Androgen-activating enzymes in the central nervous systemProceedings of Xth International Congress on Hormonal Steroids, Quebec, Canada, 17–21 June 1998.

In the rat brain, several steroids can be converted by specific enzymes to either more potent compounds or to derivatives showing new biological effects. One of the most studied enzyme is the 5-reductase (5-R), which acts on 3keto-Δ4 steroids. In males, testosterone is the main substrate and gives rise to the most potent natural androgen dihydrotestosterone. In females, progesterone is reduced to dihydroprogesterone, a precursor of allopregnanolone, a natural anxiolytic/anesthetic steroid. Other substrates are some gluco- and minero-corticoids. Two isoforms of the 5-R, with limited degree of homology, have been cloned: 5-R type 1 and type 2. The 5-R type 1 possesses low affinity for the various substrates and is widely distributed in the body, with the highest levels in the liver; in the brain, this isoform is expressed throughout life and does not appear to be controlled by androgens. 5-R type 1 in the rat brain is mainly concentrated in myelin membranes, where it might be involved in the catabolism of potentially neurotoxic steroids. The 5-R type 2 shows high affinity for the various substrates, a peculiar pH optimum at acidic values and is localized in androgen-dependent structures. In the rat brain, the type 2 isoform is expressed at high levels only in the perinatal period and is controlled by androgens, at least in males. In adulthood, the type 2 gene appears to be specifically expressed in localised brain regions, like the hypothalamus and the hippocampus.

The 5-R type 2 is present in the GT1 cells, a model of LHRH-secreting neurons. These cells also contain the androgen receptor, which is probably involved in the central negative feedback effect exerted by androgens on the hypothalamic–pituitary–gonadal axis. The physiological significance of these and additional data will be discussed.     

3D-structure of human estrogenic 17beta-HSD1: binding with various steroids.

Human estrogenic dehydrogenase (17β-HSD1) catalyses the last step in the biosynthesis of the active estrogens that stimulate the proliferation of breast cancer cells. While the primary substrate for the enzyme is estrone, the enzyme has some activity for the non-estrogenic substrates. To better understand the structure–function relationships of 17β-HSD1 and to provide a better ground for the design of inhibitors, we have determined the crystal structures of 17β-HSD1 in complex with different steroids.

The structure of the complex of estradiol with the enzyme determined previously (Azzi et al., Nature Structural Biology 3, 665–668) showed that the narrow active site was highly complementary to the substrate. The substrate specificity is due to a combination of hydrogen bonding and hydrophobic interactions between the steroid and the enzyme binding pocket. We have now determined structures of 17β-HSD1 in complex with dihydrotestosterone and 20-OH-progesterone. In the case of the C19 androgen, several residues within the enzyme active site make some small adjustments to accommodate the increased bulk of the substrate. In addition, the C19 steroids bind in a slightly different position from estradiol with shifts in positions of up to 1.4 Å. The altered binding position avoids unfavorable steric interactions between Leu 149 and the C19 methyl group (Han et al., unpublished). The known kinetic parameters for these substrates can be rationalized in light of the structures presented. These results give evidence for the structural basis of steroid recognition by 17β-HSD1 and throw light on the design of new inhibitors for this pivotal steroid enzyme.     

Two 17beta-hydroxysteroid dehydrogenases (17HSDs) of estradiol biosynthesis: 17HSD type 1 and type 7.

Two 17β-hydroxysteroid dehydrogenases (17HSDs), type 1 and type 7, are enzymes of estradiol biosynthesis, in addition to which rodent type 1 enzymes are also able to catalyze androgens. Both of the 17HSDs are abundantly expressed in ovaries, the type 1 enzyme in granulosa cells and type 7 in luteinized cells. The expression of 17HSD7, which has also been described as a prolactin receptor-associated protein (PRAP), is particularly up-regulated in corpus luteum during the second half of rodent pregnancy. A moderate or slight signal for mouse 17HSD7/PRAP mRNA has also been demonstrated in samples of placenta and mammary gland, for example. Human, but not rodent, 17HSD1 is expressed in placenta, breast epithelium and endometrium in addition to ovaries. A cell-specific enhancer, silencer and promoter in the hHSD17B1 gene participate in the regulation of type 1 enzyme expression. The enhancer consists of several subunits, including a retinoic acid response element, the silencer has a binding motif for GATA factors, and the proximal promoter contains adjacent and competing AP-2 and Sp binding sites.     

