WITHDRAWN: Profiles in Comparative Endocrinology: Carl B. Schreck Howard Bern Lecturer, Annual Meeting of Society of Integrative and Comparative Endocrinology, Seattle, January 5, 2010.

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Functional characterization and kinetic studies of an ancestral lamprey GnRH-III selective type II GnRH receptor from the sea lamprey, Petromyzon marinus.

The recently cloned lamprey GnRH receptor was shown to have several unique features, including the longest intracellular C-terminal tail (120 amino acids (aa)) of any previously described GnRH receptor. In the current study, a series of experiments were performed examining cAMP responses, binding kinetics, whole cell competitive binding assays and internalization studies of the lamprey GnRH receptor using a series of three C-terminal tail truncations (80 aa, 40 aa and 0 aa) to better describe the functional significance of this unique vertebrate GnRH receptor. Activation of the lamprey GnRH receptor was shown to stimulate cAMP production in a dose-dependent manner when treated with either lamprey GnRH-I (LogEC50 -6.57+/-0.15) or lamprey GnRH-III (LogEC(50) -8.29+/-0.09). Truncation analysis indicated that the membrane proximal 40 aa of the lamprey GnRH receptor C-terminal tail contain a motif required for cAMP accumulation. Saturation binding assays using the wild type and truncated lamprey GnRH receptors revealed that all of three truncated lamprey GnRH receptors were capable of binding lamprey GnRH-I. Competitive, intact cell-binding assays suggested that the lamprey GnRH receptor is lamprey GnRH-III selective, based on the observed pharmacological profile: lamprey GnRH-III (Inhibitory constant (Ki) 0.708+/-0.245 nM)=chicken GnRH-II (Ki 0.765+/-0.160 nM) > mammalian GnRH (Ki 12.9+/-1.96 nM) > dAla(6)Pro(9)NEt mammalian GnRH (Ki 21.6+/-9.68 nM) > lamprey GnRH-I (Ki 118.0+/-23.6). Finally, the lamprey GnRH receptor was shown to undergo rapid ligand-dependent internalization, which was significantly diminished in the tail-less truncated form. We have shown from our current and our previous structural studies that this unique lamprey GnRH receptor shares several characteristics of both type I and type II GnRH receptors which suggests that this receptor has retained ancestral characteristics that can provide insight into the function and evolution of the vertebrate GnRH receptor family.

Primary structure and biological activity of a third gonadotropin-releasing hormone from lamprey brain.

Previous studies have led to the identification of two molecular forms of GnRH (GnRH-I and II) in the brain of the sea lamprey, Petromyzon marinus. From analysis of these two forms, the primary structure of GnRH-I and the amino acid composition of GnRH-II were determined. We have now isolated a third molecular form of GnRH (GnRH-III) from the brain of this species that is different from GnRH-I and -II. The primary structure of GnRH-III is pGlu-His-Trp-Ser-His-Asp-Trp-Lys- Pro-Gly-NH2. A synthetic decapeptide with this amino acid sequence was chromatographically identical to natural GnRH-III. Intraperitoneal injection of synthetic lamprey GnRH-III (0.1 microgram/g) produced a significant (P < 0.05) elevation of plasma progesterone (31% over basal values) in female lampreys that was comparable to that produced by the same dose of lamprey GnRH-I (36% over basal). The elevation in plasma estradiol produced by lamprey GnRH-III (244% over basal) was significantly (P < 0.05) less than the elevation produced by GnRH-I (322% over basal). We propose based on the biological activity of lamprey GnRH-III in these studies and the occurrence of this peptide during metamorphosis in lampreys, that both lamprey GnRH-I and -III are neurohormones involved in reproduction in lampreys.

Characterization and localization of gonadotropin-releasing hormone receptors in the adult female sea lamprey, Petromyzon marinus.

