Lamprey GnRH-I Research Articles

Evidence that progestins play an important role in spermiation and pheromone production in male sea lamprey (Petromyzon marinus)

Progestins (progestogens, C21 steroids) have been shown to regulate key physiological activities for reproduction in both sexes in all classes of vertebrates except for Agnathans. Progesterone (P) and 15α-hydroxyprogesterone (15α-P) have been detected in sea lamprey (Petromyzon marinus) plasma, but the expression patterns and functions of putative progestin receptor genes have not yet been investigated. The first objective of this study was to determine the differences in mRNA expression levels of nuclear progestin receptor (nPR) and the membrane receptor adaptor protein ‘progesterone receptor membrane component 1’ (pgrmc1) in putative target tissues in males at different life stages, with and without lamprey GnRH-I and -III treatment. The second objective was to demonstrate the function of progestins by implanting prespermiating males (PSM) with time-release pellets of P and measuring the latency to the onset of spermiation and plasma concentrations of sex pheromones and steroids. The third objective was to measure the binding affinity of P in the nuclear and membrane fractions of the target tissues. Expression levels of nPR and pgrmc1 differed between life stages and tissues, and in some cases were differentially responsive to lamprey GnRH-I and -III. Increases in nPR and pgrmc1 gene expressions were correlated to the late stages of sexual maturation in males. The highest expression levels of these genes were found in the liver and gill of spermiating males. These organs are, respectively, the site of production and release of the sex pheromone 3 keto-petromyzonol sulfate (3kPZS). The hypothesis that pheromone production may be under hormonal control was tested in vivo by implanting PSM with time-release pellets of P. Concentrations of 3kPZS in plasma after 1week were 50-fold higher than in controls or in males that had been implanted with androstenedione, supporting the hypothesis that P is responsible for regulating the production of the sex pheromone. P treatment also accelerated the onset of spermiation. Saturation and Scatchard analyses of the target tissues showed that both nuclear and membrane fractions bound P with high affinity and low capacity (KD 0.53pmol/g testis and 0.22pmol/g testis, and Bmax 1.8 and 5.7nM, respectively), similar to the characteristics of nPR and mPR in other fish. The fact that a high proportion of P was also converted in vivo to 15α-P means that it is not yet possible to determine which of these two steroids is the natural ligand in the sea lamprey.

Pheromonal bile acid 3-ketopetromyzonol sulfate primes the neuroendocrine system in sea lamprey.

BackgroundVertebrate pheromones are known to prime the endocrine system, especially the hypothalamic-pituitary-gonadal (HPG) axis. However, no known pheromone molecule has been shown to modulate directly the synthesis or release of gonadotropin releasing hormone (GnRH), the main regulator of the HPG axis. We selected sea lamprey (Petromyzon marinus) as a model system to determine whether a single pheromone component alters the output of GnRH.Sea lamprey male sex pheromones contain a main component, 7α, 12α, 24-trihydroxy-5α-cholan-3-one 24-sulfate (3 keto-petromyzonol sulfate or 3kPZS), which has been shown to modulate behaviors of mature females. Through a series of experiments, we tested the hypothesis that 3kPZS modulates both synthesis and release of GnRH, and subsequently, HPG output in immature sea lamprey.ResultsThe results showed that natural male pheromone mixtures induced differential steroid responses but facilitated sexual maturation in both sexes of immature animals (χ2 = 5.042, dF = 1, p < 0.05). Exposure to 3kPZS increased plasma 15α-hydroxyprogesterone (15α-P) concentrations (one-way ANOVA, p < 0.05) and brain gene expressions (genes examined: three lamprey (l) GnRH-I transcripts, lGnRH-III, Jun and Jun N-terminal kinase (JNK); one-way ANOVA, p < 0.05), but did not alter the number of GnRH neurons in the hypothalamus in immature animals. In addition, 3kPZS treatments increased lGnRH peptide concentrations in the forebrain and modulated their levels in plasma. Overall, 3kPZS modulation of HPG axis is more pronounced in immature males than in females.ConclusionsWe conclude that a single male pheromone component primes the HPG axis in immature sea lamprey in a sexually dimorphic manner.

