Distinct Gonadotropins Research Articles

Molecular cloning, sequence analysis and expression of the snake follicle-stimulating hormone receptor

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control gonadal function in mammalian and many non-mammalian vertebrates through the interaction with their receptors, FSHR and LHR. Although the same is true for some reptilian species, in Squamata (lizards and snakes) there is no definitive evidence for the presence of either two distinct gonadotropins or two distinct gonadotropin receptors. Our aim was to characterize the gonadotropin receptor(s) of the Bothrops jararaca snake. Using a cDNA library from snake testis and amplification of the 5 ′-cDNA ending, we cloned a cDNA related to FSHR. Attempts to clone a cDNA more closely related to LHR were unsuccessful. Expression of FSHR mRNA was restricted to gonadal tissues. The snake FSHR is a G protein-coupled receptor with 673 amino acids, and the aminoterminal domain with 346 amino acids consists of a nine leucine-rich repeat-containing subdomain (LRR) flanked by two cysteine-rich subdomains. The β-strands in the LRR are conserved with exception of the third, a region that may be important for FSH binding. In contrast with mammalian, avian and amphibian FSHRs, the snake FSHR presents amino acid deletions in the carboxyterminal region of the extracellular domain which are also seen in fish and lizard FSHRs. cAMP assays with the recombinant protein transiently expressed in HEK-293 cells showed that the snake FSHR is more sensitive to human FSH (hFSH) than to human chorionic gonadotropin. Phylogenetic analysis indicated that the squamate FSHRs group separately from mammalian FSHRs. Our data are consistent with the apparently unique gonadotropin-receptor system in Squamata reptilian subgroup. Knowledge about the snake FSHR structure may help identify structural determinants for receptor function.

Unique expression of gonadotropin-I and -II subunit genes in male and female red seabream (Pagrus major) during sexual maturation.

Two distinct gonadotropins (GTHs) have been demonstrated in a number of teleost fishes. Although the physiological roles of GTHs have been extensively studied in salmonids, little is known about their biological functions in nonsalmonid fishes. In this study, to elucidate the role of GTH-I and GTH-II in reproduction, we cloned the alpha-glycoprotein subunit (alphaGSU) and gonadotropin beta subunits (Ibeta and IIbeta) of red seabream using the 5'- and 3'-RACE methods and used these cDNA probes to reveal changes in mRNA levels of each subunit during sexual maturation of both male and female red seabream. The nucleotide sequences of alphaGSU, Ibeta, and IIbeta are 629, 531, and 557 base pairs long, encoding peptides of 117, 120, and 146 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high homology with those of other teleosts. Northern blot analysis showed that Ibeta mRNA levels of males increase in association with gonadal development, whereas those of females remain low throughout sexual maturation, indicating sexual dimorphism in the expression pattern of Ibeta. In contrast, IIbeta mRNA levels of both sexes are maintained at high levels from the beginning of gametogenesis to spawning season. These results are different than those of salmonids and suggest that GTH-I may have important roles in male, but not female, gametogenesis. GTH-II may be involved in regulation of early and late gametogenesis in both male and female red seabream.

Brain aminergic systems in salmonids and other teleosts in relation to steroid feedback and gonadotropin release

Two distinct gonadotropins, GtH I and GtH II have been evidenced in most of the teleost species, especially in salmonids. GtH II release is controlled by a dopaminergic inhibitory tone. An oestrogenic activation of a dopaminergic preoptico-hypophysial pathway should be involved in the rainbow trout. GtH I release depends in salmonids upon a negative oestrogenic feedback, but a control by catecholamines is not evidenced. Some targets of sexual steroids on catecholaminergic neurones in fish are evoked.

