Positive Feedback Loop Between Prostaglandin E2 and EGF-Like Factors Is Essential for Sustainable Activation of MAPK3/1 in Cumulus Cells During In Vitro Maturation of Porcine Cumulus Oocyte Complexes1

Abstract

During in vitro maturation of porcine cumulus-oocyte complexes (COCs), follicle-stimulating hormone (FSH) increases both prostaglandin E2 (PGE2) production and the expression levels of EGF-like factors. The ligands act on cumulus cells by the autocrine system due to their specific receptors, EP2, EP4, or EGF receptor. When each pathway is suppressed by inhibitors, complete cumulus expansion and oocyte maturation do not occur. In this study, we examined the relationship between both of these pathways in cumulus cells of porcine COCs. When COCs were cultured with FSH, Fshr mRNA expression was immediately decreased within 5 h, whereas Ptger2, Ptger4, and Ptgs2 expression levels were significantly increased in cumulus cells in the culture containing FSH for 5 or 10 h. The PTGS2 inhibitor NS398 significantly suppressed not only PGE2 secretion at any culture time point but also Areg, Ereg, and Tace/Adam17 expression in cumulus cells at 10 and 20 h but not at 1 or 5 h. During the early culture period, phosphorylation of MAPK3 and MAPK1 (MAPK3/1) was not affected by NS398; however, at 10 and 20 h, phosphorylation was suppressed by the drug. Furthermore, down-regulations of MAPK3/1 phosphorylation and expression of the target genes by NS398 was overcome by the addition of either PGE2 or EGF. FSH-induced cumulus expansion and meiotic progression to the MII stage were also suppressed by NS398, whereas these effects were also overcome by addition of either PGE2 or EGF. These results indicated that PGE2 is involved in the sustainable activation of MAPK3/1 in cumulus cells via the induction of EGF-like factor, which is required for cumulus expansion and meiotic maturation of porcine COCs.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus–oocyte complexes

“…Next was PTX3, which was increased by FGF10 at 12 h, followed by TNFAIP6 expression, which was upregulated at 22 h of culture. TNFAIP6 was reported to be a target of PG action in mice and pigs, and either silencing of PTGS2 or disruption of PG signaling severely impaired TNFAIP6 expression (Ochsner et al 2003, Takahashi et al 2006, Yamashita et al 2011. Therefore, the effects of FGF10 on TNFAIP6 expression are probably mediated through increased PTGS2.…”

The Release of EGF Domain from EGF-like Factors by a Specific Cleavage Enzyme Activates the EGFR-MAPK3/1 Pathway in Both Granulosa Cells and Cumulus Cells During the Ovulation Process

“…In an in vitro culture system of porcine COCs, expression of Ptgs2 and Ptger2 was induced by FSH concomitant with Areg, Ereg and Tace/Adam17 expression [23]. The expression of Areg, Ereg and Tace/Adam17 was significantly downregulated by the PTGS2 inhibitor, NS398, in cumulus cells of COCs cultured for 10 h, but not in those cultured for 5 h. During in vitro maturation of porcine COCs, the cAMP level was dramatically increased within 5 h, and this level was then maintained for up to 40 h [24].…”

“…The expression of Areg, Ereg and Tace/Adam17 was significantly downregulated by the PTGS2 inhibitor, NS398, in cumulus cells of COCs cultured for 10 h, but not in those cultured for 5 h. During in vitro maturation of porcine COCs, the cAMP level was dramatically increased within 5 h, and this level was then maintained for up to 40 h [24]. However, the level of Fshr mRNA that encoded the FSH receptor was rapidly and significantly decreased in cumulus cells of COCs cultured with FSH alone [23], suggesting that other stimulators that increase the cAMP level in cumulus cells must be secreted from cumulus cells. Strikingly, the cAMP level was not changed by NS398 at 5 h, whereas it was significantly decreased at 10 h. Furthermore, NS398 dramatically suppressed MAPK3/1 after 10 h of culture but not after 5 h of culture.…”

“…Importantly, our previous study showed the downregulation of EGF-like factor expression in Ptgs2-deficient mice during administration of hCG. Interestingly, the expression of Ptgs2 was upregulated by EGF-like factors [17], strongly indicating the existence of a positive feedback system between EGF-like factors and PGE2 during the ovulation process.In an in vitro culture system of porcine COCs, expression of Ptgs2 and Ptger2 was induced by FSH concomitant with Areg, Ereg and Tace/Adam17 expression [23]. The expression of Areg, Ereg and Tace/Adam17 was significantly downregulated by the PTGS2 inhibitor, NS398, in cumulus cells of COCs cultured for 10 h, but not in those cultured for 5 h. During in vitro maturation of porcine COCs, the cAMP level was dramatically increased within 5 h, and this level was then maintained for up to 40 h [24].…”

Role of PTGS2-generated PGE2 during gonadotrophin-induced bovine oocyte maturation and cumulus cell expansion

“…Many pathways including cAMP and cAMP-dependent protein kinase A are reported to be involved in regulating this process (Downs et al, 2002;Homa, 1988). FSH stimulates germinal-vesicle breakdown (GVBD) and resumption of oocyte maturation both in vitro and in vivo (Assidi et al, 2008(Assidi et al, , 2010Sirard et al, 2007) through binding to FSH receptors on granulosa cells or cumulus cells which are G-protein-coupled receptors (Heitman and Ijzerman, 2008;Segaloff et al, 1990) and results in a signalling cascade involving increasing intracellular cAMP concentrations (Dekel, 1988) and stimulation of prostaglandin E synthesis (Shimada et al, 2006;Yamashita et al, 2011).…”