Cloning and expression of a functional estrogen receptor-alpha from African catfish (Clarias gariepinus) pituitary.

An African catfish (Clarias gariepinus) estrogen receptor-alpha (cfERalpha) cDNA fragment was amplified by RT-PCR, in combination with a modified 3'-RACE procedure, on total RNA extracted from pituitary. This cDNA fragment was used to screen an African catfish pituitary cDNA library. A clone was obtained that contained an open-reading frame coding for a 620 amino acid cfERalpha protein with a deduced molecular mass of 68.1 kDa. In addition, a partial African catfish estrogen receptor-beta (cfERbeta) cDNA fragment was amplified by RT-PCR on total RNA extracted from testis. Neighbor-joining analysis was used to infer a phylogenetic classification for cfERalpha and cfERbeta. The tree obtained indicated that there are two major clusters of vertebrate ERs: ERalpha and ERbeta. Within each cluster, teleost and tetrapod ER sister clades could be distinguished. The cfERalpha clustered with other teleost ERalphas, whereas cfERbeta clustered with other teleost ERbetas. The ligand-induced transcriptional activity of cfERalpha was demonstrated in a transient gene expression assay using cells in which an acute estrogenic response was created by co-transfecting cultures with recombinant cfERalpha cDNA expression vector constructs in the presence of an estrogen-dependent reporter plasmid. Real-time, quantitative PCR revealed that cfERalpha transcripts were most abundantly expressed in pituitary, while in all other tIssues tested the relative cfERalpha mRNA levels were less than approximately 5% of the level obtained in pituitary. Moreover, we found that, during pubertal development, the relative cfERalpha mRNA levels gradually increased in African catfish pituitary.

Both recombinant African catfish LH and FSH are able to activate the African catfish FSH receptor.

LH and FSH are heterodimeric glycoprotein hormones, composed of a common alpha-subunit non-covalently associated with a hormone-specific beta-subunit. Repeated efforts to isolate catfish FSH (cfFSH) have not been successful and only catfish LH (cfLH) has been purified from catfish pituitaries. Recently, however, we succeeded in cloning the cDNA encoding the putative cfFSHbeta; the cDNAs for the alpha- and beta-subunit of cfLH have been cloned before. Here we report the expression of biologically active cfLH and cfFSH in the soil amoeba, Dictyostelium discoideum. The biological activity of the recombinant hormones was analyzed using cell lines transiently expressing either the cfLH receptor or the cfFSH receptor. Moreover, a primary testis tIssue culture system served to study the steroidogenic potency of the recombinant hormones. Our results demonstrated that Dictyostelium produced biologically active, recombinant catfish gonadotropins, with recombinant cfLH being almost indistinguishable from its native counterpart, purified from pituitaries. Although recombinant cfFSH has significant effects in the bioassays used in this study, the specific function of native cfFSH in the control of reproduction and its expression patterns are not yet understood.

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.

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 5ß-pregnane-3α,17α-diol-20-one, 5ß-pregnane-3α,17α,20α-triol, 5ß-pregnane-3α,6α,17α-triol-20-one, 5ß-pregnane-3α,6α,17α,20ß-tetrol, 5ß-androstane-3α,17ß-diol and 5ß-androstane-3α,17ß-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: 5ß-pregnane-3α, 17α,20ß-triol, etiocholanolone, 5ß-dihydrotestosterone, 5ß-androstane-3α,11ß-diol-17-one, testosterone and estradiol. After treatment with ß-glucuronidase the following steroids could be determined in a similar way: 5ß-pregnane-3α,17α-diol-20-one, 5ß-pregnane-3α,17α,20α-triol, 5ß-pregnane-3α,17α,20ß-triol, 5ß-pregnane-3α,6α,17α-triol-20-one, 5ß-pregnane,3α,6α,17α,20ß-tetrol, 5ß-androstane-3α,17ß-diol, etiocholanolone, 5ß-dihydrotestosterone, testosterone and estradiol.The free steroids 5ß-pregnane-3α,6α,17α,20ß-tetrol and 5ß-pregnane-3α,6α,17α-triol-20-one and the steroid glucuronides of testosterone, 5ß-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.

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 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.

Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus).

A putative FSH receptor (FSH-R) cDNA was cloned from African catfish testis. Alignment of the deduced amino acid sequence with other (putative) glycoprotein hormone receptors and analysis of the African catfish gene indicated that the cloned receptor belonged to the FSH receptor subfamily. Catfish FSH-R (cfFSH-R) mRNA expression was observed in testis and ovary; abundant mRNA expression was also detected in seminal vesicles. The isolated cDNA encoded a functional receptor since its transient expression in human embryonic kidney (HEK-T) 293 cells resulted in ligand-dependent cAMP production. Remarkably, African catfish LH (cfLH; the catfish FSH-like gonadotropin has not been purified yet) had the highest potency in this system. From the other ligands tested, only human recombinant FSH (hrFSH) was active, showing a fourfold lower potency than cfLH, while hCG and human TSH (hTSH) were inactive. Human CG (as well as cfLH, hrFSH, eCG, but not hTSH) stimulated testicular androgen secretion in vitro but seemed to be unable to bind to the cfFSH-R. However, it was known that hCG is biologically active in African catfish (e.g., induction of ovulation). This indicated that an LH receptor is also expressed in African catfish testis. We conclude that we have cloned a cDNA encoding a functional FSH-R from African catfish testis. The cfFSH-R appears to be less discriminatory for its species-specific LH than its avian and mammalian counterparts.