Knaggs P, Lambert A, Proudfoot F, Nickson I, Hooper M A, Lenton E, Robertson W R
Department of Medicine, Hope Hospital, Salford, UK.
Mol Hum Reprod. 1998 Feb;4(2):147-51. doi: 10.1093/molehr/4.2.147.
The presence of 11beta-hydroxysteroid dehydrogenase (11beta-HSD; EC1.1.1.146), the enzyme responsible for the interconversion of cortisol and cortisone, in granulosa-lutein (GL) cells is associated with a poor outcome in in-vitro fertilization (IVF). We have developed a simple method of assessing the reductase component of 11beta-HSD in these cells which is sufficiently rapid to provide data on the enzyme's activity prior to embryo replacement. Cells were pooled from follicular aspirates and challenged with cortisone within 2 h of aspiration. Cortisol secretion was then measured by radioimmunoassay. Conversion of cortisone to cortisol was linear for up to 3 h and was completely inhibited by glycyrrhetinic acid, a specific 11beta-HSD inhibitor. Initial velocity rates were determined for eight cortisone concentrations (range 0.1-8 micromol/l), and the apparent Km calculated (1.6 +/- 0.4 micromol/l). There was no evidence of substrate/product inhibition and conversion of cortisone to cortisol was <2% in all experiments. In subsequent work, cells were challenged with cortisone (6 micromol/l) for 2 h. Cells challenged for 2 h immediately following purification from follicular aspirates produced varying amounts of cortisol (range 25-150 nmol/pooled follicles from each patient, n = 10 patients), while basal outputs were <6 nmol/l. Enzyme activity was also examined in cells on a per follicle basis from individual patients and found to vary considerably (e.g. 19, 53 and 36 nmol/l cortisol/1000 cells, three follicles). Having established the method for assessing 11beta-reductase activity within GL cells, we performed a small prospective study on a series of 20 patients examining the enzyme activity within 110 individual follicles. 11Beta-reductase activity varied greatly from patient to patient and from follicle to follicle ranging from <0.024-0.57 nmol cortisol/microg DNA but at present low patient numbers preclude a meaningful correlation between enzyme activity and pregnancy rate. In summary, we have developed a simple, rapid (<8 h) assay for detecting the reductase activity of 11beta-HSD in GL cells isolated from pooled or individual follicles. This procedure is sufficiently quick to aid in the choice of embryo for replacement.
11β-羟基类固醇脱氢酶（11β-HSD；EC1.1.1.146）负责皮质醇和可的松的相互转化，颗粒黄体（GL）细胞中该酶的存在与体外受精（IVF）的不良结局相关。我们开发了一种简单的方法来评估这些细胞中11β-HSD的还原酶成分，该方法足够快速，能够在胚胎移植前提供有关该酶活性的数据。从卵泡抽吸物中收集细胞，并在抽吸后2小时内用可的松进行刺激。然后通过放射免疫测定法测量皮质醇分泌。可的松向皮质醇的转化在长达3小时内呈线性，并且被11β-HSD特异性抑制剂甘草次酸完全抑制。测定了八种可的松浓度（范围为0.1 - 8微摩尔/升）的初始速度，并计算了表观Km（1.6±0.4微摩尔/升）。所有实验中均未发现底物/产物抑制的证据，可的松向皮质醇的转化率<2%。在随后的研究中，用可的松（6微摩尔/升）刺激细胞2小时。从卵泡抽吸物中纯化后立即刺激2小时的细胞产生了不同量的皮质醇（每位患者每汇集卵泡的范围为25 - 150纳摩尔，n = 10例患者），而基础产量<6纳摩尔/升。还从个体患者的每个卵泡基础上检查了细胞中的酶活性，发现差异很大（例如，三个卵泡中每1000个细胞分别为19、53和36纳摩尔/升皮质醇）。在建立了评估GL细胞中11β-还原酶活性的方法后，我们对20例患者进行了一项小型前瞻性研究，检测了110个单个卵泡中的酶活性。11β-还原酶活性在患者之间和卵泡之间差异很大，范围从<0.024 - 0.57纳摩尔皮质醇/微克DNA，但目前患者数量较少，无法确定酶活性与妊娠率之间有意义的相关性。总之，我们开发了一种简单、快速（<8小时）的检测方法，用于检测从汇集或单个卵泡中分离的GL细胞中11β-HSD的还原酶活性。该程序足够快速，有助于选择用于移植的胚胎。
