School of Veterinary Medicine, Faculty of Agriculture, Tottori University, 4-101 Koyamachou-minami, Tottori, 680-8553, Japan.
J Ovarian Res. 2009 Dec 24;2:20. doi: 10.1186/1757-2215-2-20.
During ovulation, it has been shown that LH stimulus induces the expression of numerous genes via PKA, p38 MAPK, PI3K and ERK1/2 in cumulus cells and granulosa cells. Our recent study showed that EGF-like factor and its protease (TACE/ADAM17) are required for the activation of EGF receptor (EGFR), cumulus expansion and oocyte maturation of porcine cumulus-oocyte complexes (COCs). In the present study, we investigated which signaling pathways are involved in the gene expression of EGF-like factor and in Tace/Adam17 expression in cumulus cells of porcine COC during in vitro maturation.
Areg, Ereg, Tace/Adam17, Has2, Tnfaip6 and Ptgs2 mRNA expressions were detected in cumulus cells of porcine COCs by RT-PCR. Protein level of ERK1/2 phosphorylation in cultured cumulus cells was analyzed by westernblotting. COCs were visualized using a phase-contrast microscope.
When COCs were cultured with FSH and LH up to 2.5 h, Areg, Ereg and Tace/Adam17 mRNA were expressed in cumulus cells of COCs. Areg, Ereg and Tace/Adam17 gene expressions were not suppressed by PI3K inhibitor (LY294002), whereas PKA inhibitor (H89), p38 MAPK inhibitor (SB203580) and MEK inhibitor (U0126) significantly suppressed these gene expressions. Phosphorylation of ERK1/2, and the gene expression of Has2, Tnfaip6 and Ptgs2 were also suppressed by H89, SB203580 and U0126, however, these negative effects were overcome by the addition of EGF to the medium, but not in the U0126 treatment group.
The results showed that PKA, p38 MAPK and ERK1/2 positively controlled the expression of EGF-like factor and TACE/ADMA17, the latter of which impacts the cumulus expansion and oocyte maturation of porcine COCs via the EGFR-ERK1/2 pathway in cumulus cells.
在排卵期间，已经表明 LH 刺激通过 PKA、p38MAPK、PI3K 和 ERK1/2 在卵丘细胞和颗粒细胞中诱导大量基因的表达。我们最近的研究表明，表皮生长因子样因子及其蛋白酶（TACE/ADAM17）是激活表皮生长因子受体（EGFR）、卵丘扩展和猪卵丘-卵母细胞复合体（COC）卵母细胞成熟所必需的。在本研究中，我们研究了在猪 COC 的体外成熟过程中，哪些信号通路参与了卵丘细胞中表皮生长因子样因子的基因表达和 Tace/Adam17 的表达。
通过 RT-PCR 检测猪 COC 卵丘细胞中 Areg、Ereg、Tace/Adam17、Has2、Tnfaip6 和 Ptgs2 的 mRNA 表达。通过 Westernblotting 分析培养的卵丘细胞中 ERK1/2 磷酸化的蛋白水平。使用相差显微镜观察 COC。
当 COC 用 FSH 和 LH 培养至 2.5 小时时，Areg、Ereg 和 Tace/Adam17 mRNA 在 COC 的卵丘细胞中表达。PI3K 抑制剂（LY294002）并没有抑制 Areg、Ereg 和 Tace/Adam17 的基因表达，而 PKA 抑制剂（H89）、p38MAPK 抑制剂（SB203580）和 MEK 抑制剂（U0126）则显著抑制了这些基因的表达。ERK1/2 的磷酸化以及 Has2、Tnfaip6 和 Ptgs2 的基因表达也被 H89、SB203580 和 U0126 抑制，但这些负效应可以通过向培养基中添加 EGF 来克服，但在 U0126 处理组中则没有。
结果表明，PKA、p38MAPK 和 ERK1/2 正向调控表皮生长因子样因子和 TACE/ADMA17 的表达，后者通过 EGFR-ERK1/2 途径影响猪 COC 的卵丘扩展和卵母细胞成熟。