Quantitative in vitro autoradiography was used to characterize and localize putative GnRH receptors in the anterior pituitary of the adult female sea lamprey, Petromyzon marinus. Pituitaries were sectioned at 20 microns and incubated for 3 h at 4 C with DAla6,Pro9 NEt mammalian GnRH as both the labeled and unlabeled ligand. Scatchard analysis revealed two classes of high affinity binding sites with Kds of 1.5 x 10(-12) M and 5 x 10(-9) M. Binding to the GnRH receptors was saturable, reversible, tissue specific, and time and temperature dependent. Displacement studies showed that labeled peptide could be displaced by chicken GnRH-I, chicken GnRH-II, synthetic mammal, salmon, lamprey GnRH-I, lamprey GnRH-III, DAla6,Pro9 NEt mammalian GnRH and DPhe2,6,Pro3 lamprey GnRH. The proximal pars distalis region of the anterior pituitary contained most of the GnRH binding sites with slight binding in the rostral pars distalis. These data provide direct evidence of GnRH activity on the Agnathan pituitary and are the first to demonstrate that a vertebrate pituitary contains two high affinity binding sites for GnRH.

A novel glycoprotein in the olfactory and pituitary systems of larval and adult lampreys.

A novel homodimeric glycoprotein was isolated and characterized from the pituitaries of adult sea lampreys, Petromyzon marinus, modern representatives of the earliest vertebrates. The monomer consists of 121 amino-acid residues in a sequence that has no resemblance to any known pituitary hormone. Whereas this protein is localized in most cells of the rostral pars distalis of adult lampreys, we have chosen to name it, nasohypophysial factor (NHF), because it first appears in the olfactory system of developing larval lampreys. Not only may NHF be a new pituitary hormone but a useful probe for examining the ontogenetic and phylogenetic relationships of the pituitary and olfactory systems in vertebrates.

Comparative biological properties of lamprey gonadotropin-releasing hormone in vertebrates.

The biological activities of lamprey GnRH were determined in the lamprey, chicken, sheep, and rat. Lamprey GnRH elevated plasma steroid levels and stimulated ovulation in the lamprey, but had little or no LH-releasing activity in chicken and sheep pituitary bioassays or effect on GnRH receptor-binding activity in the rat pituitary. The lamprey GnRH molecule is structurally distinct from other known vertebrate GnRHs and is the first identified molecule in this family to have different amino acids in the third and sixth positions. These data suggest that the presence of Tyr3 or Glu6 in lamprey GnRH may account for the lack of biological activity in the representatives of the two different vertebrate classes investigated in this study.

Two molecular forms of gonadotropin-releasing hormone from the brain of the frog, Rana ribibunda: purification, characterization, and distribution.

The molecular forms of GnRH in amphibia have not been characterized structurally. An antiserum to mammalian GnRH that shows appreciable cross-reactivity with chicken GnRH-I ([Gln8]GnRH), chicken GnRH-II ([His5,Trp7,Tyr8]GnRH), and salmon GnRH ([Trp7, Leu8]GnRH) was used in a RIA to detect GnRH-related peptides in an extract of the brain of the European green frog, Rana ridibunda. Two peptides were purified to apparent homogeneity, and determination of their primary structures showed that they are identical to mammalian GnRH and chicken GnRH-II. Salmon GnRH and lamprey GnRH-I ([Tyr3,Leu5,Glu6,Trp7,Lys8]GnRH) were not identified in this species of frog. Immunocytochemical analysis using specific antisera has identified regions of the frog diencephalon and telencephalon in which chicken GnRH-II and mammalian GnRH are localized to different populations of neurons. This differential distribution suggests distinct physiological roles for the two forms of GnRH in amphibia.

Relationship of gonadotropin-releasing hormone (GnRH) neurons to the olfactory system in developing lamprey (Petromyzon marinus).

