Effects of fetal alcohol exposure on brain 5 alpha-reductase/aromatase activity

The local formation of the testosterone metabolites 5 alpha-dihydrotestosterone and 17 beta-estradiol within the hypothalamic-preoptic area (HPOA) is essential for the normal sexual differentiation of the male central nervous system (CNS) during a perinatal critical period in the rat. Testosterone, the substrate for these reactions, is derived primarily from synthesis within the fetal testis. Fetal alcohol exposure (FAE) during this critical period profoundly affects fetal testicular steroidogenesis as well as the sexual differentiation of the CNS. The present study was conducted to determine whether FAE directly affects the local metabolism of androgens within the developing CNS or whether reduced androgen substrate, via a testicular lesion, is a more likely explanation for the known effects of FAE on the CNS. The enzymatic activities of 5 alpha-reductase and aromatase were simultaneously quantitated in the newborn rat HPOA following FAE. Neither the enzymatic activity of 5 alpha-reductase, aromatase nor their ratio were significantly influenced (P greater than 0.05) by FAE with respect to controls. FAE apparently does not alter the disposition of the androgens within the newborn rat HPOA. These results support the hypothesis that FAE alters the sexual differentiation of the CNS through inhibition of androgen biosynthesis at the level of the perinatal rat testis.     

Effects of exogenous steroids on androgen receptors in fetal guinea pig brain

We treated pregnant guinea pigs on Day 50 of gestation with 10 mg testosterone propionate (TP), obtaining fetuses 2, 4, 8, or 18 h later as well as after 5 days of treatment. In a second group of pregnant guinea pigs, dihydrotestosterone propionate (DHTP), estradiol benzoate (E2B), progesterone (P), or cortisol was given 2 h before obtaining fetuses. Although TP treatment elevated fetal serum T (p less than 0.05), brain cytosolic androgen receptor (ARc) content was unchanged in fetuses of either sex. In female fetuses, nuclear androgen receptors (ARn) increased 10-fold in medial-basal hypothalamus (MBH) and preoptic area (POA) at 2 and 4 h (respectively) after treatment, while fetal male ARn content was unchanged. Maternal injection of other steroids (E2B, P, or cortisol, but not DHTP) significantly increased these hormones in the fetus 2 h later (p less than 0.05). Only androgens affected fetal androgen receptor (AR) content. While TP increased ARn in female MBH, DHTP decreased ARc in fetal anterior pituitary of both sexes. In this latter case, a metabolite of DHT may mediate the effects. We conclude that T crosses the guinea pig placenta and activates ARn in POA and MBH of female fetuses; male ARn appear to be maximally occupied by endogenous T. Steroids of other classes do not induce AR responses in fetal guinea pig brain. These AR changes may represent an initial cellular mechanism in brain sexual differentiation.     

Effects of gonadectomy and steroid treatment on renal prostaglandin 9-ketoreductase activity in the rat

The effect of gonadectomy and treatment with sex-steroids on renal prostaglandin 9-ketoreductase activity in 10-11 week old male and female rats was determined. Rats were gonadectomized or subjected to sham operation at 3 weeks of age. During week 7, rats were injected s.c. twice over a 6-day interval with vehicle (peanut oil, 0.5 ml X kg-1) or with depot forms of testosterone (5 mg X kg-1), estradiol (0.02 mg X kg-1), progesterone (5 mg X kg-1), or estradiol and progesterone combined. Renal prostaglandin 9-ketoreductase activity was about 50% higher in female rats than in males. Gonadectomy decreased 9-ketoreductase activity in females, but not in males, and eliminated the gender difference in enzyme activity. Treatment with estradiol elevated 9-ketoreductase activity in males and females, while treatment with testosterone or progesterone was without effect. Progesterone did, however, antagonize the elevation in 9-ketoreductase activity produced by estradiol.     

Fast liquid chromatographic assay of androgen aromatase activity

A rapid, sensitive nonradiometric assay method for the enzymatic aromatization of androgens has been developed using reversed-phase fast liquid chromatography. The use of a 3-microns-particle, 5-cm-long column allowed analysis of androstenedione, estrone, testosterone, and estradiol within 1 min. The reliability of the method was demonstrated by measuring the aromatase activity of human placental microsomes and that of the purified reconstituted aromatase system using both substrates. The rapid analysis enabled the processing of a large number of samples in a short time, which makes the present method especially suitable for the analysis of chromatographic fractions obtained during enzyme purifications and for enzyme kinetic studies.     

