A relaxin-like gonad-stimulating peptide identified from the starfish Astropecten scoparius.

A relaxin-like gonad-stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B-chain (21 aa), C-peptide (47 aa), and A-chain (24 aa). There are three putative processing sites (Lys-Arg) between the B-chain and C-peptide, between the C-peptide and A-chain, and within the C-peptide. This structural organization revealed that the mature AscRGP is composed of A- and B-chains with two interchain disulfide bonds and one intrachain disulfide bond. The C-terminal residues of the B-chain are Gln-Gly-Arg, which is a potential substrate for formation of an amidated C-terminal Gln residue. Non-amidated (AscRGP-GR) and amidated (AscRGP-NH2 ) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP-GR and AscRGP-NH2 induced oocyte maturation and ovulation in similar dose-dependent manners. This is the first report on a C-terminally amidated functional RGP. Collectively, these results suggest that AscRGP-GR and AscRGP-NH2 act as a natural gonadotropic hormone in A. scoparius.

Molecular identification of GnIH and its potential role in reproductive physiology and male pregnancy of the lined seahorse (Hippocampus erectus).

The gonadotropin-inhibitory hormone (GnIH) plays a negative role in the hypothalamic-pituitary-gonadal (HPG) axis by inhibiting gonadotropin secretion in vertebrates. Male pregnancy and ovoviviparous behavior are unique phenomena among vertebrates. To better understand the neuroendocrine regulatory mechanisms in ovoviviparous fish with male pregnancy, we identified the orthologous GnIH gene in the lined seahorse (Hippocampus erectus). The full-length cDNA of the GnIH precursor was 658 base pairs with an open reading frame of 528 base pairs that encoded a 175-amino acid prepro-GnIH peptide. The seahorse GnIH precursor contained two putative LPXRFamide peptides. Both seahorse LPXRFa-1 and LPXRFa-2 were found to be unique among vertebrates. The synteny blocks of GnIH gene loci were conserved in mammals and teleosts. Tissue distribution analysis revealed that seahorse GnIH mRNA was mainly expressed in the hypothalamus, with relatively high levels observed in the brood pouch. The expression patterns of seahorse GnIH during different reproductive stages and pregnancy stages were also detected, and GnIH mRNA expression was significantly reduced during the early puberty stage. In addition, GnIH mRNA expression was significantly increased during the pregnancy stage compared to non-pregnancy stages. In summary, our results reveal the existence of GnIH in ovoviviparous fish and suggest its involvement in regulation of reproductive behavior and male pregnancy in the male seahorse.

Enzyme-linked immunosorbent assay of relaxin-like gonad-stimulating peptide in the starfish Patiria (Asterina) pectinifera.

A relaxin-like gonad-stimulating peptide (RGP) from starfish Patiria (Asterina) pectinifera is the first identified invertebrate gonadotropin for final gamete maturation. Recently, we succeeded in obtaining specific antibodies against P. pectinifera RGP (PpeRGP). In this study, the antibodies were used for the development of a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of PpeRGP. A biotin-conjugated peptide that binds to peroxidase-conjugated streptavidin is specifically detectable using 3,3',5,5'-tetramethylbenzidine (TMB)/hydrogen peroxide as a substrate; therefore, biotin-conjugated RGP (biotin-PpeRGP) was synthesized chemically. Similarly to PpeRGP, synthetic biotin-PpeRGP bound to the antibody against PpeRGP. In binding experiments with biotin-PpeRGP using wells coated with the antibody, a displacement curve was obtained using serial concentrations of PpeRGP. The ELISA system showed that PpeRGP could be measured in the range 0.01-10pmol per 50µl assay buffer. On the contrary, the B-chains of PpeRGP, Asterias amurensis RGP, Aphelasterias japonica RGP, and human relaxin showed minimal cross-reactivity in the ELISA, except that the A-chain of PpeRGP affected it slightly. These results strongly suggest that this ELISA system is highly specific and sensitive with respect to PpeRGP.

Characterization of carp gonadotropins: Structure, annual profile, and carp and zebrafish pituitary topographic organization.

