E. Burzawa-Gerard

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.

Effects of 17β-estradiol and carp gonadotropin on vitellogenesis in normal and hypophysectomized European silver female eel (Anguilla anguilla L.) employing a homologous radioimmunoassay for vitellogenin

The European silver eels are at the early stages of vitellogenesis before the marine reproductive migration. Vitellogenesis was induced by 17 beta-estradiol (E2) alone and by a purified carp gonadotropin (cGTH). We studied and compared their effects on plasma vitellogenin (Vg) levels and ovarian yolk contents in female normal (N) and hypophysectomized (H) eels for both treatments. To this purpose an homologous radioimmunoassay (RIA) was established. Eel Vg was purified to homogeneity on 0.1% SDS-Electrophoresis. Native Vg has a molecular weight of 340 +/- 15 kilodalton (kDa) and was partially separated into subunits. The RIA was established with a sensitivity of 1.1 ng and was specific for eel Vg. In control (N and H) silver eels, plasma Vg levels were 0.04 +/- 0.02 microgram/ml and unchanged throughout the experiment. Similarly, yolk was indetectable in control ovarian extracts. E2 treatment increased plasma Vg levels proportionally with time to 783.4 +/- 130.7 micrograms/ml in N eels. The same profile was seen in H eels but terminally the mean value was 36.7 times lower than in N eels (P less than 0.01). Yolk at 0.005 microgram/g in N eels was indetectable in H eels. cGTH treatment gave a biphasic kinetic change: plasma Vg increased within 12 days, peaked at 93.6 +/- 13.0 micrograms/ml at 20 to 24 days, and stabilized to decrease at 40.2 +/- 7.5 micrograms/ml. The gonadosomatic index (GSI) increased alongside the yolk content (980.4 +/- 153.1 micrograms Vg/g). The kinetic profile for H eels was different: a peak was not apparent, rather there was a delayed increase, and at 67 days levels were still 8.23 times lower than in N eels (P less than 0.01). The GSI increased as the yolk content to 202.7 +/- 64.8 micrograms Vg/g ovary showing an ovarian incorporation of Vg in H eels.

L'Hormone gonadotrope hypophysaire d'un poisson chondrostéen, l'esturgeon (Acipenser stellatus pall.) I. Purification

Abstract Pituitary glands of sturgeon ( Acipenser stellatus ) have been extracted and fractionated by alcoholic percolation, gel filtration on Sephadex G 100, and chromatography on DEAE-C. Biological activity was assayed in vivo on frog spermiation and in vitro on the maturation of oocytes from amphibians and sturgeon. Only one gonadotropic fraction—adsorbed on DEAE-C in glycinate buffer pH 9.4, 0.02 M —was obtained. Its biological activity was about 12 times that of the pituitary acetonic powder both on spermiation and on oocyte maturation. This material (aci-GTH) was slightly more active than carp GTH on frog spermiation.

Synthesis of vitellogenin and its uptake by the ovary in the catfish, Heteropneustes fossilis (bloch) in response to carp gonadotropin and its subunits

Abstract The gonadotropic potency of purified carp gonadotropin (c-GTH) and its α- and β-subunits was tested by evaluating their actions on synthesis of vitellogenin and its uptake by the ovary in the hypophysectomized catfish, Heteropneustes fossilis . Daily treatment with c-GTH (1, 5, 10, and 25 μg/fish or 18, 80, 160, and 470 ng/g body wt) for 11 days evoked a dose-dependent vitellogenic response leading to formation of yolky oocytes; daily treatment with c-GTH subunits (α: 25 μg/fish or 396 ng/g body wt and β: 25 μg/fish or 450 ng/g body wt) for 11 days induced a low but significant formation of vitellogenin, the egg-yolk precursor, in serum without promoting its uptake into oocytes.

Light and electron microscopic identification of gonadotrophic cells in the pituitary gland of the goldfish by means of immunocytochemistry.

Immunocytochemical techniques were used at the light and electron microscopical levels in order to localize and to characterize the gonadotrophs in the goldfish pituitary gland by means of antibodies to carp gonadotrophin (c-GTH) or its subunit (c-GTH beta). At the light microscopical level antibodies to c-GTH reacted weakly with cells located in the rostral pars distalis (RPD) and strongly with cells of the proximal pars distalis (PPD). The labeling was restricted to the proximal pars distalis when antibodies to c-GTH beta were employed. The PAP and colloidal-gold postembedding procedures demonstrated that two cell types of the PPD react with both immune sera. These cells correspond to the so-called globular and nonglobular basophils of the goldfish pituitary. The labeling was located over the small secretory granules and the large globules. A relationship was noted between the intensity of the labeling and the electron density of the globules.

