Characterization of dopamine D2 receptors in the pituitary of the African catfish, Clarias gariepinus.

Dopamine receptors in the pituitary of the African catfish, Clarias gariepinus, were characterized using [3H]spiperone as radioligand. Specific binding of [3H]spiperone to pituitary membranes reached equilibrium within 60 min of incubation. The binding of the radioligand was tissue specific since the amount of binding was linear with pituitary membrane content in the incubations. In addition, pituitary membranes were observed to bind considerably more [3H]spiperone, compared to membrane preparation of various other tissues. Saturation experiments revealed the presence of a single class of high affinity/low capacity binding sites. The binding characteristics, estimated by Scatchard analysis, were: Kd = 3.2 +/- 0.5 x 10(-9) M and Bmax = 105 +/- 5 fmol/mg protein. Specific binding was displaceable with dopamine and with various specific D2 agonists and antagonists. The nature of displacement curves resembles those observed in studies on mammalian dopamine receptors. Binding experiments with cell fractions, obtained after centrifugation of dispersed pituitary cells over a Percoll density gradient, showed that most [3H]spiperone binding was obtained in an enriched gonadotropic cell fraction. This observation indicates that the receptor characteristics, estimated with the [3H]spiperone assay, are representative for dopamine receptors on the gonadotropic cells.

Characterization of gonadotropin-releasing hormone (GnRH) binding sites in the pituitary and testis of the frog, Rana esculenta.

Frog, Rana esculenta, pituitary and testis gonadotropin-releasing hormone (GnRH) receptors were characterized by using 125I-chicken IIGnRH (cIIGnRH) as radiolabeled ligand. At 4 C equilibrium binding of 125I-cIIGnRH to pituitary homogenates was achieved after 90 min of incubation; binding of 125I-cIIGnRH to testis membrane fractions reached its maximum at 60 min of incubation. Binding of the radioligand was a function of tissue concentration, with a positive correlation over the range 0.5-2 tissue equivalents per tube. One pituitary and one testis per tube were used as standard experimental condition. Incubation of the pituitary homogenate with increasing concentrations of 125I-cIIGnRH indicated saturable binding at radioligand concentrations of 1 nM and above while for the testis membrane preparation saturation was achieved using 5 nM 125I-cIIGnRH. The binding of 125I-cIIGnRH was found to be reversible after addition of the cold analog and the displacement curves could be resolved into one linear component for both tissues. Scatchard analysis suggested the presence of one class of binding sites for both pituitary and testis (Pituitary: Kd = 1.25 +/- 0.14 nM and Bmax = 8.55 +/- 2.72 fmol/mg protein; testis: Kd = 2.23 +/- 0.89 nM and Bmax = 26.48 +/- 7.39 fmol/mg protein). Buserelin displaced the labeled 125I-cIIGnRH with a lower IC50 as compared with cIIGnRH cold standard, while Arg-vasopressin (AVP) was completely ineffective, confirming the specificity of binding.

Catecholestrogens inhibit dopamine methylation in the gonadotrops of the African catfish,Clarias gariepinus.

Isolated gonadotrops of the African catfish,Clarias gariepinus, were incubated with dopamine (DA) and/or catecholestrone and the activity of the enzyme catechol-O-methyltransferase (COMT) was determined by measuring the methylated products. From the apparent Km values for DA and catecholestrone of 0.4-1.3 µM and 17.9-25.2 µM respectively, it was concluded that catecholestrone is a better substrate for the enzyme COMT, compared to DA. Moreover, the methylation of DA is inhibited by comparatively low concentrations of catecholestrone.

