Metabolism of tibolone and its metabolites in uterine and vaginal tissue of rat and human origin.

In postmenopausal women, tibolone shows clear tissue differences in its stimulatory effects on the vagina and uterus. In rats, however, it has stimulatory effects on both tissues, with a different, more estrogenic, effect on the uterus than in humans. This may be due to differences in local metabolism. Therefore, in the present study, the metabolism of tibolone was analyzed in incubations of uterine and vaginal tissue from postmenopausal women and ovariectomized rats using radiolabeled tibolone in order to understand the tissue- and species-specific metabolism. In the rat, tibolone (50 nM) was mainly 3alpha-reduced to the estrogenic 3alpha-OH-tibolone in the uterus and vagina. The 3beta-OH tibolone can be isomerized to 3alpha-OH-tibolone with tibolone as intermediate. In contrast, in the same tissues from postmenopausal women, the progestagenic Delta4-isomer and estrogenic 3beta-OH-tibolone were the major metabolites of tibolone. The formation of the Delta4-isomer was higher in uterine tissue. The 3beta-hydroxysteroid dehydrogenase (HSD) inhibitor epostane had no effect on tibolone metabolism in human uterine and vaginal tissue microsomes and HEK293 cells expressing the human 3beta-HSD types 1 and 2 isoforms did not metabolize tibolone. Moreover, the 3beta-reduction of tibolone to 3beta-OH-tibolone was NADPH dependent, while the isomerization of tibolone to the Delta4-isomer did not require a cofactor. It was therefore concluded that human 3beta-HSD isoforms are not involved in the metabolism of tibolone, and that the 3beta-reduction and the Delta5-10 to Delta4 isomerization may be catalyzed by different enzymes. In conclusion, we showed that, in hormone therapy target tissues of the rat as compared with the human, different metabolic pathways for tibolone exist and therefore result in metabolites with different pharmacological properties. The rat is therefore a poor model to predict the effects of tibolone on the uterus in postmenopausal women.

Localization of aromatase in the brain of the male African catfish, Clarias gariepinus (Burchell), by microdissection and biochemical identification.

Aromatase activity was determined in small discrete areas of the brain of the African catfish, Clarias gariepinus, by a radiometric assay. The fore- and midbrain were divided into eighteen 500-microns transverse sections. From these sections several punches (0.3 mg of tissue) were taken and incubated with [19-3H]-androstenedione. The aromatase activity, calculated from the release of tritium label during aromatization, is expressed in pmol mg-1 tissue hour-1. The highest activity (3.7 pmol) was detected in the preoptic region. The more caudally located area tuberalis, including the nucleus lateralis tuberis and the nucleus recessus lateralis, also showed a relatively high activity (2.5 pmol). A similar activity was found in the most rostral part of the telencephalon and the dorsal parts of the mesencephalon, i.e., the tectum opticum and torus semicircularis (2.3 pmol). A moderate aromatase activity was observed in remaining parts of the brain, except the cerebellum and hindbrain, in which aromatase activity was hardly detectable (0.1-0.3 pmol). It is concluded that a high aromatase activity is present in regions known to be involved in the regulation of reproduction. Since both the torus semicircularis and the tectum opticum display a high aromatase activity, it is suggested that also these structures are involved in reproductive processes.

A quantitative study of steroid bioconversions in the testis of the African catfish, Clarias gariepinus (Burchell), under natural spawning and natural and cultivated non-spawning conditions.

