An improved in vitro bioassay method for measuring luteinizing hormone (LH) activity using mouse Leydig cell preparations.
An improved in vitro bioassay method for measuring luteinizing hormone (LH) activity using mouse Leydig cell preparations.
复制标题
一种改进的体外生物测定方法，使用小鼠 Leydig 细胞制剂测量黄体生成素 (LH) 活性。
DOI：
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发表时间：
1974
期刊：
影响因子：
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通讯作者：
E. Diczfalusy
中科院分区：
文献类型：
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作者：
M.;David Robertson;E. Diczfalusy
提出了一种改进的体外LH活性测定方法。该方法是基于在添加促性腺激素的情况下，对小鼠睾丸的“间质细胞”制剂产生的睾酮进行测定。与先前描述的使用成年小鼠脱囊睾丸的生物测定方法相比，该方法在灵敏度、精密度和实用性方面有显著提高。改进的方法对HCG的灵敏度为15 \g=m\IU，对HMG的灵敏度为50 \g=m\IU。对于HCG，该方法的有效范围为15\p=n-\260\g=m\ iu；对于HMG，该方法的有效范围为50\p=n-\900\g=m\ iu。采用3 + 3点检测设计，每个剂量四次重复，19次检测的平均精密度指数（λ̅）为0.044。人体FSH、TSH、ACTH、LTH、STH、催产素、抗利尿激素和LHRH制剂在生物样品中可能发现的水平对生物测定方法没有影响。当使用各种HCG和HMG制剂时，“间质细胞”法和脱囊睾丸法获得的估计值之间存在良好的相关性。采用该方法，每周至少可由2人检测30份样品，成本显著降低。福特基金会生殖内分泌学研究员。在之前的一篇论文（Van Damme et al. 1973）中，描述了一种用于测量LH活性的体外生物测定方法。证据表明，该方法是基于在黄体生成素制剂存在的情况下，由脱囊小鼠睾丸产生的睾酮的测定，满足公认的可靠性标准。然而，该方法有2个局限性；相对较差的精度（= 0.22），实用性有限。本文描述了对先前方法的显著改进。从被破坏的小鼠睾丸中提取的“间质细胞”*用于生物测定。这种改进大大提高了精度、灵敏度和实用性。CV =变异系数：HHG =人垂体促性腺激素；HHLH =人垂体黄体生成素；人促甲状腺激素；=精度指数；LHRH =促黄体生成素释放激素；OAAD =卵抗坏血酸耗尽试验；放射免疫测定法；WITARO =未成熟大鼠附属生殖器官总重量增加。材料：Sprague-Dawley大鼠和美国海军医学研究所（Bethesda， USA）小鼠系购自斯德哥尔摩AB Anticimex，在献祭前至少笼养24小时。大多数使用的激素在以前已经被描述过（Van Damme et al. 1973）。所使用的其他制剂已在文中说明。所有使用的玻璃器皿都被硅化（Silikonvätska MS 1107, Kebo, Sweden； 2%丙酮），并在50°C下过夜干燥。Eagle的最低必需培养基和小牛血清来自瑞典斯德哥尔摩的Statens Bakteriologiska实验室。使用前，将含有犊牛血清的培养基用o- co»（93.5:6.5）起泡几分钟。所得pH值为7.2。血清取自1-3月龄的犊牛。根据目前的“间质细胞”生物测定法，45 ul血清中LH活性未检测到。胶原酶（137 U/mg）和利马豆胰蛋白酶抑制剂来自美国新泽西州Worthington生化公司。方法：方法设计。-每个剂量水平使用3 + 3点试验，一式四次，对数剂量间隔为1.5。通常对10种未知制剂进行1 *的检测，该术语涵盖了从睾丸管间组织中获得的所有细胞类型。下载自Bioscientifica.com于11/24/2018 02:57:12PM免费访问标准。效度测试按照先前出版物（Van Damme et al. 1973）的描述进行。细胞的制备。-成年小鼠（3Vi个月）颈椎脱臼处死，睾丸脱囊，置于含有Eagle培养基+ 2°/o犊牛血清的培养皿中。通常每个实验使用3只动物。将睾丸用剪刀剪成小块，加入Eagle培养基+ 2”Vo犊牛血清至最终浓度为6睾丸/50毫升。将含有睾丸的培养基在室温下用磁力搅拌器轻轻搅拌10分钟。然后介质通过一个精细的尼龙网过滤。滤液在代谢摇床（New Brunswick Scientific Co., Inc., New Jersey， USA）中，在34°C的02/ co2（93.5:6.5）气氛中，于80 pm预孵育1小时。预孵育后，将细胞悬液置于冰中，在4°C， 130 g离心15分钟。丢弃上清，将细胞重悬于等量的新鲜Eagle培养基+ 2%小牛血清中。在台盼蓝（0.1°/o, Fluka AG，瑞士）存在下，在血细胞计数仪中计数细胞制备中的细胞数量和破碎细胞的百分比。没有尝试对存在的各种细胞类型进行分类。在任何制剂中均未发现精管。当间质细胞产生的睾酮在每个细胞中表达时，计算中使用的是存在的细胞总数，而不是认为有活力的细胞数量。孵化条件。将一部分细胞悬浮液（0.1 ml，含6个IO4细胞）加入含有0.1 ml适量促性腺激素的冰管中。在标准测定中，HCG的剂量为22-114 //IU， HMG和HHG在含有20/o小牛血清的Eagle培养基中为79-400 //IU。样品在80 r / pm的O^/COg气氛下于34°C下孵育3小时。放射免疫分析法（RIA）。孵育完成后，将试管放入冰中，加入一份（0.2 ml）含有氚标记睾酮的混合物，并加入适当稀释的抗血清。混合物在60℃下孵育10分钟，然后在30℃下孵育30分钟或在4℃下孵育过夜。平行处理的睾酮校准曲线（0-1600 pg）包含未知样品中存在的相同浓度的小牛血清。这纠正了犊牛血清中睾酮结合成分对RIA的影响。RIA方法的进一步细节（如特异性）在以前的一篇论文中提出（Van Damme el al. 1973）。剂量反应关系的线性化。当产生的睾酮（pg）与剂量的对数相对应时，剂量响应线在较低剂量水平上偏离线性（图1a）。为了扩大工作范围以包括较低剂量，尝试了各种超度量变换（例如芬尼1964年）。研究发现，通过绘制睾酮产生量的平方根（y' s）作为响应参数与剂量对数的关系，可以在很宽的剂量范围内得到一条直线（图1a）。在随后的10次分析中，线性的改善是一致的。使用Bartlett（1937）检验评估方差的同质性。采用适用于5%水平下每个剂量的低重复数的表，在有用范围内获得了稳定的方差（Pearson 8c Hartley 1954）。然而，在684个四次重复中，有17个发现了“异常值”。当如上所述进行检验时，这种“异常值”的存在导致了方差的异质性。如果“异常值”被去除，Bartlett检验表明方差的同质性。从Bioscientifica.com下载于11/24/2018 02:57:12PM免费获取睾丸素r， 440 -I [pg] 2睾丸素
