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Pharmacological PKA inhibition: all may not be what it seems.药理学上的蛋白激酶A抑制作用：情况可能并非如表面所见。
Sci Signal. 2008 Jun 3;1(22):re4. doi: 10.1126/scisignal.122re4.
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Developmentally acquired PKA localisation in mouse oocytes and embryos.小鼠卵母细胞和胚胎中发育性获得的蛋白激酶A定位
Dev Biol. 2008 May 1;317(1):36-45. doi: 10.1016/j.ydbio.2008.01.045. Epub 2008 Feb 14.
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H89对猪卵母细胞减数分裂恢复的影响。

Effect of H89 on the meiotic resumption of pig oocytes.

作者信息

Cayo-Colca Ilse Silvia, Harayama Hiroshi, Miyano Takashi

机构信息

Graduate School of Agricultural Science Kobe University 1-1 Rokkodai-cho, Nada-Ku 657-8501 Kobe Japan.

出版信息

Reprod Med Biol. 2011 Jan 20;10(2):89-96. doi: 10.1007/s12522-010-0073-2. eCollection 2011 Jun.

DOI:10.1007/s12522-010-0073-2
PMID:29699085
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5907130/
Abstract

PURPOSE

We studied the effect of H89, an inhibitor of protein kinase A (PKA), on the meiotic resumption of pig oocytes.

METHODS

Pig cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were cultured for 27 h to induce meiotic resumption. COCs and DOs were exposed to H89 for different periods. Oocyte PKA activity was assessed by in vitro kinase assay and immunocytochemistry using an antibody against fully active PKA catalytic subunits. Oocyte serine/threonine (Ser/Thr)-phosphorylated proteins were detected by Western blotting and immunocytochemistry using an anti-pSer/pThr PKA substrate antibody.

RESULTS

H89 suppressed germinal vesicle break down (GVBD) in COCs and DOs. To determine whether the suppression was due to inhibition of oocyte PKA, we analyzed oocyte PKA. Kinase assay showed that both types of oocytes possessed PKA activity throughout the culture period. Immunocytochemistry showed that fully active PKA catalytic subunits and Ser/Thr phosphorylated proteins were present in the oocytes at the GV stage and after GVBD. Western blotting indicated that both types of oocytes contained Ser/Thr phosphorylated proteins at the GV stage, and that several proteins became phosphorylated after GVBD.

CONCLUSIONS

Pig oocytes contain active PKA during the occurrence of GVBD, and H89 suppresses the GVBD.

摘要

目的

我们研究了蛋白激酶A（PKA）抑制剂H89对猪卵母细胞减数分裂恢复的影响。

方法

将猪卵丘-卵母细胞复合体（COCs）和裸卵（DOs）培养27小时以诱导减数分裂恢复。COCs和DOs在不同时间段暴露于H89。通过体外激酶测定和使用针对完全活性PKA催化亚基的抗体进行免疫细胞化学来评估卵母细胞PKA活性。使用抗pSer/pThr PKA底物抗体通过蛋白质印迹和免疫细胞化学检测卵母细胞丝氨酸/苏氨酸（Ser/Thr）磷酸化蛋白。

结果

H89抑制了COCs和DOs中的生发泡破裂（GVBD）。为了确定这种抑制是否是由于卵母细胞PKA的抑制，我们分析了卵母细胞PKA。激酶测定表明，在整个培养期间，两种类型的卵母细胞都具有PKA活性。免疫细胞化学表明，在GV期和GVBD后，卵母细胞中存在完全活性的PKA催化亚基和Ser/Thr磷酸化蛋白。蛋白质印迹表明，两种类型的卵母细胞在GV期都含有Ser/Thr磷酸化蛋白，并且在GVBD后几种蛋白质发生了磷酸化。

结论

猪卵母细胞在GVBD发生期间含有活性PKA，并且H89抑制GVBD。