Kunimasa Suzuki

Isolation and characterization of two distinct gonadotropins from chum salmon pituitary glands

Two distinct gonadotropins, GTH I and GTH II, were extracted with 35% ethanol-10% ammonium acetate, pH 6.1, from female chum salmon pituitary glands, and purified by ion-exchange chromatography on DE-52 and CM-Sephadex C-25 by stepwise elution, and gel filtration on Sephadex G-75. Gonadotropic activities of these preparations were demonstrated in vivo by stimulation of gonad growth in juvenile rainbow trout, and in vitro by enhancement of estradiol-17 beta production by amago salmon ovarian follicles. Molecular weights were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 50,000 and 36,000 for GTH I and GTH II, respectively. Both gonadotropins are glycoproteins composed of two distinct subunits with N-terminal amino acid residues of Tyr/Gly for GTH I, and Tyr/Ser for GTH II. These results suggest the presence in teleost fish of two chemically distinct gonadotropic glycoproteins.

The duality of teleost gonadotropins.

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.

Steroidogenic activities of two distinct salmon gonadotropins

The effects of salmon gonadotropins, GTH I and GTH II, on production of two major steroid hormones in female salmonid reproduction, estradiol-17 beta and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) were compared using amago salmon (Oncorhynchus rhodurus) intact ovarian follicles in vitro. In addition, the production of 17 alpha-hydroxyprogesterone (17 alpha-OHprog) by thecal layers and 17 alpha,20 beta-diOHprog by granulosa layers in response to GTH I and II was examined during oocyte maturation. Both GTHs enhanced estradiol-17 beta production by midvitellogenic ovarian follicles in a dose-dependent manner; there was no significant difference in potency between GTH I and II. In postvitellogenic follicles, GTH II appeared to be more effective in stimulating 17 alpha,20 beta-diOHprog production than GTH I. GTH II was also found to be more potent than GTH I in stimulating 17 alpha-OHprog production by thecal layers and 17 alpha,20 beta-diOHprog production by granulosa layers in the presence of 17 alpha-OHprog. Thus, GTH II appears to differ from GTH I showing a reproductively high specificity for 17 alpha,20 beta-diOHprog production during oocyte maturation.

Properties of common carp gonadotropin I and gonadotropin II

Two gonadotropins, GtH I and GtH II, were extracted with 35% ethanol-10% ammonium acetate, pH 6.1, from female common carp pituitary glands and purified by ion-exchange chromatography on a DE-52 column followed by gel filtration on a Sephadex G-75 column. Molecular weights of GtH I and GtH II as determined by SDS-PAGE were 45,000 and 35,000, respectively. Both GtHs dissociate into two subunits following reduction with beta-mercaptoethanol. These subunits contain different N-terminal amino acids (Tyr and Gly for GtH I; Tyr and Ser for GtH II). GtH I was acid stable and did not dissociate into subunits following treatment with 0.1% trifluoroacetic acid; GtH II readily dissociated into subunits by this treatment. GtH I and GtH II have distinct elution profiles on reverse-phase HPLC. The N-terminal amino acid sequence of the beta-subunit of GtH II was identical to that of common carp maturational GtH described by other workers suggesting that GtH I is a newly identified molecule. This was supported by radioimmunoassay analysis. GtH II and a common carp maturational GtH preparation (F11 cGtH; Peter et al., 1982, J. Interdiscipl. Cycle Res. 13, 229-239) had similar immunological activity in tests with antisera to the beta-subunit of maturational GtH whereas GtH I had low (less than 6%) cross-reactivity. GtH I, GtH II, and F11 cGtH were equipotent in tests with antisera to the alpha-subunit of maturational GtH suggesting these molecules contain a similar alpha-subunit. In vitro bioassays using goldfish revealed that GtH I and GtH II share the same spectrum of biological activities causing stimulation of ovarian and testicular steroidogenesis and induction of oocyte final maturation. The demonstration of two chemically distinct GtHs in common carp is similar to what has been described for chum and coho salmon.

Gonadotropins I and II in juvenile coho salmon

The present study was designed to obtain basic endocrine information on GTH I and GTH II in previtellogenic and prespermatogenic coho salmon (immature). Levels of GTH II in pituitary extracts were 6.5 ± 2.0 and 6.7 ± 2.0 pg/μg pituitary protein in male and female fish, respectively. In contrast, the pituitary content of GTH I was approximately 100-fold higher than GTH II (1.302 ± .22 and 1.173 ± .21 ng/μg pituitary protein in male and female fish, respectively). Plasma levels of GTH II in immature salmon were not detectable by RIA whereas plasma GTH I levels were approximately 0.62 ± 0.12 and 0.78 ± 0.13 ng/ml in male and female fish, respectively. Highly purified coho salmon GTH I and GTH II stimulated testicular testosterone production and ovarian estradiol productionin vitro in a similar manner, though GTH II appeared more potent than GTH I. Therefore, it appears that although the salmon pituitary contains predominantly GTH I prior to puberty, the gonad can respond to both GTH I and GTH II.

Development of salmon GTH I and GTH II radioimmunoassays.