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 [(3)H]-pregnenolone and [(3)H]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,20ß-dihydroxy-4-pregnen-3-one, 5ß-pregnane-3α, 17α-diol-20-one, 5ß-pregnane-3α,6α,17α-triol-20-one, 5ß-pregnane-3α,17α,20ß-triol and 5ß-pregnane-3α,6α,17α,20ß-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 20ß-hydroxysteroid dehydrogenase (HSD), 5ß-reductase, 3α-HSD, 6α-hydroxylase and UDP-glucuronosyltransferase. During ovulation the activity of all steroidogenic enzymes, including such key enzymes as 3ß-HSD and 17α-hydroxylase, gradually decreases.Not only 17α,20ß-dihydroxy-4-pregnen-3-one, but also the 5ß-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.

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,20ß-dihydroxy-4-pregnen-3-one and of five 5ß-reduced pregnanes. During germinal vesicle breakdown the GTH concentration remained high, the plasma level of 17α-hydroxyprogesterone tended to increase, and the levels of 5ß-pregnane-3α, 17α-diol-20-one, 5ß-pregnane-3α,17α,20ß-triol and 5ß-pregnane-3α,17α,20α-triol reached a maximum. At pre-ovulation the GTH concentration did not change, and peak levels were reached of 17α,20ß-dihydroxy-4-pregnen-3-one and 5ß-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,20ß-dihydroxy-4-pregnen-3-one and the 5ß-reduced steroids, with the exception of 5ß-pregnane-3α,17α,20α-triol, 5ß-pregnane-3α,6α,17α,20ß-tetrol and 5ß-dihydrotestosterone. The plasma concentrations of androstenedione, estrone, etiocholanolone and 5ß-androstane-3α,17ß-diol showed marginal fluctuations during oocyte maturation and ovulation. Apart from 17α,20ß-dihydroxy-4-pregnen-3-one, the 5ß-reduced pregnanes might be candidates for the function of oocyte maturation inducing hormone inC. gariepinus.

The dopaminergic regulation of gonadotropin-releasing hormone receptor binding in the pituitary of the African catfish, Clarias gariepinus.

In several teleost species, including the African catfish, dopamine acts as an endogenous inhibitor of gonadotropin-releasing hormone (GnRH)-stimulated gonadotropin (GTH) release. The present in vivo study was carried out to investigate whether this inhibitory action of dopamine can be explained by an effect on the pituitary GnRH receptors. To that end, sexually mature female catfish were treated with dopamine and the dopamine antagonist pimozide (PIM), respectively. At different time intervals after injection, the pituitaries were collected, and in a GnRH receptor assay the GnRH-binding parameters were determined. The dopamine treatment affected neither GnRH-binding capacity nor affinity. The PIM treatment resulted in a two-fold increase in pituitary GnRH-binding capacity without affecting binding affinity. The time course of this effect coincided with the potentiating effect of PIM of the GTH-releasing activity of a GnRH analog. It is concluded that the stimulatory effect of PIM on the action of GnRH might, in part, be due to an increased pituitary GnRH-binding capacity. Reversely, these results suggest that the endogenous dopaminergic inhibition of GnRH-stimulated GTH release may be mediated, at least in part, through down-regulation of the pituitary GnRH receptors.

Estrogen-2-hydroxylase in the brain of the male African catfish, Clarias gariepinus.

Estrogen-2-hydroxylase activity, involved in the biosynthesis of catecholestrogens, was localized in the brain of the male African catfish, Clarias gariepinus, by means of a radiometric assay using [2-3H]estradiol as substrate. Fore- and midbrain were divided in 18, 500-microns thick, transverse sections from which small defined areas were punched out and assayed. The estrogen-2-hydroxylase activity was calculated from the release of tritium during hydroxylation, and expressed in femtomole catecholestradiol.milligram-1 tissue.hour-1. The enzyme could be demonstrated throughout the brain. A high activity (greater than 350 fmol) was observed in the telencephalon, in particularly the rostral part and the area ventralis pars dorsalis; in the diencephalon in the preoptic region, including the magnocellular part of the preoptic nucleus and the rostral part of the anterior periventricular nucleus; and in the area tuberalis, including the nucleus lateralis tuberis, the rostral part of the nucleus anterior tuberis, the caudal part of the nucleus posterior periventricularis, and in the nucleus recessus posterioris. Also a high activity was detected in the mesencephalic tectum opticum and the dorsolateral part of the torus semicircularis. The ventral mesencephalon showed a moderate (200-350 fmol) to low (less than 200 fmol) activity, whereas the lowest activity was found in the hindbrain (118 fmol). The significance of the biosynthesis of catecholestrogens in the brain is discussed in light of the negative feedback mechanism of gonadal steroids on gonadotropin release.