Two gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), are important players in the hypothalamic-pituitary-gonadal axis of vertebrates. In the present work, we describe the construction of recombinant (r) common carp (Cyprinus carpio; c) FSH (rcFSH) and LH (rcLH) using the Pichia pastoris system, the generation of specific antibodies against their respective ß subunits, and their use in the development and validation of specific ELISAs. We produced carp rLH and rFSH as single-chain polypeptides, wherein the GTH subunit α was joined with either cLHß or cFSHß mature protein-coding sequences to form a fusion gene that encodes a yoked polypeptide, in which the GTH ß-subunit forms the N-terminal part and the α-subunit forms the C-terminal part. Competitive ELISAs were developed, using primary antibodies against rcLHß or rcFSHß, respectively, and rcLHßα or rcFSHßα for the standard curves. The standard curves for cLH paralleled those of pituitary extracts of the homologous fish and also those of other cyprinids species like the black carp (Mylopharyngodon piceus), goldfish (Carassius auratus), silver carp (Hypophthalmichthys molitrix), and grass carp (Ctenopharyngodon idella). We used the specific antibodies raised against cFSH and cLH to study the specific localization of the different GTH cells in the pituitary of carp and its taxonomic relative species - the zebrafish. Both FSH and LH cells are localized in the center of the proximal pars distalis enveloping both sides of the neurohypophysis. LH cells form a continuous population throughout the PPD, while FSH cells are more loosely distributed throughout the same area and form small aggregations. Marked annual changes were encountered in gonadosomatic index (GSI), follicle diameter, mRNA levels and protein levels of FSH and LH. From September to November, all fish had low GSI, and the ovary contained previtellogenic follicles. From December, the GSI level increased and remained high until March, the follicular diameter reached its maximum in January, where the ovary contained large fully grown follicles. Thereafter, spawning occurred through March and April and ended in May, and GSI level and follicle diameter increased again; and the ovary contained mid-vitellogenic follicles. LH pituitary content and mRNA levels were low at pre- and early vitellogenesis, increasing gradually during this process to reach a peak of LH mRNA levels in mid vitellogenic ovary and a peak of LH content in fully grown ovarian follicles. However, no significant change occurred in FSH pituitary content and mRNA levels in vitellogenic fish and in fish during final maturation stages. A dramatic difference was found in the total content of each gonadotropin in the pituitary, with higher LH than FSH. Moreover, follicle diameter was positively and significantly correlated with LH pituitary content and its transcript levels - but not with the pituitary content or mRNA levels of FSH. Taken together, these results indicate that in carp, LH alone is sufficient to regulate both vitellogenesis and final oocyte maturation while FSH may have another, yet undefined role.

In silico insights into intra- and inter-species interactions of piscine gonadotropin hormones and receptor crosstalk.

In mammals, the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are macromolecules secreted during specific reproductive phases and display strict specificity towards their cognate receptors. However, fish gonadotropins (GTH) and their receptors (GTHR) display diverse species-specific expression patterns, secretion patterns, and intra- and interspecies cross-activation. To uncover the molecular basis of this diversity, we generated and analyzed 29 in-silico models of intra- and inter-species combinations of sturgeon, carp, tilapia, and human gonadotropins with piscine receptors and analyzed the resulting receptor activation and signal transduction of these GTHR-GTH complexes in-vitro. Our results suggest that unlike humans, the surface charge on piscine FSH/LH ß-seatbelt and N107huLHCGR/K104hFSHR homologs does not necessarily determine binding specificity. Instead, sequence and structural variations allow piscine GTHs significant conformational flexibility when binding to the receptor extracellular domain, thereby enabling cross-activation. The resulting diversity may support various reproductive strategies in different environmental niches.

Evolutionary origin of a functional gonadotropin in the pituitary of the most primitive vertebrate, hagfish.

Hagfish, which lack both jaws and vertebrae, are considered the most primitive vertebrate known, living or extinct. Hagfish have long been the enigma of vertebrate evolution not only because of their evolutionary position, but also because of our lack of knowledge on fundamental processes. Key elements of the reproductive endocrine system in hagfish have yet to be elucidated. Here, the presence and identity of a functional glycoprotein hormone (GPH) have been elucidated from the brown hagfish Paramyxine atami. The hagfish GPH consists of two subunits, alpha and beta, which are synthesized and colocalized in the same cells of the adenohypophysis. The cellular and transcriptional activities of hagfish GPHalpha and -beta were significantly correlated with the developmental stages of the gonad. The purified native GPH induced the release of gonadal sex steroids in vitro. From our phylogenetic analysis, we propose that ancestral glycoprotein alpha-subunit 2 (GPA2) and beta-subunit 5 (GPB5) gave rise to GPHalpha and GPHbeta of the vertebrate glycoprotein hormone family, respectively. The identified hagfish GPHalpha and -beta subunits appear to be the typical gnathostome GPHalpha and -beta subunits based on the sequence and phylogenetic analyses. We hypothesize that the identity of a single functional GPH of the hagfish, hagfish GTH, provides critical evidence for the existence of a pituitary-gonadal system in the earliest divergent vertebrate that likely evolved from an ancestral, prevertebrate exclusively neuroendocrine mechanism by gradual emergence of a previously undescribed control level, the pituitary, which is not found in the Protochordates.