Quantitative aspects of bioconversions in the testes of the African catfish (Clarias gariepinus) were studied in vitro by incubation of tissue with [3H]pregnenolone or [3H]androstenedione. During the breeding period, spawning and non-spawning animals were collected from their natural habitat, the Hula nature reserve, in northern Israel. In the same period, non-spawning animals were collected from a fish pond in the same region. It was shown that spawning was accompanied by significant changes in steroid bioconversions, i.e. a reduction in androgen synthesis, especially of 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone and an increase in the production of C21-steroids, especially progesterone, 17 alpha-hydroxyprogesterone and a pregnenolone ester. These changes resulted from a decreased contribution of the cytochrome P-450 enzymes 17 alpha-hydroxylase, C17-20-lyase and 11 beta-hydroxylase. A rise in plasma gonadotrophin concentration was observed only in spawning catfish. In the absence of such an increase in plasma gonadotrophin, steroid synthesis in the testes of non-spawning feral and pond catfish was primarily directed towards the production of 11-oxygenated androgens and 5 beta-pregnane-3 alpha,17 alpha,20 alpha-triol. It is suggested that spawning is induced by gonadotrophin and the ensuing change in steroidogenesis. It is possible that husbandry conditions inhibit the necessary increase in gonadotrophin release.

Steroid glucuronides as male pheromones in the reproduction of the African catfish Clarias gariepinus--a brief review.

After ovulation, female African catfish are strongly attracted by the odor of male conspecifics. This attraction depends on the presence of the seminal vesicle, a part of the male reproductive organs. Removal of the seminal vesicle illustrates this fact. A low dose of seminal vesicle fluid, added to the water, appears to be highly attractive for catfish which have ovulated. Fractionation of the fluid and testing of the different fractions shows that steroid glucuronides could be responsible for the attraction. These steroid glucuronides can be identified with gas chromatographic-mass spectrometric analysis. A mixture of glucuronides, prepared to resemble the composition of the seminal vesicle fluid, evokes a dose-dependent attraction. The most potent odorant, observed by measuring electrical responses from the olfactory epithelium and from the olfactory tract appears to be 3 alpha,17 alpha-dihydroxy-5 beta-pregnan-20-one-3 alpha-glucuronide.

Distribution and regulation of estrogen-2-hydroxylase in the quail brain.

The anatomical distribution and endocrine regulation of the estrogen-2-hydroxylase activity were investigated in the brain of adult male and female Japanese quail. Significant levels of enzymatic activity were detected in all brain regions that were studied, but the highest levels were observed in preoptic and hypothalamic brain nuclei that are known to contain high levels of aromatase activity. These data are consistent with previous results suggesting that the placental aromatase is also responsible for the estrogen-2-hydroxylase activity. However, there is a marked sex difference and a control by T of aromatase activity in the quail brain, and no such difference in 2-hydroxylase activity could generally be detected except in the VMN. Further studies will be needed to know whether the previously published conclusions concerning the human placenta also apply to the brain. The present data are consistent with the idea that estrogens formed locally in the brain by testosterone aromatization could affect reproduction by interfering with the catecholaminergic transmission after being metabolized into catechol-estrogens.

Cortisol affects testicular development in male common carp, Cyprinus carpio L., but not via an effect on LH secretion.

Previous work showed that prolonged elevated cortisol levels, implicated in the stress adaptation, inhibits testicular pubertal development in male common carp, as well as an impairment of the synthesis of the 11-oxygenated androgens. This may be a direct effect of cortisol on the testis or via the gonadotropin secretion by the pituitary. The aim of the present study was to investigate whether cortisol has an effect on pituitary LH secretion. Juvenile common carp were fed with cortisol containing food pellets. Elevated cortisol levels blocked the increase in testosterone levels and pituitary LH content, but induced higher plasma LH levels at the end of puberty. The in vitro LH release capacity was correlated to the pituitary LH content. At the final stage of pubertal development, when a significant difference in pituitary LH content was observed, sGnRHa-induced LH release was also decreased. Testosterone has been shown to induce development of pituitary gonadotrophs, leading to an increase in LH content and GnRH-inducible LH release, but a decrease in plasma LH levels. We observed decreased plasma testosterone levels as a consequence of prolonged cortisol treatment. It is hypothesised that cortisol inhibits the testicular testosterone secretion and thereby, prevents LH storage. In vitro, this leads to a reduced GnRH-inducible LH release, but in vivo to increased LH plasma levels. It is very unlikely that the impaired testicular development is due to an effect of cortisol on LH secretion.

The impact of hospital discharge on inappropriate hospital stay.

