Pituitary Glycoprotein Hormones Research Articles

Cloning and Expression of FSHb Gene and the Effect of FSHβ on the mRNA Levels of FSHR in the Local Chicken

Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that is encoded by separate alpha- and betasubunit genes. It plays a key role in stimulating and regulating ovarian follicular development and egg production in chicken. FSH signal transduction is mediated by the FSH receptor (FSHR) that exclusively interacts with the beta-subunit of FSH, but characterization of prokaryotic expression of the FSHb gene and its effect on the expression of the FSHR gene in local chickens have received very little attention. In the current study, the cDNA fragment of the FSHb gene from Dagu chicken was amplified using reverse transcription polymerase chain reaction (RT-PCR), and inserted into the pET-28a (+) vector to construct the pET-28a-FSHb plasmid. After expression of the plasmid in E. coli BL21 (DE3) under inducing conditions, the recombination protein, subunit, was purified and injected into the experimental hens and the effect on the mRNA expression levels of the FSHR gene was investigated. Sequence comparison showed that the coding region of the FSHb gene in the local chicken shared 99%-100% homology to published nucleotides in chickens; only one synonymous nucleotide substitution was detected in the region. The encoded amino acids were completely identical with the reported sequence, which confirmed that the sequences of the chicken FSHb gene and the peptides of the subunit are highly conserved. This may be due to the critical role of the normal function of the FSHb gene in hormonal specificity and regulation of reproduction. The results of gene expression revealed that a recombinant protein with a molecular weight of about 19 kDa was efficiently expressed and it was identified by Western blotting analysis. After administration of the purified protein, significantly higher expression levels were demonstrated in uterus, ovary and oviduct samples (p protein possesses biological activity, and has a potential role in regulation of reproductive physiology in chickens.

Thyroid hormone and reproduction in goldfish

Event Abstract Back to Event Thyroid hormone and reproduction in goldfish Hamid R. Habibi1, 2*, Erik R. Nelson1, 2 and Euan R O. Allan1, 2 1 University of Calgary, Department of Biological Sciences, Canada 2 Duke University Medical Center, Pharmacology and Cancer Biology, United States As in many vertebrates, fish reproduction is regulated by environmental cues such as photoperiod and temperature. During reproductive season a significant amount of metabolic energy is diverted to development of ovary and testis through precise regulation of brain-pituitary-peripheral axis by a number of key hormones, including pituitary gonadotropins and gonadal steroids. Since thyroid hormones are important regulator of metabolism and development in vertebrates, we investigated the possibility that thyroid hormones may mediate a change from gonadotropic to somatotropic state. Multiple experiments were performed across the four reproductive seasons to test the effects of triiodothyronine (T3) on estrogen and androgen receptors in the liver, testis and ovary as well as secretion of gonadal steroids and expression of pituitary glycoprotein hormone subunits (TSHβ, LHβ & FSHβ) and aromatase. Previous studies suggested that thyroid hormones influence reproduction in vertebrates, but little information is available on the mechanisms. In the present study, we test the hypothesis that thyroid hormones regulate pituitary hormone and gonadal steroid production in fish. The results demonstrate that thyroid hormone, (T3) regulates estrogen level as well as estrogen receptor subtypes (ER alpha, beta-1 and beta-II) in the testis and ovary of goldfish. We also observed that T3 influence pituitary gonadotropin and gonadal aromatase gene expression in goldfish. Collectively, it appears that T3 acts to diminish gonadotropic response by influencing hormone production as well as decreasing sensitivity to estrogen by down-regulating the estrogen receptor subtypes. We propose that thyroid hormones play a role in switching energy expenditure from reproductive to growth in goldfish. Funded by NSERC grants to HRH. Keywords: comparative endocrinology Conference: 25th Conference of the European Comparative Endocrinologists, Pécs, Hungary, 31 Aug - 4 Sep, 2010. Presentation Type: Conference Presentation Topic: Comparative endocrinology Citation: Habibi HR, Nelson ER and Allan EO (2010). Thyroid hormone and reproduction in goldfish. Front. Endocrinol. Conference Abstract: 25th Conference of the European Comparative Endocrinologists. doi: 10.3389/conf.fendo.2010.01.00006 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Aug 2010; Published Online: 29 Aug 2010. * Correspondence: Dr. Hamid R Habibi, University of Calgary, Department of Biological Sciences, Calgary, Canada, habibi@ucalgary.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Hamid R Habibi Erik R Nelson Euan R O Allan Google Hamid R Habibi Erik R Nelson Euan R O Allan Google Scholar Hamid R Habibi Erik R Nelson Euan R O Allan PubMed Hamid R Habibi Erik R Nelson Euan R O Allan Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Low-molecular regulators of polypeptide hormones receptors containing LGR-repeats

