Yves-Alain Fontaine

Stimulation of gonadotropin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol

In freshwater or seawater female silver eel, the release of gonadotropin (GTH) accumulated in the pituitary under estradiol (E2) influence could be stimulated by a conjugated treatment with a mammalian gonadoliberin agonist (GnRH-A = des-Gly10, (D-Ala6)-LH-RH ethylamide) and a blocker of dopamine receptor (pimozide). Furthermore, despite the GTH release, no reduction or even a significant increase in pituitary GTH levels were noted, indicating a stimulation of GTH synthesis. In consequence of the endogenous GTH release, a stimulation of ovarian development was induced, as demonstrated by the gonadosomatic index and histological study. Similar results were obtained with a combined treatment with GnRH-A and an inhibitor of catecholamine synthesis (L-alpha-methyl-3,4-dihydroxyphenylalanine). In contrast, no effect was produced by GnRH-A, pimozide, or L-alpha-methyl-DOPA, given alone. The results suggest that a double neuroendocrine mechanism (a lack of GnRH production and a dopaminergic inhibition of GnRH action) is involved in the prepubertal blockage of eel gonadotropic function before the reproductive migration.

Chromatographic and immunological evidence for mammalian GnRH and chicken GnRH II in eel (Anguilla anguilla) brain and pituitary

Gonadotropin-releasing hormone (GnRH) peptides in the brain and pituitary of the European eel (Anguilla anguilla) were investigated by reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. Two GnRH molecular forms were demonstrated in brain and pituitary extracts. One form eluted in the same position as synthetic mammalian GnRH on HPLC and was recognized by antibodies directed against the NH2 and COOH termini of mammalian GnRH as well as by antibodies to the middle region. The second form eluted in the same position as synthetic chicken GnRH II and was recognized by specific antibodies to this molecule. Salmon GnRH and chicken GnRH I were not detected. The occurrence of mammalian GnRH in teleost fish suggests that this molecular form is more ancient than was previously suspected and arose earlier than in primitive tetrapods, or that it has arisen in the eel through random mutation of salmon GnRH. The lack of salmon GnRH in the eel brain indicates that this molecular form is not common to all teleost species. The finding in eel brain of chicken GnRH II, which has previously been described in species of Mammalia, Aves, Reptilia, Amphibia, Osteichthyes, and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early evolved and conserved form of GnRH.

Esquisse de l'evolution des hormones gonadotropes et thyreotropes des vertébrés☆

Results from comparative endocrinology, from structural studies on mammalian hormones, and classical data of vertebrate phylogeny have been used to outline the evolution of thyrotropic (TSH) and gonadotropic (GTH) hormones. The main hypotheses are as follows. A common ancestral molecule gave rise, by genic duplication, to two subunits α and β. The first hormone (α-β) was gonadotropic but later had both functions (TSH and GTH). Duplication of β then gave rise to β1 (with gonadotropic, LH-type, potentialities) and β2 (with thyrotropic potentialities). Finally the FSH β-type subunit originated later from a duplication of β2. Along these lines, hormonal specializations and diversifications occurred, due to genic modifications and associated structural changes in both α and β subunits.

Development of a heterologous radioimmunoassay for eel (Anguilla anguilla) gonadotropin

A heterologous radioimmunoassay (RIA) for eel gonadotropin (GTH) was developed, using an antiserum to the beta subunit of carp GTH (cGTHbeta). Partially purified eel GTH preparations or crude eel pituitary extracts produced partial cross-reactions: the inhibition curves reached a plateau before complete inhibition of the binding of the tracer and the slopes were lower than those for cGTH. However, the parallelism between the inhibition curves produced by all the eel samples allowed estimation of their GTH activity relative to an eel GTH standard; under these conditions, the RIA exhibited good reproducibility within and between assays. Three different RIA systems for cGTHbeta using different antisera gave similar estimations of immunoreactive GTH (irGTH) content of eel GTH preparations and pituitary extracts. The specificity of teh heterologous RIA for eel GTH was first assessed by comparing RIA and bioassay data: as with carp pituitary extracts, no significant difference was found between immunological and biological estimations of GTH activity for partially purified or crude pituitary extracts from normal female eel. Further validation of the specificity of the RIA for eel GTH was provided by the variation of the eel pituitary irGTH content under different hormonal treatments: a significant increase in female eel pituitary irGTH content was produced by treatment with estradiol-17 beta or with carp pituitary extract, a result in agreement with previous histological reports of Olivereau and collaborators. The heterologous RIA for eel GTH, in view of its sensitivity, reproducibility, and specificity, is a useful tool for physiological studies in the eel.