Immunoreactivity of gonadotrophs (FSH and LH Cells) and gonadotropin subunit gene expression in the male chub mackerel Scomber japonicus pituitary during the reproductive cycle.

The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are heterodimers composed of a common α subunit (GPα) and a unique ß subunit (FSHß or LHß); they are synthesized in and secreted from gonadotrophs (FSH and LH cells) in the pituitary. Little is known about the roles of FSH and LH during spermatogenesis in perciform fishes. In this study, we examined immunoreactive changes in FSH and LH cells, and changes in the gene expression of the three gonadotropin subunits in the pituitary of male chub mackerel Scomber japonicus during testicular development. FSHß-immunoreactive (ir) and LHß-ir cell area were measured immuno-histochemically based on the FSH and LH cell-occupying area in the proximal pars distalis. The FSHß-ir cell area increased significantly during spermiation, while FSHß mRNA levels, already high at the beginning of spermatogenesis, increased further, peaking during spermiation. In contrast, LHß-ir cell area and LHß mRNA levels, which were low at the beginning of spermatogenesis, increased significantly during late spermatogenesis, peaking during spermiation. For both FSH and LH, GtHß-ir cell area and GtHß mRNA levels decreased until gonadal resting. GPα mRNA levels showed similar changes to LHß mRNA levels. These results suggest that in the chub mackerel, FSH may play an important role in the early and late phases of spermatogenesis, and that LH may play a role during late spermatogenesis and spermiation. Moreover, our results demonstrate that changes in GtHß-ir cell area were accompanied by similar changes in the expression of the FSHß and LHß genes, both of which increased during testicular development.

An invertebrate [hydroxyproline]-modified neuropeptide: further evidence for a close evolutionary relationship between insect adipokinetic hormone and mammalian gonadotropin hormone family.

An octapeptide of the adipokinetic hormone (AKH) peptide family is identified in the corpora cardiaca of the stink bug, Nezara viridula, by ESI-MS(N) (electrospray ionization multistage MS). This is the second AKH in N. viridula and it has a hydroxyproline residue at position 6, whereas the major AKH (known as Panbo-RPCH) has Pro as the sixth amino acid residue. The correct sequence assignment of [Hyp(6)]-Panbo-RPCH is confirmed by retention time and MS spectra of the synthetic peptide. Various extraction procedures were followed to ascertain whether the hydroxylation is an artefact of extraction, or whether it is due to a true post-translational modification at the prohormone level. The proline hydroxylation is unique for invertebrate neuropeptides, while it has been described in the vertebrate gonadotropin-releasing hormone (GnRH). The current finding is another piece of evidence that AKH and GnRH form a peptide superfamily and are closely related evolutionarily. Biologically, [Hyp(6)]-Panbo-RPCH is active in vivo as an AKH, causing hyperlipaemia in the stink bug at low doses, indicating again that it is an endogenous, mature and functional hormone in this insect species.

Novel pathways in gonadotropin receptor signaling and biased agonism.

Gonadotropins play a central role in the control of male and female reproduction. Selective agonists and antagonists of gonadotropin receptors would be of great interest for the treatment of infertility or as non steroidal contraceptive. However, to date, only native hormones are being used in assisted reproduction technologies as there is no pharmacological agent available to manipulate gonadotropin receptors. Over the last decade, there has been a growing perception of the complexity associated with gonadotropin receptors' cellular signaling. It is now clear that the Gs/cAMP/PKA pathway is not the sole mechanism that must be taken into account in order to understand these hormones' biological actions. In parallel, consistent with the emerging paradigm of biased agonism, several examples of ligand-mediated selective signaling pathway activation by gonadotropin receptors have been reported. Small molecule ligands, modulating antibodies interacting with the hormones and glycosylation variants of the native glycoproteins have all demonstrated their potential to trigger such selective signaling. Altogether, the available data and emerging concepts give rise to intriguing opportunities towards a more efficient control of reproductive function and associated disorders.

Molecular characterization of marbled eel (Anguilla marmorata) gonadotropin subunits and their mRNA expression profiles during artificially induced gonadal development.