Effects of lamprey PQRFamide peptides on brain gonadotropin-releasing hormone concentrations and pituitary gonadotropin-β mRNA expression

Within the RFamide peptide family, PQRFamide peptides that include neuropeptide FF and AF possess a C-terminal Pro–Gln–Arg–Phe–NH2 motif. We previously identified PQRFamide peptides, lamprey PQRFa, PQRFa-related peptide (RP)-1 and -RP-2 by immunoaffinity purification in the brain of lamprey, one of the most ancient vertebrate species [13]. Lamprey PQRFamide peptide precursor mRNA was expressed in regions predicted to be involved in neuroendocrine regulation in the hypothalamus. However, the putative function(s) of lamprey PQRFamide peptides (PQRFa, PQRFa-RP-1 and PQRFa-RP-2) were not examined nor was the distribution of PQRFamide peptides examined in other tissues besides the brain. The objective of this study was to determine tissue distribution of lamprey PQRFamide peptide precursor mRNA, and to examine the effects of PQRFamide peptides on brain gonadotropin-releasing hormone (GnRH)-I, -II, and -III protein concentrations, and pituitary gonadotropin (GTH)-β mRNA expression in adult lampreys. Lamprey PQRFamide peptide precursor mRNA was expressed in the eye and the brain. Lamprey PQRFa at 100μg/kg increased brain concentrations of lamprey GnRH-II compared with controls. PQRFa, PQRFa-RP-1 and PQRFa-RP-2 did not significantly change brain protein concentrations of either lamprey GnRH-I, -III, or lamprey GTH-β mRNA expression in the pituitary. These data suggest that one of the PQRFamide peptides may act as a neuroregulator of at least the lamprey GnRH-II system in adult female lamprey.

A role of Histidine 151 in the lamprey gonadotropin-releasing hormone receptor-1 (lGnRHR-1): Functional insight of diverse amino acid residues in the position of Tyr of the DRY motif in GnRHR from an ancestral type II receptor

The highly conserved DRY motif located at the end of the third transmembrane of G-protein-coupled receptors has been described as a key motif for several aspects of GPCR functions. However, in the case of the vertebrate gonadotropin-releasing hormone receptor (GnRHR), the amino acid in the third position in the DRY motif is variable. In the lamprey, a most basal vertebrate, the third amino acid of the “DRY” in lamprey (lGnRHR-1) is His, while it is most often His/Gln in the type II GnRHR. To investigate the functional significance of the substitution of DRY to DRH in the GnRHR-1, second messenger signaling, ligand binding and internalization of the wild-type and mutant lGnRH receptors were characterized with site-directed mutagenesis. Treatment of the DRE 151 and DRS 151 mutant receptors with lamprey GnRH-I significantly reduced inositol phosphate compared to wild-type (DRH 151) and DRY 151 receptors. The Log IC 50 of wild-type receptor (−9.554 ± 0.049) was similar to the Log IC 50 of DRE 151, DRS 151 and DRX 151 mutants, yet these same mutants were shown to significantly reduce cell-surface expression. However, the DRY 151 mutant compared to the wild-type receptor increased cell-surface expression, suggesting that the reduction of IP production was due to the level of the cell-surface expression of the mutant receptors. The rate of internalization of DRX 151 (35.60%) was reduced compared to wild-type and other mutant receptors. These results suggest that His 151 of the lamprey GnRH receptor-1 may play a critical role in the retention of a certain level of cell-surface expression for subsequent cellular second messenger events.