Immunohistochemical labeling of androgen receptors in the brain of rat and monkey.

Androgen receptor antibodies have recently been developed using fusion proteins containing fragments of human prostatic androgen receptor. We have used a polyclonal antibody raised in rabbits to label androgen receptors in brain sections from male and female rats and monkeys. Free-floating frozen sections were incubated in primary antibody, and processed by the peroxidase-avidin-biotin complex method using biotinylated anti-rabbit IgG. Nickel intensified diaminobenzidine was used as the chromagen, and neurons were labeled in the amygdala, hippocampus, bed nucleus of stria terminalis, septum, preoptic area, in several hypothalamic nuclei including the supraoptic and paraventricular nuclei, in several brain stem motor nuclei and in cerebral cortex. Staining was most intense in cell nuclei but also occurred in cytoplasm and in some neuronal processes. Labeling was more restricted in monkey than in rat brain. Omitting the primary antibody or pre-incubating the primary antibody with rat prostatic cytosol for control purposes demonstrated the specificity of staining.     

Estrogen synthesis in fetal sheep brain: effect of maternal treatment with an aromatase inhibitor

The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8-1.4 pmol/h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02-0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The Michaelis-Menten dissociation constant (K(m)) for aromatase in the fetal sheep brain was 3-4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development.     

Low aromatase activity and gene expression in human fetal testes

Because of previous indications that estradiol (E2) plays a role in the regulation of testicular testosterone (T) production in some species, the production of E2 and aromatase gene expression in human fetal testes were investigated. Testicular minces from 14 fetuses (fetal age 15-23 weeks) were incubated with and without 200 ng/ml highly purified hCG, and the production of E2 and T was measured by RIA. Basal T production was high at 15-18 weeks of gestation and decreased thereafter. Estradiol production was low in all testes. Aromatase mRNA (P-450 arom messenger ribonucleic acid) was not detectable in fetal testicular tissues when studied by Northern and dot blot techniques. Placenta and fetal liver expressed aromatase mRNA, but fetal ovary contained only miniscule amounts. HCG significantly stimulated the production of both T and E2 in the testes of older fetuses (19-23 weeks), but the testicular E2 production of the youngest fetuses (15-18 weeks) did not increase significantly after hCG stimulation. These results indicate that aromatase activity and gene expression are very low in human fetal testes. These findings suggest that E2 may not play a major role in testicular T production in the human fetus.     

Ontogeny of androgen receptors in fetal guinea pig brain

Sexual differentiation of the guinea pig brain is androgen dependent. To understand the cellular mechanisms of androgen action, we studied the ontogeny of cytosolic (ARc) and nuclear (ARn) androgen receptors in the brains and anterior pituitaries of fetal, neonatal, and adult guinea pigs. Using cytosol from the hypothalamus-preoptic area-amygdala-septum of 60- to 65-day fetuses and nuclear preparations from 6-day-old neonates treated with testosterone propionate, validation studies revealed an AR with an apparent Kd of 1.9 +/- 1.1 (mean +/- SEM, n = 3) x 10(-10) M (ARc) and 3.4 +/- 3.2 (n = 3) x 10(-10) M (ARn). The cytosolic receptors were highly specific for androgens. After assay validation, AR content was determined from specific brain regions of fetuses obtained on Days 30, 40, 50, and 59 of gestation and on Days 6 and 120 postpartum. ARc differed significantly (p less than 0.05) between brain regions and times of gestation, but no sex differences were apparent. In contrast, ARn showed little difference between tissues or with gestational age, but there were significant differences between males and females, especially in late gestation and early postnatal life, with males having greater ARn binding (p less than 0.05). These data demonstrate the presence of ARc and ARn in the fetal brain and pituitary gland during the critical period of sexual differentiation (Days 30-37 of gestation), thus establishing the identity of cellular structures involved in androgen action.     

