An improved in vitro bioassay method for measuring luteinizing hormone (LH) activity using mouse Leydig cell preparations.

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