Gonadotropin-releasing hormone (GnRH) and a GnRH analog induce ovarian maturation in the giant freshwater prawn, Macrobrachium rosenbergii

Gonadotropin-releasing hormone (GnRH) is a highly conserved peptide that plays a role in regulating reproduction in both vertebrates and invertebrates. The present study investigated the effects of lamprey (l)GnRH-I, lGnRH-III, octopus (oct)GnRH and buserelin, a GnRH analog (GnRHa), on ovarian maturation and spawning in the giant freshwater prawn, Macrobrachium rosenbergii. We demonstrated that the times for ovarian maturation in prawns treated with octGnRH (at 50 and 500 ng/g BW), lGnRH-I and lGnRH-III (at 500 ng/g BW), and GnRHa (at 1,000 ng/g BW) were significantly shorter (23.50 ± 2.12 and 22.50 ± 1.15, 25.50 ± 4.04 and 25.50± 4.03, and 26.67 ± 4.04 days) than the controls (40.0 ± 3.40 days). On day 22 post-treatment, the gonadosomatic index (GSI) values of the prawns treated with octGnRH (at 50 and 500 ng/g BW), lGnRH-I and lGnRH-III (at 500 ng/g BW), and GnRHa (at 1,000 ng/g BW) were significantly greater (2.50 ± 0.72 and 4.64 ± 0.95, 6.07 ± 1.29 and 8.90 ± 1.04, and 3.88 ± 1.34%) than the controls (0.45 ± 0.15%). Vitellin protein was first detected in the ovary on day 15 and had increased significantly by day 22 in prawns treated with GnRHs at 500 ng/g BW, while it was not detected in the controls. The prawns treated with the GnRHs and GnRHa showed similar numbers and percentages of spawned and fertilized eggs to those of the control group. These findings indicate that GnRH controls ovarian maturation and spawning in this prawn, as in other species.

The existence of gonadotropin-releasing hormone (GnRH) immunoreactivity in the ovary and the effects of GnRHs on the ovarian maturation in the black tiger shrimp Penaeus monodon

Immunocytochemical localization using antibodies against five isoforms of gonadotropin-releasing hormone (GnRH), namely, luteinizing hormone-releasing hormone (LHRH), salmon (s)GnRH, octopus (oct)GnRH, lamprey (l)GnRH-I, and lGnRH-III, showed that only lGnRH-I immunoreactivity (ir-lGnRH-I) was localized in follicular cells of proliferative, vitellogenic, and mature ovaries. The effects of exogenous GnRHs on the ovarian maturation cycle of Penaeus monodon were compared by treating female broodstocks with LHRH, sGnRH, and lGnRH-I. The cycle of ovarian maturation in both eyestalk-ablated and eyestalk-intact shrimp administered with the three isoforms of GnRH was significantly shorter than that of the control animals. Moreover, administrations of all GnRH isoforms showed similar numbers of spawned eggs and the percentage of successful fertilization as in the control animals. These findings suggest that GnRHs may be highly conserved peptides that play an important role in inducing the ovarian maturation in the shrimp.

Neuroendocrine and behavioral responses to weak electric fields in adult sea lampreys ( Petromyzon marinus)

We characterized the behavioral and neuroendocrine responses of adult sea lampreys ( Petromyzon marinus) to weak electric fields. Adult sea lampreys, captured during upstream spawning migration, exhibited limited active behaviors during exposure to weak electric fields and spent the most time attached to the wall of the testing arena near the cathode (−). For adult male sea lampreys, exposure to weak electric fields resulted in increased lamprey (l) GnRH-I mRNA expression but decreased lGnRH-I immunoreactivities in the forebrain, and decreased Jun (a neuronal activation marker) mRNA levels in the brain stem. Similar effects were not observed in the brains of female sea lampreys after weak electric field stimulation. The influence of electroreception on forebrain lGnRH suggests that electroreception may modulate the reproductive systems in adult male sea lampreys. The changes in Jun expression may be associated with swimming inhibition during weak electric field stimulation. The results for adult sea lampreys are the opposite of those obtained using parasitic-stage sea lampreys, which displayed increased activity during and after cathodal stimulation. Our results demonstrate that adult sea lampreys are sensitive to weak electric fields, which may play a role in reproduction. They also suggest that electrical stimuli mediate different behaviors in feeding-stage and spawning-stage sea lampreys.