Gonadotropin releasing-hormone (GnRH) regulates the hypothalamo-pituitary-gonadal axis in vertebrates. The regulation of GnRH is intimately related to information from the olfactory system. Additionally, GnRH neurons are thought to be derived from progenitor cells in medial olfactory placodes. The present experiments were conducted to characterize the earliest development of GnRH neurons in lamprey and to determine their relationship to cells and fibers derived from the olfactory system. Eggs from fertile adult sea lamprey were fertilized in the laboratory, and larvae were maintained for up to 100 days. GnRH neurons were visualized within the lamprey preoptic area and hypothalamus as soon as GnRH was detectable (22 days after fertilization). The number of neurons increased with age through day 100. GnRH neurons were never seen within the olfactory system. The cells and fibers of the olfactory system were identified using the lectin, Grifonia Simplicifolia-1 (GS-1). Overlap between the olfactory and GnRH systems were at the level of fiber projections. GS-1 reactive cells of apparent placodal origin did not enter the region of the preoptic area or hypothalamus that contained GnRH neurons. Recently divided cells were labeled with the thymidine analog, bromodeoxyuridine (BrdU). The positions of BrdU-labeled cells after different survival times suggest a predominant medial-lateral radial neuron migration with a small number in positions suggestive of migration between the olfactory epithelium and the telencephalic lobes. Regardless of survival time, these cells were always found close to their entry point into the brain, suggesting minimal rostral-caudal migration. Based on these results, we hypothesize that GnRH neurons in developing lamprey originate within proliferative zones of the diencephalon and not in the olfactory system. Based on the overlap of olfactory- and GnRH-containing fibers from prolarval stages to metamorphosis, olfactory stimuli may play a major role in the regulation of GnRH secretion in lamprey.

Multiple transcripts encoding lamprey gonadotropin-releasing hormone-I precursors.

The cDNA encoding lamprey prepro-gonadotropin releasing hormone-I (lamprey GnRH-I) has been isolated and sequenced in an agnathan, the sea lamprey, Petromyzon marinus. The lamprey GnRH-I precursor is the first identified in an ancient lineage of vertebrates and has the same overall tripartite structure as other vertebrate GnRH precursors. The amino acid sequence of lamprey GnRH-I and the processing site (Gly-Lys-Arg) are highly conserved during 500 million years of evolution with 60-70% identity compared with those of tetrapod and teleost GnRH precursors. In contrast, the GnRH associated peptide regions are markedly divergent, with less than 20% identity compared with all identified vertebrate precursors. Unlike all other known vertebrate GnRH precursors, which typically have one and in a single case two transcripts, three distinct transcripts were isolated and sequenced in lampreys. These lamprey GnRH-I transcripts, termed GAP49, GAP50 and GAP58, differed in the length of the GAP coding sequence and were demonstrated to be the products of a single gene. Analysis of the lamprey GnRH-I gene intron-2 splice junction demonstrated that alternate splicing produces the different lamprey GnRH-I transcripts. Lamprey GnRH-I is the first GnRH gene demonstrated to utilize splice sequence variants to produce multiple transcripts, which may reflect an ancestral gene regulatory mechanism.

Limb malformations and abnormal sex hormone concentrations in frogs.

Declines in amphibian populations, and amphibians with gross malformations, have prompted concern regarding the biological status of many anuran species. A survey of bullfrogs, Rana catesbeiana, and green frogs, Rana clamitans, conducted in central and southern New Hampshire showed malformed frogs at 81% of the sites sampled (13 of 16 sites). Brain gonadotropin-releasing hormone (GnRH) and the synthesis of androgens and estradiol, hormones essential to reproductive processes, were measured from limb-malformed and normal (no limb malformation) frogs. Normal frogs had significantly higher concentrations (nearly 3-fold) of in vitro produced androgens and of brain GnRH than malformed frogs. Because most malformations are thought to occur during development, we propose that environmental factors or endocrine-disrupting chemicals that may cause developmental abnormalities also act during early development to ultimately cause abnormally reduced GnRH and androgen production in adult frogs. The consequences of reduced GnRH and androgens on anuran reproductive behavior and population dynamics are unknown but certainly may be profound and warrant further research.