The effect of gonadectomy and aromatase inhibition on the excretion of 19-nordeoxycorticosterone in rats

19-Nordeoxycorticosterone (19-norDOC) is a powerful mineralocorticoid, which has been postulated to be involved in the pathogenesis of some forms of hypertension. The urinary excretion of 19-norDOC by female rats is up to 20 times that of males. To demonstrate the influence of the gonads on the excretion of 19-norDOC, we measured the excretion of 19-norDOC in intact and gonadectomized male and female rats with and without replacement with testosterone (40 mg testosterone enanthate s.c.) or estrogen (4 mg estradiol valerate s.c.) and in intact animals receiving the aromatase inhibitor, 10-propargyl androstenedione (10-pA) (10 mg s.c.). Orchiectomy produced a significant increase in the urinary excretion of 19-norDOC in males. Testosterone treatment decreased 19-norDOC excretion by castrated males to below intact values, while estrogen administration increased its excretion. Oophorectomy had no consistent effect on 19-norDOC excretion. In oophorectomized females, testosterone administration significantly suppressed 19-norDOC excretion and estrogen replacement increased excretion slightly. 10-pA had little effect on the excretion of 19-norDOC in intact rats of either sex. In conclusion, it appears that 19-norDOC production is inhibited by testosterone, but is affected only slightly by estrogens.     

Organizing effects of sex steroids on brain aromatase activity in quail

Preoptic/hypothalamic aromatase activity (AA) is sexually differentiated in birds and mammals but the mechanisms controlling this sex difference remain unclear. We determined here (1) brain sites where AA is sexually differentiated and (2) whether this sex difference results from organizing effects of estrogens during ontogeny or activating effects of testosterone in adulthood. In the first experiment we measured AA in brain regions micropunched in adult male and female Japanese quail utilizing the novel strategy of basing the microdissections on the distribution of aromatase-immunoreactive cells. The largest sex difference was found in the medial bed nucleus of the stria terminalis (mBST) followed by the medial preoptic nucleus (POM) and the tuberal hypothalamic region. A second experiment tested the effect of embryonic treatments known to sex-reverse male copulatory behavior (i.e., estradiol benzoate [EB] or the aromatase inhibitor, Vorozole) on brain AA in gonadectomized adult males and females chronically treated as adults with testosterone. Embryonic EB demasculinized male copulatory behavior, while vorozole blocked demasculinization of behavior in females as previously demonstrated in birds. Interestingly, these treatments did not affect a measure of appetitive sexual behavior. In parallel, embryonic vorozole increased, while EB decreased AA in pooled POM and mBST, but the same effect was observed in both sexes. Together, these data indicate that the early action of estrogens demasculinizes AA. However, this organizational action of estrogens on AA does not explain the behavioral sex difference in copulatory behavior since AA is similar in testosterone-treated males and females that were or were not exposed to embryonic treatments with estrogens.     

Sexual differentiation of behavior in the zebra finch: effect of early gonadectomy or androgen treatment

Treatment of nestling zebra finches with estradiol benzoate (EB) has been shown to masculinize singing in females and demasculinize copulatory behavior in males, suggesting that sexual differentiation of these behaviors is under hormonal control such that testicular hormones induce the capacity for song and ovarian hormones suppress the capacity for mounting. Two experiments were carried out to obtain a more complete picture of sexual differentiation in this species. In Experiment 1, nestlings were injected daily for the first 2 weeks after hatching with testosterone propionate (TP), dihydrotestosterone propionate (DHTP), or a combination of DHTP and EB. As adults, birds were gonadectomized and implanted with TP prior to testing, then tested again after implantation with EB. Singing was not increased in females by any of the treatments. The only effect of either TP or DHTP given alone was defeminization of female proceptive behavior by DHTP. Thus androgens appear to have less influence than estrogens on sexual differentiation of behavior in this species. The combination of DHTP and EB demasculinized mounting in males. In Experiment 2, nestlings were gonadectomized at 7-9 days of age and implanted with TP prior to testing in adulthood. Early gonadectomy had little effect on later behavior; early castrated males sang, danced, and copulated normally and early ovariectomized females neither sang nor mounted.     

Increased peripheral aromatase activity in prepubertal children with partial androgen insensitivity syndrome

It has been suggested that rate of estrogen formation was higher in patients with androgen insensitivity syndrome (AIS). This work was designed to find out if peripheral aromatase activity could be related to a defect in androgen action in prepubertal children with male pseudohermaphroditism. Fibroblast estrogen production was assayed by a highly specific enzymatic determination. Foreskin fibroblast strains were raised from 17 children with partial androgen insensitivity (PAIS) as defined by dihydrotestosterone binding activity in cells. Results are expressed as pmol estrogens/mg proteins synthetized/day when cultured fibroblasts are incubated with D4-androstenedione. In normal prepubertal boys (n = 19), aromatase activity ranged between 5 and 10 pmol estrogens/mg proteins/day, while in postpubertal boys it varied between 15 and 34 pmol estrogens/mg proteins/day. In prepubertal boys with PAIS (n = 17) aromatase activity is highly elevated: 19.4 +/- 8.4 pmol/mg proteins/day. These results show that (a) peripheral aromatase activity is low before puberty and (b) fibroblast estrogen synthesis is significantly (p less than 0.001) increased in prepubertal children with PAIS. Our data suggest that low utilization of androgens by target cells stimulates the production of estrogen. Peripheral aromatase activity can thus be considered as a 'marker' of androgen insensitivity in prepubertal children with male pseudohermaphroditism.     