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.

Identification of sea lamprey GTHβ-like cDNA and its evolutionary implications

We have identified the first and perhaps only gonadotropin β-like protein by cDNA cloning in sea lamprey, a member of the oldest lineage of vertebrates, the agnathans. Two pituitary gonadotropins (GTHs: follicle-stimulating hormone (FSH) and luteinizing hormone (LH)) have been identified in representative species of all classes of vertebrates except the agnathans. The present study was undertaken to identify GTH in sea lamprey, Petromyzon marinus, to gain a further understanding of the origin and evolution of reproductive pituitary hormones and their respective genes in vertebrates. Sea lamprey preGTHβ-like cDNA was cloned from a plasmid cDNA library using an expressed sequence tag analysis. The preGTHβ-like cDNA encoded 150 amino acids, in which the GTHβ-like protein consisted of 134 amino acid residues. Sea lamprey GTHβ-like protein contained 12 Cys residues and two N-glycosylation sites at homologous positions to those of FSHβ and LHβ. The region of the molecule that has been proposed to control receptor binding specificity (i.e., the region between the 10th and 12th Cys residues) suggests that the proposed heterodimer would be more like a FSH than a LH. Sea lamprey GTHβ-like protein-producing cells were identified immunocytochemically in the ventral part of the proximal pars distalis of pituitary using antiserum prepared against a synthetic peptide of preGTHβ-like protein (52–68). Intraperitoneal administration of sea lamprey GnRH-I and -III at 100 μg/g body weight (twice at a 24 h interval) increased expression of GTHβ-like protein in the pituitary of adult female sea lamprey during the final maturational period. Thus, these results are the first to demonstrate the presence of a single GTH-like system in lampreys. Because the sea lamprey GTHβ-like protein is a clear out-group compared to those of the LH and FSH family based on phylogenic analysis, we propose that an ancestral glycoprotein hormone gave rise to only one GTH in lampreys and to the glycoprotein hormone family that gave rise to LH, FSH, and TSH during the early evolution of gnathostomes.

Cloning and Characterization of a Functional Type II Gonadotropin-Releasing Hormone Receptor with a Lengthy Carboxy-Terminal Tail from an Ancestral Vertebrate, the Sea Lamprey

A full-length transcript encoding a functional type II GnRH receptor was cloned from the pituitary of the sea lamprey, Petromyzon marinus. The current study is the first to identify a pituitary GnRH receptor transcript in an agnathan, which is the oldest vertebrate lineage. The cloned receptor retains the conserved structural features and amino acid motifs of other known GnRH receptors and notably includes a C-terminal intracellular tail of approximately 120 amino acids, the longest C-terminal tail of any vertebrate GnRH receptor identified to date. The lamprey GnRH receptor was shown to activate the inositol phosphate (IP) signaling system; stimulation with either lamprey GnRH-I or lamprey GnRH-III led to dose-dependent responses in transiently transfected COS7 cells. Furthermore, analyses of serially truncated lamprey GnRH receptor mutants indicate perturbations of the C-terminal tail disrupts IP accumulation, however, the tailless lamprey GnRH receptor was not only functional but was also capable of stimulating IP levels equal to wild type. Expression of the receptor transcript was demonstrated in the pituitary and testes using RT-PCR, whereas in situ hybridization showed expression and localization of the transcript in the proximal pars distalis of the pituitary. The phylogenetic placement and structural and functional features of this GnRH receptor suggest that it is representative of an ancestral GnRH receptor. In addition to having an important role in lamprey reproductive processes, the extensive C-terminal tail of this lamprey GnRH receptor may have great significance for understanding the evolutionary change of this vital structural feature within the GnRH receptor family.

