Assay system for simultaneous measurement of three steroidogenic enzyme activities in rat and human testis--effect of human chorionic gonadotropin.
An assay system that measures the enzymatic activities (17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase) in the delta 4 pathway of testosterone biosynthesis using rat and human testicular homogenate was examined. This system involves the simultaneous separation of the steroid intermediates by a three-step TLC procedure. The observed Rf values were 0.78 for progesterone (P), 0.59 for 17 alpha-hydroxyprogesterone (17 alpha-HP), 0.70 for androstenedione (A), 0.5 for testosterone, 0.64 for dihydrotestosterone, and 0.45 for 3 alpha, 17 beta-androstanediol. The identification of these steroid intermediates was further accomplished by acetylation and rechromatography of the representative samples along with the authentic standards and by recrystallization to constant specific activity until three consecutive crystallizations were within +/- 5% of the mean value. Incubation time up to 30 min and increasing protein concentrations showed a linear relationship with respect to these three enzymatic activities. The optimum temperature for these enzymatic activities varied from 32 to 34 degrees C, with a sharp decline between 37 and 40 degrees C. The Michaelis constants (Km) for the rat testis homogenate samples were 0.17 microM for P, 0.22 microM for 17 alpha-HP, and 2.5 microM for A, while for the human testis the Km values were 1.2, 2.2, and 2.3 microM, respectively, for these substrates. The concentrations of the endogenous steroid substrates present in these homogenate samples did not alter the Km or Vmax values. The effect of human chorionic gonadotropin (hCG) in vitro on these steroidogenic enzyme activities was also studied. In the rat testis, 10 IU of hCG produced a significant rise in all the three enzyme activities whereas in the human testis 10 and 30 IU of hCG showed no significant change in any of these enzymatic activities. However, 100 IU of hCG resulted in a significant increase in 17 alpha-hydroxylase and 17,20-desmolase activities in the human testis. These studies suggest that this assay system for the measurement of these enzymatic activities using a testicular homogenate sample provides consistent and reproducible results. Based on the sensitivities of the measurements and our experience with testicular biopsy technique, we conclude that a routine testicular biopsy in the human should provide sufficient tissue to run these enzymatic assays.
D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000368:Aged; D000446:Aldehyde-Lyases / Q000378:metabolism*; D000818:Animals; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013254:Steroid 17-alpha-Hydroxylase / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013696:Temperature; D013737:Testis / Q000201:enzymology*; D013739:Testosterone / Q000096:biosynthesis; D013997:Time Factors
D006063:Chorionic Gonadotropin; D013739:Testosterone; D015067:17-Hydroxysteroid Dehydrogenases; D013250:Steroid Hydroxylases; D013254:Steroid 17-alpha-Hydroxylase; D000446:Aldehyde-Lyases