Three cDNA sequences encoding the gonadotropin subunits, common glycoprotein alpha subunit (GTHalpha), FSHbeta and LHbeta subunits were isolated from marbled eel. The cDNA of GTHalpha encodes 116 amino acids with a signal peptide of 24 amino acids and a mature peptide of 92 amino acids. The FSHbeta subunit consists of 127 amino acids with a 22 amino acid signal peptide and a 105 amino acid mature peptide, while the LHbeta subunit consists of 140 amino acids with a 24 amino acid signal peptide and a 116 amino acid mature peptide. Comparison of the deduced amino acid sequences of marbled eel GTHalpha, FSHbeta, and LHbeta with that of other fishes shows a high degree of conservation in the number of cysteine residues and potential N-linked glycosylation sites. The mRNA of GTHalpha, FSHbeta and LHbeta were not only detected in pituitary, but also in ovary and testes by RT-PCR. Quantitative realtime PCR analysis revealed that the GTHalpha and LHbeta transcriptional levels in pituitaries of female and male eels gradually increased during the artificially inducing gonadal development, and peaked at late vitellogenic stage and spermiation stage, respectively. FSHbeta mRNA in the pituitaries of female eels maintained a high level at previtellogenic stage, early vitellogenic stage as well as mid-vitellogenic stage but declined sharply at late vitellogenic stage and migratory nucleus stage. In male eels, the mRNA levels of FSHbeta in the pituitaries were higher at early spermatogenesis stage than at both late spermatogenesis stage and spermiation stage. These results suggested that FSH would be in control of initiation and maintenance of gonadal growth and gametogenesis, whereas LH would be involved in the final gonadal maturation and spermiation/ovulation in the tropic eel Anguilla marmorata.

Expression of three gonadotropin subunits in Southern catfish gonad and their possible roles during early gonadal development.

The three gonadotropin (GtH) subunit cDNAs, GtHalpha, FSHbeta and LHbeta, which contain complete open reading frames were isolated from Southern catfish (Silurus meridionalis Chen) ovary. RT-PCR revealed that GtHalpha, FSHbeta and LHbeta mRNA were expressed in ovary, female and male pituitaries, but not in testis. Ontogeny study showed that GtHalpha and FSHbeta expressed in ovary from 25 dah (days after hatching) and LHbeta expressed from 40 dah onwards. The expression levels of these genes in all-female Southern catfish gonad were down-regulated after treatment with tamoxifen from 5 to 25 dah when measured at 65 dah. These results indicated the involvement of the three subunits in gonadal development and sexual differentiation.

The role of O-linked and N-linked oligosaccharides on the structure-function of glycoprotein hormones: development of agonists and antagonists.

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common alpha and a hormone specific beta subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the alpha and beta subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (alpha, FSHbeta and hCGbeta) asparagine-linked (N-linked) oligosaccharides. CGbeta subunit is distinguished among the beta subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure-function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.

An introduction to mass spectrometry based proteomics-detection and characterization of gonadotropins and related molecules.

This review introduces fundamental aspects of mass spectrometry (MS) based proteomics and illustrates how MS is an effective tool for the analysis of glycoprotein hormones. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) MS are complementary approaches that have been applied for the analysis of gonadotropins, e.g. to characterize differences in the oligosaccharide distribution of commercial human chorionic gonadotropin preparations, for isolated nicked beta-subunit, and identification of a metabolite of placental transforming growth factor in pharmaceutical hCG preparations. Immunoaffinity trapping and concentration of digested sample extract prior to MS analysis confers analytical sensitivity akin to immunoassay. A desirable objective would be to develop for clinical purposes a rapid procedure for MS detection and characterization of gonadotropins. Refinement of on-target immobilization and digestion for subsequent ionization by MALDI may eventually help to provide this capability. The advent of hybrid mass spectrometers will further advance the characterization of these complex molecules.

Ligand-selective determinants in gonadotropin receptors.

In mammals, the interactions between gonadotropins and their cognate receptors are highly specific; unintended cross-reactivity under normal physiological conditions has not been observed. This paper summarizes the comparative structure-function studies that aim at elucidating the molecular basis of the ligand selectivity.

Single-chain, triple-domain gonadotropin analogs with disulfide bond mutations in the alpha-subunit elicit dual follitropin and lutropin activities in vivo.