In situ Characterization of Gonadotropin- Releasing Hormone-I, -III, and Glutamic Acid Decarboxylase Expression in the Brain of the Sea Lamprey, Petromyzon marinus

The distribution of lamprey gonadotropin-releasing hormone (GnRH)-I and -III has been extensively characterized by immunocytochemistry in the forebrain of the sea lamprey, Petromyzon marinus. However, the cellular location of lamprey GnRH-III mRNA expression by in situ hybridization in the lamprey brain has not been determined. We show for the first time the location of expression of lamprey GnRH-III, as well as provide a more comprehensive in situ study of lamprey GnRH-I and glutamic acid decarboxylase (GAD; GABA-synthesizing enzyme) mRNA expression in the brain of the lamprey in different reproductive life stages. Colorimetric and dual-label fluorescent amplification methods of in situ hybridization were used on brain tissue sections of adult, juvenile, and larval sea lamprey. In each life stage of the lamprey, expression of lamprey GnRH-I was shown in the preoptic area (POA) and the hypothalamus forming the characteristic arc-like cell population extending from the preoptic nucleus (NPO) to the neurohypophysis. Lamprey GnRH-III expression was also seen in the POA of each life stage in close proximity to lamprey GnRH-I mRNA containing neurons. GAD expression was shown in distinct cell clusters in and around the POA, in the olfactory bulb, in the dorsal thalamus beneath the habenular region, and also in the ventral-medial hypothalamus stretching from the periventricular region to the anterior portion of the rhombencephalon. Using dual-label in situ hybridization, we have shown that lamprey GnRH-I and -III mRNA are colocalized in the same cells in the POA in adult lampreys. Dual-label in situ hybridization also showed close proximity of GAD mRNA containing neurons and GnRH containing neurons in the POA. These data suggest that γ-aminobutyric acid (GABA) may directly affect GnRH release in the brain of the sea lamprey.

Cloning and analysis of the lamprey GnRH-III cDNA from eight species of lamprey representing the three families of Petromyzoniformes

The lamprey, which are divided into three families, including the Petromyzonidae, Geotriidae, and Mordaciidae, have been shown to regulate the reproductive axis through a functional hypothalamic–pituitary–gonadal axis. To date, two forms of gonadotropin-releasing hormone (GnRH) have been identified in the sea lamprey ( Petromyzon marinus), lamprey GnRH-I (decapeptide and cDNA) and lamprey GnRH-III (decapeptide), both of which have been shown to be expressed in the preoptic-anterior hypothalamic region and both forms have been demonstrated to regulate reproductive function (i.e. steroidogenesis and gametogenesis). The objective of this study was to isolate the cDNA encoding the prepro-lamprey GnRH-III from eight species of lamprey using a PCR based subcloning procedure. A degenerate primer designed to the lamprey GnRH-III decapeptide was used to amplify the 3′ end of each transcript, while gene specific primers were used to amplify the 5′ ends. Phylogenetic analysis using the prepro-lamprey GnRH-III amino acid sequences was performed, in which the lamprey GnRH-III sequences divided into three groups, supporting the current view of the lamprey lineage at the family level. Finally, a phylogenetic analysis of these newly identified deduced amino acid sequences together with 64 previously described GnRH sequences suggests that the lamprey GnRHs are unique, as they group together separately from the three previously described paralogous lineages of the GnRH family.