During the last years the low-molecular non-peptidic regulators of the polypeptide hormones receptors containing LGR-repeats were identified. In the review the data on the structure and the molecular mechanisms of action of these regulators as agonists and antagonists of the luteinizing, follicle-stimulating and thyrotropin hormones are analyzed and systematized. The regulators interact with the serpentine domain of LGR-receptor and trigger the signaling cascades coupled with the receptor. Low-molecular agonists and antagonists of the LGR-receptors are considered as a new generation of the drugs that regulates the functional activity of sensitive to pituitary glycoprotein hormones signaling systems with high efficiency and selectivity. These regulators are more accessible compared to the hormones and can be use orally.

The highly related LIM factors, LMO1, LMO3 and LMO4, play different roles in the regulation of the pituitary glycoprotein hormone α-subunit (αGSU) gene

LMO1, LMO3 and LMO4 were cloned from the adult porcine pituitary cDNA library. Amino acid sequences of porcine LMO1, LMO3 and LMO4 were highly conserved among mammalian species. Transfection assay of the pituitary-derived cell line L beta T2 was carried out using the pituitary alpha GSU (glycoprotein hormone alpha-subunit) promoter (-1059/+12 b) fused to pSEAP2-Basic vector as a reporter gene. The results demonstrated that, whereas LMO4 showed no apparent effect, alpha GSU promoter activity was markedly repressed by LMO1 but activated by LMO3, indicating the different roles of the three highly homologous proteins, LMO1, LMO3 and LMO4. Knockdown assay by LMO siRNAs (small interfering RNAs) confirmed the above results for LMO1 and LMO3, whereas that by LMO4 siRNA increased the expression, indicating different modes of action. RT-PCR (reverse transcription-PCR) for total RNAs of several cell lines showed that LMO1 and LMO4 mRNAs were present ubiquitously in all cell lines, except for LMO1 in L929 cells. In contrast, LMO3 mRNA was abundant only in L beta T4 and GH3 cells with only small amounts in L beta T2 and MtT/S cells, indicating the cell-type-specific function of this protein. Real-time analyses of porcine pituitary ontogeny revealed that the three LMO genes are expressed during the fetal period and decline immediately afterwards, followed by a remarkably low level of LMO3 and LMO4 after birth. RT-PCR of the porcine tissues examined showed ubiquitous expression of LMO4, whereas LMO1 and LMO3 are expressed tissue specifically. Thus the present study demonstrated that three highly related LIM cofactors, LMO1, LMO3 and LMO4, have different effects on alpha GSU gene expression in the pituitary glands.

Restriction fragment length polymorphisms at the ovine locus for the α-subunit of pituitary glycoprotein hormones

Restriction fragment length polymorphisms at the ovine locus for the α-subunit of pituitary glycoprotein hormones

Expression of the gene for ancestral glycoprotein hormone β subunit in the nerve cord of amphioxus

Amphioxus belongs to the subphylum cephalochordata, a clade of chordates phylogenetically placed at the most basal position. Despite many studies on the endocrine system of amphioxus, there were no confident lines of evidence on the presence of pituitary hormones, whereas recent amphioxus genome analysis reported that amphioxus has no pituitary hormone except for thyrostimulin, which is a glycoprotein hormone in the pituitary, brain, and other organs of vertebrates. In the present study, we cloned cDNA for one glycoprotein hormone β subunit (GPB) from amphioxus, AmpGPB5, and phylogenetically indicated that AmpGPB5 is the ancestral molecule of glycoprotein hormone β subunits of vertebrates including pituitary glycoprotein hormones. Synteny analyses showed conservation of chromosomal location of genes near GPB genes from amphioxus through human. The AmpGPB5 gene was expressed in a restricted region of the dorsal part of the nerve cord, glandular atrial cells of gills, and pre-vitellogenic oocytes in amphioxus. However, expression was not detected in the Hatschek’s pit which is considered to be a primitive pituitary gland. On the basis of present results, we hypothesize that a portion of vertebrate pituitary hormones might be derived from an ancestral glycoprotein hormone of amphioxus that functions as a neuroendocrine hormone.

Efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones

Complete dissociation into subunits was attained by incubating Chinese hamster ovary (CHO)-derived or native human thyrotropin, follitropin and lutropin overnight at 37°C in acetic acid. The α-and β-subunits of the pituitary glycoprotein hormones were rapidly and quantitatively isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). A dissociation efficiency of >98% was obtained on the basis of mass determinations of the heterodimers and subunits carried out via mass spectrometry. CHO-derived or native subunits were isolated on a C4 column (80–90% total recovery) and characterized comparatively for purity, hydrophobicity, molecular mass and charge distribution by HPLC, mass spectrometry, sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Thyrotropin was used as a model for showing that, after subunit reassociation, the in vivo bioactivity of the hormone was completely restored. The method described is mild, practical, flexible, and can be adapted to dissociate microgram amounts of native or recombinant glycoprotein hormones, allowing characterization of each subunit.

Regulation of Porcine Pituitary Glycoprotein Hormone Alpha Subunit Gene with LIM-Homeobox Transcription Factor Lhx3

The aim of this study was to characterize the promoter activity of the porcine pituitary glycoprotein hormone common alpha gene (Cga) promoter (-1059/+12) and the role of LIM homeodomain transcription factor Lhx3. A transfection assay using Chinese hamster ovary (CHO) cells showed that the -1059/-101 region of the Cga promoter definitely responds to Lhx3 and that the -1059/-240 region exhibits a high basal transcriptional level in a pituitary-derived cell line, LbetaT2. A DNA binding and DNase I footprinting assay demonstrated that Lhx3 has seven binding sites in the -1059/+12 region of Cga, including a pituitary glycoprotein hormone basal element (PGBE) known as a LIM homeodomain factor-binding site. A transfection assay of the sequence of Lhx3-binding sites fused with minimal promoter vector confirmed their Lhx3-dependent stimulations in LbetaT2 cells. RT-PCR analysis of porcine pituitary ontogeny demonstrated that porcine Lhx3 showed striking changes of expression in both sexes during the fetal period but a stable high level of expression after birth. Thus, the porcine Cga promoter is regulated by Lhx3 through seven sites in the distal and proximal regions.

Proteomic Analysis of the Mouse Ovary in Response to Two Gonadotropins, Follicle-Stimulating Hormone and Luteinizing Hormone

Functional and structural changes in the mammalian ovary are coordinately regulated by the pituitary glycoprotein hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), leading to follicular development, ovulation and transformation of follicles into corpus lutea. To investigate protein profiles during these processes of the mouse ovarian cycle, we applied combined methods (two-dimensional gel electrophoresis [2-DE] for separation and visualization of proteins plus matrix laser desorption/ionization time-of-flight mass spectrometry [MALDI-TOF/MS] analysis for protein identification) for comparative proteomic analysis using immature mice at 3 weeks of age. Protein profiles were obtained from proteins extracted from intact ovaries that had been collected from pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed immature mice at 0 (no PMSG), 24 and 48 h post PMSG, as well as at 10 and 20 h post hCG. The results showed that 1028 common protein spots were found in representative gels that had been separated in the 3 to 11 pH range and the 15-200 kDa range, 253 protein spots (24.6%) of which were differentially expressed (p<0.05) during the mouse ovarian cycle. Of these 253 protein spots, 99 were identified by MALDI-TOF/MS. This comparative proteomic approach to identifying proteins that were potentially involved in the complex process of the ovarian cycle could contribute to our understanding of the molecular basis of functional and structural changes in the ovary in response to gonadotropins. Furthermore, the interesting ovarian proteins identified in this study may eventually serve as diagnostic biomarker candidates of ovarian function.

