, no homo- or hetero- mature dimers of GDF9 were detected in any of the species studied.  In summary, GDF9 was predominantly present as promature and cleaved mature forms both within the oocyte and in the secretions from the oocyte. Overall, these results indicated that the promature form was present in higher levels than the cleaved mature form. With the exception of the rat, there were no detectable species differences in the levels of the GDF9 forms within or secreted from the oocyte using Western blotting methodologies.</p>