In vitro and in vivo effects of GABA, muscimol, and bicuculline on lamprey GnRH concentration in the brain of the sea lamprey ( Petromyzon marinus)

γ-Aminobutyric acid (GABA) is a neurotransmitter with a demonstrated neuroregulatory role in reproduction in most representative species of vertebrate classes via the hypothalamus. The role of GABA on the hypothalamus–pituitary axis in lampreys has not been fully elucidated. Recent immunocytochemical and in situ hybridization studies suggest that there may be a neuroregulatory role of GABA on the gonadotropin-releasing hormone (GnRH) system in lampreys. To assess possible GABA–GnRH interactions, the effects of GABA and its analogs on lamprey GnRH in vitro and in vivo were studied in adult female sea lampreys ( Petromyzon marinus). In vitro perfusion of GABA and its analogs at increasing concentrations (0.1–100 μM) was performed over a 3-h time course. There was a substantial increase of GnRH-I and GnRH-III following treatment of muscimol at 100 μM. In in vivo studies, GABA or muscimol injected at 200 μg/kg significantly increased lamprey GnRH concentration in the brain 0.5 h after treatment compared to controls in female sea lampreys. No significant change in lamprey GnRH-I or GnRH-III was observed following treatment with bicuculline. These data provide novel physiological data supporting the hypothesis that GABA may influence GnRH in the brain of sea lamprey.

Evidence for Lamprey GnRH-I and -III-like Molecules in the Brains of the Southern Hemisphere Lampreys Geotria australis and Mordacia mordax

The present study has characterized gonadotropic releasing hormone (GnRH)-like molecules in the brains of representatives of the two southern hemisphere families of lampreys, Geotriidae and Mordaciidae. Chromatographic and immunocytochemical evidence showed that the brains of Geotria australis and Mordacia mordax contain two forms of GnRH-like molecules. These two forms correspond to lamprey GnRH-I and -III, which were first sequenced from the brain of the anadromous sea lamprey Petromyzon marinus, a representative of the family Petromyzontidae that is found only in the northern hemisphere. In chromatographic studies (HPLC) using lamprey GnRH-I and -III antiserum, two early eluting GnRH forms coeluted with synthetic lamprey GnRH-I and -III standards. Our studies thus indicate that, despite their apparently long period of separation, the three families of extant lampreys have each retained both of the lamprey GnRH (-I and -III forms) molecules. Moreover, immunocytochemical localization of lamprey GnRH indicated that the pattern of its distribution in the adult brain of at least one of these southern hemisphere lampreys (G. australis) is similar to that previously described for P. marinus. Distribution of GnRH in the brain of larval G. australis was not as extensive as that in larval P. marinus, which may account for the later gonadal development in the former species. The fact that lamprey GnRH-I and -III are the dominant GnRH forms in all three families of lampreys implies that these neurohormones have an ancient origin.

The effects of lamprey GnRH-I, -III and analogs on steroidogenesis in the sea lamprey ( Petromyzon marinus)

The objective of this study was to examine the in vivo and in vitro effects of lamprey gonadotropin-releasing hormone (GnRH)-I, -III or analogs on steroidogenesis in the adult sea lamprey for the purpose of identifying putative potent agonists and antagonists. In the in vivo studies, the effect on steroid production was examined by injecting males with lamprey GnRH-I, -III and analogs at 8°C and 16°C. The following peptides and analogs were tested: lamprey GnRH-I and -III, [D-Glu 6] lamprey GnRH-I, cyclo [Glu 6-Trp 7-Lys 8] lamprey GnRH-I, cyclo [D-Glu 6-Trp 7-Lys 8] lamprey GnRH-I, [Gly 6] lamprey GnRH-I, [D-Phe 2,6, Pro 3] lamprey GnRH-I, [Phe 2] lamprey GnRH-I, [Trp 3] lamprey GnRH-I, and [Gly 6] lamprey GnRH-III. All peptides tested in vivo, except [Trp 3] lamprey GnRH-I, effectively stimulated plasma oestradiol after 4 h in lampreys held at 8°C or 16°C. In the in vitro studies, lamprey GnRH-I and -III significantly stimulated the pituitary to release a putative gonadotropin capable of stimulating the ovaries to release oestradiol when incubated at 18°C. [D-Glu 6] lamprey GnRH-I at all doses suppressed the putative pituitary response on the testis at 14°C, whereas cyclo [Glu 6-Trp 7-Lys 8] lamprey GnRH-I only suppressed the pituitary at a dose of 100 and 1000 ng/ml. It is suggested from these studies that the actions and differences between the in vivo and in vitro studies on lamprey GnRH-I and -III and analogs are dependent on temperature and/or stage of reproduction likely reflecting differences in metabolic turnover or degradation rates of GnRH, GTH, and/or their receptors. From these studies, proposed putative agonists/antagonists have been identified that may be used to enhance reproduction in lampreys. Agonists/antagonists will be tested further to determine their ability to inhibit spermatogenesis without destroying the mating competitiveness of males. This would be a valuable tool in a sterile-male release program in the Great Lakes.