Isolation and Characterization of Two Distinct Gonadotropins from the Pituitary Gland of Mediterranean Yellowtail,Seriola dumerilii(Risso, 1810)

Two gonadotropins, GTH I and GTH II, were isolated and chemically characterized from the pituitary of Mediterranean yellowtail. They were extracted with 35% ethanol–10% ammonium acetate, separated by ion-exchange chromatography on a DE-52 column, and purified by reversed-phase high-performance liquid chromatography on Asahipak C4P-50 and subsequently by gel filtration chromatography on Superdex 75. The molecular weights were estimated at 47 kDa for GTH I and 29 kDa for GTH II by SDS–PAGE and at 49 kDa for GTH I and 42 kDa for GTH II by gel filtration. GTH II was completely dissociated, while GTH I was partially dissociated into α- and β-subunits by treatment with 0.1% trifluoroacetic acid. The complete amino acid sequences of GTH α-, GTH Iβ-, and GTH IIβ-subunits were determined. The GTH α-subunit consisted of 91 amino acid residues. The GTH Iβ and GTH IIβ consisted of 105 and 115 amino acid residues, respectively, and had a 28% sequence identity to each other. They had the highest sequence identity with the respective gonadotropin subunits of bonito, tuna, and striped bass: 81–83% for GTH α, 67–71% for GTH Iβ, and 91–93% for GTH IIβ. The sequence identity of the GTH α-subunit with those of other teleosts and human and bovine LH and FSH was 57–67%. The GTH Iβ-subunit showed a low sequence identity with other known fish GTH Iβs (36–51%) and was more similar to human and bovine FSH βs (34% identity) than to human and bovine LH βs (29% identity). The sequence identity of the GTH IIβ-subunit with those of other teleosts was higher (60–73%), being more similar to LH βs (43% identity) than FSH βs (38% identity). Thus, two distinct gonadotropins, GTH I and GTH II, homologous to mammalian FSH and LH, respectively, are synthetized by M. yellowtail pituitary glands.

The chinook salmon gonadotropin II beta subunit gene contains a strong minimal promoter with a proximal negative element.

The salmon pituitary expresses two distinct gonadotropins, gonadotropin I (GTHI) and gonadotropin II (GTHII). These two hormones are synthesized in distinct pituitary cells and secreted at different stages during the reproductive cycle. To study the transcriptional regulation of the hormone-specific beta-subunit of GTHII (sGTHII beta) gene, approximately 3.5 kilobases of the 5'-flanking region was characterized and sequenced. The pituitary specificity of sGTHII beta was examined by analyzing sGTHII beta promoter activity in homologous primary pituitary cells derived from spawning rainbow trout and in a collection of heterologous cell lines. Various lengths of the 5'-flanking region of the sGTHII beta gene were ligated into a vector encoding the bacterial chloramphenicol acetyltransferase (CAT) gene, and the resulting sGTHII beta/CAT chimeric constructs were analyzed using transient expression systems. Several constructs (-3500CAT, -1260CAT, -563CAT, and -39CAT) displayed readily detectable CAT activity in the pituitary cells derived from spawning male and female rainbow trout. In contrast, three of the constructs (-3500CAT, -1260CAT, and -563CAT) were expressed only at background levels in a variety of heterologous cell lines, suggesting that the 5'-flanking sequence of sGTHII beta contains information dictating its pituitary specificity. A silencer sequence (-95 to -35, pSil) was identified, which might function to repress sGTHII beta gene expression in the nongonadotropes or in the gonadotropes at developmental stages that precede final maturation and spawning.

Purification and characterization of gonadotropin I and II from pituitary glands of tuna (Thunnus obesus)

The duality of teleost pituitary gonadotropins was established in an advanced marine fish, the tuna (Thunnus obesus). Two different molecular forms of gonadotropins, designated tGTH I and tGTH II, were isolated from an alcoholic extract of pituitary glands following ion-exchange chromatography and reversed-phase HPLC. Both tGTH I and tGTH II stimulated estradiol-17 beta and testosterone production in tuna ovarian follicles in vitro, although responses to tGTH II were significantly greater than those to tGTH I. Each gonadotropin consisted of alpha- and beta-subunits. tGTH I was stable in acidic conditions, whereas tGTH II dissociated into two subunits after acid treatment. Alpha subunits of tGTH I and tGTH II had identical amino acid sequences of 94 amino acid residues. The tGTH I beta and tGTH II beta consisted of 102 and 115 amino acid residues, respectively, and showed 35% sequence identity. tGTH I beta is structurally more similar to salmon GTH I beta than to salmon GTH II beta, whereas tGTH II beta is more similar to salmon GTH II beta. Thus it is evident that the tuna pituitary gland produces two chemically distinct gonadotropins.