Appropriate hospital stay should be effective, efficient and tailored to patient needs. Previous studies have found that on average 20 per cent of hospital stay is inappropriate. Within obstetrics, inappropriate hospital stay consists mostly of delays in hospital discharge. The specific goals of this study were to reduce inappropriate hospital stay by fine-tuning patient logistics, increasing efficiency and providing more comfortable surroundings. New policies using strict discharge criteria were implemented. Total inappropriate hospital stay decreased from 13.3 to 7.2 per cent. The delay in discharge procedures halved. P-charts showed a decrease in inappropriate hospital stay, indicating the current process to be stable. Concludes that a significant reduction in inappropriate hospital stay was found following the implementation of innovative hospital discharge policies, indicating greater efficiency and accessibility of hospital services.

Appropriate length of extended day care.

Extended day care (EDC) is a one-day admission spending one night in hospital. Many EDC patients do not need hospital care over night, so probably they could be transferred to a day surgery setting, resulting in decreased costs and increased efficiency. The objectives of the study were to assess the appropriate length of extended day care (ALED) and a possible transfer to day surgery. ALED was defined as the time between the start of the surgical procedure and the final moment appropriate hospital care was provided. About 80 per cent of the patients could possibly have been treated in day surgery. The other patients could not be transferred, because of a prolonged ALED. With the implementation of new policies on admission to and discharge from the hospital and the use of altered types of operation room scheduling or patient logistics the transfer of most EDC patients to day surgery would be possible.

Hypocorticism and interrenal hyperplasia are not directly related to masculinization in XX mas(-1)/mas(-1) carp, Cyprinus carpio.

This study reports on a homozygous XX male strain of common carp (E5), which fail to mount a normal cortisol stress response. Earlier classical genetic analysis had indicated that masculinization of E5 fish was caused by a putative recessive mutation (mas(-1)/mas(-1)). Hypocorticism in E5 fish was studied to investigate if it was related to masculinization. Head-kidney tissues isolated from E5 fish showed a low cortisol-producing capacity in vitro, and also demonstrated a reduced sensitivity to stimulation with ACTH, when compared with an isogenic XY male carp strain (STD). There was no strain difference in androgen production by head-kidney tissues in vitro. E5 fish exhibited significant hyperplasia of the interrenal tissue (adrenal homologue of teleost fish) located in the head-kidney. Conversion of pregnenolone was significantly lower in E5 head-kidney homogenates, compared to STD homogenates, however, no strain difference was found in the conversion of 17alpha-hydroxyprogesterone into cortisol. Gonad homogenates incubated with pregnenolone showed no strain difference in conversion to corticosteroids and androgens. Results indicate that the interrenal hyperplasia and hypocorticism in this strain of carp may be due to a dysfunction of the 17alpha-hydroxylase activity of the enzyme P450c17 in the interrenal, but that this defect may not be the primary factor resulting in masculinization of these XX genotypes.

Steroid metabolism in the seminal vesicles of the African catfish, Clarias gariepinus (Burchell), during the spawning season, under natural conditions, and kept in ponds.

Steroid metabolism in the seminal vesicles of Clarias gariepinus, collected in the Hula nature reserve and in a fish pond in North Israel during the spawning season, was studied in vitro by homogenate and tissue incubations with [3H]pregnenolone, [3H]dehydroepiandrosterone, or [3H]androstenedione as precursor. The seminal vesicles were able to synthesize androgens, 11-oxygenated androgens, and 5 beta-reduced C19- and C21-steroids; a pregnenolone ester was also formed. Furthermore a potent synthesis of steroid glucuronides, i.e., the glucuronides of testosterone, 5 beta-dihydrotestosterone, 5 beta-androstan-3 alpha, 17 beta-diol, and etiocholanolone, was observed. When reared in ponds, the African catfish fails to spawn. This might be caused by a change in steroid glucuronide (pheromone) production by the seminal vesicles, as it appeared that the synthesis of glucuronides differs in "wild" and "pond" animals. Etiocholanolone glucuronide is completely absent in pond animals.

