LAUSR

The release of a fertilizable oocyte from the ovary is dependent upon the expansion of the cumulus cells. The expansion requires cooperation between epidermal growth factor (EGF) family peptide‐activated mitogen‐activated protein kinase (MAPK)3/1 and oocyte paracrine factor‐activated‐Sma‐ and Mad‐related protein (SMAD)2/3 signaling in cumulus cells. However, the mechanism underlying (MAPK)3/1 signaling is unclear. In the present study, the EGF‐activation of EGF receptor (EGFR) induced cyclic adenosine 3′,5′‐monophosphate (cAMP) response element‐binding protein (CREB) phosphorylation in cumulus cells, and the interruption of CREB functional complex formation by naphthol AS‐E phosphate (KG‐501) completely blocked the EGF‐stimulated expansion‐related gene expression. EGF‐stimulated phosphorylation of CREB was completely inhibited by MAPK3/1 inhibitor U0126, suggesting that EGF‐activated MAPK3/1 results in the activation of CREB for cumulus expansion. Also, the role of EGF‐stimulated calcium signaling was studied. Calcium‐elevating reagents ionomycin and sphingosine‐1‐phosphate mimicked, but calcium chelators bis‐(o'aminophenoxy)‐ethane‐N,N,N,N‐tetraacetic acid, tetra(acetoxymethyl)‐ester, and 8‐(N,N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate abolished the activity of EGF on CREB phosphorylation, cumulus expansion, and expansion‐related gene expression. Furthermore, EGF‐induced cumulus expansion was inhibited by calmodulin (CaM)‐dependent protein kinase II (CaMKII) inhibitors, KN‐93 and autocamtide‐2‐related inhibitory peptide. However, the inhibition of SMAD2/3 activity by removal of oocyte from cumulus–oocyte complexes did not affect the EGF‐induced CREB phosphorylation, indicating that EGF‐activated CREB is independent of oocyte‐activated SMAD2/3 signaling. Therefore, EGF‐induced CREB activity by MAPK3/1 and Ca2+/CaMKII signaling pathways promotes the expansion‐related gene expression and consequent cumulus expansion.