AbstractsBiology & Animal Science

Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-secreted factors with well documented effects on ovarian follicular development and ovulation-rate. The aims of these studies were to: (i) identify the molecular forms of BMP15 and GDF9 that are produced and secreted by both the ovine and bovine oocyte using highly specific monclonal antibodies; (ii) assess the biological activity of some recombinant molecular forms of BMP15 and GDF9; (iii) visualise the various molecular forms using protein modelling techniques and; (iv) provide a hypothetical model of how oocyte-secreted form(s) of BMP15, GDF9 and their cell surface receptors may interact. Using genetic modifications and transformations of HEK293 cells, recombinant forms of ovine (o) BMP15, including a BMP15 (S356C) mutant capable of forming covalent dimers, and oGDF9 were produced. The bioactivity of these proteins was established using a rat granulosa cell proliferation bioassay. The specificity of the monoclonal antibodies MN2-61A (anti-BMP15) and 37A (anti-GDF9) used in these studies, and determination of the forms they recognise, was examined by Western blotting. The recombinant forms of oBMP15 were further interrogated by purification using both immobilised metal affinity chromatography (IMAC) and reverse phase HPLC. The BMP15 and GDF9 proteins produced and/or secreted by ovine and bovine oocytes, before and after in vitro incubation, were identified and compared with the molecular forms(s) of recombinant oBMP15 or oGDF9 using Western blotting under non-reducing, reducing and cross-linking conditions. The molecular forms of recombinant oBMP15 and oGDF9 comprise mainly mature monomers with a lesser amount of the uncleaved pro-mature form. Mature domains, in the dimeric mature form, were detected for oGDF9 and oBMP15 (S356C), but not oBMP15. These mature domains were almost entirely located within high molecular weight multimeric complexes, which likely also contain the pro-region. In contrast, BMP15 and GDF9 secreted from ruminant oocytes under in vitro conditions were found mainly in an unprocessed promature form, along with some fully processed mature domains that did not interact to form detectable mature homodimers or heterodimers. Throughout ovarian follicular development, BMP15 and GDF9 are co-expressed and it has been established that these two factors have synergistic effects on granulosa cell proliferation both in vitro and in vivo and also on follicular maturation and ovulation-rate in vivo. Moreover, the recombinant proteins oBMP15 and oGDF9 generated for this study, when added together, also demonstrated a synergistic effect in the granulosa cell proliferation assay but this was not observed for oBMP15 (S356C) and oGDF9. Currently, no adequate model has been proposed to explain how interactions between the cell membrane and forms of oocyte-derived BMP15 and GDF9 achieve their synergistic effects. To investigate this, two homology models of the promature BMP15 and GDF9 proteins were generated using… Advisors/Committee Members: McNatty, Kenneth, Pitman, Janet.