Analyses of the Involvement of PKA Regulation Mechanism in Meiotic lncon〕petence of Porcine Growing Oocytes.
Analyses of the Involvement of PKA Regulation Mechanism in Meiotic lncon〕petence of Porcine Growing Oocytes.
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PKA调控机制参与猪卵母细胞减数分裂失常的分析。
DOI：
10.1095/biolreprod.112.101279
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发表时间：
2013
影响因子：
3.6
通讯作者：
Takanori Nishimura
中科院分区：
文献类型：
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作者：
An-Dih Yu;Tadanori Kurosawa;Mitsuru Ueda;Wen-Chang Chen;戸江哲理;Takanori Nishimura
哺乳动物生长卵母细胞（GO）缺乏恢复减数分裂的能力，尽管这种限制的分子机制尚不完全清楚。在本研究中，我们从猪卵母细胞中克隆了cAMP依赖性蛋白激酶（PKA）亚基的cDNA，并在分子水平上分析了PKA调节机制在GO减数分裂不全中的作用。我们使用我们建立的猪 PKA 测定系统，在整个体外培养过程中发现 GO 中存在不依赖于 cAMP 的高 PKA 活性，并且通过注射 PKA 催化亚基 (PKA-C) 的反义 RNA 抑制该活性，诱导 GO 中减数分裂恢复。然后我们检查了以下可能性：由于两个 PKA-R（PRKAR1A 和 PRKAR2A）的过表达，能够结合并抑制 PKA-C 的 PKA 调节亚基（PKA-R）的量不足以抑制 GO 中的 PKA 活性。我们发现它们都不影响 PKA 活性并诱导 GO 减数分裂恢复，尽管 PRKAR2A 可以抑制 PKA 活性并诱导 cAMP 处理的成熟卵母细胞 (FGO) 减数分裂。最后，我们分析了 PKA 亚基的亚细胞定位，发现在减数分裂停滞期间，所有亚基都定位在细胞质中，并且 FGO 中 PKA-C 和 PRKAR2A（而不是 PRKAR1A）在减数分裂恢复前进入细胞核，而在整个培养期间，所有亚基都保留在 GO 中的细胞质中。我们的研究结果表明，持续的高PKA活性是猪GOs减数分裂无能的主要原因，并且这种PKA活性不仅仅是由PKA-R表达水平不足引起的，还可以归因于更复杂的时空调节机制。
Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. In the present study, we cloned cDNAs of cAMP-dependent protein-kinase (PKA) subunits from porcine oocytes and analyzed the involvement of the PKA regulation mechanism in the meiotic incompetence of GOs at the molecular level. We found a cAMP-independent high PKA activity in GOs throughout the in vitro culture using a porcine PKA assay system we established, and inhibition of the activity by injection of the antisense RNA of the PKA catalytic subunit (PKA-C) induced meiotic resumption in GOs. Then we examined the possibility that the amount of the PKA regulatory subunit (PKA-R), which can bind and inhibit PKA-C, was insufficient to suppress PKA activity in GOs because of the overexpression of two PKA-Rs, PRKAR1A and PRKAR2A. We found that neither of them affected PKA activity and induced meiotic resumption in GO although PRKAR2A could inhibit PKA activity and induce meiosis in cAMP-treated full-grown oocytes (FGOs). Finally, we analyzed the subcellular localization of PKA subunits and found that all the subunits were localized in the cytoplasm during meiotic arrest and that PKA-C and PRKAR2A, but not PRKAR1A, entered into the nucleus just before meiotic resumption in FGOs, whereas all of them remained in the cytoplasm in GOs throughout the culture period. Our findings suggest that the continuous high PKA activity is a primary cause of the meiotic incompetence of porcine GOs and that this PKA activity is not simply caused by an insufficient expression level of PKA-R, but can be attributed to more complex spatial-temporal regulation mechanisms.