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.

Characterization of the receptor for gonadotropin-releasing hormone in the pituitary of the African catfish,Clarias gariepinus.

Receptors for gonadotropin-releasing hormone (GnRH) were characterized using a radioligand prepared from a superactive analog of salmon GnRH (sGnRH), D-Arg(6)-Pro(9)-sGnRH-NEt (sGnRHa). Binding of(125)I-sGnRHa to catfish pituitary membrane fractions reached equilibrium after 2 h incubation at 4°C. Displacement experiments with several GnRH analogs as well as other peptides, demonstrated the specificity of(125)I-sGnRHa binding. Specific binding was enhanced in the presence of the cation chelator ethylene bis (oxyethylenenitrilo) tetra-acetic acid (EGTA), indicating an inhibitory effect of cations on GnRH-receptor binding. The binding of(125)I-sGnRHa to pituitary membranes was found to be saturable at radioligand concentrations of 5 nM and above. A Scatchard analysis of the saturation data suggested the presence of a single class of high-affinity binding sites (Ka=0.901±0.06×10(9)M(-1), Bmax=1678±150 fmol/mg protein). A comparative study on(125)I-sGnRHa binding to pituitary membrane fractions of male and female catfish, indicated that there were no differences in binding affinity and binding capacity between both sexes. The results demonstrate the presence of specific, saturable GnRH receptors in the African catfish pituitary.

Immunocytochemical evidence for peptidergic (GnRH) and dopaminergic innervation of the gonadotropic cells in the pituitary of the African catfish, Clarias gariepinus.

The proximal pars distalis (PPD) of the pituitary of the African catfish, Clarias gariepinus, was studied with immunocytochemical methods at the ultrastructural level. Anti-serum raised against synthetic mammalian luteinizing hormone-releasing hormone (LHRH) was applied on Lowicryl-embedded pituitaries and the antigenic sites were visualized with protein A-gold. In nerve fibers contacting the gonadotropic cells, granulated vesicles with a diameter of 90-120 nm were labeled after this procedure, whereas the glandular cells were not labeled. For the immunocytochemical demonstration of dopaminergic fibers, the preembedding method was performed on Vibratome sections, using highly specific antibodies against dopamine. Immunoreactivity was restricted to fibers containing granulated vesicles with a diameter of approximately 80 nm and terminating on gonadotropic cells. The present data support the results of earlier in vivo and in vitro studies on the catfish pituitary, indicating a dual neuroendocrine regulation of the gonadotropic cells.

The skin of the male African catfish, Clarias gariepinus: a source of steroid glucuronides.