Hormone measures in finger-prick blood spot samples: New field methods for reproductive endocrinology

Comparative endocrine studies have notably advanced understanding of ecological factors that contribute to variation in human reproductive function. Such research has relied on methodological advances that permit hormone determinations in samples that are easily and safely collected, stored, and transported, most recently on measurement of steroids in saliva. This report seeks to further expand the scope of endocrine research by demonstrating the value of blood spot samples collected by finger prick. As a sampling strategy, finger-prick blood spot collection offers the advantages of short collection time, low invasiveness, repeatability, absence of postcollection processing, low biohazard risk, and ease of sample storage and transport. We document good sample stability and present sensitive assay methods for a range of steroids and proteins (FSH, LH, PRL, T, E2, DHEAS, androstenedione, cortisol, SHGB) in blood spots that require sample volumes of 3–12 μl and display good reliability, specificity, precision, accuracy, and convertibility of results to plasma/serum equivalent concentrations. Laboratory evaluation was augmented by a feasibility study at a remote site in Papua New Guinea that confirmed validity and stability of blood spot collections under field conditions. Research applications of blood spot sampling are illustrated with a series of studies, including cross-sectional surveys for developmental and life span endocrinology, a longitudinal, population-based developmental epidemiologic study of puberty, and serial sampling in a dynamic study of neuroendocrine response to suckling. We conclude that the sampling features and wide range of measurable biomolecules of blood spots do constitute a methodological advance for endocrine research. Am J Phys Anthropol 104:1–21, 1997. © 1997 Wiley-Liss, Inc.     

Styles of male social behavior and their endocrine correlates among low-ranking baboons

We have previously studied the relationship between social subordinance (by approach-avoidance criteria) and physiology among male olive baboons (Papio anubis) living freely in a national park in Africa. In stable hierarchies, subordinate individuals have elevated basal glucocorticoid concentrations and a blunted glucocorticoid response to stress, as well as a prompt suppression of testosterone concentrations during stress. These facets have been interpreted as reflecting the chronic stress of social subordinance. In the present report, we find that these endocrine features do not mark all subordinate individuals. Instead, endocrine profiles differed among subordinate males as a function of particular stylistic traits of social behavior. A subset of subordinate males was identified who had significantly high rates of consortships, a behavior usually shown only by high-ranking males. Such behavior predicted the beginning transition to dominance, as these males were significantly more likely than other subordinates to have moved to the dominant half of the hierarchy over the subsequent 3 years. In keeping with this theme of emerging from subordinance, these individuals also had significantly larger glucocorticoid stress-responses, another feature typical of dominant males. However, these subordinate males also had significantly elevated basal glucocorticoid concentrations; it is suggested that this reflects that stressfulness of their overt and precocious strategy of reproductive competition. In support of this, subordinate males with high rates of covert “stolen copulations” did not show elevated basal glucocorticoid concentrations. A second subset of subordinate males were the most likely to initiate fights or to displace aggression onto a third party after losing a fight. These males had significantly or near-significantly elevated testosterone concentrations, compared to the remaining subordinate cohort. Moreover, these males had significantly lower basal glucocorticoid concentrations; this echoes an extensive literature showing that the availability of a displacement behavior (whether aggressive or otherwise) after a stressor decreases glucocorticoid secretion. In support of this interpretation suggesting that it was the initiation of these aggressive acts which attenuated glucocorticoid secretion, there was no association between glucocorticoid concentrations and participation (independent of initiation) in aggressive interactions. Thus, these findings suggest that variables other than rank alone may be associated with distinctive endocrine profiles, and that even in the face of a social stressor (such as subordinance), particular behavioral styles may attenuate the endocrine indices of stress. Am. J. Primatol. 42:25–39, 1997. © 1997 Wiley-Liss, Inc.     

A comparison of sex steroid hormone excretion and metabolism by psittacine species

The metabolism, excretory rates, and excretory patterns of carbon 14 (¹⁴C) radiolabeled estradiol (E₂) and testosterone (T) were studied in female budgerigars (Melopsittacus undulatus) and orange‐winged Amazon parrots (Amazona amazonica). Radiolabeled E₂ and T were injected intramuscularly into six budgerigars and two orange‐winged Amazon parrots. Serial fecal/urine samples were collected for 168 h post‐radiolabel injection. Peak radiolabeled E₂ excretion was observed at 4 h post‐injection, and by 24 h, 93.3 ± 6.3 and 65.9% (range, 59.1–72.7%) of the injected radiolabel was recovered in the fecal/urine matter of budgerigars and orange‐winged Amazon parrots, respectively. Similarly, peak radiolabeled T excretion was observed at 4 h post‐injection with 92.7± 3.6 and 66.2% (range, 57.5–75.2%) of the injected radiolabel recovered in the fecal/urine matter by 24 h in the budgerigars and orange‐winged Amazon parrots, respectively. High‐performance liquid chromatography (HPLC) analysis of the fecal/urine material revealed that both parrot species excreted >80% of the radiolabel in the form of complex steroid conjugates. Immunoreactive E₂ and T metabolites were detected using estrone (E₁) and C‐21/C‐19 conjugate enzyme immunoassays, respectively. Hydrolysis of the E₂ metabolites and HPLC analysis of the ether extracts revealed that E₂ and E₁ were the major steroid moieties. Hydrolysis of the T metabolites and HPLC analysis of the ether extracts revealed two and three major unconjugated peaks for the budgerigars and the orange‐winged Amazon parrots, respectively. Zoo Biol 18:247–260, 1999. © 1999 Wiley‐Liss, Inc.     