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.

The Distribution of Lamprey GnRH-III in Brains of Adult Sea Lampreys (Petromyzon marinus)

In the sea lamprey, Petromyzon marinus, two forms of GnRH, lamprey GnRH-I and -III, have been demonstrated to be neurohormones regulating the pituitary–gonadal axis. The objective of the present study was to determine the distribution of lamprey GnRH-III in the brains of adult sea lampreys and to compare it to the distribution of lamprey GnRH-I. For this purpose, two kinds of immunostaining were employed: one was a single immunostaining by one of two GnRH antibodies using two successive sections; the other was double immunostaining of a single section. A dense accumulation of neuronal cells immunoreactive (ir) to antisera against either lamprey GnRH-I or -III was found in the arc-shaped preoptico-anterior hypothalamic area. Additional smaller numbers of irGnRH cells were found in the periventricular zone of the posterior hypothalamus. In the above-mentioned locations, the distribution of both irGnRH-I and -III cells was intermixed and very similar, but the cells exhibiting GnRH-III immunoreactivity were distinctly different from those exhibiting GnRH-I immunoreactivity. The relative numbers of irGnRH-III cells were larger than those of irGnRH-I cells in the preoptico-anterior hypothalamic area, and more than 90% of GnRH cells in the posterior hypothalamus were irGnRH-III cells. Both irGnRH-I and -III cells projected their fibers primarily into the neurohypophysis. The relative densities of the accumulated irGnRH-III fibers were similar to those of irGnRH-I fibers in the anterior neurohypophysis but higher than those of irGnRH-I fibers in the posterior neurohypophysis. The present study provides further immunocytochemical data to the already compelling physiological evidence that indicates that both lamprey GnRH-I and -III act through the hypothalamic–pituitary–gonadal axis to modulate reproductive processes in the sea lamprey.

Effects of Five Natural Gonadotropin-Releasing Hormones on Cell Suspensions of Marine Bivalve Gonad: Stimulation of Gonial DNA Synthesis

Gonadotropin-releasing hormones (GnRH) constitute a family of neuropeptides which are important regulators of reproduction in vertebrates. The effect of mammalian GnRH (mGnRH), salmon GnRH, chicken GnRH-I, chicken GnRH-II, and lamprey GnRH-I on [3H]thymidine incorporation into DNA of dissociated gonadal cells of marine bivalves has been studied. The incorporation of [3H]thymidine is linear between 1.5 and 8 h of incubation. All five GnRHs significantly increased DNA synthesis in gonial cells ofCrassostrea gigas.The maximal activation was about of 135–140% above control. The activation is dose dependent, over the range 10−11to 10−6M, but is modulated by the physiological condition of the cells and the stage of sexual maturity of the gonad. mGnRH has also a mitogenic effect in dissociated mantle cells ofMytilus edulis.The effect of mGnRH is blocked by a GnRH antagonist ([D-pGlu1,D-Phe2,D-Trp3,6]GnRH, 5× 10−6M) inC. gigasas well as inM. edulis,suggesting that the GnRH action in the gonad is mediated by specific receptors for GnRH or GnRH-like peptides. The existence of GnRH-immunoreactive neurons and fibers in the cerebral and pedal ganglia ofM. eduliswas demonstrated by immunocytochemistry. They are located principally in the anterior internal area of the cerebral ganglia, close to the cerebral commissure and in the posterior part of the pedal ganglia. The presence of GnRH-responsive cells and GnRH-like immunoreactive material suggests that peptides of the GnRH-like family are present and functional in bivalve molluscs.