Ultrastructural characteristics of two distinct gonadotropes (GTH I- and GTH II-cells) in the pituitary of rainbow trout Oncorhynchus mykiss

The salmonid pituitary produces two chemically distinct gonadotropins (GTHI and GTHII). Ultrastructural characteristics of GTHI- and GTHII-producing cells were studied in the trout pituitary with electronmicroscopic immunocytochemistry using antisera against salmon GTHIβ- and IIβ-subunits. In pituitaries from vitellogenic fish, GTHI-cells were characterized by numerous dilated cisternae of the granular endoplasmic reticulum (GER) and a small number of Iβ-positive granules (diameter, 100-300 nm), whereas GTHIIβ-immunoreactivity was found on granules (diameter, 200-400 nm) and large globules (diameter, 500-4000 nm) in apparently different cells (GTHII-cells). Distinct cellular distributions of GTHI and GTHII were maintained during gametogenesis, although morphological characteristics of GTHI- and GTHII-cells overlapped each other due to changes in number and size of the granules, globules and cisternae of the GER. Interestingly, the globules in the GTHI-cells were immunonegative for GTHIβ, although in the GTHII-cells they were always stained with GTHIIβ-antiserum. These results confirm that GTHIβ and GTHIIβ are synthesized in distinctly different cell-types in the salmonid pituitary and indicate that morphological characteristics cannot be used to distinguish these two cell-types.

Isolation and characterization of two distinct gonadotropins, GTHI and GTHII, from bonito (Katsuwonus plelamis) pituitary glands

Two distinct glycoproteins homologous to chum salmon GTHI and GTHII were isolated from the pituitary glands of a marine fish, the bonito (Katsuwonus plelamis), and characterized by amino acid sequence analysis in order to obtain additional evidence for duality of teleost GTHs. Glycoproteins were extracted from the pituitary glands, and intact GTHI and GTHII, consisting of two distinct subunits, were purified by ion-exchange chromatography on DEAE-cellulose, rpHPLC on Asahipak C4P-50 in alkaline buffer, and gel filtration on Superdex 75. The association of the subunits was stable in GTHI (39 kD) and unstable in GTHII (30 kD) in acidic conditions. Immunoblotting revealed that antisera against beta subunits of chum salmon GTHs reacted with GTHII, but not with GTHI. In addition, none of the GTHs was stained with antiserum against human TSH beta. Sequence analysis demonstrated that bonito GTHI beta is homologous to salmon GTHI beta with 43% sequence identity, and bonito GTHII beta is homologous to salmon GTHII beta with 67% identity. Sequence identity between bonito GTHI beta and GTHII beta was only 28%. Thus, it is evident that the bonito pituitary gland produces two chemically distinct gonadotropins homologous to chum salmon GTHs.

Histochemical and immunocytochemical identification of the pituitary cell types in three sciaenid fishes: Atlantic croaker ( Micropogonias undulatus), spotted seatrout ( Cynoscion nebulosus), and red drum ( Sciaenops ocellatus)

The adenohypophysial cell types in three species of sciaenid teleosts were identified using a combination of classical histochemical techniques and immunocytochemistry with antisera raised against piscine and human pituitary hormones. In general greater specificity for the different cell types was observed with antisera to the piscine pituitary hormones than with antisera raised against the human hormones. The distribution of the cell types did not differ significantly among the three species, Atlantic croaker ( Micropogonias undulatus), spotted seatrout ( Cynoscion nebulosus), and red drum ( Sciaenops ocellatus), and was similar to that reported in several other teleosts. A major portion of the rostral pars distalis was composed of acidophilic prolactin cells which showed specific binding to antisera to carp, coho salmon, and chinook salmon prolactins. The acidophilic corticotrops formed cords of cells which bordered the prolactin cells and showed strong immunoreactivity with anti-human ACTH. This antiserum also cross-reacted with the lead hematoxylin-positive cells in the pars intermedia. The acidophilic growth hormone cells were restricted to the dorsal part of the proximal pars distalis in close contact with the neurohypophysis and were specifically bound by anti-carp and anti-chum salmon growth hormone. The basophilic thyrotrops were located in the dorsal-anterior part of the proximal pars distalis and showed strong immunoreactivity to antiserum to the human thyrotropin β subunit. This antiserum also bound weakly to the basophilic gonadotrops which were concentrated in the ventral part of the proximal pars distalis and also extended posteriorly to form a border around the pars intermedia. Antiserum to croaker maturational gonadotropin, which recognizes two distinct gonadotropins in sciaenids, bound strongly and specifically to the gonadotrops. The gonadotrops were also bound specifically by antisera to the α and β subunits of human luteinizing hormone. Antisera to the β subunits of two recently isolated coho salmon gonadotropins, GTH-I and GTH-II, failed to bind the gonadotrops strongly or differentially in the three sciaenid species. Consequently, the number of gonadotropin cell types in sciaenid fishes remains unresolved.