Catechol-O-methyltransferase in the brain of the male African catfish,Clarias gariepinus; distribution and significance for the metabolism of catecholestrogens and dopamine.

Catechol-O-methyltransferase, involved in the methylation of catechol substrates, was localized in the brain of the male African catfish,Clarias gariepinus, by means of a radiometric assay using [Methyl-(3)H]S-adenosylmethionine as methyldonor and catecholestrone as substrate. Fore- and midbrain were divided into eighteen, 500 µm thick, transverse sections. With a hollow needle (diameter 1 mm), specific areas of the brain were punched out and assayed. The catechol-O-methyltransferase activity was calculated from the amount of radioactive methoxyestrone formed from catecholestrone and expressed in pmol.mg(-1) tissue.h(-1).The enzyme could be demonstrated throughout the brain. Although the enzyme activity did not differ very much between the various brain regions (max. 15.4; min. 7.5 pmol), there were some areas in the brain with a more than average activity,i.e., the lateral telencephalon (10.3 pmol), the nucleus preopticus (13.1 pmol), nucleus lateralis tuberis (11.0 pmol) and nucleus recessus posterioris (12.0 pmol) of the diencephalon, the tectum opticum (15.4 pmol) and torus semicircularis (13.6 pmol) of the mesencephalon, and the caudal cerebellum of the metencephalon (10.8 pmol). The lowest activity was detected in the caudal metencephalon (7.5 pmol).The presence of the enzyme catechol-O-methyltransferase in the brain of the African catfish and the observation that both catecholestrogens and dopamine can be methylated by this enzyme suggest that catecholestrogens can influence the methylation (inactivation) of dopamine. Incubations of forebrain homogenates with dopamine and catecholestrone or catecholestradiol confirmed that both catecholestrogens can inhibit the methylation of dopamine. Lineweaver-burk plots with various concentrations of the catecholestrogens indicated that the inhibition is competitive. Dixon plots from the inhibition studies gave inhibition constants of 1.4 and 0.6 µM for catecholestrone and catecholestradiol, respectively, indicating that catecholestradiol is a two times stronger inhibitor than catecholestrone.The significance of the inhibition of the methylation of dopamine by the catecholestrogens in the brain is discussed in the light of the negative feedback of gonadal steroids on the central regulation of reproductive processes.

Gas chromatographic-mass spectrometric analysis of steroids and steroid glucuronides in the seminal vesicle fluid of the African catfish, Clarias gariepinus.

Gas chromatographic-mass spectrometric analysis was carried out to identify steroids and steroid glucuronides in the seminal vesicle fluid of African catfish, Clarias gariepinus, collected in the Hula nature reserve (Israel) during the breeding season. Full mass spectra of 5 beta-pregnane-3 alpha, 17 alpha-diol-20-one and cholesterol were obtained. After treatment with beta-glucuronidase the following steroid glucuronides were determined by full mass spectra of the corresponding free steroids: etiocholanolone, 5 beta-androstane-3 alpha, 17 beta-diol-11-one, 5 beta-pregnane-3 alpha, 17 alpha-diol-20-one, and cholesterol. Furthermore, after selected ion monitoring the following steroids and steroid glucuronides could be detected by the presence of at least two characteristic ions at the expected retention time: 5 beta-androstane-3 beta, 17 beta-diol, 5 beta-androstane-3 alpha, 17 beta-diol, etiocholanolone, 5 beta-androstane-3 alpha, 17 beta-diol-11-one, testosterone, 5 beta-androstane-3 alpha, 17 beta-diol-glucuronide, and testosterone-glucuronide. These results agree with the hypothesis that steroid glucuronides, synthesized by the seminal vesicles, are excreted with the seminal vesicle fluid into the external environment, where they might function as sex pheromones.

Measurement of aromatase activity in the brain of the African catfish, Clarias gariepinus--a comparison of two assay methods.