Steroid metabolism in the skin of mature male African catfish, Clarias gariepinus, reared in the laboratory, was studied in vitro by tissue incubations with [3H]pregnenolone, [3H]dehydroepiandrosterone, [3H]17 alpha-hydroxyprogesterone, [3H]androstenedione, [14C]11 beta-hydroxyandrostenedione, and [3H]testosterone as precursors. While pregnenolone was not converted to any other steroid, dehydroepiandrosterone was transformed mainly to 5-androstene-3 beta, 17 beta-diol. The products of 17 alpha-hydroxyprogesterone incubations were 5 beta-pregnane-3 alpha,17 alpha-diol-20-one, 5 beta-pregnane-3 alpha,17 alpha, 20 beta-triol, and 5 beta-pregnan-17 alpha-o1-3,20-dione. The major steroids of androstenedione incubations were etiocholanolone, testosterone, and androsterone. Testosterone was converted mainly to etiocholanolone and androstenedione, and only small quantities of 11 beta-hydroxytestosterone, 11-ketotestosterone, and 11-ketoandrostenedione were the metabolites found in 11 beta-hydroxyandrostenedione incubation. These results demonstrated the presence of the enzymes 5 alpha- and 5 beta-reductases and 3 alpha-, 11 beta-, 17 beta-, and 20 beta-hydroxysteroid dehydrogenases in the skin. From enzymehistochemical results it appeared that the steroid conversions take place in the epithelial cells. Moreover, the presence of UDP-glucose dehydrogenase, an enzyme involved in the synthesis of glucuronic acid, in these cells indicates the possibility of steroid glucuronide formation. Indeed significant amounts of water-soluble steroid conjugates, particularly 5 beta-dihydrotestosterone- and testosterone-glucuronide, were found in the incubations with androstenedione and testosterone, indicating the presence of the UDP-glucuronosyl transferase in the catfish skin. In the light of these results, a role of the skin of African catfish in the production of semiochemicals having pheromonal properties is discussed.

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.

Gonadotropin production and release in female goldfish (Carassius auratus) after administration of pimozide and an LHRH analogue as studied by electron microscopy and radioimmunoassay.

The effect of pimozide and an LHRH-analogue (LHRH-A) on gonadotropic cells of the goldfish pituitary gland were described qualitatively and quantitatively. A scale of four categories was devised to reflect various ultrastructural appearances of the cells. Experimental animals were divided into a control group, a group injected with LHRH-A alone, pimozide alone, and groups receiving these two substances in combination. Fish injected with the single substance were killed 12 h after injection while the groups receiving the combined treatments were killed at 4, 12 and 48 h. Serum levels of gonadotropin measured by radioimmunoassay were used to indicate whether an increase in hormone release had occurred. An immunocytochemical technique, the protein A-gold procedure, assured that the cells studied were gonadotropes. The control group showed variation in the profiles of gonadotropic cells. The single treatment groups showed some increase in secretory inclusions. At 4 h after injection the combined treatment caused a significant increase in hormone granules; at 12 and 48 h there was a gradual decrease in content of secretory products, and an increase in vacuolization. The results indicate that the combined pimozide and LHRH-A treatment stimulated gonadotropin production as well as release.

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.

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.

Gonadotropic hormone-releasing hormone (GnRH) bioactivity in the brain of the immature rainbow trout, Salmo gairdneri: the effect of testosterone.

The effect of exogenous testosterone on the synthesis and release of a bioactive gonadotropic hormone-releasing hormone (GnRH)-like substance has been studied in juvenile male rainbow trout. Long-term testosterone treatment caused an increased gonadotropic hormone (GTH) release and precocious sexual maturity. Tel- and diencephalon extracts from long-term testosterone-treated juvenile rainbow trout showed significantly higher GTH-releasing activity than extracts from control animals when injected in short-term testosterone-pretreated juvenile rainbow trout. It was concluded that long-term testosterone treatment caused an increased synthesis and release of GnRH-like material.

The dopaminergic inhibition of the gonadotropin-releasing hormone-induced gonadotropin release: an in vitro study with fragments and cell suspensions from pituitaries of the African catfish, Clarias gariepinus (Burchell).

Pituitary fragments and pituitary cell suspensions of the African catfish in a perifusion system released gonadotropic hormone (GTH) spontaneously. After a few hours of perifusion a baseline of GTH release was established. The dopamine (DA) agonist apomorphine (APO) caused a decreased in the release from pituitary fragments but not from pituitary cell suspensions. APO abolished the luteinizing hormone-releasing hormone analog (LH-RHa)-induced GTH release from both pituitary fragments and cell suspensions. It was concluded that DA inhibits the GTH release induced by gonadotropin-releasing hormone.