The human glycoprotein hormones chorionic gonadotropin (CG), TSH, LH, and FSH are heterodimers composed of a common alpha-subunit and a hormone-specific beta-subunit. The subunits assemble noncovalently early in the secretory pathway. LH and FSH are synthesized in the same cell (pituitary gonadotrophs), and several of the alpha-subunit sequences required for association with either beta-subunit are different. Nevertheless, no ternary complexes are observed for LH and FSH in vivo, i.e. both beta-subunits assembled with a single alpha-subunit. To address whether the alpha-subunit can interact with more than one beta-subunit simultaneously, we genetically linked the FSHbeta- and CGbeta-subunit genes to the common alpha-subunit, resulting in a single-chain protein that exhibited both activities in vitro. These studies also indicated that the bifunctional triple-domain variant (FSHbeta-CGbeta-alpha), is secreted as two distinct bioactive populations each corresponding to a single activity, and each bearing the heterodimer-like contacts. Although the data are consistent with the known secretion events of gonadotropins from the pituitary, we could not exclude the possibility whether transient intermediates are generated in vivo in which the alpha-subunit shuttles between the two beta-subunits during early stages of accumulation in the endoplasmic reticulum. Therefore, constructs were engineered that would direct the synthesis of single-chain proteins completely devoid of heterodimer-like interactions but elicit both LH and FSH actions. These triple-domain, single-chain chimeras contain the FSHbeta- and CGbeta-subunits and an alpha-subunit with cystine bond mutations (cys10-60 or cys32-84), which are known to prevent heterodimer formation. Here we show that, despite disrupting the intersubunit interactions between the alpha- and both CGbeta- and FSHbeta-subunits, these mutated analogs exhibit both activities in vivo comparable to nonmutated triple-domain single chain. Such responses occurred despite the absence of quaternary contacts due to the disrupted bonds in the alpha-subunit. Thus, gonadotropin heterodimer assembly is critical for intracellular events, e.g. hormone-specific posttranslational modifications, but when heterodimers are present in the circulation, the alpha/beta-contacts are not a prerequisite for receptor recognition.

Structure-based design and protein engineering of intersubunit disulfide bonds in gonadotropins.

Pairs of cystine residues were introduced in the alpha- and beta-subunits of human choriogonadotropin at positions with optimal geometries for the formation of disulfide bonds. Using the homology with luteinizing hormone and follicle stimulating hormone, similar mutations were carried out in these glycoprotein hormones. In nearly all mutants the corresponding disulfide bonds were formed leading to a non-natural, covalent linkage between the alpha- and beta-subunits. The mutants typically display wild-type receptor binding and bioactivity. The mutants with non-natural intersubunit disulfide bonds display enhanced thermostabilities relative to the corresponding heterodimeric glycoprotein hormones, rendering them candidates for long acting gonadotropins with enhanced shelf lives.

Clinical Applications of Gonadotropins in the Male.

The pituitary gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) play a pivotal role in reproduction. The synthesis and secretion of gonadotropins are regulated by complex interactions among several endocrine, paracrine, and autocrine factors of diverse chemical structure. In men, LH regulates the synthesis of androgens by the Leydig cells, whereas FSH promotes Sertoli cell function and thereby influences spermatogenesis. Gonadotropins are complex molecules composed of two subunits, the α- and ß-subunit, that are noncovalently associated. Gonadotropins are decorated with glycans that regulate several functions of the protein including folding, heterodimerization, stability, transport, conformational maturation, efficiency of heterodimer secretion, metabolic fate, interaction with their cognate receptor, and selective activation of signaling pathways. A number of congenital and acquired abnormalities lead to gonadotropin deficiency and hypogonadotropic hypogonadism, a condition amenable to treatment with exogenous gonadotropins. Several natural and recombinant preparations of gonadotropins are currently available for therapeutic purposes. The difference between natural and the currently available recombinant preparations (which are massively produced in Chinese hamster ovary cells for commercial purposes) mainly lies in the abundance of some of the carbohydrates that conform the complex glycans attached to the protein core. Whereas administration of exogenous gonadotropins in patients with isolated congenital hypogonadotropic hypogonadism is a well recognized therapeutic approach, their role in treating men with normogonadotropic idiopathic infertility is still controversial. This chapter concentrates on the main structural and functional features of the gonadotropin hormones and how basic concepts have been translated into the clinical arena to guide therapy for gonadotropin deficit in males.

Secondary structure prediction of beta-subunits of the gonadotropin-thyrotropin family from its aligned sequences using environment-dependent amino-acid substitution tables and conformational propensities.