Polygenic expression of somatostatin in lamprey.

Previous studies have led to the identification of three biosynthetically related molecular forms of somatostatin (somatostatin-14, -34 and -37) from the pancreas of the sea lamprey (Petromyzon marinus). We have now isolated from the brain of the same species a second form of somatostatin-14 that is identical to mammalian somatostatin-14 and differs from lamprey pancreatic somatostatin-14 by the substitution Ser12 to Thr. Larger forms of somatostatin were not identified in lamprey brain in this study. These data suggest that the two molecular forms of lamprey somatostatin-14 are the products of different genes that are expressed in a tissue-specific manner.

Multiple molecular forms of gonadotropin-releasing hormone in the brain of an elasmobranch: evidence for IR-lamprey GnRH.

These studies investigated brains of skate, Raja erinacea (order Rajiformes, class Chondrichthyes), for gonadotropin-releasing hormone (GnRH) peptides by chromatograph and immunoreactivity with region-specific antisera raised against mammalian GnRH and lamprey GnRH. The region-specific antibody to lamprey GnRH-I was produced following conjugation to bovine serum albumin using the bis-diazotized benzidine method. This antibody was characterized by assaying a range of increasing dilutions of the known vertebrate GnRHs, as well as analogs to lamprey GnRH-I. Two analogs, lamprey [Phe2]GnRH-I and lamprey [Leu7]GnRH-I, were synthesized by solid phase peptide synthesis using a benzhydrylamine resin as the supporting medium and purified by chromatography. This antibody demonstrated less than 0.01% cross-reactivity with all GnRH peptides tested, suggesting a highly specific antibody with a region of amino acids 2-8 that appears essential for binding. In the skate brain, five immunoreactive (IR) GnRH forms were detected, four of which eluted in the same positions as synthetic mammal and chicken GnRH-I (which coelute): lamprey GnRH-I, salmon and chicken GnRH-II, and one that was an unidentified form. A minor peak coeluted with lamprey GnRH-III. The major form in the skate brain is considered to have eluted with synthetic mammalian GnRH. These studies confirm an earlier report of an IR-mammalian GnRH peptide and provide new evidence for IR-lamprey GnRH in the brain of an elasmobranch.

Polygenic expression of gonadotropin-releasing hormone (GnRH) in human?

A non-mammalian lamprey-like gonadotropin-releasing hormone (lGnRH) has been detected in human hypothalami using a combination of immunocytochemistry, high performance liquid chromatography and radioimmunoassay. The hypothalamic distribution of immunopositive lGnRH neurons is similar to that observed for those containing the mammalian gonadotropin-releasing hormone (mGnRH), indicating a possible role for this newly identified peptide in the regulation of pituitary function. Our data suggest the existence of a separate gene for lamprey-like GnRH in humans. Confirmation of the exact nature and role of this newly detected form of GnRH will require future isolation and sequence analysis. The possibility that polygenic expression of a given peptide may be a common phenomenon even in higher mammals is discussed.

Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain.

