T. Tsuruhara

Radioimmunoassay of plasma testosterone

Abstract Radioimmunoassay of testosterone in plasma extracts purified by thin layer chromatography has been applied to the development of a highly sensitive and specific method for measurement of plasma testosterone. Antibodies to testosterone coupled to bovine gamma globulin via the 3 position were raised in sheep, and an antiserum of high association constant ( K = 4.5 × 10 9 M −1 ) was employed for assay. Isolation of plasma testosterone prior to assay was performed by thin layer chromatography, and extraction losses were corrected for recovery of tritiated testosterone indicator. For assay, antiserum (1:2500) and tritiated testosterone (40 000 dpm) were incubated at 4° for 16 h, followed by separation of bound and free tracer with 500 μg dextrancharcoal. The sensitivity of the assay was 10 pg, between-assay precision was ± 12% and testosterone recovery was 95–100%. Plasma testosterone levels in individual samples were similar to those obtained by competitive binding assay, and the levels observed in males (709 ± 143; 450–1100 ng/100 ml) and females 54 ± 22; 25–80 ng /100 ml ) were in agreement with those reported by other methods. The combination of radioimmunoassay and thin layer chromatography provides a sensitive and reliable method for the specific determination of testosterone in small plasma samples.

Interaction of glycoprotein hormones with agarose-concanavalin A

Abstract The ability of agarose-coupled concanavalin A to bind carbohydrate and glycoprotein molecules has been applied to chromatography of glycoprotein hormones. Human chorionic gonadotrophin and subunits were bound strongly by Sepharose-concanavalin A, and could be displaced by 0.2 M methyl α- d -glucopyranoside. Desialylated human chorionic gonadotrophin was eluted more slowly than the intact hormone, and asialo-agalacto-human chorionic gonadotrophin was not readily dissociable from combination with concanavalin A. Human luteinizing hormone and follicle stimulating hormone were also adsorbed by Sepharose-concanavalin A columns, and eluted with methyl α- d -glucopyranoside. Prior combination with specific antibody did not prevent 125I-labeled human chorionic gonadotrophin binding to concanavalin A, and subsequent elution with methyl glucopyranoside released the antigen-antibody complex in high yield. Chromatography of human chorionic gonadotrophin on Sepharose-concanavalin A was applied to preparation of 125I-labeled human chorionic gonadotrophin with improved binding affinity for antibody and gonadal receptor sites, and to purification and isolation of human chorionic gonadotrophin from crude urine extracts and human pregnancy plasma. Comparison of plasma and urinary human chorionic gonadotrophin revealed an elution profile consistent with partial desialylation of the urinary hormone. These experiments have shown that the group-specific interactions of glycoprotein hormones with agarose-concanavalin A are of potential value for the purification and isolation of such hormones and their derivatives, and for the preparation of radioiodinated gonadotrophins of improved binding characteristics.

Retention of in vitro biological activities by desialylated human luteinizing hormone and chorionic conadotropin

Summary The marked reduction of biological activity after desialylation of human luteinizing hormone and chorionic gonadotropin is not accompanied by a corresponding loss of activity in vitro. The desialylated gonadotropins show increased affinity for gonadal binding sites, and retain a considerable degree of steroidogenic activity when incubated with the rat testis in vitro. These findings indicate that the sialic acid residues of gonadotropic hormones are not essential for biological activity at the target cell level.

Gonadotrophin stimulation of testosterone production by the rat testis in vitro

Measurement of testosterone production by the rat testis in vitro provides an extremely sensitive response system for the biological activities of luteinizing hormone (LH) and human chorionic gonadotrophin (HCG) activity in vitro. The sensitivity of the response to gonadotrophins depends upon the structural integrity of the isolated testis, as physical disruption by teasing apart the testis tubules results in substantial loss of the steroidogenic response to trophic hormones. The intact testis responds to as little as 0.2 ng HCG or 1 ng ovine LH, and shows maximal stimulation with 2 ng HCG or 10 ng ovine LH. Testosterone production in vitro was also markedly enhanced by Sepharose-coupled ovine LH and by dibutyryl cyclic AMP, consistent with the view that LH acts upon cell-membrane receptors to stimulate steroidogenesis in the testis via activation of adenyl cyclase. Desialylated gonadotrophins retained considerably greater biological activity in vitro than in vivo, indicating that the sialic acid residues are not essential for interaction of gonadotropins with their tissue receptor sites.

Gonadotrophin binding sites of the rat testis

Abstract Gonadotrophin binding sites with high affinity and specificity for human chorionic gonadotrophin and luteinizing hormone have been demonstrated in rat testis homogenates and subcellular fractions. The binding of 125I-labeled chorionic gonadotrophin to the rat testis homogenate is temperature dependent, with limited capacity (10−12 mole/g) and high affinity (Ka = 2.4·1010 M−1) for chorionic gonadothrophin at 24°C. Gonadotrophin binding is not significantly affected by variations in calcium concentration within the physiological range, and is associated with membrane fragments during differential centrifugation of testis homogenates labeled with 125I-chorionic gonadotrophin. These properties of the chorionic gonadotrophin/luteinizing hormone binding sites present in rat testis homogenate are consistent with those of a specific hormone receptor for gonadotrophins which activate testicular steroidogenesis.