In the brain of the African catfish aromatase activity was demonstrated with two different methods using [7-3H]androstenedione and [19-3H]androstenedione as substrates. Kinetic analysis of estrogen formation following incubations of a cell-free fraction of brain homogenates showed that the apparent Km is the same for both substrates (0.03 micro M), but the Vmax is smaller with [19-3H]androstenedione as substrate. This indicates a so-called isotope effect. A time course study showed that after an incubation of 4 hr the aromatase activity is still linearly time dependent. Comparing the amount of estrogens formed from both precursors showed that the value of the isotope effect is 3.4 Incubation of symmetric punches of telencephalon and diencephalon with the two substrates likewise showed an isotope effect of 3.4

Steroid metabolism in the testes of the African catfish, Clarias gariepinus (Burchell), during spawning season, under natural conditions and kept in ponds.

Steroid metabolism in the testes of Clarias gariepinus, collected in the Hula nature reserve and a fish pond in North Israel during the spawning season, was studied in vitro by homogenate and tissue incubations with [3H]pregnenolone or [3H]androstenedione as precursors. It could be demonstrated that [3H]pregnenolone was mainly converted via progesterone and 17 alpha-hydroxyprogesterone into androstenedione, 11 beta-hydroxyandrostenedione, and 11 beta-hydroxytestosterone. The final products from [3H]androstenedione incubations, 11 beta-hydroxyandrostenedione, and 11 beta-hydroxytestosterone, confirm these findings. The major difference in steroid metabolism between wild and pond animals was a reduction in androgen synthesis in the first group, resulting in higher amounts of C21-steroids. Moreover, the synthesis of two steroid esters, including pregnenolone ester, could be established with tissue incubations of wild animals only. Steroid conjugates were hardly synthesized at all in the testes of C. gariepinus.

Pregnenolone, testosterone, and estradiol in the migratory locust Locusta migratoria; a gas chromatographical-mass spectrometrical study.

Homogenates of ovaria as well as testes from the locust Locusta migratoria were analyzed for the presence of pregnenolone, testosterone, and estradiol by gas chromatography followed by mass spectrometry. Selected ion monitoring analyses revealed that the molecular ion and two characteristic fragment ions of pregnenolone, the molecular ion with its isotopes and two characteristic mass fragments of testosterone, and the molecular ion, its isotopes and four characteristic fragment ions of estradiol, were present at the proper retention times and with the correct abundance ratios.

Synthesis and metabolism of vertebrate-type steroids by tissues of insects: a critical evaluation.

This review covers the synthesis and the metabolism of vertebrate-type steroids (progesterone, testosterone, estradiol, corticosteroids) by insect tissues and discusses the significance of the reactions for insect physiology. Biosynthesis of vertebrate-type steroids from cholesterol hitherto has been demonstrated in only two insect species, i.e. the water beetle Acilius sulcatus (Coleoptera) and the tobacco hornworm Manduca sexta (Lepidoptera). In Acilius, steroid synthesis is associated with exosecretion (chemical defense). Nothing, however, is known about a physiological role of the C21 steroid conjugate present in ovaries and eggs of Manduca. No synthesis of vertebrate-type steroids was observed in any other insect investigated to date. Most metabolic conversions of steroids by insects concerned oxidoreduction of oxygen groups (hydroxysteroid dehydrogenase activity) and (polar and apolar) conjugate formation. All important enzymatic steps involved in synthesis and catabolism, as known from studies with tissues of vertebrates, were not, or hardly observed. The conclusion is drawn that typical vertebrate-type (C21, C19 and C18) steroids probably do not act as physiologically active substances in insects.

Aromatase activity in brain and pituitary of immature and mature Atlantic salmon (Salmo salar L.) parr.

Brain homogenates from Atlantic salmon parr converted tritiated androstenedione to testosterone, estrone, and 17 beta-estradiol. The formation of estrogens was markedly higher in homogenates of whole brains from mature parr males than from immature parr males. The highest estrogen synthesis was found in the telencephalon and diencephalon. In both of these parts the estrogen production was higher in mature males than in immature males. There was no difference in estrogen formation by pituitaries from immature female and immature male parr, whereas the formation of estrogens and testosterone was higher in the mature male parr.