Phosphorylation processes mediate rapid changes of brain aromatase activity

The enzyme aromatase (also called estrogen synthase) that catalyzes the transformation of testosterone (T) into estradiol plays a key limiting role in the action of T on many aspects of reproduction. The distribution and regulation of aromatase in the quail brain has been studied by radioenzyme assays on microdissected brain areas, immunocytochemistry, RT-PCR and in situ hybridization. High levels of aromatase activity (AA) characterize the sexually dimorphic, steroid-sensitive medial preoptic nucleus (POM), a critical site of T action and aromatization for the activation of male sexual behavior. The boundaries of the POM are clearly outlined by a dense population of aromatase-containing cells as visualized by both immunocytochemistry and in situ hybridization histochemistry. Aromatase synthesis in the POM is controlled by T and its metabolite estradiol, but estradiol receptors alpha (ER) are not normally co-localized with aromatase in this brain area. Estradiol receptor beta (ERβ) has been recently cloned in quail and localized in POM but we do not yet know whether ERβ occurs in aromatase cells. It is therefore not known whether estrogens regulate aromatase synthesis directly or by affecting different inputs to aromatase cells as is the case with the gonadotropin releasing hormone neurons. The presence of aromatase in presynaptic boutons suggests that locally formed estrogens may exert part of their effects by non-genomic mechanisms at the membrane level. Rapid effects of estrogens in the brain that presumably take place at the neuronal membrane level have been described in other species. If fast transduction mechanisms for estrogen are available at the membrane level, this will not necessarily result in rapid changes in brain function if the availability of the ligand does not also change rapidly. We demonstrate here that AA in hypothalamic homogenates is rapidly down-regulated by exposure to conditions that enhance protein phosphorylation (addition of Ca²⁺, Mg²⁺, ATP). This inhibition is blocked by kinase inhibitors which supports the notion that phosphorylation processes are involved. A rapid (within minutes) and reversible regulation of AA is also observed in hypothalamic explants incubated in vitro and exposed to high Ca²⁺ levels (K⁺-induced depolarization, treatment by thapsigargin, by kainate, AMPA or NMDA). The local production and availability of estrogens in the brain can therefore be rapidly changed by Ca²⁺ based on variation in neurotransmitter activity. Locally-produced estrogens are as a consequence available for non-genomic regulation of neuronal physiology in a manner more akin to the action of a neuropeptide/neurotransmitter than previously thought.     

Parallel evolution between aromatase and androgen receptor in the animal kingdom

There are now many known cases of orthologous or unrelated proteinsin different species that have undergone parallel evolutionto satisfy a similar function. However, there are no reportedcases of parallel evolution for proteins that bind a commonligand but have different functions. We focused on two proteinsthat have different functions in steroid hormone biosynthesisand action but bind a common ligand, androgen. The first protein,androgen receptor (AR), is a nuclear hormone receptor and thesecond one, aromatase (cytochrome P450 19 [CYP19]), convertsandrogen to estrogen. We hypothesized that binding of the androgenligand has exerted common selective pressure on both AR andCYP19, resulting in a signature of parallel evolution betweenthese two proteins, though they perform different functions.Consistent with this hypothesis, we found that rates of aminoacid change in AR and CYP19 are strongly correlated across themetazoan phylogeny, whereas no significant correlation was foundin the control set of proteins. Moreover, we inferred that genomictoolkits required for steroid biosynthesis and action were presentin a basal metazoan, cnidarians. The close similarities betweenvertebrate and sea anemone AR and CYP19 suggest a very ancientorigin of their endocrine functions at the base of metazoanevolution. Finally, we found evidence supporting the hypothesisthat the androgen-to-estrogen ratio determines the gonadal sexin all metazoans.     