Androgen‐metabolizing enzymes show region‐specific changes across the breeding season in the brain of a wild songbird

The Lapland longspur (Calcarius lapponicus) is an arctic‐breeding songbird that shows rapid behavioral changes during a short breeding season. Changes in plasma testosterone (T) in the spring are correlated with singing but not territorial aggression in males. Also, T treatment increases song but not aggression in this species. In contrast, in temperate‐zone breeders, song and aggression are highly correlated, and both increase after T treatment. We asked whether regional or temporal differences in androgen‐metabolizing enzymes in the longspur brain explain hormone‐behavior patterns in this species. We measured the activities of aromatase, 5α‐reductase and 5β‐reductase in free‐living longspur males. Aromatase and 5α‐reductase convert T into the active steroids 17β‐estradiol (E₂) and 5α‐dihydrotestosterone (5α‐DHT), respectively. 5β‐Reductase deactivates T via conversion to 5β‐DHT, an inactive steroid. We examined seven brain regions at three stages in the breeding season. Overall, aromatase activity was high in the hypothalamus, hippocampus, and ventromedial telencephalon (containing nucleus taeniae, the avian homologue to the amygdala). 5β‐Reductase activity was high throughout the telencephalon. Activities of all three enzymes changed over time in a region‐specific manner. In particular, aromatase activity in the rostral hypothalamus was decreased late in the breeding season, which may explain why T treatment at this time does not increase aggression. Changes in 5β‐reductase do not explain the effects of plasma T on aggressive behavior. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 176–188, 1999     

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO_KF1 from Comamonas testosteroni KF1 and found that it had an apparent k_cat/K_m for phthalate of 0.58 ± 0.09 μM⁻¹s⁻¹, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α₃ trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO_KF1 larger than that of other characterized ROs. Complexes of PDO_KF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.     

Corticosterone induces steroidogenic lesion in cultured adult rat Leydig cells by reducing the expression of star protein and steroidogenic enzymes

The present study was designed to investigate the dose-dependent direct effect of corticosterone on adult rat Leydig cell steroidogenesis in vitro. Leydig cells were isolated from the testis of normal adult male albino rats, purified on discontinuous Percoll gradient and plated in culture plates/flasks overnight at 34 degrees C in a CO(2) incubator under 95% air and 5% CO(2) using DME/F12 medium containing 1% fetal bovine serum. After the attachment of cells, serum-containing medium was removed and cells were exposed to different doses (0, 50, 100, 200, 400, and 800 nM) of corticosterone using serum-free fresh medium for 24 h at 34 degrees C. At the end of exposure period, cells were utilized for assessment of the activities and mRNA expression of steroidogenic enzymes (cytochrome P(450) side chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and cytochrome P(450) aromatase) and steroidogenic acute regulatory protein gene expression. Testosterone and estradiol production were also quantified. Activities of cytochrome P(450) side chain cleavage enzyme, 3beta- and 17beta-hydroxysteroid dehydrogenases were declined significantly in a dose-dependent manner after corticosterone exposure, while their mRNA expression were significantly reduced at higher doses of corticosterone exposure. The activity and mRNA expression of cytochrome P(450) aromatase registered a significant increase at 100 nM dose of corticosterone whereas at 200-800 nM doses both the activity as well as the mRNA levels was significantly reduced below the basal level. StAR protein gene expression was significantly inhibited by higher doses of corticosterone employed. At all doses employed, corticosterone significantly reduced the production of testosterone by Leydig cells, while estradiol level registered a significant increase at 50 and 100 nM doses but at higher doses, it registered a significant decrease when compared to basal level. It is concluded from the present in vitro study that the molecular mechanism by which corticosterone reduces the production of Leydig cell testosterone is by reducing the activities and mRNA expression of steroidogenic enzymes and steroidogenic acute regulatory protein.