Mechanisms of FSH- and Amphiregulin-Induced MAP Kinase 3/1 Activation in Pig Cumulus-Oocyte Complexes During Maturation In Vitro

Abstract

The maturation of mammalian oocytes in vitro can be stimulated by gonadotropins (follicle-stimulating hormone, FSH) or their intrafollicular mediator, epidermal growth factor (EGF)-like peptide-amphiregulin (AREG). We have shown previously that in pig cumulus-oocyte complexes (COCs), FSH induces expression and the synthesis of AREG that binds to EGF receptor (EGFR) and activates the mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. However, in this study we found that FSH also caused a rapid activation of MAPK3/1 in the cumulus cells, which cannot be explained by the de novo synthesis of AREG. The rapid MAPK3/1 activation required EGFR tyrosine kinase (TK) activity, was sensitive to SRC proto-oncogene non-receptor tyrosine kinase (SRC)-family and protein kinase C (PKC) inhibitors, and was resistant to inhibitors of protein kinase A (PKA) and metalloproteinases. AREG also induced the rapid activation of MAPK3/1 in cumulus cells, but this activation was only dependent on the EGFR TK activity. We conclude that in cumulus cells, FSH induces a rapid activation of MAPK3/1 by the ligand-independent transactivation of EGFR, requiring SRC and PKC activities. This rapid activation of MAPK3/1 precedes the second mechanism participating in the generation and maintenance of active MAPK3/1-the ligand-dependent activation of EGFR depending on the synthesis of EGF-like peptides.

Keywords: FSH; amphiregulin; cumulus cells; epidermal growth factor receptor; mitogen-activated protein kinase 3/1; signal transduction.

Figure 1

Time course of MAPK3/1 activation in cumulus-enclosed oocytes (COCs) stimulated with follicle-stimulating hormone (FSH) or amphiregulin (AREG). The panels show representative results of immunoblotting of phosphorylated and total MAPK3/1 in samples of 25 COCs cultured in vitro for the indicated periods of time. The experiments shown in panels A and B were repeated twice with the same results. The experiments shown in panels (C–F) were repeated three times. Quantification of the activated MAPK3/1 was performed by densitometry and is shown in the graphs as proportions of phosphorylated and total MAPK3/1 and expressed in arbitrary units as the fold increase over the proportion found in unstimulated COCs at the beginning of the culture. (A) Activation of MAPK3/1 in FSH-stimulated COCs during the long-term culture. (B) Activation of MAPK3/1 in AREG-stimulated COCs during the long-term culture. (C) Activation of MAPK3/1 in FSH-stimulated COCs during the short-term culture. (D) Activation of MAPK3/1 in AREG-stimulated COCs during the short-term culture. (E) Activity of MAPK3/1 in COCs cultured for short-term period in control medium without FSH and AREG. (F) Activity of MAPK3/1 in COCs cultured for long-term period in control medium without FSH and AREG. The different superscripts above the columns indicate significant differences (p < 0.05).

Figure 2

Effect of protein kinase and proteinase inhibitors on FSH- and AREG-induced rapid activation of MAPK3/1 in pig COCs. The panels show representative results of the immunoblotting of phosphorylated and total MAPK3/1 in samples of 25 COCs cultured in vitro for 10 min. The experiments were repeated three times. Quantification of the activated MAPK3/1 was performed by densitometry and is shown in the graphs as proportions of phosphorylated and total MAPK3/1, and expressed in arbitrary units as the fold increase over the proportion found in unstimulated COCs at the beginning of the culture. (A,B) show COCs stimulated by FSH; (C,D) show COCs stimulated by AREG. The different superscripts above the columns (D) or superscripts with no common letters (B) indicate significant differences (p < 0.05). AG: AG1478; CalC: calphostin C; Gal: galardin.

Figure 3

Effect of protein kinase and proteinase inhibitors on maintenance of FSH- and AREG-induced activation of MAPK3/1 in pig COCs. The panels show representative results of immunoblotting of phosphorylated and total MAPK3/1 in samples of 25 COCs cultured in vitro for 16 h. The experiments were repeated three times. Quantification of the activated MAPK3/1 was performed by densitometry and is shown in the graphs as proportions of phosphorylated and total MAPK3/1 and expressed in arbitrary units as the fold increase over the proportion found in unstimulated COCs at the beginning of the culture. (A,B) show COCs stimulated by FSH; (C,D) show COCs stimulated by AREG. The different superscripts above the columns (D) or superscripts with no common letters (B) indicate significant differences (p < 0.05). AG: AG1478; CalC: calphostin C; Gal: galardin.

Figure 4

Ligand-independent and ligand-dependent transactivation of epidermal growth factor receptor (EGFR)/MAPK3/1 pathway in pig cumulus cells by FSH. The binding of FSH to the G_q class of G-protein coupled receptors (GPCRs) results in the activation of protein kinase C (PKC) and SRC that phosphorylates EGFR in its intracellular domain. It is not clear whether this phosphorylation leads to receptor dimerization [26]. The SRC-mediated phosphorylation is associated with activation of the RAS/RAF/MEK1 pathway and rapid activation of MAPK3/1, but it may not lead to full phosphorylation of the intracellular domain and activation of all pathways known to be associated with EGFR TK activity [31,32]. FSH also binds to the G_s class of GPCRs and activates adenylyl cyclase, which leads to an increase in cAMP and activation of PKA. This PKA activity is required for the synthesis of the AREG pro-peptides [13], which are cleaved at the plasma level by TACE/ADAMs metalloproteinases and released outside the cumulus cells into the extracellular space. The active forms of AREG serve as ligands for EGFR, which leads to dimerization of the receptor, full phosphorylation of its intracellular domain and activation of downstream signaling pathways, including the MAPK3/1, PKC and PI3K pathways. The transcription- and ligand synthesis-dependent process of EGFR/MAPK3/1 activation requires 1–2 h to be activated [13] and operates in cumulus cells for an extended period of time. MAPK3/1 may thus be involved in events occurring in minutes after the exposure of follicular cells to gonadotropins, such as gap junction closure through connexin phosphorylation [15] and the regulation of cGMP level in follicular cells [41,42,43], as well as in delayed events such as the regulation of steroidogenesis and cumulus expansion [20]. MEK: MEK1/2; MAPK: MAPK3/1; MMP: matrix metalloproteinases; AC: adenylyl cyclase.