Metabolic studies on eel (Anguilla anguilla L.) hepatocytes in primary culture: effect of 17β-estradiol and growth hormone

Previous studies demonstrated that native and recombinant growth hormone from mammalian and fish species potentiate the estrogenic induction of vitellogenin synthesis by cultured eel hepatocytes. In the present study, the metabolic competence (respiratory activity and estradiol catabolism) of cultured hepatocytes and their functional capacity to synthesize a specific protein, vitellogenin, in the presence of estradiol and/or bovine growth hormone was investigated. In addition, we examined the possible role of insulin-like growth factors as mediators of growth hormone. Hepatocytes retain a high level of metabolic activity under the primary culture conditions applied. Estradiol has a half life of several hours in the hepatocyte culture, and is metabolized into conjugated forms. Estradiol and/or growth hormone had no effects on respiratory activity of the cultured hepatocytes. Moreover, the estradiol catabolic parameters were not affected by growth hormone. Finally, human and trout recombinant insulin-like growth factors do not potentiate vitellogenin synthesis induced by estradiol.

L'hormone gonadotrope hypophysaire d'un poisson chondrostéen, l'esturgeon (Acipenser stellatus Pall.). II. Propriétés biochimiques☆

Abstract Biochemical properties of the aci-GTH prepared by gel filtration and DEAE-C chromatography were studied. This material had a sedimentation coefficient of 2.4–2.8. In analytical electrophoresis acrylamide gel at pH 8.9 it gave two main bands, a and b ( R f = 0.27 and 0.31) both of them active in the frog spermiation test. Preparative acrylamide electrophoresis led to the separation of two active fractions, of which one contained only band a. Amino acid composition of this fraction was determined. Dissociation agents strikingly modified the properties of aci-GTH. Sedimentation rate was decreased to a value of 1.5. Biological activity was less than one-tenth of that of the native hormone. Analytical electrophoresis showed the formation, from band a, of two more acidic bands ( R f = 0.53 and 0.59). These results strongly indicate that aci-GTH was, in these conditions, dissociated into two subunits. Aci-GTH is different from carp GTH in amino acid composition, electrophoretic behavior, and level of the equilibrium between the hormone and its subunits in several experimental conditions.

Pituitary gonadotropic hormone from a chondrostean fish, starred sturgeon (Acipenser stellatus Pall.): III. Polymorphism

Four biologically active fractions of gonadotropic hormone (aci-GTH-A, -B, -C, -D) were isolated and purified from acetonized pituitaries of the starred sturgeon (Acipenser stellatus Pall.). Their separation was achieved by DEAE-cellulose chromatography. Disc-electrophoresis and especially isoelectric focusing in polyacrylamide gel showed that each fraction contained several components. Not less than 15 different components as a whole with isoelectric points ranging from 4.5 to 7.0 could be counted in four aci-GTH preparations. All these components were active in toad oocyte maturation test. Only two of four preparations (aci-GTH-A and -D) were practically free of common components. All aci-GTH preparations were shown to be homogeneous and identical by molecular weight, sedimentation coefficient, sialic acid content, and some immunological properties. N-terminal amino acid analysis revealed tyrosine and leucine in all aci-GTH preparations, with the only exception of aci-GTH-D that contained an additional polypeptide with N-terminal glycine. No differences in the spectra of aci-GTH isoforms were found when pituitary extract, newly purified or 3 years older hormone preparations were submitted to isoelectric focusing.

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.

Effects of carp gonadotropin on ovarian maintenance, maturation, and ovulation in hypophysectomized catfish, Heteropneustes fossilis (Bloch) ☆

Abstract The gonadotropic potency of purified carp gonadotropin (c-GTH) was tested by evaluating ovarian maintenance, maturation, and ovulation during the spawning season in the hypophysectomized gravid catfish, Heteropneustes fossilis . Daily treatment with c-GTH (0.5, 1.0, and 2.5 μg/fish or 10, 20, and 40 ng/g body weight) for 7 days significantly maintained the yolky oocytes in the gravid ovaries. c-GTH also evoked a dose-dependent maturation and ovulation response; catfish injected with 50 or 100 μg (or 0.55 or 1 μg/g body weight) ovulated a large number of ripe eggs. The present data show that c-GTH acts as a potent gonadotropin in the catfish.