Oral Presentation ESA-SRB Conference 2015

Cumulin, an oocyte-secreted heterodimer of the transforming growth factor-ß family, is a potent activator of granulosa cells and improves oocyte quality (#98)

David G Mottershead 1 2 , Satoshi Sugimura 1 3 , Sara L Al-Musawi 4 , Jing-Jie Li 1 5 , Dulama Richani 6 , Melissa A White 1 , Georgia A Martin 1 , Andrew Trotta 1 , Lesley J Ritter 1 , Junyan Shi 1 , Thomas D Mueller 7 , Craig A Harrison 4 , Robert B Gilchrist 1 6
  1. School of Paediatrics & Reproductive Health, Robinson Research Institute, University of Adelaide, Adelaide, SA, Australia
  2. Mottasis Oy Ltd, Helsinki, Finland
  3. Institute of Agriculture, Department of Biological Production, Tokyo University of Agriculture and Technology, Tokyo, Japan
  4. Hudson Institute of Medical Research, Clayton, Vic, Australia
  5. Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
  6. School of Women’s & Children’s Health, University Of New South Wales, Sydney, NSW, Australia
  7. Julius-von-Sachs Institute , University of Wuerzburg, Wuerzburg, Germany

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions, and their effects on granulosa cells (GC) and oocytes. By generating a covalent BMP15 homodimer that cannot heterodimerize with GDF9, we found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Modelling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (cumulin). Both pro-cumulin and cumulin exhibited highly potent bioactivity on GC: activating both SMAD2/3 and SMAD1/5/8 signaling pathways in human granulosa COV434 cells, and promoting mouse GC proliferation (ED50: 4ng/ml and 0.6ng/ml, respectively) and expression of a set of genes (Ptx3, Has2, Tnfaip6, Ptgs2) associated with oocyte-regulated GC differentiation towards the cumulus cell phenotype. In all cases cumulin was more potent than pro-cumulin, pro-GDF9, pro-BMP15 or the two combined on GC. However, on cumulus-oocyte complexes, pro-cumulin was more effective than all other growth factors at improving porcine oocyte quality using a low developmental competence model. Pro-cumulin increased subsequent blastocyst development 2.3-fold from 28% to 63%. Our results support a model of activation for human GDF9 dependant on cumulin formation through heterodimerization with BMP15. Oocyte-secreted cumulin is likely to be a central regulator of fertility in mono-ovular mammals.