INRA, UR85 Physiologie de la Reproduction et des Comportements, Nouzilly, France.
Mol Reprod Dev. 2013 Feb;80(2):166-82. doi: 10.1002/mrd.22148. Epub 2013 Jan 30.
In vitro maturation (IVM) of immature oocytes is widely used in assisted reproduction technologies in cattle, and is increasingly used to treat human infertility. The development competence of IVM oocytes, however, is lower than preovulatory, in vivo-matured oocytes. During maturation, cumulus cells (CC) are metabolically coupled with an oocyte and support the acquisition of its developmental potential. Our objective was to identify genes and pathways that were affected by IVM in bovine CC. Microarray transcriptomic analysis of CC enclosing in vitro- or in vivo-mature oocytes revealed 472 differentially expressed genes, including 28% related to apoptosis, correlating with twofold higher cell death after IVM than in vivo, as detected by TUNEL. Genes overexpressed after IVM were significantly enriched in functions involved in cell movement, focal adhesion, extracellular matrix function, and TGF-beta signaling, whereas under-expressed genes were enriched in regulating gene expression, energy metabolism, stress response, and MAP kinases pathway functions. Differential expression of 15 genes, including PAG11 (increased) and TXNIP (decreased), which were never detected in CC before, was validated by real-time RT-PCR. Moreover, protein quantification confirmed the lower abundance of glutathione S-transferase A1 and prostaglandin G/H synthase 2, and the higher abundance of hyaluronan synthase 2 and SMAD4, a member of TGF-beta pathway, in CC after IVM. Phosphorylation levels of SMAD2, MAPK3/1, and MAPK14, but not MAPK8, were higher after IVM that in vivo. In conclusion, IVM provokes the hyper-activation of TGF-beta and MAPK signaling components, modifies gene expression, leads to increased apoptosis in CC, and thus affects oocyte quality.
体外成熟（IVM）的不成熟卵母细胞在牛的辅助生殖技术中被广泛应用，并且越来越多地用于治疗人类不育症。然而，IVM 卵母细胞的发育能力低于排卵前、体内成熟的卵母细胞。在成熟过程中，卵丘细胞（CC）与卵母细胞代谢偶联，并支持其获得发育潜能。我们的目的是鉴定在牛 CC 中受 IVM 影响的基因和途径。对体外或体内成熟卵母细胞包裹的 CC 的微阵列转录组分析显示，有 472 个差异表达基因，其中 28%与凋亡有关，与 IVM 后细胞死亡增加两倍有关，这是通过 TUNEL 检测到的。IVM 后过表达的基因显著富集在与细胞运动、焦点黏附、细胞外基质功能和 TGF-β信号相关的功能中，而下调的基因则富集在调节基因表达、能量代谢、应激反应和 MAP 激酶途径功能中。15 个基因的差异表达，包括 PAG11（增加）和 TXNIP（减少），这些基因以前从未在 CC 中检测到，通过实时 RT-PCR 得到验证。此外，蛋白质定量证实，在 IVM 后 CC 中，谷胱甘肽 S-转移酶 A1 和前列腺素 G/H 合酶 2 的丰度降低，透明质酸合酶 2和 SMAD4（TGF-β途径的成员）的丰度升高。SMAD2、MAPK3/1 和 MAPK14 的磷酸化水平在 IVM 后高于体内，而 MAPK8 则没有。总之，IVM 引发 TGF-β和 MAPK 信号通路组件的过度激活，改变基因表达，导致 CC 中凋亡增加，从而影响卵母细胞质量。