Differential regulation of aromatase and androgen receptor in granulosa cells

During follicular development, androgen acts in three distinct ways. During the early stage of follicular differentiation, androgen acts as an enhancer of FSH-stimulated follicular differentiation. As follicular differentiation progresses, this effect is decreased and androgen is mainly utilized as a substrate for estrogen synthesis under increasing stimulation of FSH and LH. These two events are mediated by androgen receptor (AR) and aromatase (P450arom), respectively. In the rat and marmoset monkey, AR and P450arom are predominantly expressed in granulosa cells, and both are developmentally regulated. The expression of AR is highest in preantral/early antral follicles and gradually decreases as follicles mature, whereas expression of P450arom is increased as follicular differentiation progresses. We propose that differential regulation of these two androgen-utilizing factors contributes to the smooth transition of developing follicles from the early stage of differentiation to the fully mature ovulatory status. A failure of this transition due to improper androgen stimulation might result in follicular atresia.     

Effect of gonadectomy on brain catecholamines during the postnatal period

The effect produced by gonadectomy on dopamine (DA) and noradrenaline (NA) levels in the diencephalon and the rest of the brain of male and female rats during postnatal development has been studied. DA and NA metabolism or biosynthesis seems to be regulated by the ovarian hormones, directly or by means of hypophysary hormones, since both catecholamine levels rise acutely during postnatal development when ovariectomy is performed. In contrast with controls, the NA level is not stable at 45 days, but continues rising to day 60. Orchidectomy also acutely increases the level of diencephalic DA, but in contrast with females, its concentration progressively decreases, being at day 60 the same as in the controls. The reasons that cause this normalization, in the absence of testicular androgens, are unknown. In the same way, the extirpation of the testicles increases the diencephalic concentration of NA, the concentration change is similar to the control one: however, the level is higher. There is also a clear difference from the gonadectomized females, in which the diencephalic NA rises during 45-60 days. Gonadectomy does not significantly alter the level of DA or NA in the rest of the brain.     

Cyclic AMP phosphodiesterase activity in fetal and adult muscle of the rhesus monkey.

Total cyclic AMP phosphodiesterase activity of voluntary skeletal muscle of the rhesus monkey was highest in the 100-day fetal series, decreased near term, and was lowest in the adult series. Kinetic data indicated the existence of two cyclic AMP phosphodiesterase enzymes in both the fetal and adult muscle. The apparent K_m values for the high-affinity phosphodiesterase were similar in the 100-day fetal and adult skeletal muscle, whereas those for the low-affinity enzyme were twofold higher in the fetal series. The V_max of the high K_m enzyme was tenfold higher in the fetal than in the adult series and the low K_mV_max was fourfold higher in the fetal series. Both caffeine and theophylline were competitive inhibitors of the low K_m phosphodiesterase activity and noncompetitive inhibitors of the high K_m enzyme. No difference was observed in the sensitivity of the fetal and adult enzyme preparations to the methylxanthines or to Ro20-1724.     

Steroid sulfatase and sulfuryl transferase activity in monkey brain tissue

Dehydroepiandrosterone and its sulfated form are commonly known as modulators of gamma-aminobutyrate A and N-methyl-D-aspartate receptors. In spite of poor permeability of the blood-brain barrier for sulfated steroids, high concentrations of dehydroepiandrosterone and also its sulfate have been found in brain tissue. Physiological concentrations of these neuromodulators are maintained by two enzymes present in the blood and many peripheral tissues, including the brain, namely, steroid sulfatase and neurosteroid sulfuryl transferase (NSST). This prompted us to investigate activities of these enzymes in primate brain tissue. Rather low neurosteroid sulfuryl transferase activity was detectable in in vitro incubations of cytosol fractions from male and female Macaca mulatta brains, dissected to cerebral cortex, subcortex, and cerebellum. In male monkeys, the highest activity was found in the cerebellum followed by cortex and subcortex. On the other hand, in female monkeys, the highest activity was determined in the cortex followed by subcortex and cerebellum. Steroid sulfatase activity was determined in in vitro microsomal samples from each of the above-mentioned brain regions. Specific activities in female cerebral regions declined in the order: cerebellum, cortex, and subcortex. In male monkeys, no significant difference among the studied regions was observed. Using dehydroepiandrosterone sulfate as a substrate, the apparent kinetic characteristics of steroid sulfatase were determined as follows: K(M) 36.10 +/- 8.33 microM, V(max) 8.38 +/- 1.68 nmol/h/mg protein. These results will serve as a basis for further studies concerning the pathophysiology of human brain tumors.