Brain and Pituitary Hormones of Lampreys, Recent Findings and their Evolutionary Significance

SYNOPSIS. Lampreys of the Class Agnathan are of particular importance in understanding endocrinological relationships since they represent one of the oldest lineages of extant vertebrates, which evolved over 550 million years ago. In this review, agnathans are considered to be paraphyletic in origin, with the modern agnathans classified into two groups, myxinoids (hagfish) and petromyzonids (lamprey). Recent paleontological analysis of modern groups suggests that the jawed vertebrates are more closely related to lampreys than either is to the hagfishes. It has been proposed that there have been two periods of gene duplication during the course of vertebrate evolution, one of which occurred close to the origin of the vertebrates. This paper will summarize the most recent information on the structure and function of newly identified hormones and genes of the brain (lamprey GnRH-I and -III, somatostatin-14; neuropeptide Y-related peptide and a lamprey tachykinin) and pituitary (ACTH, MSH-A, MSH-B, NHF-nasohypophysial factor, POM, and POC) in lampreys. In addition, these data provide further information for the prediction of gene duplication during the early development of vertebrates and increases our understanding of the molecular evolution and functional diversity of these hormones.

Characteristics of GnRH Binding in the Gonads and Effects of Lamprey GnRH-I and -III on Reproduction in the Adult Sea Lamprey

In the present study, both lamprey GnRH-I and -III stimulated steroidogenesis and induced ovulation in adult female sea lampreys during their final reproductive stage. One injection of lamprey GnRH-III at 0.1 or 0.2 μg/g lamprey stimulated plasma estradiol levels in lampreys held at each of three water temperatures, 13°, 17°, and 19°, corresponding to increasing stages of maturation. Four successive injections, 3 to 4 days apart, of lamprey GnRH-III at 0.1 or 0.2 μg/g body weight induced ovulation in 100 or 88% of lampreys, respectively, compared to 21% in controls by Day 31. Lamprey GnRH-III also had a direct stimulatory effect on estradiol production in the sea lamprey gonadsin vitro.Lamprey GnRH-III at 100 or 1000 ng/ml stimulated estradiol levels in media incubated with either lamprey ovaries or testes. In contrast to a previous finding in which lamprey GnRH-III was more potent than lamprey GnRH-I in inducing spermiation in adult male sea lampreys (Deragon and Sower, 1994), the results from the present study indicate that lamprey GnRH-I and -III are equally potent in inducing ovulation and stimulating steroidogenesis in female sea lampreys. In addition, GnRH binding sites have been demonstrated for the first time in both the testis and the ovary of the adult sea lamprey using an analog of mammalian GnRH ([D-Lys6] mammalian GnRH) as a labeled ligand. Scatchard analysis suggested the presence of a high affinity binding site in both the testis and the ovary. In summary, lamprey GnRH-III is biologically active in stimulating the pituitary–gonadal axis in adult female sea lampreys. This is the first report demonstrating the presence of a GnRH binding site in the gonads of an Agnathan. The evidence for a direct stimulatory effect of lamprey GnRH in the gonads, the presence of GnRH binding site, and the absence of GnRH in the plasma suggest that, like other vertebrates including rat, rabbit, teleost fish, and human, there may be a GnRH-like factor produced in the gonads of the lamprey and it may act as a paracrine/autocrine modulator of gonadal function. This study further strengthens the paracrine regulatory role of GnRH peptides in the gonads of vertebrates, which appear to be evolutionarily conserved.