An improved in vitro bioassay method for the measurement of LH activity is presented. The method is based on the assay of testosterone produced by "Leydig cell" preparations from mouse testes in the presence of added gonadotrophin. The method is significantly improved in terms of sensitivity, precision and practicability when compared to the previously described bioassay method employing decapsulated testes from adult mice. The sensitivity of the improved method is 15 \g=m\IUfor HCG and 50 \g=m\IU for HMG. The useful range of the method is 15\p=n-\260\g=m\IUfor HCG and 50\p=n-\900\g=m\IUfor HMG. Using a 3 + 3 point assay design with each dose in quadruplicate, a mean index of precision (λ̅)of 0.044 was obtained in 19 assays. Human FSH, TSH, ACTH, LTH, STH, oxytocin, vasopressin and LHRH preparations did not influence the bioassay method at levels likely to be found in biological samples. A good correlation was found between estimates obtained by the "Leydig cell" method and by the method using decapsulated testes when various HCG and HMG preparations were used. With the proposed method at least 30 samples can be assayed each week by 2 persons, with a marked reduction in cost. 0 Ford Foundation Fellow in Reproductive Endocrinology. Downloaded from Bioscientifica.com at 11/24/2018 02:57:12PM via free access In a previous paper (Van Damme et al. 1973) an in vitro bioassay method for measuring LH activity was described. Evidence was presented indicating that the method, which is based on the assay of testosterone produced by decapsulated mouse testes in the presence of LH preparations, fulfils the recognised criteria of reliability. There were, however, 2 limitations of the method; the relatively poor precision ( = 0.22) and a limited practicability. The present paper describes a significantly improved modification of the previous method. "Leydig cell"* preparations from disrupted mouse testes are used in the bioassay. This modification results in a considerable increase in precision, sensitivity and practicability. MATERIALS Abbreviations and trivial names CV = coefficient of variation: HHG = human hypophyseal gonadotrophin; HHLH = human hypophyseal luteinizing hormone; HTSH = human thyroid stimulating hormone; = index of precision; LHRH = luteinizing hormone releasing hormone: OAAD = ova¬ rian ascorbic acid depletion assay; RIA = radioimmunoassay; WITARO = weight in¬ crease of the total accessory reproductive organs in intact immature rats. Materials Sprague-Dawley rats and mice of the Naval Medical Research Institute strain (Bethesda, USA) were purchased from AB Anticimex, Stockholm, and caged for a minimum of 24 h prior to sacrifice. The majority of the hormones used have been described previously (Van Damme et al. 1973). Additional preparations used are indicated in the text. All glassware used was siliconized (Silikonvätska MS 1107, Kebo, Sweden; 2% in acetone) and dried at 50°C overnight. Eagle's Minimum Essential Medium and calf serum were obtained from Statens Bakteriologiska Laboratorium, Stockholm, Sweden. The medium containing the calf serum was bubbled with Oo-CO» (93.5:6.5) for several minutes before use. The re¬ sulting pH was 7.2. The serum was obtained from calves aged 1-3 months. As assessed by the present "Leydig cell" bioassay method LH activity in 45 ul of serum was non-detectable. The collagenase (137 U/mg) and the Lima bean trypsin inhibitor were obtained from Worthington Biochemical Co., New Jersey, USA. METHODS Proposed method Assay design. — A 3 + 3 point assay is used in quadruplicate for each dose level with a log dose interval of 1.5. Usually 10 unknown preparations are assayed against 1 * This term covers all cell types obtained from the intertubular tissue of the testes. Downloaded from Bioscientifica.com at 11/24/2018 02:57:12PM via free access standard. Validity tests are performed as described in a previous publication (Van Damme et al. 1973). Preparation of cells. — Aduli mice (3Vi months) are sacrificed by cervical dislocation, the testes decapsulated and placed in a Petri-dish containing Eagle's medium + 2 °/o