Positive feedback control by the gonads on gonadotropin (GTH) and gonadoliberin (GnRH) levels in experimentally matured female silver eels,Anguilla anguilla

Treatment of sham-operated female silver eels with carp pituitary extract stimulated ovarian development and induced increases in pituitary gonadotropin (GTH) and gonadoliberin (GnRH) contents. Both effects of carp pituitary extract were abolished in ovariectomized eels, indicating the involvement of the gonads. Endogenous sexual steroids, the secretion of which was increased during sexual maturation, should be responsible for the stimulation of GTH and GnRH levels. Ovariectomy itself had no significant effect on pituitary GTH and GnRH contents, reflecting the fact that, at the silver stage, sexual steroid levels are too low to exert any significant effect on pituitary GTH and GnRH. The positive feedback control exerted by the gonads on GTH and GnRH levels during sexual maturation, in the eel as well as in some other teleosts, would produce an amplification of the pubertal stimulation of the hypothalamo-pituitary-gonadal axis.

Cloning and sequence analysis of the cDNA for the pituitary glycoprotein hormone α-subunit of the European eel

A cDNA library constructed using mRNAs isolated from pituitary glands of estradiol-treated eels was screened with a cDNA fragment for the rat glycoprotein hormone alpha-subunit. Three out of 10,000 cDNA clones were revealed and subcloned in pUC13 for characterization and sequencing. All three had the same nucleotide sequence except for a single, silent change in the coding sequence for one of them, and for the location of the poly(A) tail. Analysis of the deduced amino acid sequence strongly suggests that these cDNA clones encode the precursor for the eel common glycoprotein hormone alpha-subunit. This precursor would therefore consist of a 93 amino acid apoprotein preceded by a 24 amino acid long signal peptide. Alignment with glycoprotein hormone alpha-subunits from fish and mammals reveals high homology, ranging from 60 to 90%. Particularly, the ten cysteines and the two putative N-linked glycosylation sites were at the same position. Comparison between fish and mammals shows also that two regions are highly conserved, comprising about half of the protein length. This high conservation rate through evolution argues for the importance of these regions in the conservation of biological properties of the alpha-subunits. In contrast, other regions are highly variable and could be responsible for the immunological specificity. Northern blot analysis of pituitary RNA from control and estradiol-treated eels showed that estradiol treatment strongly increases the pituitary content of mRNA encoding the glycoprotein hormone alpha-subunit.

Relations immunologiques entre les hormones glycoprotéiques hypophysaires de poissons et de Mammifères ainsi qu'entre leurs sous-unités α et β☆

Abstract Immunological relationships among pituitary glycoproteic hormones and their subunits from fishes and mammals were studied with antisera (IS) raised in rabbits against carp gonadotropin (c-GTH), its α subunit (c-GTH α), and its β subunit (c-GTH β). Properties of the following preparations were compared: c-GTH and its subunits; gonadotropins (GTH) from two other Teleosts (indian catfish and eel), and a Chondrostean (sturgeon); and mammalian hormones (bovine LH and TSH, ovine FSH) and their subunits. In the α radioimmunoassays (RIAs) (IS c-GTH α, [ 125 I]c-GTH α) or (IS c-GTH, [ 125 I]c-GTH α) only the GTH from Cypriniform Teleosts (carp and indian catfish) cross-reacted: thus these α RIAs showed a high zoological specificity. However a weak immunological relatedness between c-GTH α and mammalian α subunits (b-LH α, b-TSH α, o-FSH α) could be shown in binding studies; in contrast, even at high concentration, the IS c-GTH α did not bind mammalian β subunits. The β RIA (IS c-GTH β, [ 125 I]c-GTH β) exhibited a much weaker zoological specificity: not only all fish GTHs studied but also b-LH and b-LH β were able to compete with [ 125 I]c-GTH β; furthermore, the degree of cross-reactivity (reflected by the slopes of the regressions) followed phylogenetic relationships. No cross-reaction was observed with the other mammalian preparations. The c-GTH RIA (IS c-GTH, [ 125 I]c-GTH) showed an intermediate zoological specificity. Binding studies carried out with 125 I-labeled mammalian preparations confirmed these data: b-LH and b-LH β were highly bound by IS c-GTH and IS c-GTH β whereas very low or no binding was observed with o-FSH or o-FSH β. A weak binding of b-TSH and b-TSH β by IS c-GTH and IS c-GTH β indicated a relationship between mammalian TSH β and TSH β or GTH β itself from the carp. Even at high concentration the IS c-GTH β did not bind mammalian α subunits. Thus the comparison of antigenic determinants gives additional evidence for homology among fish and mammal pituitary glycoproteins, indicating evolutionary relationship between the subunits of the same type (α or β). In the case of the β subunits, c-GTH β appears more closely related to mammalian LH β than to FSH β. Finally, due to the relative lack of immunological species specificity of the IS c-GTH β, the β RIA may be a useful tool to assay specifically GTHs from other species of fish.