The secondary structures of beta-subunits of the glycoprotein hormone family, LH (luteinizing hormone), CG (chorionic gonadotropin), FSH (follicle stimulating hormone), TSH (thyroid stimulating hormone), and GTH I/GTH II (two types of fish gonadotropins), are predicted by comparing an amino-acid substitution pattern at equivalent sites in their aligned sequences with environment-dependent amino-acid substitution tables and conformational propensities calculated from other protein families whose three-dimensional structures are known. According to the prediction results, together with other structural information obtained from experiments, the following points come up as important structural features of the beta-subunits of this family; The regions assigned to regular secondary structures (one alpha-helix and three beta-strands) are considered to constitute a core of the beta-subunits. They involve interaction sites with carbohydrate and alpha-subunit. Out of the six disulfide bonds formed in the beta-subunit, four are located together on one side of the core, and the other two on the opposite side. The two regions assumed to be a receptor binding region from experiments (therefore, species-specific regions) are predicted as loops located on the same side of the beta-subunit in this study. Some of the predicted loops are rich in proline residues. While the positions of proline residues are conserved in the family generally, there are hormone- or species-specific ones in the loop that is assumed to take part in receptor binding. The possible importance of proline residues in hormone or species specificity is discussed. (After submitting the manuscript the X-ray crystal structure of human CG was published. In order to evaluate the prediction, the original manuscript is kept intact and a comparison has been made between the prediction results and the crystal structure in an appendix).

Catfish gonadotrophins: cellular origin, structural properties and physiology.

Gonadotrophins (GTHs) play a central role in the regulation of gametogenesis and spawning. The structural duality of the GTHs [luteinising hormone (LH) and follicle-stimulating hormone (FSH)] is established in fishes with the exception of ancestral vertebrates. Most studies indicate that, in teleosts, the GTHs are secreted in separate cells. Phylogenetic analysis shows that the common α-subunit of the GTHs (and also of thyroid-stimulating hormone) and LHß are highly conserved in fishes, as in tetrapods. However, FSHß shows considerable divergence in teleosts. There may be 12 or 13 cysteine residues, with an additional one near the N-terminus. There may be one or two N-linked glycolsyation sites. In catfishes, there are 13 cysteine residues and one N-linked glycosylation site. In an extreme situation, a potential glycosylation site is lacking in some fishes. Both FSH and LH receptors are characterised in teleosts. The FSH receptor is promiscuous and can be cross-activated by LH. By contrast, the LH receptor is highly selective, being activated by its natural ligand or by heterologous ligands (e.g. human chorionic gonadotrophin). Consequently, teleosts show different patterns of LH and FSH secretion. In catfishes, in the absence of native FSH protein, LH controls all aspects of reproduction, from early gametogenesis to spawning.

Comparative mapping of human thyrotropin, gonadotropins, and free subunits with antipeptide antibodies.

To compare the structural topology of the human TSH to that of the structurally related gonadotropins, 10 peptides covering the entire primary sequence of the alpha- and beta-subunits of TSH were synthesized and used as antigens for the preparation of polyclonal antibodies. The alpha-subunit was synthesized as 4 nonoverlapping peptides (1-25, 26-51, 49-73, 72-92) while the beta-subunit was segmented in 6 overlapping sequences (2-18, 10-38, 31-51, 53-76, 77-96, 92-112). Most of the peptide sequences were predicted to contain a putative antigenic determinant. All antipeptide antisera were found to bind to the corresponding synthetic sequence in an enzyme-linked immunosorbent assay as well as to denatured TSH subunits after Western blotting. The N-terminal half of the alpha-subunit was found differentially accessible in TSH and gonadotropins compared to the free subunit: antipeptide-alpha 1-25 antibodies exhibited variable affinity for the four glycoprotein hormones whereas anti-alpha 26-51 displayed a remarkable recognition of free alpha-subunit. Four peptides proved to be accessible in the TSH beta-subunit: the N-terminal peptide (beta 2-18) elicited antibodies that bound to free TSH-beta and poorly to the dimer while antibodies against the C-terminal sequence (beta 92-112) recognized equally well free beta-subunit and TSH. Antipeptide-beta 31-51 antibodies proved to be specific for TSH while the beta 53-76 contiguous peptide appeared accessible in both TSH and gonadotropins. The current findings therefore demonstrate that most of the sequences predicted to contain antigenic sites in the alpha- or the beta-subunits are indeed accessible at the surface of these proteins. Additionally, both subunits appear to contain amino acid sequences that are differentially expressed in TSH and gonadotropins as well as in free and combined subunits.