Shitsukawa K, Andersen C B, Richard F J, Horner A K, Wiersma A, van Duin M, Conti M
Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317, USA.
Biol Reprod. 2001 Jul;65(1):188-96. doi: 10.1095/biolreprod65.1.188.
In the preovulatory follicle, oocyte meiotic resumption occurs soon after the LH surge and is associated with a decrease in cAMP. Inhibition of cAMP degradation blocks germinal vesicle breakdown as well as activation of meiotic promoting factor, both hallmarks of reentry into the cell cycle. In situ and pharmacological analysis of rodent ovaries suggested the presence of a phosphodiesterase 3 (PDE3) in the germ cell but not the somatic cell compartment. Here we have investigated the structure and properties of the PDE form expressed in mouse oocytes. Polymerase chain reactions using a mouse oocyte cDNA library as a template, and primers based on the conserved sequence of rat and human PDE3As, yielded partial fragments corresponding to mouse PDE3A. Further screening of the mouse oocyte cDNA library and subsequent ligation of individual cDNA clones yielded PDE3A cDNA containing the entire coding region of mouse PDE3A. To determine the kinetic properties of this PDE, the cDNAs encoding the full-length PDE3A and NH(2)-truncation forms Delta 1 (Delta346aa) and Delta 2 (Delta608aa) were expressed in mouse Leydig tumor cells. Whereas the full-length recombinant protein was always found in the particulate fraction, the Delta 1 and Delta 2 truncated PDE3As were recovered mostly in the soluble fraction. The Michaelis constant values for hydrolysis of cAMP of PDE3A Delta 1 and PDE3A Delta 2 were similar to those of intact full-length PDE3A or oocyte PDE (0.2-0.5 microM). More importantly, there was good correlation between the rank of potency of selective and nonselective compounds in inhibiting recombinant PDE3A or PDE activity derived from cumulus-oocyte complexes and in blocking resumption of meiosis. These data provide evidence that the PDE expressed in the oocyte is a soluble form of PDE3A and that activity of this enzyme is involved in the control of resumption of meiosis.
在排卵前卵泡中，促黄体生成素（LH）峰后不久卵母细胞减数分裂恢复，并伴有环磷酸腺苷（cAMP）水平下降。抑制cAMP降解可阻止生发泡破裂以及减数分裂促进因子的激活，这两者都是重新进入细胞周期的标志。对啮齿动物卵巢的原位和药理学分析表明，生殖细胞而非体细胞区室中存在磷酸二酯酶3（PDE3）。在此，我们研究了在小鼠卵母细胞中表达的PDE形式的结构和特性。以小鼠卵母细胞cDNA文库为模板，使用基于大鼠和人PDE3A保守序列的引物进行聚合酶链反应，得到了与小鼠PDE3A对应的部分片段。进一步筛选小鼠卵母细胞cDNA文库，并随后连接各个cDNA克隆，得到了包含小鼠PDE3A完整编码区的PDE3A cDNA。为了确定该PDE的动力学特性，将编码全长PDE3A和氨基端截短形式Delta 1（Delta346aa）和Delta 2（Delta608aa）的cDNA在小鼠睾丸间质细胞瘤细胞中表达。全长重组蛋白总是存在于颗粒部分，而Delta 1和Delta 2截短的PDE3A大多在可溶部分中回收。PDE3A Delta 1和PDE3A Delta 2水解cAMP的米氏常数与完整全长PDE3A或卵母细胞PDE的米氏常数相似（0.2 - 0.5 microM）。更重要的是，在抑制重组PDE3A或来自卵丘 - 卵母细胞复合体的PDE活性以及阻断减数分裂恢复方面，选择性和非选择性化合物的效力等级之间存在良好的相关性。这些数据提供了证据，表明卵母细胞中表达的PDE是PDE3A的可溶形式，并且该酶的活性参与减数分裂恢复的调控。
