Testosterone and sport: current perspectives.

Testosterone and other anabolic-androgenic steroids enhance athletic performance in men and women. As a result, exogenous androgen is banned from most competitive sports. However, due to variability in endogenous secretion, and similarities with exogenous testosterone, it has been challenging to establish allowable limits for testosterone in competition. Endogenous androgen production is dynamically regulated by both exercise and winning in competition. Furthermore, testosterone may promote athletic performance, not only through its long-term anabolic actions, but also through rapid effects on behavior. In women, excess production of endogenous testosterone due to inborn disorders of sexual development (DSD) may convey a competitive advantage. For many years, female competitors have been subject to tests of sexual genotype and phenotype known as gender verification. Although gender verification has not identified any normal man competing as a woman, this process has identified women athletes with DSD. As understanding of DSD has expanded in recent years, women with DSD are increasingly able to continue athletic competition.

Genetic aspects of epitestosterone formation and androgen disposition: influence of polymorphisms in CYP17 and UGT2B enzymes.

OBJECTIVE: Testosterone is a commonly abused androgen in sports and in the gym culture of the society. Its abuse is conventionally disclosed by urinary assay of the testosterone/epitestosterone (T/E) glucuronide ratio, which should not exceed 4. A noteworthy number of athletes, however, have higher natural ratios than 4, most likely because of decreased excretion of epitestosterone glucuronide. Falsely positive doping test results are of great concern for the legal rights of the sportsman. Our objective was to study the genetic aspects of epitestosterone formation, and to elucidate the impact of genetic variation in androgen-metabolizing enzymes. METHODS: Urine from different study populations was analysed for androgen glucuronides by gas chromatography-mass spectrometry. All men were genotyped for the uridine diphospho-glucuronosyltransferase (UGT) 2B17 deletion polymorphism and single nucleotide polymorphisms in the cytochrome P-450c17alpha (CYP17), UGT2B15 and UGT2B7 genes. Expression of UGT2B15 mRNA in human liver samples was analysed using real-time PCR. RESULTS: A T>C (A1>A2) promoter polymorphism in the CYP17 gene was associated with the urinary glucuronide levels of epitestosterone and its putative precursor androstene-3beta, 17alpha-diol, resulting in 64% higher T/E ratios in A1/A1 homozygotes. Individuals devoid of UGT2B17 had significantly higher UGT2B15 mRNA levels in liver than individuals carrying two functional UGT2B17 alleles. CONCLUSION: The CYP17 promoter polymorphism may partly explain high natural (>4) T/E ratios. Our data indicate that 5-androstene-3beta, 17alpha-diol is an important precursor of epitestosterone and that CYP17 is involved in its production. In addition, we found that lack of the UGT2B17 enzyme may be compensated for by increase in UGT2B15 transcription.

Characterization of 17alpha-hydroxysteroid dehydrogenase activity (17alpha-HSD) and its involvement in the biosynthesis of epitestosterone.

BACKGROUND: Epi-testosterone (epiT) is the 17alpha-epimer of testosterone. It has been found at similar level as testosterone in human biological fluids. This steroid has thus been used as a natural internal standard for assessing testosterone abuse in sports. EpiT has been also shown to accumulate in mammary cyst fluid and in human prostate. It was found to possess antiandrogenic activity as well as neuroprotective effects. So far, the exact pathway leading to the formation of epiT has not been elucidated. RESULTS: In this report, we describe the isolation and characterization of the enzyme 17alpha-hydroxysteroid dehydrogenase. The name is given according to its most potent activity. Using cells stably expressing the enzyme, we show that 17alpha-HSD catalyzes efficienty the transformation of 4-androstenedione (4-dione), dehydroepiandrosterone (DHEA), 5alpha-androstane-3,17-dione (5alpha-dione) and androsterone (ADT) into their corresponding 17alpha-hydroxy-steroids : epiT, 5-androstene-3beta,17alpha-diol (epi5diol), 5alpha-androstane-17alpha-ol-3-one (epiDHT) and 5alpha-androstane-3alpha,17alpha-diol (epi3alpha-diol), respectively. Similar to other members of the aldo-keto reductase family that possess the ability to reduce the keto-group into hydroxyl-group at different position on the steroid nucleus, 17alpha-HSD could also catalyze the transformation of DHT, 5alpha-dione, and 5alpha-pregnane-3,20-dione (DHP) into 3alpha-diol, ADT and 5alpha-pregnane-3alpha-ol-20-one (allopregnanolone) through its less potent 3alpha-HSD activity. We also have over-expressed the 17alpha-HSD in Escherichia coli and have purified it by affinity chromatography. The purified enzyme exhibits the same catalytic properties that have been observed with cultured HEK-293 stably transfected cells. Using quantitative Realtime-PCR to study tissue distribution of this enzyme in the mouse, we observed that it is expressed at very high levels in the kidney. CONCLUSION: The present study permits to clarify the biosynthesis pathway of epiT. It also offers the opportunity to study gene regulation and function of this enzyme. Further study in human will allow a better comprehension about the use of epiT in drug abuse testing; it will also help to clarify the importance of its accumulation in breast cyst fluid and prostate, as well as its potential role as natural antiandrogen.