Thyrotropin in teleost fish

Thyrotropin (TSH), a pituitary glycoprotein hormone that stimulates the thyroid gland, has been cloned and sequenced from over a dozen teleost fish species. Although TSH is established as a primary driver of systemic thyroid status in mammals, its importance in the regulation of fish thyroid function is still uncertain. We review recent studies indicating that TSH structure is highly conserved across species representing six teleost families. These studies have found TSH messenger RNA consistently expressed in teleost pituitary tissue, although ectopic expression, particularly in gonads, has also been observed. They have also provided evidence for negative feedback inhibition of TSH expression by thyroid hormones, as well as stimulation by hypothalamic peptides. Descriptive studies have found increased TSHβ expression associated with life history events thought to be promoted by thyroid hormones. These results, coupled with the discovery of a G-protein coupled TSH receptor in several teleost species, supports an active and conserved role for TSH in the regulation of teleost thyroid function. The relative importance of central pathways in regulating thyroid hormone provision to targets and the identity of a proposed thyrotropin-inhibiting factor in teleost fish are still unanswered questions whose resolution will be facilitated by development of methods to measure circulating TSH and its secretion from the pituitary gland.

Glycoprotein Hormone Receptors in the Sea Lamprey Petromyzon marinus

Secretion of the pituitary glycoprotein hormones (GpH) follitropin, lutropin, and thyrotropin in vertebrates is the main mechanism by which neuroendocrine signals are propagated at the level of the peripheral glands, gonads and thyroid. Receptors of these hormones (glycoprotein hormone receptors, GpH-R) evolved from a common ancestor through gene duplication and subsequent functional divergence during the split of gnathostomes from their agnathan ancestors. Here we review the properties of two novel receptors closely related to gnathostome GpH-Rs identified in the sea lamprey. Although these are the oldest members of this family of receptors described so far in vertebrates, their overall structural features are remarkably close to their mammalian counterparts. However, they cannot be classified unequivocally as either gonadotropin (FSH-R, LH-R) or as thyrotropin receptors (TSH-R) since they share characteristics with both these groups. This may indicate that lamprey receptors reflect in part properties of the ancestral molecule(s) from which all vertebrate GpH-Rs originated. Molecular phylogenetic relationships among gnathostome GpH-Rs are heavily dependent on the functional domain used in analysis. This suggests large variation in functional constraints acting at the level of different segments of the receptor molecule.

Sexually dimorphic expression of pituitary glycoprotein hormones in a sex‐changing fish (Pseudolabrus sieboldi)

It is widely accepted that the hypothalamic-pituitary-gonadal axis is involved in gonadal sex change in socially controlled sex-changing fish. However, the specific secretion profiles of pituitary gonadotropins (GtHs) in this type of fish are not known. To address this fundamental question, we demonstrated that the diurnal secretion patterns of GtHs differ distinctly between males and females in a socially controlled sex-changing fish. We analyzed the pituitary mRNA levels of glycoprotein hormone subunits (i.e., the common alpha-subunit and specific beta-subunits follicle-stimulating hormone beta, luteinizing hormone beta, and thyroid-stimulating hormone beta) in the wrasse Pseudolabrus sieboldi, which is a model fish that exhibits accurate diurnal rhythms of gametogenesis in both males and females. Northern blots clearly showed that each subunit gene exhibits a diurnal rhythm of expression in the pituitary and that the expression patterns differ distinctly between the sexes. Our results suggest that oogenesis and spermatogenesis in this hermaphroditic fish are regulated differentially through the distinct secretion patterns of pituitary glycoprotein hormones. This study also provides direct evidence of the sexual plasticity of pituitary GtH secretion in a socially controlled sex-changing fish.

Estrogen Receptor α Signaling Pathways Differentially Regulate Gonadotropin Subunit Gene Expression and Serum Follicle-Stimulating Hormone in the Female Mouse

Estrogen, acting via estrogen receptor (ER)alpha, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ERalpha signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ERalpha DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and -independent estrogen signaling in estrogen regulation of LH, FSH, prolactin, and activin/inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LHbeta mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary glycoprotein hormone alpha-subunit, FSHbeta, and activin-betaB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ERalpha signaling suppresses serum LH via reduced secretion, not synthesis. Additionally, ERE-dependent and ERE-independent ERalpha pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.

