Mondschein J S, Hersey R M, Dey S K, Davis D L, Weisz J
Endocrinology. 1985 Dec;117(6):2339-46. doi: 10.1210/endo-117-6-2339.
Formation of the catechol estrogens 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from estradiol by pig blastocysts was studied using a direct product isolation assay for estrogen-2/4-hydroxylase (E-2/4-H). Blastocyst E-2/4-H activity was characterized biochemically using homogenates of blastocysts obtained on day 12 of pregnancy. This information was used to establish appropriate incubation conditions for the assay of E-2/4-H activity in blastocysts during the preimplantation period. Catechol estrogen formation was linear with time for up to 30 min and with blastocyst protein concentrations of up to 100 micrograms in a reaction volume of 150 microliters. The E-2/4-H activity of pig blastocysts was maximal at pH 7.9 and was not affected by the nonionic detergent Tween-80. The E-2/4-H activity was dependent on nicotinamide cofactor, with NADPH preferred over NADH for 2-OHE2 formation. The predominant catechol estrogen formed was 2-OHE2: maximum velocities (Vmax) for the formation of 2- and 4-OHE2 were 1570 and 174 pmol/mg protein . 30 min, respectively. The apparent Km values with respect to estradiol for 2- and 4-OHE2 were similar, 4.39 and 4.27 microM, respectively. Blastocyst E-2/4-H activity was detectable in one of two samples of blastocysts from day 10 of pregnancy (4.4 pmol 2-OHE2/mg protein . 30 min), increased to a maximum on days 12 and 13 (628 +/- 153 and 516 +/- 227 pmol 2-OHE2/mg protein . 30 min, respectively), and declined by day 14 (63.2 +/- 32.9 pmol 2-OHE2/mg protein . 30 min). The activity of E-2/4-H was positively correlated with aromatase activity assayed in the same tissue samples from days 10-14 of pregnancy. The surge in E-2/4-H activity coincides with several of the critical events that occur near the time of implantation. Our findings are consistent with the hypothesis that catechol estrogens mediate some of the actions of estrogens in early pregnancy in the pig.
采用雌激素 - 2/4 - 羟化酶（E - 2/4 - H）直接产物分离测定法，研究了猪囊胚将雌二醇转化为儿茶酚雌激素2 - 羟基雌二醇和4 - 羟基雌二醇（2 - OHE2和4 - OHE2）的过程。利用妊娠第12天获得的囊胚匀浆，对囊胚E - 2/4 - H活性进行了生化特性分析。这些信息用于确定在着床前期测定囊胚中E - 2/4 - H活性的合适孵育条件。在150微升反应体积中，儿茶酚雌激素的形成在长达30分钟的时间内与时间呈线性关系，并且在囊胚蛋白浓度高达100微克时也呈线性关系。猪囊胚的E - 2/4 - H活性在pH 7.9时最高，不受非离子去污剂吐温 - 80的影响。E - 2/4 - H活性依赖于烟酰胺辅因子，在形成2 - OHE2时，NADPH比NADH更受青睐。形成的主要儿茶酚雌激素是2 - OHE2：2 - OHE2和4 - OHE2形成的最大反应速度（Vmax）分别为1570和174 pmol/mg蛋白·30分钟。2 - OHE2和4 - OHE2相对于雌二醇的表观Km值相似，分别为4.39和4.27 microM。在妊娠第10天的两个囊胚样本中，有一个检测到囊胚E - 2/4 - H活性（4.4 pmol 2 - OHE2/mg蛋白·30分钟），在第12天和第13天增加到最大值（分别为628±153和516±227 pmol 2 - OHE2/mg蛋白·30分钟），到第14天下降（63.2±32.9 pmol 2 - OHE2/mg蛋白·30分钟）。在妊娠第10 - 14天相同组织样本中测定的E - 2/4 - H活性与芳香化酶活性呈正相关。E - 2/4 - H活性的激增与着床期附近发生的几个关键事件相吻合。我们的研究结果与儿茶酚雌激素介导猪妊娠早期雌激素某些作用的假设一致。
