272. Signalling pathways involved in mouse GDF9 and BMP15 stimulated thymidine uptake by rat granulosa cells

Abstract

The oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are essential for ovarian follicular growth and development. Understanding the molecular mechanisms of these factors could assist with the development of future products for fertility control. Thymidine uptake by rat granulosa cells is stimulated cooperatively by GDF9 and BMP15. Inhibitors of the activin receptor-like kinase (ALK) 4,5,7 and the nuclear factor kappaB (NF_KB) second messenger pathways block ovine GDF9 and BMP15 stimulated thymidine incorporation. The ALK 4,5,7 receptor pathway is known to be essential for the cooperative effects of mouse (m)GDF9 and mBMP15 on thymidine incorporation but the role of other pathways has yet to be determined, which was the focus of this study. Inhibitors of NF_KB (Sn50; 10µg/mL), ALK 2,3,6 receptor (Dorsomorphin; 1µM), p38 mitogen-activated protein kinase (p38 MAPK; SB239063; 5 µM) and c-Jun-N-terminal kinase (JNK; TAT-TI-JIP153–163; 5 µM) pathways were each cultured with recombinant mGDF9 (25 ng/mL) and mBMP15 (6 ng/mL) in a rat granulosa cell [³H]-thymidine bioassay. The p38 MAPK inhibitor caused partial inhibition of thymidine uptake but this appeared to be non-specific as a similar level of suppression was observed in the control cultures. Neither the ALK 2,3,6 receptor nor the NF_KB pathway inhibitors had any effect on mGDF9 and mBMP15 stimulated thymidine uptake. The JNK inhibitor showed a 1.7-fold increase in stimulation above the mGDF9 and mBMP15 effect (P < 0.01) but a similar stimulation was also observed in some controls. This differs from the results observed with ovine GDF9 and BMP15 where thymidine uptake was completely blocked by the NF_KB inhibitor and the JNK inhibitor had no effect. In conclusion, the molecular mechanisms of GDF9 and BMP15 function are dependent on the species of origin of the growth factor and therefore caution is needed when extrapolating findings from one species to another.