Effects of Pituitary Glycoprotein Hormones and Thyroid Hormones on In-Vitro Vitellogenin Incorporation into Organ-Cultured Oocytes in the Japanese Eel, Anguilla japonica

The objectives of the present study were to establish a long-term culture system for previtellogenic ovarian fragments of the Japanese eel (Anguilla japonica) and to identify the effects of salmon pituitary glycoprotein fraction (SPG), thyroxine (T4), and 3,5,3'-triiodothyronine (T3) on the uptake of vitellogenin (VTG) by cultured ovarian fragments evidenced by the appearance of yolk globules (YGs) within the oocytes. Yolk globules first appeared in the oocytes incubated in media containing only VTG (VTG-only group) after 9 days, whereas YGs began to accumulate in the oocytes of ovarian fragments cultured in media containing VTG+SPG (SPG group) following only 3 days of incubation. Furthermore, the occurrence of vitellogenic oocytes (%VO) and proportion of YGs within oocytes (%YG area) were significantly higher in follicles cultured in 30 ng/ml SPG throughout the culture period. No such stimulatory effects of T4 on VTG uptake were observed. Incubation of ovarian fragments with VTG and T3 (T3 group; 50 ng/ml) resulted in an increased %VO compared to follicles in the VTG group by day 9 of culture, and from day 10 onwards, both %VO and %YG area became significantly higher in follicles of the T3 group. Interestingly, SPG stimulated VTG incorporation and YG accumulation even in small oocytes (approximately 150 microm), whereas T3 showed these effects only in larger sized oocytes (> 180 microm). These results suggest that both SPG and T3 can accelerate VTG incorporation, but the mechanisms whereby this is achieved may differ between these hormone preparations.

Functional specificity of the rainbow trout (Oncorhynchus mykiss) gonadotropin receptors as assayed in a mammalian cell line

In vertebrates, gonadotropins (GTHs) (FSH and LH) are two circulating pituitary glycoprotein hormones that play a major role in the regulation of gonadal functions, including gonadal cell proliferation/differentiation and steroidogenesis. In mammals, it is well known that their biological effects are mediated by highly specific membrane-bound receptors expressed preferentially on the somatic cells of the gonads. However, in fish, binding and functional studies have shown that cross-reactivity may occur in GTH receptors depending on the species. To understand the molecular mechanisms involved in GTH actions, functional characterization of trout GTH receptors and their gonadal gene expression pattern has been carried out. The present study describes the presence of two distinct GTH receptors in trout showing similarities with those of higher vertebrates but also differences in their structural determinants. In vitro functional studies demonstrate that rtLH specifically activates its cognate receptor (EC(50) = 117 ng/ml), whereas purified rainbow trout FSH (rtFSH) activates FSHR but also LHR at supraphysiological doses (EC(50) = 38 vs 598 ng/ml for FSHR and LHR respectively). The high doses of rtFSH required to activate LHR put into question the physiological relevance of this interaction. The use of heterologous chinook GTHs confirms the strong preference of each hormone for its cognate receptor. The gonadal expression pattern of the GTH receptor genes suggests that FSH may play an important role in regulating gonadal functions, not only at the early stages but also at the final stages of the male and female reproductive cycles, in addition to the LH pathway.