calf serum. Usually 3 animals are used in each experiment. The testes are cut with scissors into small pieces and Eagle's medium + 2 "Vo calf serum are added to a final concentration of 6 testes/50 ml. The medium containing the testes is gently stirred by a magnetic stirrer for 10 min at room temperature. The medium is then filtered through a fine nylon mesh. The filtrate is pre-incubated for 1 h at 34°C in an 02/C02 (93.5:6.5) atmosphere in a Metabolyte shaker (New Brunswick Scientific Co., Inc., New Jersey, USA) at 80 r. p. m. After pre-incubation the cell suspension is placed in ice and centrifuged at 4°C at 130 g for 15 min. The supernatant is discarded and the cells are resuspended in the same volume of fresh Eagle's medium + 2 % calf serum. The number of cells in the cell preparation and the percentage of broken cells are assessed by counting in a haemocytomcter in the presence of Trypan Blue (0.1 °/o, Fluka AG, Switzerland). No attempts were made to classify the various cell types present. Seminiferous tubules were not found in any preparation. When the testos¬ terone produced by the Leydig cells was expressed per cell, the total number of cells present was used in the calculations and not the number of cells considered viable. Incubation conditions. A portion (0.1 ml containing 6 IO4 cells) of cell suspension is added to tubes in ice containing 0.1 ml of the appropriate amount of the gonado¬ trophic hormone. In the standard assay the doses are 22-114 //IU HCG and 79-400 ,«IU HMG and HHG in Eagle's medium containing 20/o calf serum. The samples are incubated at 34°C for 3 h under an O^/COg atmosphere at 80 r. p. m. Radioimmunoassay method (RIA). At the completion of the incubation, the tubes are placed in ice and a portion (0.2 ml) of a mixture containing tritium labelled testos¬ terone and the appropriate dilution of the antiserum is added. The mixture is in¬ cubated at 60°C for 10 min followed by incubation at 30°C for 30 min or at 4°C overnight. A testosterone calibration curve (0-1600 pg) which is processed in parallel, contains the same concentration of calf serum present in the unknown samples. This corrects for the influence of the testosterone binding components in the calf serum on the RIA. Further details of the RIA method (e. g. specificity) are presented in a previous paper (Van Damme el al. 1973). Linearization of the dose response relationship. — When the testosterone produced (pg) was plotted against the logarithm of the dose, the dose response line deviated from linearity at the lower dose levels (Fig. 1 a). In an attempt to extend the working range to include lower doses, various metametric transformations (e. g. Finney 1964) were tried. It was found that by plotting the square root of the amount of testosterone produced (y''s) as the response metameter against the logarithm of the dose, a straight line is obtained over a wide range of doses (Fig. 1 a). This improvement in linearity was consistent when 10 subsequent assays were analysed. The homogeneity of variances was assessed by the use of Bartlett's (1937) test. A stable variance was obtained over the useful range employing the tables applicable to low numbers of replicates at each dose at the 5% level (Pearson 8c Hartley 1954). However, in 17 out of 684 quadruplicates, "outliers" were found. The presence of such "outliers" resulted in heterogeneity of variances when tested as described above. If the "outliers" were removed, Bartlett's test indicated homogeneity of variances. Downloaded from Bioscientifica.com at 11/24/2018 02:57:12PM via free access TESTOSTERONE r ,4 40 -I [pg] 2 TESTOSTERONE