Yamashita Yasuhisa, Kawashima Ikko, Gunji Yosuke, Hishinuma Mitsugu, Shimada Masayuki
School of Veterinary Medicine, Faculty of Agriculture, Tottori University, Tottori, Japan.
J Reprod Dev. 2010 Jun;56(3):315-23. doi: 10.1262/jrd.09-199h. Epub 2010 Feb 18.
During in vitro maturation of porcine cumulus-oocyte complexes (COCs), progesterone was secreted from cumulus cells and acted on the cumulus cells themselves, which required for cumulus expansion and oocyte maturation. EGF-like factor (amphiregulin, AREG; epiregulin, EREG) and its protease, TACE/ADAM17, are also expressed in cumulus cells, and thereby, soluble EGF domain was acted on the EGF receptor expressed on cumulus cells. In this study, we examined the relationship between progesterone function and EGF-like factor stimuli in cumulus cells of porcine COCs. When COCs were cultured with FSH and LH, Areg, Ereg and Tace/Adam17 were expressed in cumulus cells. Treatment with a progesterone receptor (PGR) antagonist, RU486, did not affect the Areg and Ereg mRNA expression levels at any culture time points. However, the Tace/Adam17 mRNA level, protein level and its activity were significantly suppressed by RU486 at the 30 or 40 h time point. At 20 h of culture, phosphorylation of ERK1/2 and the expressions of target genes (Has2, Tnfaip6 and Ptgs2) were not suppressed by RU486; however, at 40 h, ERK1/2 phosphorylation and the target gene expression levels were significantly downregulated by RU486 in cumulus cells. Furthermore, the negative effects of RU486 at 40 h were overcome by the addition of EGF. These results indicated that the level of TACE/ADAM17 in cumulus cells was regulated by the progesterone-PGR pathway during in vitro maturation of porcine COCs. Therefore, we concluded that the progesterone-induced TACE/ADAM17 leads to production of soluble EGF domain from cumulus cells, which enhances functional changes of cumulus cells and progresses meiotic maturation of oocytes during in vitro maturation of porcine COCs.
在猪卵丘-卵母细胞复合体（COCs）的体外成熟过程中，孕酮由卵丘细胞分泌并作用于卵丘细胞自身，这是卵丘扩展和卵母细胞成熟所必需的。表皮生长因子样因子（双调蛋白，AREG；表皮调节素，EREG）及其蛋白酶TACE/ADAM17也在卵丘细胞中表达，从而使可溶性表皮生长因子结构域作用于卵丘细胞上表达的表皮生长因子受体。在本研究中，我们检测了猪COCs卵丘细胞中孕酮功能与表皮生长因子样因子刺激之间的关系。当COCs与促卵泡激素（FSH）和促黄体生成素（LH）一起培养时，Areg、Ereg和Tace/Adam17在卵丘细胞中表达。用孕酮受体（PGR）拮抗剂RU486处理，在任何培养时间点都不影响Areg和Ereg mRNA的表达水平。然而，在30或40小时时间点，RU486显著抑制了Tace/Adam17 mRNA水平、蛋白质水平及其活性。在培养20小时时，RU486没有抑制ERK1/2的磷酸化以及靶基因（Has2、Tnfaip6和Ptgs2）的表达；然而，在40小时时，RU486显著下调了卵丘细胞中ERK1/2的磷酸化和靶基因表达水平。此外，添加表皮生长因子可克服RU486在40小时时的负面影响。这些结果表明，在猪COCs体外成熟过程中，卵丘细胞中TACE/ADAM17的水平受孕酮-PGR途径调控。因此，我们得出结论，孕酮诱导的TACE/ADAM17导致卵丘细胞产生可溶性表皮生长因子结构域，这在猪COCs体外成熟过程中增强了卵丘细胞的功能变化并促进了卵母细胞的减数分裂成熟。