A highly specific antiserum against lamprey gonadotropin-releasing hormone (GnRH) was used to localize 1-GnRH in areas of the rat brain associated with reproductive function. Immunoreactive 1-GnRH-like neurons were observed in the ventromedial preoptic area (POA), the region of the diagonal band of Broca and the organum vasculosum lamina terminalis, with fiber projections to the rostral wall of the third ventricle and the organum vasculosum lamina terminalis. Another population of 1-GnRH-like neurons was localized in the dorsomedial and lateral POA, with nerve fibers projecting caudally and ventrally to terminate in the external layer of the median eminence. Other fibers apparently projected caudally and circumventrically to terminate around the cerebral aqueduct in the mid-brain central gray. By using a highly specific antiserum directed against mammalian luteinizing hormone-releasing hormone (m-LHRH), the localization of the LHRH neuronal system was compared to that of the 1-GnRH system. There were no LHRH neurons in the dorsomedial or the lateral region of the POA that contained the 1-GnRH neurons. As expected, there was a large population of LHRH neurons in the ventromedial POA associated with the diagonal band of Broca and organum vasculosum lamina terminalis. In both of these regions, there were many more LHRH neurons than 1-GnRH neurons and the LHRH neurons extended more dorsally and laterally than the 1-GnRH neurons. The LHRH neurons seemed to project to the median eminence in the same areas as those that were innervated by the 1-GnRH neurons. Absorption studies indicated that 1-GnRH cell bodies were eliminated by adding 1 microg of either 1-GnRH-I or 1-GnRH-III, but not m-LHRH to the antiserum before use. Fibers were largely eliminated by the addition of 1 microg 1-GnRH-III to the antiserum. No chicken GnRH-II neurons or nerve fibers could be visualized by immunostaining. Because the antiserum recognized GnRH-I and GnRH-III equally, we have visualized an 1-GnRH system in rat brain. The results are consistent with the presence of either one or both of these peptides within the rat hypothalamus. Because 1-GnRH-I has only weak nonselective gonadotropin-releasing activity, whereas 1-GnRH-III is a highly selective releaser of follicle-stimulating hormone, and because 1-GnRH neurons are located in areas known to control follicle-stimulating hormone release selectively, our results support the hypothesis that 1-GnRH-III, or a closely related peptide, may be mammalian follicle-stimulating hormone-releasing factor.

Purification of a neuropeptide Y-related peptide from the brain of the sea lamprey and its effect on steroidogenesis.

A peptide with neuropeptide Y-like immunoreactivity was identified by radioimmunoassay in an extract of the brain of the sea lamprey, Petromyzon marinus using an antiserum raised against the conserved COOH-terminal region of mammalian neuropeptide Y. Purification of the peptide and determination of its primary structure showed that it was identical to peptide methionine-tyrosine (PMY), previously isolated from the intestine of the same species. Intraperitoneal injection of synthetic PMY (0.15 micrograms/g) into female lampreys undergoing final maturation before spawning produced a significant (P < 0.05) decrease in plasma concentrations of estradiol compared with control lampreys injected with vehicle only. These data suggest the hypothesis that the observed decrease in the concentration of PMY-containing cells in the intestines of lampreys during upstream migration may correlate with the increase in circulating estradiol concentrations and final maturational processes.

A new family member for gonadotropin-releasing hormone.

The two living representatives of the most ancient vertebrates, Agnathans, are lamprey and hagfish. Using immunological methods, we identified gonadotropin-releasing hormone (GnRH)-like molecules in the lamprey brain, but not hagfish. The lamprey GnRH was detected poorly by antisera directed at the C-terminus, suggesting that a C-terminal amino acid substitution may have occurred in the lamprey molecule compared with mammalian GnRH. In spite of this, lamprey and mammalian GnRH-like molecules have the same retention time on an isocratic HPLC system and parallel inhibition of mammalian 125I-GnRH in a radioimmunoassay. The lamprey GnRH-like molecule has a distinct HPLC elution pattern compared with dogfish shark, salmon, trout and probably birds. Thus lamprey GnRH represents another member of the growing family of GnRH molecules. Additionally, lamprey GnRH may be a stem molecule in the vertebrate evolution of GnRH.

In vivo effects of lamprey GnRH-I and cyclized analogs: a structure-activity study.

Lamprey GnRH-I and lamprey GnRH-III are the only two members of the GnRH family to have substitutions in the sixth position, Glu6 and Asp6, respectively; all other GnRH peptides have Gly in the sixth position suggesting a different conformational structure. Thus, a structure-activity study of lamprey GnRH-I or analogs that were cyclized or with sixth position substitutions were determined in vivo in adult female sea lamprey, Petromyzon marinus. The following analogs which were tested, ([D-Glu6]-GnRH-I; cyclo-[D-Glu6-Trp7-Lys8]-GnRH-I; or cyclo-[Glu6-Trp7-Lys8]-GnRH-I), significantly elevated plasma estradiol compared to controls. However, [D-Glu6]-lamprey GnRH-I was the only analog to significantly stimulate ovulation while another analog [Gly6]-lamprey GnRH-I significantly delayed ovulation. These data suggest that the sixth position of lamprey GnRH is critical for function.

