Tsutsumi O, Satoh K, Sakamoto S
Nihon Naibunpi Gakkai Zasshi. 1982 Oct 20;58(10):1321-32. doi: 10.1507/endocrine1927.58.10_1321.
The steroid hormone has an important role in the early stages of reproduction. There has been abundant histochemical evidence that oocytes contain steroid hormones and are able to synthesize these hormones. But there have been few methods of analyzing one oocyte biochemically because it is too small and light. In order to study steroidogenesis in the oocyte, a microassay method sensitive enough to analyze the enzyme activities in one oocyte was developed using enzymatic cyling for amplifying the reaction product to 10,000-fold. An oil-well technique and a microtube method were applied in the assay for achieving the reaction in a medium as small as 1.0 to 5.0 microliters under a stereomicroscope. Immature Wistar rats were superovulated by PMS-hCG administration. Oocytes were collected by puncturing the follicle and flushing the tube. They were freeze-dried after washing to remove cumulus cells. The dry weight of one oocyte was 51.2 +/- 6.2 ng in a quartz fiber fishpole balance. The activity of 3 beta hydroxysteroid dehydrogenase (3 beta HSD) (picomol/oocyte/hr, substrate:pregnenolone) in the PMS-treated oocyte was 2.66 +/- 0.59, which corresponds to 3 times the activity of the ovarian homogenate as control, indicating the high capacity of oocytes to produce progesterone. The activity increased significantly (P less than 0.01) by hCG administration up to 4.17 +/- 0.29 after ovulation, suggesting that gonadotropin regulates steroidogenesis in the oocyte. The activities of G6PD and 6PGD were 8.41 +/- 1.09 picomol/oocyte/min and 3.85 +/- 2.02 picomol/oocyte/hr, respectively. The high activity of G6PD (more than 10 times that of the ovarian homogenate) suggests that the pentose phosphate shunt concerned with steroidogenesis is active in the oocyte. HCG decreased the activities of both G6PD and 6PGD. The present results show that steroidogenesis in the oocyte is very active under the control of gonadotropin, suggesting that steroid hormones may play an important role in oocyte maturation, ovulation and fertilization.
类固醇激素在生殖早期阶段发挥着重要作用。已有大量组织化学证据表明，卵母细胞含有类固醇激素并能够合成这些激素。但由于卵母细胞体积过小且重量过轻，几乎没有能够对单个卵母细胞进行生化分析的方法。为了研究卵母细胞中的类固醇生成，我们开发了一种微分析方法，该方法灵敏度足以分析单个卵母细胞中的酶活性，通过酶循环将反应产物放大10000倍。在测定中应用了油井技术和微管方法，以便在实体显微镜下于仅1.0至5.0微升的培养基中实现反应。通过注射孕马血清促性腺激素（PMS）-人绒毛膜促性腺激素（hCG）对未成熟的Wistar大鼠进行超排卵。通过穿刺卵泡和冲洗输卵管收集卵母细胞。冲洗后将其冻干以去除卵丘细胞。在石英纤维鱼竿天平上，单个卵母细胞的干重为51.2±6.2纳克。经PMS处理的卵母细胞中3β羟类固醇脱氢酶（3βHSD）（皮摩尔/卵母细胞/小时，底物：孕烯醇酮）的活性为2.66±0.59，相当于作为对照的卵巢匀浆活性的3倍，表明卵母细胞产生孕酮的能力很强。排卵后通过注射hCG，该活性显著增加（P＜0.01），达到4.17±0.29，表明促性腺激素调节卵母细胞中的类固醇生成。葡萄糖-6-磷酸脱氢酶（G6PD）和6-磷酸葡萄糖脱氢酶（6PGD）的活性分别为8.41±1.09皮摩尔/卵母细胞/分钟和3.85±2.02皮摩尔/卵母细胞/小时。G6PD的高活性（超过卵巢匀浆活性的10倍）表明与类固醇生成相关的磷酸戊糖途径在卵母细胞中很活跃。hCG降低了G6PD和6PGD的活性。目前的结果表明，在促性腺激素的控制下，卵母细胞中的类固醇生成非常活跃，这表明类固醇激素可能在卵母细胞成熟、排卵和受精中发挥重要作用。
