W.G.E.J. Schoonen

A dual function of alcohol dehydrogenase in Drosophila

Alcohol dehydrogenase (ADH) of Drosophila not only catalyzes the oxidation of ethanol to acetaldehyde, but additionally catalyzes the conversion of this highly toxic product into acetate. This mechanism is demonstrated by using three different methods. After electrophoresis the oxidation of acetaldehyde is shown in an NAD-dependent reaction revealing bands coinciding with the bands likewise produced by a conventional ADH staining procedure. In spectrophotometric measurements acetaldehyde is oxidized in an NAD-dependent reaction. This activity is effectively inhibited by pyrazole, as specific inhibitor of ADH. By means of gas chromatographic analysis a quick generation of acetate from ethanol could be demonstrated. Our conclusion is further supported by experimental results obtained with either purified ADHF enzyme or genotypes with or without ADH, aldehyde-oxidase, pyridoxal-oxidase and xanthine-dehydrogenase activity. These results are discussed in relation to ethanol tolerance in the living organism in particular with respect to differences found between ADH in Drosophila melanogaster and D. simulans, and in relation to the possible implications for the selective forces acting on ADH-polymorphism.

The chemical nature of sex attracting pheromones from the seminal vesicle of the African catfish, Clarias gariepinus

Abstract In attraction tests, ovulated female African catfish, Clarias gariepinus , were attracted by the administration of 2.0 ml/l seminal vesicle fluid to the aquarium water. Lower doses (0.1 and 0.5 ml/l) were ineffective, and a high concentration (8.0 ml/l) seemed to have a repulsive effect. After fractionation of the fluid, an isolated steroid conjugate fraction appeared to contain the attractant. No other stimuli beyond this fraction were required to attract groups of female catfish after ovulation. Other constituents of the fluid such as polysaccharides, proteins, phospholipids and steroids failed to attract the females. GCMS analysis of the steroid conjugate fraction revealed the presence of eight different steroid glucuronides. After removal of these steroid glucuronides, the steroid conjugate fraction lost its attractive effect. A mixture of seven available synthetic steroid glucuronides was composed according to the quantitative results of the GCMS analysis. This mixture appeared to have a powerful dose-dependent attractive effect on ovulated females. It is concluded that a combination of various steroid glucuronides, present in seminal vesicle fluid, functions as a sex attractant, bringing male and female African catfish together shortly before spawning.

Dual function of the alcohol dehydrogenase of Drosophila melanogaster: ethanol and acetaldehyde oxidation by two allozymes ADH-71k and ADH-F.

SummaryUntil recently the alcohol dehydrogenase of Drosophila melanogaster was thought to act only in the first step of primary alcohol oxidation, producing an aldehyde. Instead, acetic acid is the main product of a two-step process.A rapid procedure was developed for the isolation and purification of two allozymes. The thermostability of the purified enzymes was found to be very different, t1/2 at 35°C, being 45 min and 130 min for ADH-F and ADH-71k respectively.The kinetic parameters of ethanol oxidation by the two purified allozymes were determined within physiological substrate and coenzyme ranges. The use of artificial electron acceptors has a notable influence on the ethanol oxidation: the apparent Michaelis constants increase: the oxidation rate with ADH-71k increases, whereas it decreases with ADH-F.Purified ADH is shown to be able to catalyze the oxidation of acetaldehyde solely in the presence of NAD+, and PMS and MTT as artificial electron acceptors.From the kinetic data the relative in vivo oxidation rates of ethanol by both ADH allozymes were calculated. ADH-F turned out to be somewhat less effective (30%–40%) than ADH-71k. The physiological consequences of these differences are discussed.

Quantitative analysis of steroids and steroid glucuronides in the seminal vesicle fluid of feral spawning and feral and cultivated nonspawning African catfish, Clarias gariepinus.

The seminal vesicles of African catfish, Clarias gariepinus, can produce at least seven different steroid glucuronides, especially during the breeding season. In the present experiments quantification of the seven compounds and their free steroids was carried out by means of gas chromatography-mass spectrometry in particular by selected ion monitoring, using seminal vesicle fluid of African catfish collected in the Hula nature reserve (Israel) and from a nearby fish farm. Some of the feral fish were in spawning condition; the others and the pond fish were in nonspawning condition. Compared to the nonspawning groups, in the feral spawning animals a significantly higher level was found especially of 5 beta-pregnane- 3 alpha,17 alpha-diol-20-one-glucuronide, but also of 5 beta-androstane-3 alpha,17 beta-diol-glucuronide, 5 beta-androstane-3 alpha,17 beta-diol-11-one-glucuronide, and 5 beta-pregnane-3 alpha,17 alpha-diol-20-one. The feral and pond nonspawning groups mainly differed in a signficantly higher concentration of 5 beta-pregnane-3 alpha,17 alpha-diol-20-one-glucuronide in the feral nonspawning fish. For the levels of etiocholanolone, testosterone, 5 beta-androstane-3 beta,17 beta-diol, and their glucuronides no significant differences were observed between the three groups, and 5 beta-dihydrotestosterone and its glucuronide could not be detected in the seminal vesicle fluid of any of the animals. The increase in the concentration of three 5 beta-reduced steroid glucuronides may point to a pheromonal function of one or more of these compounds, as demonstrated for the entire steroid glucuronide fraction of the seminal vesicle fluid. Experiments are being carried out to verify this hypothesis.

