Metabolism of exogenous 4- and 2-hydroxyestradiol in the human male

The metabolic fate of the isomeric catecholestrogens 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) was studied to elucidate possible differences in their metabolism as an explanation for their different bioactivities. Healthy young men (n = 3 each) were infused (90 min) with 4-OHE2 (60 micrograms/h) or 2-OHE2 (100 micrograms/h). The main metabolites were determined in plasma and urine before, during and after infusion. Unconjugated and conjugated steroids, the latter after hot acid hydrolysis, were subjected to chromatography on LH-20 columns and measured by specific RIAs. During the infusion 4-OHE2 reached significant plasma concentrations whereas 2-OHE2 was so rapidly metabolised that its plasma levels remained virtually undetectable in spite of a higher infusion rate. The metabolism of 4-OHE2 was dominated by direct conjugation, that of 2-OHE2 by methyl ether formation. These findings were corroborated by the urinary excretion rates: during the infusion and the first hours afterwards, 4-OHE2 was mainly excreted as 4-OHE2 and 4-hydroxyestrone, while 2-OHE2 was predominantly excreted as 2-hydroxyestradiol 2-methyl ether and 2-hydroxyestrone 2-methyl ether.     

Testosterone metabolic clearance rate and production rate in the male infant rhesus monkey

The male infant rhesus monkey (Macaca mulatta) undergoes a period of testicular activation similar to that seen in the human infant. Plasma testosterone (T) concentrations rise after birth, reaching levels of about 500 ng/dl at 1-3 mo of age and then fall to approximately 50 ng/dl at 60 mo. The plasma T metabolic clearance rates (MCRT) and production rates (PRT) were measured in two rhesus infants at 1 and 6 mo of age to determine the mechanism of the observed increase in plasma T. While there was little change in the MCRT between 1 and 6 mo, PRT was much higher at 1 mo than at 60 mo of age. These observations are consistent with the hypothesis that the increased plasma testosterone levels in infant rhesus monkeys reflect an increased production of testosterone rather than an altered metabolic disposition of the hormone.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Intraspecific diversity in the fungal speciesChaunopycnis alba: Implications for microbial screening programs

Intraspecific variation among 84 isolates of the anamorphic fungusChaunopycnis alba from 26 different geographical locations was analyzed by investigating optimal growth temperatures, differences in the production of secondary metabolites and presence or absence of the cyclosporin synthetase gene. The genetic diversity was assessed using random amplified polymorphic DNA (RAPD). Analysis of these data showed high genetic, metabolic and physiological diversity within this species. Isolates from the Antarctic represented the most homogeneous group withinC. alba and together with isolates from the Arctic these polar strains differed from alpine, temperate and tropical strains by low optimal growth temperatures and by low production of secondary metabolites. Isolates from tropical climes were characterized by high optimal growth temperatures and by the production of comparatively diverse metabolite spectra. Most of the isolates that were similar in the combination of their physiological and metabolic characters were also genetically related. Isolates from different geographical origins did not show many similarities, with the exception of the cyclosporin A-producing isolates, and large diversity could be observed even within a single habitat. This leads us to the suggestion that for pharmaceutical screening programs samples should be collected from a diversity of different geographical and climatic locations. For the selection of strains for screening the RAPD assay seems to be the most powerful tool. It reflected the highest intraspecific diversity and the results corresponded well with the other characteristics.     

The effects of temperature on the activity of testicular steroidogenic enzymes

Decreased sperm counts and impaired sperm motility are present in a substantial proportion of men with varicocele. Elevations in the temperature of the affected testis, and increased spermatic vein estradiol (E2) concentrations have been found in some of these patients. To investigate the possibility that increases in temperature lead to a pattern of testicular steroidogenesis that results in increased E2 synthesis, we have examined the effects of temperature changes on the activities of four important testicular steroidogenic enzymes. 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17-hydroxylase (17-OH), 17,20-desmolase (17,20-D) and aromatase activities were measured in the microsomal fraction of rat, pig and horse testes. Incubations were performed at 34 degrees C, 36 degrees C, and 38 degrees C. The activities of all 4 enzymes increased with each 2 degrees C temperature elevation in roughly proportional amounts. We conclude that minor elevations in incubation temperature are associated with increases in the in vitro activity of four key testicular steroidogenic enzymes.     

Direct effect of the luteinizing hormone releasing hormone analog D-Trp6-Pro9-Net-LHRH on rat testicular steroidogenesis

The luteinizing hormone releasing hormone analog D-Trp⁶-Pro⁹-Net-LHRH (LHRH_a) inhibits rat testicular testosterone secretion. To determine whether LHRH_a decreases serum testosterone concentrations solely by inhibiting gonadotropin secretion or, in addition, by influencing directly testicular testosterone biosynthesis, we examined the effects of LHRH_a on the activities of 5 key testicular steroidogenic enzymes. Thirty hypophysectomized, hCG treated rats were given either LHRH_a (1 μg sc/day) or saline during 7 days. The LHRH_a treated animals exhibited a significant decrease of serum testosterone when compared to the control group (498 ± 37 ng/dl vs 2044 ± 105 ng/dl, mean ± SEM, P 〈0.001). 17-Hydroxyprogesterone serum levels were also decreased in the LHRH_a treated rats (61 ± 6 ng/dl vs 93 ± 7 ng/dl, P 〈0.005), while serum progesterone levels were similar in both groups of animals. These changes in steroid concentrations were associated with decreases in the musomal enzyme activities of 17-hydroxylase (37 ± 9 vs 654 ± 41 pmol/mg protein/min, P 〈0.001), 17, 20-desmolase (103 ± 9 vs 522 ± 47 pmol/mg protein/min, P 〈0.001), 3β-hydroxysteroid dehydrogenase (1.7 ± 0.02 vs 4.1 ± 0.1 nmol/mg protein/min, P 〈0.001), aromatase (95 ± 7 vs 228 ± 6 pmol/mg protein/ min, P 〈0.001) and 17-ketosteroid reductase (167 ± 9 vs 290 ± 18 pmol/mg protein/min, P 〈0.01) in the LHRH_a treated animals. These findings indicate that LHRH_a can inhibit directly rat testicular testosterone biosynthesis.     

Solid phase method for measurement of the binding capacity of testosterone-estradiol binding globulin in human serum

A solid phase method for measuring the binding capacity of serum testosterone-estradiol binding globulin (TeBG) is described and compared with other methods. TeBG, a glycoprotein, is adsorbed from serum or plasma onto a solid phase matrix of concanavalin-A, a carbohydrate-specific adsorbent. The TeBG binding capacity is determined by Scatchard analysis of the binding of radioactive testosterone at physiologic pH, in standard test tubes, and without the addition of albumin. Transcortin binding of testosterone is inhibited by the addition of cortisol.The levels of TeBG binding capacity determined with this solid phase method showed an excellent correlation with levels determined by procedures using equilibrium dialysis (with added cortisol) or ammonium sulphate precipitation. The serum TeBG binding capacity was 0.798±0.064 (mean±SE) μg/100 mL in men (n=32), 1.06±0.13 in women (n=10), 2.18±0.19 in women taking oral contraceptives (n=4), 6.2±2.9 in hyperthyroid women (n=2), and 11.6±3.1 in pregnant women (n=5). The serum TeBG binding capacity determined in heparinized plasma did not differ from that determined in serum. The within-assay variation is 9.6% and the between-assay variation is 11.2%.This solid phase method for measurement of serum TeBG binding capacity is simple, precise, and reproducible, and gives values which correlate well with those determined by other methods.