Gonadotropin-releasing hormone containing neurons and olfactory fibers during development: from lamprey to mammals.

Gonadotropin releasing-hormone (GnRH) regulates the hypothalamo-pituitary-gonadal axis in all vertebrates. The vast majority of GnRH neurons are thought to be derived from progenitor cells in medial olfactory placodes. Several antibodies and lectins that recognize cell surface carbohydrates have been useful for delineating the migratory pathway from the olfactory placodes and vomeronasal organ, through the nasal compartment, and across the cribriform plate into the brain. In rats, alpha-galactosyl-linked glycoconjugates (immunoreactive with the CC2 monoclonal antibody) are expressed on fibers along the GnRH migration pathway and approximately 10% of the GnRH neuronal population. In lamprey, the alpha-galactosyl binding lectin, Grifonia simplicifolia-I (GS-1), identifies cells and fibers of the developing olfactory system. In contrast to the CC2 immunoreactive GnRH neurons in rats, the GS-1 does not label a subpopulation of presumptive GnRH neurons in lamprey. Results from these and other experiments suggest that GnRH neurons in developing lamprey do not originate within the olfactory placode, but rather within proliferative zones of the diencephalon. However, the overlap of olfactory- and GnRH-containing fibers from prolarval stages to metamorphosis, suggest that olfactory stimuli may play a major role in the regulation of GnRH secretion in lamprey throughout life. By contrast, olfactory fibers are directly relevant to the migration of GnRH neurons from the olfactory placodes in mammalian species. Primary interactions between olfactory fibers and GnRH neurons are likely transient in mammals, and so in later life olfactory modulation of GnRH secretion is likely to be indirect.

Isolation of a peptide structurally related to mammalian corticostatins from the lamprey Petromyzon marinus.

Peptides in an extract of skin from the agnathan Petromyzon marinus (sea lamprey) were purified by reversed-phase high-performance liquid chromatography and characterized by Edman degradation. The primary structure of a cysteine- and arginine-rich peptide (termed lamprey corticostatin-related peptide [LCRP]) was established as Cys-Pro-Cys-Gly-Arg-Arg-Arg-Cys-Cys-Val-Arg-Gly-Leu-Asn-Val-Tyr-Cys-Cys- Phe. Mass spectrometry indicated that all cysteine residues are intramolecularly linked. This amino acid sequence shows structural similarity to rat corticostatin R4 and rabbit corticostatin R1. In particular, LCRP contains the polyarginine sequence at the N-terminus of the peptide that is believed to mediate both the inhibition of ACTH stimulated steroidogenesis and the antimicrobial (defensin-like) actions of the corricostatins.

Update: brain and pituitary hormones of lampreys.

Lampreys and hagfish of the class Agnatha are of particular importance in understanding endocrinological relationships since they represent the oldest lineages of extant vertebrates which evolved over 550 million years ago. This review briefly summarizes the latest findings on the reproductive endocrinology of the sea lampreys. Since the First International Symposium of Fish Endocrinology in 1988, when virtually little was known of the hypothalamic-pituitary-gonadal axis, substantial new biochemical, molecular, physiological and immunological evidence has now clearly shown that lamprey reproduction is controlled by the neuroendocrine axis. In addition, five brain and six pituitary hormones of lampreys have been identified mainly by Sower and Kawauchi and colleagues between 1986 and 2000. We now hypothesize that lamprey reproduction is a highly synchronized process that is initiated or mediated by a coordination of complex integration of environmental cues and hormonal mechanisms which is broadly similar to that exhibited by gnathostome vertebrates.