5β-Pregnane-3α,6α,17α,20β-tetrol and 5β-pregnane-3α,6α,17α-triol-20-one: Steroids of ovarian origin in the African catfish, Clarias gariepinus, during oocyte maturation

Abstract At the stage of oocyte maturation, three very polar steroids were demonstrated by in vitro incubations with tritiated pregnenolone in the ovaries of the African catfish, Clarias gariepinus . Two of these compounds could be identified by chromatography, derivatization, and oxidation as 5β-pregnane-3α,6α,17α,20β-tetrol and 5β-pregnane-3α,6α,17α-triol-20-one. Blood plasma analysis by means of gas chromatography followed by mass spectrometry confirmed the presence of these steroids with selected ion monitoring. The occurrence of the five most characteristic ions of each steroid was demonstrated at the proper retention times and with the correct abundance ratios. These very polar steroids, which were identified in vitro and in vivo , might have a function in maturation and ovulation induction.

Quantitative studies of steroid bioconversions in the seminal vesicles of spawning male African catfish, Clarias gariepinus (Burchell), under natural conditions, and of non-spawning catfish under natural and fish farm conditions

1. Steroid bioconversions in the seminal vesicles of Clarias gariepinus were studied quantitatively in vitro by tissue incubations with [3H]pregnenolone, [3H]androstenedione and [14C]11 beta-hydroxyandrostenedione, respectively. 2. Spawning and non-spawning catfish, collected in the Hula nature reserve in northern Israel during the spawning period, and non-spawning animals, collected from a fish pond in the same region during the same period, were studied. 3.Spawning animals showed a significantly higher production of 17 alpha-hydroxyprogesterone, 5 beta-pregnan-17 alpha-ol-3,20-dione and 5 beta-reduced androgens than non-spawning feral and pond catfish, as a result of a significantly increased contribution of the enzymes 5 beta-reductase and 3 alpha-hydroxysteroid dehydrogenase (HSD). 4. In spawning catfish the concentration of gonadotropin in blood plasma were also significantly higher than in the plasma of non-spawning feral and pond catfish. This increase in gonadotropin level might have induced the rise in enzyme activity of 5 beta-reductase and 3 alpha-HSD. 5. It is concluded that the absence of a shift in steroidogenesis towards the production of 5 beta-reduced steroids may be among the factors preventing spontaneous spawning in male African catfish under husbandry conditions.

Steroidogenesis during induced oocyte maturation and ovulation in the African catfish,Clarias gariepinus.

Changes in ovarian steroidogenesis accompanying oocyte maturation and ovulation were studied in the African catfish,Clarias gariepinus. Laboratory-reared females with postvitellogenic ovaries were treated with pimozide and LHRH-analogue. The plasma gonadotropin levels were determined by means of a homologous radioimmunoassay, the condition of the ovaries was studied by histological examination of the follicles, and the steroidogenetic capacity of the ovaries was analyzed byin vitro incubation of tissue fragments for 3 h with [3H]-pregnenolone and [3H]androstenedione as precursors. Data were collected at regular intervals between 0 and 16 h after pimozide-LHRH analogue administration.Until 4 h after the beginning of the experiments the plasma gonadotropin levels did not rise above 25 ng/ml, the ovaries remained in the stage of postvitellogenesis, and testosterone was the main end product of steroidogenesis. Four hours later the gonadotropin concentration in the blood had risen to more than 150 ng/ml, and the ovaries had entered the stage of germinal vesicle migration. At the same time steroidogenesis shifted towards the production of 17α,20β-dihydroxy-4-pregnen-3-one, 5β-pregnane-3α, 17α-diol-20-one, 5β-pregnane-3α,6α,17α-triol-20-one, 5β-pregnane-3α,17α,20β-triol and 5β-pregnane-3α,6α,17α,20β-tetrol. During the subsequent stage of germinal vesicle breakdown the plasma gonadotropin level remained high, and the synthesis of the C21-steroids showed a further increase. Simultaneously, the production of some C19-steroid glucuronides was enhanced. The preovulation and especially the postovulation stages were accompanied by a gradual decrease in steroidogenic capacity of the ovaries, even though the plasma gonadotropin level remained high. It is concluded that the prematuration surge of gonadotropin influences the activity of enzymes involved in steroidogenesis, leading to a reduced C17,20-lyase and to an augmented activity of the enzymes 20β-hydroxysteroid dehydrogenase (HSD), 5β-reductase, 3α-HSD, 6α-hydroxylase and UDP-glucuronosyltransferase. During ovulation the activity of all steroidogenic enzymes, including such key enzymes as 3β-HSD and 17α-hydroxylase, gradually decreases.Not only 17α,20β-dihydroxy-4-pregnen-3-one, but also the 5β-reduced pregnanes may be involved in inducing oocyte maturation and/or ovulation. The very polar triol and tetrol products may function, together with the steroid glucuronides as sex pheromones.