Mise en évidence et propriétés des sites de liaison spécifique pour la gonadotropine de carpe dans des préparations membranaires d'ovaire d'Anguille (Anguilla anguilla L.)

Abstract Membrane preparations from silver eel ovary bind specifically labeled carp gonadotropin (cGTH 125 I); similar preparations from kidney, liver, brain, and muscle exhibit either no or negligible specific binding. cGTH and partially purified eel pituitary extract, but neither hCG nor bTSH, inhibit cGTH 125 I specific binding in a dose-dependent way. However, a high membrane concentration can bind hCG 125 I and this binding is partly inhibited by excess hCG, thus suggesting the existence of a relatively small number of sites able to recognize this hormone. Studies of either cGTH inhibition of cGTH 125 I specific binding or cGTH 125 I dose-dependent binding have permitted, according to Scatchard, the binding parameters to be determined. Specific binding seems to have two components: one with low affinity and high capacity, and the other with high affinity ( K d = 1.2 10 −10 M ) and low capacity ( N = 0.2 fmol/mg ovary).

Influence de la température sur l'adényl cyclase ovarienne et sa stimulation hormonale. Étude comparée chez le rat et un poisson téléostéen (Carassius auratus)

Summary Basal adenyl cyclase (AC), as measured in homogenates of rat or goldfish ovary, with an ATP concentration between one and two apparent Km, increases from 5 to 37°C. Arrhenius curves show a break at about 26°C. Below this temperature the apparent Q10 is not different in the two species; above 26°C it is lower in goldfish than in rat. The AC activation by NaF increases with temperature. The maximal stimulation produced by FSH, in the rat, is not different at 10, 20 and 37°C; in the goldfish, the maximal stimulation produced by c-GTH is lower at 37°C than at 20 or 10°C. FSH acts mainly by increasing the Vmax of AC but also lowers its Km; these effects do not significantly vary with temperature and the same is true for the c-GTH-induced action on the Km. On the contrary, the low positive c-GTH action on the Vmax, which is observed at either 10 or 20°C, disappears at 37°C. The apparent affinity of receptor-AC systems for the hormone varies with temperature. It increases in the rat ovary (for FSH) from 10 to 37°C, whereas it gets through a maximum at 20°C in the goldfish ovary (for c-GTH). Hormonal stimulation of the ovarian AC of the goldfish, a poikilotherm, and of the ovarian AC of the rat, a homeotherm, do not differentiate from each other by a lesser efficiency in the rat at low temperature (10, 20°C) but by a reduced efficiency in the goldfish at a high temperature (37°C).

Existence d'une activité protéine kinase stimulable par le 3′,5′ — adénosine monophosphate cyclique dans le tissu adipeux d'un poisson téléostéen, la truite (Salmo gairdnerii R.)

Summary A cAMP sensitive protein kinase activity was demonstrated in the soluble extract of adipose tissue from a teleost fish, the rainbow trout ( Salmo gairdnerii ). Two enzymes with different K m (0.2 and 1.6.10 −6 M) for ATP were apparently present; cAMP doubled the total V max without extensive effects on the K m s; the apparent K a of the enzymatic preparation for cAMP was 0.05 × 10 −6 M; cGMP was about 150 times less active than cAMP. Cyclic AMP and protein kinase metabolisms may therefore be targets for a possible hormonal regulation of the activity of fish adipose tissue. However no significant difference in adipose tissue protein kinase activity was observed between two groups of trouts which differed by the relative importance of the adipose deposits. Moreover theophylline, which does not increase lipolysis when incubated with teleost adipose tissue, clearly stimulated the protein kinase activity in a homogenate of this tissue.