Gonadal steroidogenesis and the possible role of steroid glucuronides as sex pheromones in two species of teleosts.

In general, female zebrafish,Brachydanio rerio, ovulate only in the presence of males. The stimulant must be pheromonal as even male holding water is capable of inducing ovulation. After ovulation the mating phase begins. During this phase the male follows the female and oviposition as well as fertilization takes place. Both the ovulation and the mating are controlled by pheromones synthesized by the gonads. Ovulation can be induced by testicular homogenates. After the lipid material has been extracted from the testicular homogenates, the remaining aqueous phase can still induce ovulation. However, when the aqueous phase is treated with the enzymeß-glucuronidase, it loses the ability to induce ovulation. This is an indication that glucuronides, probably steroid glucuronides, are the compounds responsible.During the mating phase, ovulated female zebrafish become attractive to males. It was found that, after ovulation, ovarian extracts contain the compounds responsible for attracting males. The attractant consists of a mixture of steroid glucuronides.After incubation of the gonads with(3)H-precursors seven steroid glucuronides have been identified in the testis and five in the ovary.Under fish culture conditions the African catfish,Clarias gariepinus, can produce postivitellogenic oocytes throughout the year. However, in capitivity neither males nor females spawn. In female catfish maturation and ovulation can be induced by treatment with gonadotropins. It might be possible that, analogous to the zebrafish, some reproductive processes in the catfish have to be induced by pheromones. It has been demonstrated that pheromonal compounds released by the seminal vesicles are involved in the attraction of female conspecifics. The steroid glucuronide synthesizing capability of the testes and the seminal vesicles of the male catfish are examined, as well as that of the ovary before and after ovulation of the female catfish. Both testes and seminal vesicles appear to be capable of steroid biosynthesis but only the latter synthesizes steroid glucuronides. Six of these conjugates have been isolated and identified. In the female catfish the ovaries are capable of synthesizing seven steroid glucuronides, but only after ovulation.

Effects of natural androgens and corticosteroids on gonad differentiation in the rainbow trout, Salmo gairdneri.

Rainbow trout fry were treated with equimolar quantities of cortisol, cortisone, androstenedione, and 11 beta-hydroxyandrostenedione, added to the aquarium water during a 4-week period. All four steroids inhibited ovarian growth. Androstenedione did not influence gonadal sex differentiation; the other steroids pushed the sex ratio in the male direction. However, a near to normal sex ratio was observed 300 days after the 11 beta-hydroxyandrostenedione treatment. When added to the food in two different doses during 8 weeks, 11 beta-hydroxyandrostenedione had a pronounced masculinizing effect, and androstenedione did not modify the gonads at all. It is suggested that the two exogenous corticosteroids were probably converted into 11-oxygenated androstenedione derivatives, and that these 11-oxygenated androstenedione derivatives are particularly important in sustaining the differentiation and early development of the testes in rainbow trout. Testosterone does not seem to be indispensable for these processes, because two different doses of testosterone-blocking cyproterone acetate, added to the food for 8 weeks, failed to affect early gonad development.

Isolation of gonadotrops from the pituitary of the African catfish, Clarias lazera. Morphological and physiological characterization of the purified cells.

Dispersed pituitary cells from male African catfish, Clarias lazera, were fractionated in a density gradient of Percoll. Five fractions were isolated, consisting of about 6, 19, 39, 95 and 83% gonadotrops, respectively. The gonadotrops were identified by their ultrastructural characteristics, by immunocytochemistry, and by measuring their hormone content. After one day in culture, in each fraction the secretion of gonadotropin could be stimulated by a luteinizing hormone-releasing hormone analogue, indicating that the cells had retained their functional integrity. Since the regulatory mechanisms of different cell types from the pituitary have some similarity, purification of the gonadotrops provides a model to study the regulation of gonadotropin secretion.