Steroids and steroid glucuronides in the ovarian fluid of the African catfish,Clarias gariepinus, between ovulation and oviposition

As part of a series of experiments concerning a possible pheromonal function of steroids and steroid glucuronides excreted by the sex organs of the African catfish,Clarias gariepinus, qualitative and quantitative studies, using GCMS, were carried out to examine the presence of the steroids, that can be synthesized by the ovary during oocyte maturation and ovulation, and of the corresponding steroid glucuronides, in the fluid surrounding the eggs in the ovarian cavity shortly after ovulation.Full mass spectra were obtained of 5β-pregnane-3α,17α-diol-20-one, 5β-pregnane-3α,17α,20α-triol, 5β-pregnane-3α,6α,17α-triol-20-one, 5β-pregnane-3α,6α,17α,20β-tetrol, 5β-androstane-3α,17β-diol and 5β-androstane-3α,17β-diol-11-one. After selected ion monitoring the following steroids could be detected by the presence of at least two characteristic ions at the expected retention time: 5β-pregnane-3α, 17α,20β-triol, etiocholanolone, 5β-dihydrotestosterone, 5β-androstane-3α,11β-diol-17-one, testosterone and estradiol. After treatment with β-glucuronidase the following steroids could be determined in a similar way: 5β-pregnane-3α,17α-diol-20-one, 5β-pregnane-3α,17α,20α-triol, 5β-pregnane-3α,17α,20β-triol, 5β-pregnane-3α,6α,17α-triol-20-one, 5β-pregnane,3α,6α,17α,20β-tetrol, 5β-androstane-3α,17β-diol, etiocholanolone, 5β-dihydrotestosterone, testosterone and estradiol.The free steroids 5β-pregnane-3α,6α,17α,20β-tetrol and 5β-pregnane-3α,6α,17α-triol-20-one and the steroid glucuronides of testosterone, 5β-dihydrotestosterone and estradiol appeared to be the most abundant of these compounds. The results indicate that very polar steroids and steroid glucuronides, synthesized in the ovary, can be excreted via the ovarian fluid shortly before and during oviposition, and possibly function as sex attractants, inducing reproductive behaviour in male conspecifics.

Plasma profiles of fourteen ovarian steroids before, during and after ovulation in African catfish,Clarias gariepinus, determined by gas chromatography and mass spectrometry

The plasma concentrations of fourteen ovarian steroids were measured in postvitellogenic African catfish,Clarias gariepinus, which had been injected with pimozide and LHRHa. Postvitellogenesis persisted for at least four hours after pimozide and LHRHa administration. During this stage a limited rise in the plasma gonadotropin (GTH) level was accompanied by an increase in the testosterone concentration. The estradiol level was high and remained high except for a passing drop during the stage of germinal vesicle migration. At the stage of germinal vesicle migration a strong increase in the plasma GTH level coincided with a maximum in the testosterone concentration and a concomitant increase in the levels of 17α,20β-dihydroxy-4-pregnen-3-one and of five 5β-reduced pregnanes. During germinal vesicle breakdown the GTH concentration remained high, the plasma level of 17α-hydroxyprogesterone tended to increase, and the levels of 5β-pregnane-3α, 17α-diol-20-one, 5β-pregnane-3α,17α,20β-triol and 5β-pregnane-3α,17α,20α-triol reached a maximum. At pre-ovulation the GTH concentration did not change, and peak levels were reached of 17α,20β-dihydroxy-4-pregnen-3-one and 5β-pregnane-3α,6α,17α-triol-20-one. Shortly after ovulation the GTH concentration slightly decreased together with a sharp decline in the concentrations of 17α,20β-dihydroxy-4-pregnen-3-one and the 5β-reduced steroids, with the exception of 5β-pregnane-3α,17α,20α-triol, 5β-pregnane-3α,6α,17α,20β-tetrol and 5β-dihydrotestosterone. The plasma concentrations of androstenedione, estrone, etiocholanolone and 5β-androstane-3α,17β-diol showed marginal fluctuations during oocyte maturation and ovulation. Apart from 17α,20β-dihydroxy-4-pregnen-3-one, the 5β-reduced pregnanes might be candidates for the function of oocyte maturation inducing hormone inC. gariepinus.

Evidence for a multiple function of the alcohol dehydrogenase allozyme ADH71k of Drosophila melanogaster

Alcohol dehydrogenase of Drosophila melanogaster catalyzes the oxidation of many primary and secondary alcohols. We show that sarcosine, choline and dihydroorotate are substrates of ADH in vitro. The first two substrates are regular substrates of the choline shunt, and the latter of the de novo pyrimidine synthesis. Differences in oxidative ability towards sarcosine and dihydroorotate between two ADH allozymes, ADH71k and ADHF, are observed. The catalytic activity of ADH71k towards sarcosine and dihydroorotate might be responsible for its allelic fixation in Notch8 mutant stocks, in which Notch females have a decreased level of the regular enzymes for these substrates. Their oxidation by ADH71k might act as a bypass, which restores at least part of the decreased activity of enzymes encoded by the Notch locus.