Tsutsumi O, Satoh K, Sakamoto S
Nihon Sanka Fujinka Gakkai Zasshi. 1982 Oct;34(10):1755-62.
A microassay method sensitive enough to analyze the enzyme activities in one oocyte was developed using enzymatic cycling for amplifying the reaction product to 10,000 fold. An oil-well technique was applied in the assay for achieving the reaction in the medium as small as 1.0 to 5.0 microliter. Immature Wistar rats were superovulated by PMS-hCG administration. Oocytes were collected by the puncture of the follicle and the flushing of the tube. They were freeze-dried after washing to remove cumulus cells. The dry weight was about 50ng on a quartz fiber fishpole balance. The activity of hexokinase was 1.75 +/- 0.14 picomol/oocyte/hr corresponding to one-tenth of the ovarian homogenate as control, indicating low capacity of glucose utilization in the oocyte. The activities of G6PD, LDH, and MDH were 8.41 +/- 0.34, 35.7 +/- 2.89. 11.1 +/- 2.5 picomol/oocyte/min, respectively. High activity of G6PD suggests the pentose phosphate shunt concerned with steroidogenesis is active in the oocyte. HCG increased the activities of hexokinase and MDH and decreased that of G6PD. The activity of LDH remained unchanged.
利用酶循环将反应产物放大10000倍，开发了一种灵敏度足以分析单个卵母细胞中酶活性的微量测定方法。在测定中应用油井技术，以实现仅1.0至5.0微升培养基中的反应。通过注射孕马血清促性腺激素（PMS）-人绒毛膜促性腺激素（hCG）使未成熟的Wistar大鼠超排卵。通过卵泡穿刺和输卵管冲洗收集卵母细胞。冲洗后将其冻干以去除卵丘细胞。在石英纤维鱼竿天平上，干重约为50纳克。己糖激酶的活性为1.75±0.14皮摩尔/卵母细胞/小时，相当于作为对照的卵巢匀浆的十分之一，表明卵母细胞中葡萄糖利用能力较低。葡萄糖-6-磷酸脱氢酶（G6PD）、乳酸脱氢酶（LDH）和苹果酸脱氢酶（MDH）的活性分别为8.41±0.34、35.7±2.89、11.1±2.5皮摩尔/卵母细胞/分钟。G6PD的高活性表明与类固醇生成有关的磷酸戊糖途径在卵母细胞中活跃。hCG增加了己糖激酶和MDH的活性，降低了G6PD的活性。LDH的活性保持不变。