The in vitro biosynthesis of epitestosterone and testosterone from C19 steroid precursors in the testis of the lizard Tiliqua rugosa.

The metabolism of androgens in the testis of the lizard Tiliqua rugosa has been studied in vitro by incubating cellular homogenates with radiolabeled C19-steroid substrates. The identification 17 beta-oxidoreductase and 3 beta-hydroxysteroid dehydrogenase/isomerase activities. Aromatase, 5 alpha-reductase, and 17 alpha/beta-epimerase activities were not detected. The 17 alpha-oxidoreductase activity was temperature dependent (maximal at 32 degrees), while the 17 beta-oxidoreductase activity was temperature independent. Time yield and dual-label studies indicated that testosterone biosynthesis mainly involves the 4-ene pathway (via androstenedione), whereas the formation of epitestosterone uses both the 4-ene and 5-ene (via 5-androstene-3 beta, 17 alpha-diol) pathways. The function of alternative pathways in androgen biosynthesis is discussed, as is the role of temperature in the intratesticular regulation of androgen production.

High activity of 17 alpha-oxidoreductase in neonatally grafted mouse testes in adult female mice.

Male and female (WB-C57BL/6)F1 hybrid mice were used. Two testes from neonatal mice were grafted into the spleen of adult male and female mice, and the grafted testes were removed 30 and 60 days after grafting. Normal testes from 30- and 60-day old mice were also used. Testicular homogenates were incubated with [14C]4-androstene-3,17-dione or [3H]progesterone, and enzyme activities per g wet tissue and progesterone metabolism were examined. Activity of 17 alpha-oxidoreductase in the grafted testes in females (20 nmol/g/h) was approx. 10 times the activity in the grafted testes in males or in the normal testes, whereas 17 beta-oxidoreductase activity in the grafted testes in females was the lowest among these testes. The bilateral ovariectomy performed 1 month before the grafting of neonatal testes, artificial cryptorchidism performed at 20 days of age, and estrogen treatment for 10 days by diethylstilbestrol pellets resulted in no significant changes in 17 alpha-oxidoreductase activities in 30- and 60-day old grafted, cryptorchid or normal testes. The major 17-hydroxy-C19-steroids formed in vitro from progesterone by the grafted testes in female mice were testosterone and 17 alpha-hydroxy-4-androsten-3-one (epitestosterone), but the formation of epitestosterone was insignificant in the normal testes. The present results demonstrate for the first time that epitestosterone is formed as one of major C19-steroids in neonatally grafted mouse testes in females but not in those in males or in normal mouse testes. However, the mechanisms remain unexplained.

Testosterone metabolism in the uropygial gland of the quail.

The metabolism of testosterone in the uropygial gland of the quail principally results in the production of 17 alpha, 5 beta derivatives. Moreover, an unusually small amount of testosterone is converted to 5 alpha-dihydrotestosterone. These results question the role played by intracellular 5 alpha-reduction in the response of the gland to testosterone stimulation.