524. SPECIES DIFFERENCES IN THE REQUIREMENT OF SMAD2/3 AND MAPK SIGNALLING FOR CUMULUS CELL EXPANSION

Abstract

Cumulus cell expansion is necessary for female fertility. In the mouse, cumulus expansion is dependent on activation of two cumulus signalling pathways: 1) mitogen-activated protein kinase (MAPK3/1) by FSH/EGF and 2) Smad2/3 signalling by oocyte-secreted factors. This study was conducted to determine the involvement of these two signalling pathways in pig cumulus expansion. Abattoir-collected pig COC and eCG-primed mouse COC were cultured for either 24 or 20 hours respectively in the presence of FSH and treated with increasing doses of either a MAPK 3/1 inhibitor (U0126), it's inactive analogue (U0124), or kinase inhibitors SB431542 and dorsomorphin that prevent phosphorylation of Smad2/3 and Smad1/5/8, respectively, or a vehicle control. Morphological cumulus expansion was measured using an established subjective scoring system. At a dose of 10µM, U0126 completely abolished mouse expansion (0±0, p<0.05) and caused severe impairment of cumulus expansion in porcine COC (1.23±0.09, p<0.05). U0124 and dorsomorphin were unable to inhibit cumulus expansion in either species. SB431542 was also ineffective at blocking pig cumulus expansion (2.6±0.16 p<0.05) despite using a dose 4 times higher (8µM) than that required to ablate mouse COC expansion. To determine if pig oocytes activate cumulus cell Smad2/3 or 1/5/8, a Smad-luciferase reporter assay was undertaken. In contrast to mouse oocytes which only activated the Smad2/3 pathway, pig oocytes activated both pathways. This suggests that the failure of SB431542 to prevent pig cumulus expansion is not due to inadequate oocyte activation of Smad2/3. These results demonstrate that while activation of both MAPK3/1 and Smad2/3 pathways are required for mouse cumulus cell expansion, pig COC expansion only requires the MAPK3/1 pathway. Neither species require Smad1/5/8 pathway activation. This study provides new insight into the differing signalling mechanisms required for mammalian cumulus cell expansion.