NEUROENDOCRINOLOGY OF FISH METAMORPHOSIS AND PUBERTY: EVOLUTIONARY AND ECOPHYSIOLOGICAL PERSPECTIVES

Metamorphosis and puberty are two major events of the postembryonic development in Vertebrates. Based on some examples from fish species, we review the definition, role and regulation of these events, analyze their common and different features, as well as their impact on the evolution and diversity of life cycles. The term of puberty, firstly defined in humans, has been subsequently extended to the first acquisition of the capacity to reproduce in all mammalian and non-mammalian vertebrates as well as in invertebrates. By definition, puberty occurs only once in the life cycle. However, some similarities may be found with other events, such as annual re-activation of the reproductive function in seasonal breeders or sex-change in adults, as observed in some fish species. Metamorphosis allows the transition from one developmental stage in a specific environment to the next stage in a different environment, and includes a migration between the two habitats. Metamorphosis corresponds to drastic changes in body shape, physiology and behavior, and, unlike puberty, is encountered only in some phyla/species. In Vertebrates, the most described metamorphosis is the transformation in Amphibians of the aquatic larva (tadpole) into the terrestrial juvenile. Larval metamorphosis is also encountered in some other Vertebrates, such as lampreys and some teleosts (Elopomorphes and Pleuronectiformes). Less drastic morphological, physiological and behavioral changes occur in juveniles of some migratory teleosts. This is the case of smoltification in salmons and silvering in eels, which are referred to as ”secondary metamorphoses”. Investigations on the regulation of puberty and metamorphoses in Vertebrates reveal the crucial roles of the neuroendocrine axes. In all Vertebrates, puberty is triggered by the activation of the gonadotropic axis, constituted of brain neuropeptide (gonadotropin-releasing hormone, GnRH), pituitary glycoprotein hormones (gonadotropins: luteinizing hormone, LH and follicle stimulating hormone, FSH) and gonadal steroids. Sex steroids induce the morpho-physiological and behavioral transformations characteristic of puberty. Metamorphosis in Amphibians is triggered by the thyrotropic axis, constituted of brain neuropeptide corticotropin-releasing hormone (CRH), instead of thyrotropin-releasing hormone, (TRH), pituitary glycoprotein hormone (thyrotropin, TSH) and thyroid hormones (TH: thyroxine, T4 and triiodothyronine, T3), which play a key-role in the induction of morpho-physiological and behavioral changes. A similar control is suggested for larval metamorphosis in teleosts. Studies on smoltification also indicate an important role of thyroid hormones in secondary metamorphoses in teleosts, even though other hormones such as growth hormone (GH) and corticosteroids may be of prime importance. In contrast, recent investigations in the eel reveal that the gonadotropic axis, and ultimately sex steroids would be the major triggering control of silvering. The similarities of the morpho-physiological and behavioral changes between the two species indicate remarkable evolutionary convergences in the morphogenetic roles and target tissues of TH and sex steroids for the induction of secondary metamorphoses. In all cases, the possible synergistic role of cortisol is highlighted. Comparison of puberty and metamorphosis may also favor our understanding of the internal and environmental triggering signals of these postembryonic developmental events. In teleosts, the large plasticity in the occurrence and timing of metamorphosis and puberty, which contributes to the high diversity of fish life cycles, may provide new and relevant models to such investigations.

Pituitary glycoprotein hormone receptors in non-endocrine organs

Although glycoprotein hormones are usually regarded as pituitary-endocrine signals, their receptors can be found in non-endocrine tissues. High expression of selected receptors in the pituitary-endocrine axis is key to mammalian endocrine regulation. We hypothesize that peripheral receptor distribution during development and in secondary organs reflects older but still-applicable functions, with their concentration in the pituitary a more recent evolutionary advancement. We extrapolate additional functions of these receptors by analogy of homologous receptors in older phyla, with emphasis on the bony fishes (teleosts). Studies of the multiple roles of the glycoprotein hormone receptors are likely to uncover novel endocrine functions and axes, and highlight the potential of these receptors as novel therapeutic targets.

A Novel α-Helix in the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Is Critical for N-Glycan Polysialylation

Polysialic acid is a developmentally regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule, NCAM. Polysialylated NCAM is critical for brain development and plays roles in synaptic plasticity, axon guidance, and cell migration. The first fibronectin type III repeat of NCAM, FN1, is necessary for the polysialylation of N-glycans on the adjacent immunoglobulin domain. This repeat cannot be replaced by other fibronectin type III repeats. We solved the crystal structure of human NCAM FN1 and found that, in addition to a unique acidic surface patch, it possesses a novel alpha-helix that links strands 4 and 5 of its beta-sandwich structure. Replacement of the alpha-helix did not eliminate polysialyltransferase recognition, but shifted the addition of polysialic acid from the N-glycans modifying the adjacent immunoglobulin domain to O-glycans modifying FN1. Other experiments demonstrated that replacement of residues in the acidic surface patch alter the polysialylation of both N- and O-glycans in the same way, while the alpha-helix is only required for the polysialylation of N-glycans. Our data are consistent with a model in which the FN1 alpha-helix is involved in an Ig5-FN1 interaction that is critical for the correct positioning of Ig5 N-glycans for polysialylation.

Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone α subunit gene expression

Recently, we have reported that a Prophet of Pit-1 homeodomain factor, Prop-1, is a novel transcription factor for the porcine follicle-stimulating hormone beta subunit (FSHbeta) gene. This study subsequently aimed to examine the role of Prop-1 in the gene expression of two other porcine gonadotropin subunits, pituitary glycoprotein hormone alpha subunit (alphaGSU), and luteinizing hormone beta subunit (LHbeta). A series of deletion mutants of the porcine alphaGSU (up to -1059 bp) and LHbeta (up to -1277 bp) promoters were constructed in the reporter vector, fused with the secreted alkaline phosphatase gene (pSEAP2-Basic). Transient transfection studies using GH3 cells were carried out to estimate the activation of the porcine alphaGSU and LHbeta promoters by Prop-1, which was found to activate the alphaGSU promoter of -1059/+12 bp up to 11.7-fold but not the LHbeta promoter. Electrophoretic mobility shift assay and DNase I footprinting analysis revealed that Prop-1 binds to six positions, -1038/-1026, -942/-928, -495/-479, -338/-326, -153/-146, and -131/-124 bp, that comprise the A/T cluster. Oligonucleotides of six Prop-1 binding sites were directly connected to the minimum promoter of alphaGSU, fused in the pSEAP2-Basic vector, followed by transfecting GH3 cells to determine the cis-acting activity. Finally, we concluded that at least five Prop-1 binding sites are the cis-acting elements for alphaGSU gene expression. The present results revealed a notable feature of the proximal region, where three Prop-1-binding sites are close to and/or overlap the pituitary glycoprotein hormone basal element, GATA-binding element, and junctional regulatory element. To our knowledge, this is the first demonstration of the role of Prop-1 in the regulation of alphaGSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHbeta gene, confirm that Prop-1 modulates the synthesis of FSH at the transcriptional level. On the other hand, the defects of Prop-1 are known to cause dwarfism and combined pituitary hormone deficiency accompanying hypogonadism. Accordingly, the present observations provide a novel view to understand the hypogonadism caused by Prop-1 defects at the molecular level through the regulatory mechanism of alphaGSU and FSHbeta gene expressions.

Molecular cloning and histological localization of LH-like substances in a bottlenose dolphin (Tursiops truncatus) placenta

All mammals exhibit pituitary-specific expression of LH and FSH, whereas placental expression of gonadotropins has been reported only in primates and equids. Some cetaceans, such as dolphins, have a long gestational period and a sexual cycle of about 27 days almost comparable with that of humans. Histologically, dolphins have an epitheliochorial placentae that resembles placentas of Perissodactyla including horses. In the present study, we cloned cDNAs encoding gonadotropins and observed their immunohistochemical localization in the placenta of bottlenose dolphin. The cDNAs obtained encoded 120 amino acids for the alpha-subunit (including 96 amino acids of mature proteins), and 141 amino acids for the beta-subunit (including 121 amino acids of mature proteins). The sequence of the alpha-subunit was similar to that in the pig (Artiodactyla) pituitary glycoprotein hormone [96.7% homology at amino acids (aa) level], and the sequence of the beta-subunit was similar to that of luteinizing hormone (LH) in the pig [94.3% homology at aa level] and white rhinoceros (Perissodactyla) [93.3% homology at aa level]. Of interest, dolphin LHbeta lacks carboxyl-terminal-peptides (CTP). This fact suggests that CTP are not essential for placental expression of gonadotropin in dolphins. Immunohistochemical observations employing anti-ovine LHbeta antibody revealed positive staining in the villositycal tissue. Our observations suggest placental expression of gonadotropin homologues in cetaceans and possible evolutionary conservation of placentae-derived hormonal control of ovarian functions during pregnancy.