Radioimmunoassays (RIAs) for the measurement of two gonadotropins, GTH I and GTH II, in the plasma and pituitary of salmonid fish were developed using a rabbit antiserum to beta-subunits of chum salmon GTH I and GTH II. Intact GTH I and GTH II were used as standards and radioactive competitors. The displacement curves for plasma of salmonids including chum salmon, amago salmon, and rainbow trout were parallel to chum salmon GTH I and GTH II standards. Parallel displacement curves were obtained for pituitary extracts of chum salmon and amago salmon. The cross-reactivities of growth hormone, prolactin, and proopiomelanocortin (POMC)-related hormones were less than 1% in both RIAs. However, cross-reactivities of GTH I in the GTH II RIA and GTH II in the GTH I RIA were 10 and 12%, respectively. Plasma concentrations of both GTHs from salmonids at various stages of reproductive development were compared. In immature rainbow trout of both sexes (males: average (AV) gonadosomatic index (GSI) = 0.05; females: AV GSI = 0.24), plasma levels of GTH I and GTH II were low (less than 2 ng/ml). During prematurational stages of spermatogenesis and vitellogenesis in rainbow trout (males: AV GSI = 0.43; females: AV GSI = 2.8), the predominant GTH in the pituitary and plasma was GTH I. In contrast, plasma concentrations of GTH II were significantly higher than those of GTH I in postovulatory amago and chum salmon. Similarly, pituitary concentrations of GTH II were significantly higher than those of GTH I in postovulatory and spermiating amago salmon and postovulatory chum salmon.(ABSTRACT TRUNCATED AT 250 WORDS)

Salmonid pituitary gonadotrophs. I, Distinct cellular distributions of two gonadotropins, GTH I and GTH II

Using antisera specific for the beta subunits of two distinct coho salmon gonadotropins, GTH I and GTH II, an immunocytochemical study of rainbow trout and Atlantic salmon pituitaries was done. Cells which immunostained with anti-GTH I beta were distributed in the periphery of the glandular cords of the proximal pars distalis (PPD), in close association with somatotrophs. On the other hand, cells immunostained with anti-GTH II beta were located in the central parts of the glandular cords of the PPD. Neither the GTH I-producing nor the GTH II-producing cells stained with antisera against chum salmon growth hormone or the beta subunit of human thyroid-stimulating hormone. Moreover, GTH I and GTH II were localized in distinctly different cells. In no case was colocalization of these GTHs in the same cell observed. Finally, it was concluded that classification of GTH cells as globular and vesicular forms does not reflect the type of hormone produced by the cell, but may reflect differences in the physiological conditions of the cells.

Isolation and characterization of subunits from two distinct salmon gonadotropins

Two distinct gonadotropins, GTH I and GTH II, isolated from female chum salmon pituitary glands, were separated into subunits by acid treatment and subsequent fractionation on reversed-phase high-performance liquid chromatography. GTH II was completely dissociated in 0.1% trifluoroacetic acid, while GTH I was partially dissociated. The acid-stable form of GTH I exhibited a potency identical to that of GTH I in stimulating estradiol-17 beta production in vitro. Both GTH I and GTH II consist of two dissimilar subunits. One subunit (alpha) is common to both GTHs, has Tyr as its N-terminal residue, and a molecular weight (Mr) of 22K by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction. The other subunit (beta) has a Mr of 17K and an N-terminal residue of Gly for GTH I, whereas GTH II beta is 18K and has an N-terminal residue of Ser, after reduction.

The complete amino acid sequences of β-subunits of two distinc chum salmon GTHs

Abstract The complete amino acid sequences of β-subunits of chum salmon ( Oncorhynchus keta ) gonadotropin (GTH) I and GTH II were determined. Both GTH Iβ and GTH IIβ were treated with neuraminidase, and then reduced and carboxymethylated. Both of the modified subunits were digested separately with several endopeptidases. The resulting fragments were purified by reversed-phase high-performance liquid chromatography and subjected to sequence analysis by manual Edman degradation. GTH Iβ was a single component consisting of 113 amino acid residues including 12 half-cystine residues. GTH IIβ was composed of two variants, both of which consisted of 119 amino acid residues including 12 half-cystine residues and differed from each other by substitutions at only two amino acid residues. The presence of an N-linked glycosylation consensus sequence (Asn-X-Thr) in both GTH Iβ and GTH IIβ is also proved. GTH Iβ has only a 31% sequence identity with GTH IIβ and less than 40% with human LH β and FSH β, and differed from these β-subunits in the disulfide alignments. It appeared that GTH IIβ is almost identical to the molecule previously characterized as the β-subunits of maturational GTH from chinook and carp, and more similar to human LHβ (48% identity) than to human FSHβ (38% identity). Thus, the elucidation of the complete amino acid sequences for GTH Iβ and GTH IIβ firmly establishes that the chum salmon pituitary gland secretes two chemically distinct molecules homologous to LH and FSH of the tetrapods.

The complete amino acid sequences of α subunits of chum salmon gonadotropins

Abstract The complete amino acid sequences of α subunits of chum salmon ( Oncorhynchus keta ) gonadotropins (GTHs), GTH I and GTH II, were determined. Subunits were separated by reversed-phase high-performance liquid chromatography. Two α subunits, I α1 and I α2, were obtained from GTH I, whereas only one α subunit was obtained from GTH II. Sequence analysis revealed that GTH I α2 is identical to GTH II α, but distinct from GTH I α1. The I α1 and II α consist of 95 and 92 amino acid residues, respectively. GTH I α1 has 72% sequence identity with GTH I α2. The two α subunits show approximately 65% sequence identity with bovine α subunit. This is the first finding of two chemically distinct α subunits from one species.