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.

During in vitro maturation of porcine cumulus cell-oocyte complexes and in vitro luteinization of porcine granulosa cells, FSH induces the expression of the protease TNFα-converting enzyme/A disintegrin and metalloproteinase domain 17 (TACE/ADAM17) and the epidermal growth factor (EGF)-like factors, which activate the EGF receptor (EGFR)-MAPK3/1 pathway in both cumulus and granulosa cells. FSH is known to activate not only protein kinase A and p38MAPK pathways in both cell types but also activates protein kinase C (PKC). Because PKC-induced association of cellular-Sarcoma (c-Src) and TACE/ADAM17 is required for TACE/ADAM17 enzyme activation in some cancer cells, we hypothesized that PKC and c-Src impact TACE/ADAM17-mediated activation of EGFR signaling pathway in porcine granulosa and cumulus cells. When granulosa cells or cumulus cell-oocyte complexes were cultured with FSH, PKC activity and c-Src phosphorylation increased and were associated with increased TACE/ADAM17 enzyme activity. The PKC inhibitor calphostin C (CalC) and the c-Src inhibitor (4 amino 5 (4 chlorophenyl) 7 (t butyl)pyrazolo[3,4 d]pyrimidine [PP2]) suppressed TACE/ADAM17 enzyme activity, whereas these inhibitors did not affect Tace/Adam17 mRNA expression. Immunoprecipitation analysis showed that FSH mediated the association of c-Src with TACE/ADAM17 via a PKC-dependent mechanism. Either CalC or PP2 suppressed EGFR downstream signaling pathway (MAPK3/1) in these ovarian cell types and reduced cumulus expansion, meiotic maturation of oocytes, and progesterone production. The negative effects were overcome by the addition of amphiregulin. Collectively, these results indicate that activation of TACE/ADAM17 via a PKC-induced c-Src-dependent manner mediates proteolytic activation of the EGF-like factors that are involved in the induction of granulosa cell differentiation, cumulus expansion, and meiotic maturation of porcine oocytes in vitro.

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Japanese patients with type 2 diabetes mellitus present a different responsiveness in terms of insulin secretion to glucose and body mass index (BMI) from other populations. The genetic background that predisposes Japanese individuals to type 2 diabetes mellitus is under study. Recent genetic studies demonstrated that the locus mapped in macrophage erythroblast attacher (MAEA) increases the susceptibility to type 2 diabetes mellitus in East Asians, including Japanese individuals. MAEA encodes a protein that plays a role in erythroblast enucleation and in the normal differentiation of erythroid cells and macrophages. However, the contribution of MAEA to type 2 diabetes mellitus remains unknown. In this study, to overexpress MAEA in the mouse liver and primary mouse hepatocytes, we generated a MAEA-expressing adenovirus (Ad) vector using a novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-MAEA). Blood glucose and insulin levels in Ad-MAEA-treated mice were comparable to those in control Ad-treated mice. Primary mouse hepatocytes transduced with Ad-MAEA showed lower levels of expression of gluconeogenesis genes than those transduced with the control Ad vector. Hepatocyte nuclear factor-4α (HNF-4α) mRNA expression in primary mouse hepatocytes was also suppressed by MAEA overexpression. These results suggest that MAEA overexpression attenuates hepatic gluconeogenesis, which could potentially lead to improvement of type 2 diabetes mellitus.