Involvement of gonadotropin in the uptake of vitellogenin into vitellogenic oocytes of the rainbow trout, Oncorhynchus mykiss

The effects of two fully characterized, structurally distinct gonadotropins, GtH I and GtH II, on the uptake of vitellogenin (VTG) into oocytes of the rainbow trout, Oncorhynchus mykiss, were investigated both in vivo and in vitro. GtH I, injected into maturing vitellogenic females at a dose of 10 μg · kg body wt −1, increased the rate of [ 3H]VTG uptake into oocytes by more than two-fold, effectively doubling their rate of growth. Ovaries from females similarly treated with GtH II sequestered VTG at rates similar to controls. In vitro, GtH I stimulated VTG uptake in a dose-dependent manner. At a GtH I concentration of 100 ng · ml −1 and above, the rate of VTG uptake was significantly greater than that of the controls and at 1000 ng · ml −1 the rate of uptake was more than doubled. GtH II did not significantly increase VTG sequestration into isolated oocytes at concentrations up to, and including, 1000 ng · ml −1. These data provide the first evidence that GtH I has a primary function in stimulating VTG uptake and strongly support the contention that at least two functionally distinct GtHs occur in fish.

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-17β 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.

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 acidstable form of GTH I exhibited a potency identical to that of GTH I in stimulating estradiol-17β production in vitro. Both GTH I and GTH II consist of two dissimilar subunits. One subunit (α) is common to both GTHs, has Tyr as its N-terminal residue, and a molecular weight ( M r) of 22K by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction. The other subunit (β) has a M r of 17K and an N-terminal residue of Gly for GTH I, whereas GTH II β is 18K and has an N-terminal residue of Set, after reduction.

Separation of two distinct gonadotropins from the pituitary gland of the bullfrog Rana catesbeiana.

Chemical fractionation of pituitary glands from the bullfrog Rana catesbeiana resulted i n the purification of gonadotropic activity and separation of two distinct types of activity. One fraction was highly potent in tests in hypophysec-tomized lizards (Anolis) and in spermiation tests in frogs. A second fraction was potent in ovulation tests with excised anuran ovaries and in 32P uptake by chick testes. There was essentially no overlap in biological activities between the two fractions when tested in Anolis or ovulation assays. These results provide evidence for separate FSH- and LH-like gonadotropins in the anuran pituitary. The behavior of the two types of gonadotropins in fractionation closely resembles the respective hormones from mammalian and reptilian pituitaries. (Endocrinology 94: 1587, 1974)

Separation of two distinct gonadotropins from the pituitary gland of the snapping turtle ( Chelydra serpentina)

Two chemical fractionation schemes were used in conjunction with various non-mammalian bioassays to isolate gonadotropin from the pituitary gland of the snapping turtle ( Chelydra serpentina). Ammonium sulfate fractionation, effective in separating mammalian FSH from LH, and gel filtration on Sephadex G-100 concentrated all the turtle gonadotropin into a single fraction. A second fractionation method employing ion exchange chromatography on Amberlite IRC-50 and DEAE-cellulose effected considerable purification of gonadotropin and also resulted in the distinct separation of two different kinds of gonadotropic activities from the turtle glands. The two kinds of gonadotropic activities were distinguished by tests that are relatively specific for mammalian FSH (an Anolis lizard assay) and LH (an anuran in vitro ovulation assay). Both of the turtle fractions were active in frog spermiation and the uptake of 32P by cockerel tests which is consistent with the lack of specificity for mammalian FSH and LH shown by these assays. The turtle hormones had low potencies in mammalian assays. Growth hormone and thyrotropin were also purified during fractionation, but the latter activity was not effectively separated from the gonadotropins. These results support the existence of two separate gonadotropins having FSH- and LH-like biological and chemical properties in the turtle pituitary.